UNIT-IV : Complexation and protein binding by Abhijit Debnath

COMPLEXATION AND PROTEIN BINDING Seletted Definitions • •

Complex compounds: Those molecules in which most bonding structures can be described by classical valence theories between atoms, but one or more of these bonds are somewhat anomalous. Complexation: It is the association between two or more molecules to form a non bonded entity with a welldefined stoichiometry

•

Ligands: The ligand is a molecule that interacts with another molecule ( the substrate) by co-ordinate bonds and form a complex.

•

Metal Complex: It consists of a central metal atom or ion that is bonded to one or more ligands.

•

Coordination

•

Chelatation: It is the process of formation of two or more separate coordinate bonds between a polydentate ligand and a single central atom.

•

Inclusion complexes: These are the compounds in which one of the components is trapped in the open lattice or cage like crystal structure of the other. Clatharates: It is a cage like complex in which the coordinating compound is entrapped. Protein Binding: This is the phenomenon of complex formation of drugs with proteins

• •

Number: It is defined as the total number of ligands attached to a central metal ion/atom.

7. 1 INTRODUCTION Complex compounds are defined as those molecules in which most bonding structures can be described by classical valence theories between atoms, but one or more of these bonds are somewhat anomalous. Complexation is the association between two or more molecules to form a non bonded entity with a well-defined stoichiometry. In broad terms, complexation is used to characterize covalent or non-covalent interactions between two or more compounds that are capable of independent existence. The intermolecular forces involved in complex formation are the covalent bond, the vanderwall forces, the dipole-dipole interaction, and the hydrogen bond etc. Complexes are formed because of the donor acceptor mechanism. Donor is the neutral molecule or ion of non metallic substance that can donate the lone pair of electrons. Acceptor is the metallic ion or sometimes it might a neutral atom.

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Different steps involved are . I. The filled ligand orbital overlaps the empty metal ion orbital.

: 2.'Âˇ The ligand (Lewis base) donates the electron pair, ".3.. The metal ion accepts it 4. Form one of the covalent bonds of the complex ion. S. Such a bond, in which one atom in the bond contributes both electrons, is called a coordinate covalent bond.

7.2 CLASSIFICATION OF COMPLEXATION The complexes are classified as 1. Metal complexex or Coordination complexes a. Inorganic type b. Chelates c. Olefin type d. Aromatic type 2. Organic molecular complexes a. Quinhydrone type b. Caffeien complex c. Picric acid type d. Polymeric complex 3. Inclusion or occlusion Complexex a. Clathrate complex b. Channel lattice c. Layer type d. Monomolecular

7.2.1 Metal complexes A metal complex consists of a central metal atom or ion that is bonded to one or more ligands. Ligands are ions or molecules that contain one or more pairs of electrons that can be shared with the metal. Metal complexes can be neutral, positively charged, or negatively charged. Electrically charged metal complexes are sometimes called complex ions. A coordination compound contains one or more metal complexes. The total number of ligands attached to a central metal ion/atom is called the Coordinate Number of that particular ion. Example: In complex ion [Cu(NH))4t2, 4 ammonia ligand are attached to the central metal ion Cu. Hence the coordination number of Cu+2 ion in above complex ion is 4.

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Ligands: These are of following different types. (8) Unldentate:

Ammonia, which has single pair of electrons (basic group) for bonding with metal ion, is called unidentate ligand. Example: NH3,H20,Cr, Br",N02Âˇ etc. (b) Bidentate : Ethylenediamine has two.basic groups called Bidentate . Example: Oxalate anion, ethylenediamine etc.

-~ r C-C

Figure 7.1 : Oxalate anion

H2N~ NH2 Figure 7.2: Ethylenediamine

(c) Multidentate or Polydentate : Ligands with multiple binding sites. (d) Hexadentate - Ethylenediaminetetraacetic acid (EDTA) has a total of six points (4 oxygen and 2 Nitrogen donor group) for attachment of metal ions.

o ~

C-OH

H2C

:N-CH2

C-CH2

H2C-N:

HO-C

CH2-C

/ \

CH2

/ ~

Flgure7.3: EDTA (Ethylene Diamine Tetracetic Acid)

If the same metal ions binds with two or more sites. The complex form is called CHELATE.

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7.2.1.1 Inorganic type In inorganic metal complexes, the ligand provides only one site for binding with metal.

I I

Central atom

~igandS

(unidentate)

[CoCl(NH3)s] Cl2

Ionization sphere

Coordination sphere Figure 7.4 Inorganic complex

In a solution this compound ionise to form [CoCI(NH3)5]2+ and 2Cr. The chloride ion in the coordinate sphere can't precipitated by silver nitrate. Cl and NH3 in coordinate sphere are called ligands. Metal and ligands bonded to each other by electrostatic or covalent bond. Coordinate number is the total number of ligands attached to a central metal ion/atom in coordinate sphere. It is usually an even number. For Co, coordination number is 6.

The coordination number of Cobalt is 6. Ground-state electronic configuration for the trivalent cobalt ion, Co(III) is 3d

ClDCDCDCDCD

000

In complexation, electrons from half filled orbitals shift to other orbital and create vacant orbitals. Then ligands donate electron pairs to vacant orbitals of metal ion and form complexes. 3d

ClDClDClD 188 8 8881 d2 Sp3Octahedral and thus the d2sp3 or octahedral structure is predicted as the structure of this complex.

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7.2.1.2 Chelates: Chelatation is the process of formation of two or more separate coordinate bonds between a polydentate ligand and a single central atom

HOOC-H2C\

/CH2-COOH N-CH2-CH2-N

HOOC-H2C

CH2-COOH

Figure: 7.S EDTA

For example Ethylene diarnine tetraacetic acid (EDTA) has two donor nitrogen and acts as a bidentate (two-toothed) Ligand. When a drug forms a metal chelate, the solubility and absorption of both drug and metal ion may be affected, and drug chelation can lead to either increased or decreased absorption. Example citric acid,tartrates and EDTA

Figure 7.6: EDTA complex with metal ion

Application of chelating agents 1. EDTA is used to remove calcium ion from hard water 2. Oxidative degradation of drug preparations and ascorbic acid in fruit juice can be prevented by chelation with EDTA 3. EDTA is also used as In vitro anticoagulant. 4. EDTA is used to detoxify poisonous metal agents, such as mercury, arsenic, and lead 5. EDTA is used to remove colour impurities from antibiotic preparations 7.2.1.3 Olefin Complexes: Interaction of aqueous solutions of metal ions (such as iron, mercury, silver etc. ) with olefin (such as ethylene) form Olefin Complexes. These complexes are water soluble. These types of complexes are generally used as catalysts in the manufacture of bulk drugs and also in the analysis of drugs.

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7.2.1.4 Aromatic complexes: Interaction of metal ion with aromatic molecule (such as benzene, toluene and xylene) form aromatic complexes. If complex is formed by pie bond between metal ion and aromatic molecule, then it is called 1(- bond complex. If complex is formed by sigma bond between metal ion and aromatic molecule, then it is called sigma- bond complex.

7.2.2 Organic Molecular Complex: These type of complexes are formed by noncovalent interaction between ligand and substrate and held together by weaker forces or hydrogen bonding.

Classification of organic molecular complex 7.2.2.1 Quinhydrone type: They are formed when the alcohol solution of benzoquinone and alcoholic solution of the hydroquinone is mixed in equimolar concentration.

H0-o-~

Hydroqu~one (electron nch)

: Overlap (rt cloud)

=0= ,

Benzoquinone (electron deficient)

Figure 7.7 Quinhydrone

Complex is formed by overlapping of 1t cloud of electron deficient benzoquinone with electron rich hydroquinone. These are used as an electrode to determine pH.

cloud of

7.2.2.2 Drug and caffeine complexes: A number of acidic drugs are known to form complexes with caffeine. Drugs such as benzocaine, procaine and tetracaine form complexes with caffeine.

Figure 7.8: Caffiene

The mechanism behind this is (a) hydrogen bonding between polarized carbonyl group of caffiene and hydrogen atom of acidic drug. (b) Induced dipole-dipole force between carboxy oxygen of benzoocaine and electrophillic nitrogen of caffiene.

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Caffeine drug complexes a. may enhance or inhibit solubility. b. Also these complexes are used to mask bitter tasteof drug. c. improve stability of drug d. improve absorption and bioavailability of drugs. 7.2.2.3 Picric acid Complexes: picric acid being a strong acid, forms organic molecular complexes with weak:bases.

N02

NH2

Figure 7.9: Butesin picrate complex

The complex is indication of magnitude of carcinogenic activity. 7.2.2.4 Polymer complexes: Polymeric material such as polyethylene glycols (PEGs), carbowaxes, pluronics carboxymethyl cellulose (CMC) contain nucleophilic oxygen and form complexes with various drugs. These type of interaction produces Incompatibilities in suspensions, emulsions, suppositories and ointments. Also it may lead to precipitation, flocculation, delayed biologic absorption, loss of preservative action and other undesirable physical, chemical and pharmacological effects. Example: 1. Dissolution rate of ajmaline is enhanced by complexation with PVP due to the aromatic ring of ajmaline and the amide groups of PVP to yield a dipole dipole induced complex 2. Polyolefin container interact with drugs which can result in loss of the active component in liquid dosage forms

7.2.3 Inclusion complexes These complexes are also called occlusion compounds in which one of the components is trapped in the open lattice or cage like crystal structure of the other. 7.2.3.1 Channel type: The channels are formed by crystallization of the host molecules, the guest : component is usually limited to long, unbranched linear chain compounds. Choleic acids form

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these types of complexes. Deoxycholeic acid molecules can form channels in which molecules will get inside.

Host molecule (urea)

o Hexagonal channel

~O/CH3 S-S Guest molecule (Methyl a-lipoate)

Figure 7.10 channel complex of methyl alpha IIpoate with urea

7 _2.3.2 Layer type: The guest molecule is entrapped in the layers. Compounds such as clay montmorillonite, the main constituent of bentonite, can trap hydrocarbons, alcohols and glycols between the layers of their lattices. As a a result alternate monomolecular layers of guest and host are formed. However, these may be useful for catalysis due to a larger surface area. 7.2.3.3 Clatharates: It's a cage like complex in which the coordinating compound is entrapped. It is available as white crystalline powder. For example: Cage like structure formed through hydrogen bonding of hydroquinone molecules. Small molecules in cage get entrapped and form clatharates. Hydrogen bonding (hydrogen atoms not shown)

Figure 7.11: cage like complex form through hydrogen bonding of hydroquinone

Complexation and Protein Binding 7.2.3.4 Monomolecular

complexes

133 and macromolecular

complexes

They are the inclusion compounds. Monomolecular inclusion compounds involve the entrapment of a single guest molecule in the cavity of one host molecule. Most of the host molecules are cyclodextrins. The entrance of the cavity is hydrophilic in nature and the interior of the cavity is relatively hydrophobic. /")E--

Hydrophilic exterior Hydrophobic interior Cavity for encapsulating hydrophobic drugs Entrapped aspirin molecule

Figure 7.12 monomolecular

complex of cyclodextrin

7.3 APPLICATION OF COMPLEXATION IN PHARMACY 1. Complex formation has been used to alter the physicochemical & biopharmaceutical properties of drug. 2. In various types of poisonings: chelating agents are used as antidote in hevy metal poisoning. For example Dimercaprol in case of mercury and arsenic poisoning. And CaN a2EDT A is used in case of lead poisoning. 3. In drug absorption & bioavailability from various dosage form: Heparin,an ionized drug of rather high molecular weight is not absorbed from G.I.T. However, in the presence of EDTA or surfactant such as SLS or dioctyl sodium sulfosuccinate, intestinal absorption of heparin is increased. 4. Complexation is used in solubilisation: Adrenochrome monosemicarbazone. Adrenochrome (active) as such unstable in solution & semicarbazone has only limited solubility at the pH at which it stable. However, the stable product can be prepared by the addition of sodium salicylate which complexes with adrenochrome & increases appearent solubility by 10 folds. Another example is The complexation of caffeine by sodium benzoate increases solubility of caffeine 5. Stability of product: Example Hydrolysis of Local anesthetic esters can be decreased by complexation with caffeine. Another example is the half life for procaine in the solution get increase from 26 Hrs to about 46Hrs in the presence of 2 % caffiene & to about 71 Hrs in the presence of 5% caffiene. 6. As therapeutic agents such as complex of Iron with simple salt like, Ferrous sulphate & carbonate are used to reduce the GIT irritation, Increase the absorption, after oral administration. And less irritation at the site of injection. 7. In diagnosis: Complex of Technetium 90 (a radionuclide) with citrate. This complex is used in diagnosis of kidney function & GFR.

134 8.

As therapeutic

tool: Both EDTA & CITRATES

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are used in Preservation

of blood. They

are used as anticoagulant. 9.

Assay of drug:

Complexometric

titration are used to assay of drug containing

such as magnesium trisilicate. 10. Development of novel drug delivery system: Complexation

metal ion

. of drug with polymers used

in formulation of sustained drug delivery devices. 11. Partition coefficient: The study of complexes is essential to assess drug action because complexes

influence

partition

coefficient.

Example

By complexation

pennagnate

ion

transferred to benzene phase from water.

7.4 METHODS OF ANALYSIS Complexes

are determined

acceptor. A quantitative

by the stoichiometric

expression

ratio of ligand to the metal or donor to the

of the stability constant for complex formation are important

in the study of complexes and equation is written as

[DC] [D] [C] (eq 7.1)

Where [DC] is concentration

of drug-complex

[D] is solubility of uncomplexed [C] is concentration

drug

of uncomplexed

complexing

agent.

Several methods of estimation of complexes have been developed as follows: 1. Method of continuous variation 2. pH Titration method 3. Distribution

Method

4. Solubility Method 5. Spectroscopy 6. Miscellaneous

and Charge Transfer complexation method

7.4.1 Method of continuous variation This is also known stoichiometric

as JOB's

Method

of continuous

variation.

This process

determines

the

ratio of complex based on estimation of certain additive properties of the complex

exation and Protein Bindin

like dielectric constant,

135

absorbance,

this process when two components

spectrophotometric

extinction

coefficient

etc. According

of a complex are mixed and if no interaction

occurs between

them, then the value of the property is additive. The value is given as the mean of the values of the individual species in the mixture.

Indication of 1: 1 complex

......

.......t·..... . .. .•. • ..

Curve for no complex

0.25 0.5 Mole fraction

0.75

1.0

Figure 7.13: Plot of dielectric constant vs mole fraction

If additive

property

relationship

is observed in case if no complexation

such as dielectric

constant

is plotted

vs mole fraction,

then a linear

occurs between the two components

(dotted

line as shown in figure) If the solution of two species A and B (of equal molar concentration) are mixed. If complex is formed between them, then the value of the additive property will go from the maximum. For a constant total concentraion of A & B the complex is at highest concentraion at point where the species A & B are combined- in the ratio in which they occur in the complex. The line shows a change in slope occurs at the molar fraction. The slope change occurs in a mole fraction indicated a type of complex.

7.4.2 pH Titration method This method is used when the complexation is achieve by change in pH. This method is considered as most reliable method for studying complexation. Eg. chelation of cupric ion by glycine. (eq 7.2) As the two proton are formed in the reaction of equation , the addition of glycine to a solution containing cupric ion should result in a decrease in pH. Titration curve is obtained by adding a strong base (sodium hydroxide) to solution of glycine & to another solution containing glycine & cupric ion. When we plot the graph of pH vs no. of ml strong base added. The curve for glycinemetal (copper) complex is well below that for the glycine alone. The decreased in pH show that complexation occur is throughout most of the neutralization range.

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14 Glycine

8 pH

Âť>:

Glycine and copper complex

MLofNaOH Figure 7.14: Titration curves of glycine and copper-glycine complex

The distance between two curves gives amount of alkali consumed in reaction. This concentration of sodium hydroxide i.e equal to concentration

of ligand bound at that pH. The average number of

ligand group bound per metal ion present is given by

total concentration of ligand bound

(eq 7.3)

total concentration of metal ion

The concentration concentration

of free glycine [G] at any pH is considered as the difference between total

of glycine [G]o and concentration

of added sodium hydroxide

(eq 7.4)

or P[A] = pKa - pH -log

([G]o - [NaOH]

o~----~----~----+---~ 4

6 P[A]

Figure 7.1S: plot of n Vs p[A]

(eq 7.5)

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When we plot n Vs p[A], formation curve is obtained. It is seen that value of n is reach up to certain value which indicate that maximum no of glycine mole that can combine with Cu ion and stability constant can be obtained by using values of nand p[A]. The overall stability constant ~ can be calculated by following equation p[A] = Y2 log ~

(eq 7.6)

or log ~ = 2

* p[A]

(eq 7.7)

p[A) can be calculated from titration curve when n= 1.

7.4.3 Distribution method: This method describe the distribution of a solute between to immiscible solvents used to determines the stability constant for certain complexes. For example: Complexation of iodine with potassium iodide. (eq 7.8) Higuchi investigate the complexing action of caffeine, glycols on number of acidic drugs using this method.

1------1l---i Figure 7.16: The distribution

of iodine between water and carbon disulfide

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Solved problem Exercise 7.1 : When iodine is distributed between water and carbon

the distribution constant K(o/w) = ColCw is found to be 625. When it is distributed between a 0.1250 M solution of potassium iodide and carbon disulfide, the concentration of iodine in the organic solvent is found to be 0.1699 molelliter. When the aqueous KI solution is analyzed, the concentration of iodine is found to be 0.02535 mole/liter.

Solution:

h in the aqueous layer (free + complexed iodine) = 0.02535molelliter Total concentration of KI in the aqueous layer (free + complexed KI): 0.1250 molelliter Total concentration

of I, in the CS2layer

Concentration Distribution

coefficient,

(free): 0.1699 molelliter

0 I w) = [[12]]0 = 625 12 w

The concentration

of free iodine in the aqueous phase is obtained as follows: 0.1699 625

To obtain the concentration

2.7

10-4 mole I liter

of iodine in the complex

[I2]complex = [12]w(total)-

Ans.

[I2]W(free)

= 0.02535

= 0.025

- 0.000271

molelliter

0.025 molelliter

From equation concentrations

+ K +I - ~

K + I:;

it is clear that 12 and KI combine in equimolar

to form the complex. Therefore, [KI]complex= [12]complex = 0.025 molelliter

KI is insoluble in CS2 and remains entirely in the aqueous phase. The concentration thus [KI] And equilibrium

free

= [KI]

= 0.1

stability constant of complex can be calculated by K

= [complex] * [12]rree

Ans.922

total- [KI]complex = 0.1250 - 0.025

[KI] free

0.025 0.000271

0.1

922

molelliter

oi free KI is

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7.4.4 Solubility method: Higuchi and Lach used this method for detection of complex. In this method the complex formation is based on the solubility of the components in presence of a complexing agent. The excess amount of drug along with the solution of complexing agent is placed in a container with closure system. A series of solution of different concentration of complexing agent is prepared. The bottles are agitated at constant temperature until the equilibrium is achieved. Then a liquid portion is removed & analyzed for complex formation. For example: Complexation of paminobenzoic acid (PABA) by caffeine. In this p-arnino benzoic acid(PABA) is drug & caffeine is complexing agent. above experiments is plotted as molar conc.of PABA Vs. molar conc. of caffeine.

The results of

-><

~ •.•...

Saturation point (B)

--...,.+-- All excess solid acid converted to complex (C)

c: 0

.~ Q C1)

u Q

•...

'" 0 2;

Figure 7.17: solubility of p-amino benzoic acid in presence of caffeine

The point A at which the line crosses the vertical axis show the solubility of drug in water. On addition of caffeine, there is increase in solubility of PABA linearly owing to complexion. At point B, the solution become saturated with respect to the complex and to the drug itself. The complex continues to form and to precipitate from the saturated system as more caffeine is added. At point C, all the excess solid P ABA goes into solution and converted to the complex. Although all excess solid drug has been used up for complexation. And the solution is no longer saturated, some of the PABA remains uncomplexed in solution. Further it combines with caffeine to form higher or secondary complexes The complex formation is therefore written as PABA + caffeine and the stability constant for this complex is

K =

= PABA-

[PABA- caffeine] [PABA]

[caffeine]

caffeine

(eq 7.9)

(eq 7.10)

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7.4.5 Spectroscopy and Charge Transfer complexation Absorption spectroscopy both in the visible and U. V region of the spectrum is most commonly used for the analysis of the charge transfer complexes. When iodine is analyzed in a different noncomplexing solvent such as CCI4, a curve with single peak at 520nm is obtained. A iodine solution in benzene exhibits a maximum shift to 475nm & peak appears at 300nm. A solution of iodine in diethyl ether shows a still greater shift to lower wavelength & the new solution appeared. In benzene & ether iodine is electrons accepter and organic solvent is donor in CClt, no complex is formed. The shift towards the U.V. region becomes greater as the electron donor solvents becomes a strong electron releasing agents. The more easily a donor release its electrons as measured by its ionization potential. The complexation constant K can be obtained between the donor D & accepter A is given as

D+A

by use of UV spectroscopy.

k d=DA k.,

The association

(eq7.11)

Where K =~

k_,

is the equilibrium constant for complexation

(stability constant) and

k, and k., are the interaction rate constants

K is readily obtained from the Benesi-Hildebrand equation which is written as Ao 1 1 1 -=-+-A E x, Do

(eq 7.12)

Where Ao and Do are initial concentrations

of the acceptor and donor species, respectively( in molelliter), is the molar absorptivity of the charge transfer complex at its particular wavelength, and K, the stability constant (in liter/mole). â&#x201A;Ź

7.4.6 Miscellaneous methods Several other methods are available spectroscopy, polarography, circular diffraction.

for the analysis of complexes like NMR and I.R dicrornism, kinetics, X-ray diffraction and electron

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7.5 COMPLEXATION AND DRUG ACTION Complexes can alter the pharmacologic activity of the agent by inhibiting interactions with receptors. The action of the drugs to remove toxic metal ions from human bodies is through complexation reaction. In some instances, complexation also can lead to poor solubility or decreased absorption of drugs in the body: Aqueous solubility of tetracycline decreases substantially when it complexes with calcium ions and coadministration of some drugs with antacids decreases absorption from the gastrointestinal tract. Drug complexation with hydrophilic compounds also can enhance excretion. For example: 1. Cisplatin and carboplatin are platinum ITcomplexes that have prove to be the most useful agents in treatment of cancer. This is a coordination compound 2. Povidone Iodine complex: Here polyvinylpyrollidone (PVP) is water soluble polymer and form water soluble complex with iodine. As complex is soluble in water, drug can be removed easily from application site. It has antibacterial action. 3. Complexation of a drug in the GIT fluids may alter rate and extent of drug absorption. 4. Intestinal mucosa + Streptomycin = poorly absorbed complex 5. Calcium + Tetracycline = poorly absorbed complex (Food-drug interaction) 6. Carboxyl methylcellulose (CMC) + Amphetamine = poorly absorbed complex 7. Polar drugs + complexing agent = well-absorbed lipid soluble complex

7.6 PROTEIN BINDING The phenomenon of complex formation of drugs with proteins is called protein binding. A protein bound drug is neither metabolized nor excreted hence it is pharmacologically inactive. It remains confined to a particular tissue for which it has greater affinity. Binding of drugs to proteins is generally of reversible & irreversible. Reversible binding generally involves weak chemical bond such as: Hydrogen bonds, Hydrophobic bonds, Ionic bonds and Van der waal's forces. While Irreversible drug binding, though rare, arises as a result of covalent binding and is often a reason for the carcinogenicity or tissue toxicity of the drug. The order of binding of drugs: albumin> ul-acid glycoprotein> lipoproteins> globulins. Table 7.1: Binding of drug to protein DRUGS THAT BIND

PROTEINS Human serum albumin

All types

a I-acid gl ycoprotein

Basic drugs, imipramine, lidocaine

Lipoproteins

Basic, lipophilic drugs.chlorpromazine

al- globulin

Steroids

a,. globulin

Vitamins A, D, E, & K

Haemoglobin

Phenytoin, pentobarbital, phenothiazines

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7.6.1 Binding of drug to Human serum albumin: The Molecular weight of albumin is 65,000 - 69,000. It is synthesized in the liver. It is major component of plasma proteins. Albumin is distributed in the plasma and in the extracellular fluids of skin , muscle ,and various tissues. In Intestinal fluid, albumin concentration is about 60% of that in the plasma. Elimination half life of albumin is 17-18 days . Albumin concentration is 3.5-5.5% (w/v) or 4.5 mg/dl. Albumin is responsible for maintaining osmotic pressure of the blood and for the transport of endogenous and exogenous substances. Many weak acidic drugs bind to albumin by electrostatic and hydrophobic bonds. Salicylates, phenylbutazone and penicillin are highly bound to albumin.

I. Warfarine & Azapropazone

11.Diazepam Site

~ IV. Tamoxifen Site

Ill. Digoxine Site

i.> ~

SITE I: To this specific site a large population of drugs bind like Non-Steroidal AntiInflammatory Drugs mainly phenylbutazone, indomethacin, many sulfonarnides e.g.; sulfamethoxine, sulfarnethizole, and even many anti-epileptic drugs like phenytoin etc. this site is also called as Warfarin binding site or as Azapropazone binding site. SITE 0: This is actually said to be Diazepam binding site. Benzodiazepines, medium chain fatty acids, ibuprofen, ketoprofen, etc. bind extensively at this site. This is due to structural changes the following drugs have high and specific affinity for this site. SITE 01: This site is called as Digitoxin binding site SITE IV: This is referred as Tarnoxifen binding site.

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7.6.2 Binding of drug to ai-acid glycoprotein They are also called as orosomucoid. The molecular weight of ai-acid glycoprotein is 44,000. They are bound by Hydrophobic bonds E.g. : Basic Drugs such as Imipramine, Amytriptyline , Lidocaine, nortriptyline, Propranolol, Quinidine and disopyramide cound to ai-acid glycoprotein 7.6.3 Binding of drug to Lipoproteins They are bound by hydrophobic bond. The molecular weight of lipoprotein is 2-3 lakhs to 34 lakhs. Bound drug dissolve in lipid core. Example acidic drug (diclofenac), Neutral (cyclosporin) and Basic drug (chlorpromazine) Table 7.2 :Binding of drug to Globulin Globulin

DRUGS THAT BIND

al- globulin

Steroids

a2- globulin

Vitamins A, D, E, & K

~l globulin

Ferrous ions

~2 globulin

Carotenoids

o globulin

Antigens

7.6.4 Binding Of Drugs To Blood CeUs Red Blood Cells (RBC's) are the major blood cells which rates about 40% of total blood. The red blood corpuscles constitute 95% of the total blood cells concentration in the body. Major portion of red blood cells to which drugs can bind are: i)

ii) Hi)

Hemoglobin: The weight & structural is similar to that of HSA but the concentration is much higher than of albumins in blood. Examples of drugs that bind are phenytoin, pentobarbital etc. Carbonic Anhydrase Inhibitors: Carbonic anhydrase inhibitors mainly bind to the site like chlorthaizine. Red Blood cell membrane: basic drugs like imipramine are known to bind to RBC membrane.

144

7.7 SIGNIFICANCE a. b. c. d.

e. f.

Physical Pharmaceutics-I

OF PROTEIN BINDING

Absorption: Protein binding disturb Absorption equilibrium. Distribution: Protein binding decrease distribution of drug because protein bound drug does not cross Blood Brain Barrier, placental barrier etc. binding decreases the metabolism of drugs & enhances the biological half life Elimination: Protein binding prevent the entry of drug to the metabolizing organ (liver) & to glomerulus filtration. Only the unbound drug is capable of being eliminated. Example: Tetracycline is eliminated mainly by glomerular filtration Drug action: Protein binding inactivates the drugs because sufficient concentration of drug can not be build up in the receptor site for action In diagnosis: The chlorine atom of chloroquine replaced with radiolabeled 1- 131. It can be used to visualize-melanomas of eye & disorders of thyroid gland.

Metabolism: Protein

7.8 FACTORS AFFECTING PROTEIN BINDING 7.8.1 Factors relating to the drug It include a. Physicochemical properties of drug: The binding affinity depends on physical and chemical properties of drug. Increase in lipophilicity increases the extent of binding . b. Concentration of drug: Alteration in the concentration of drug substance as well as the protein molecules cause alteration in the protein binding. At low concentrations, most drugs may be bound to proteins. At high concentrations, more free drugs may be present owing to saturation of binding sites on protein c. Affinity of drug for binding component: This depend on affinity of protein molecule to bind to drug . For example Digoxin has more affinity for proteins of cardiac muscles than those of skeletal muscles

7.8.2 Factors relating to protein: a. Physicochemical properties of protein: lipoproteins bind with lipophilic drugs b. Concentration of protein: Disease states affect the concentration of proteins in blood c. Number of binding sites: Albumin has a large number of binding sites as compared to other proteins. Indomethacin binds to 3 sites on albumin

7.8.3 Drug interactions a. Displacement reactions: The competition

between Drugs for the Binding Sites. e.g. warfarin and phenyl butazone. Interactions will result when: The displaced drug (E.g. warfarin) is more than 95% bound, has a small volume of distribution, shows a rapid onset of therapeutic or adverse effects and has a narrow therapeutic index. While the displacer drug (E.g. phenyl butazone) has a high degree of affinity as the drug to be

Compiexation and Protein Binding

145

displaced competes for the same binding sites, the drug/protein concentration ratio i high, and it shows a rapid and large increase in plasma drug concentration. b. Competition between drugs and normal body constituents: It cause interaction with free fatty acids. Free fatty acid levels are increased during fasting, diabetes etc. Eg.: Interaction of sodium salicylate with bilirubin in neonates. c. AUosteric changes in protein molecules: It involves alteration of the protein structure by the drug or metabolite modify binding capacity. Example: Aspirin acetylation of albumin; modify the binding capacity of NSAIDs (increased affinity)

7.8.4 Patient Related Factors a. Age: Neonates have very low level of albumin and change in albumin content affects the drugs binding. b. Disease States: Disease state alter portein drug binging affinity. For example Hypoalbuminemia severely impair protein-drug binding

7.9 KINETICS OF PROTEIN - DRUG BINDIN~ If "P" represents protein and "0" the drug then applying law of mass action to reversible proteinbinding, the equation will be as follows Protein [P] + Drug [DJ ~

Drug-Protein-Complex

[PD]

(eq 7.13)

The association constant K can be expressed as [PD] Ka=-[P] [D]

(eq 7.14) Or

[PD]

= Ka [ P]

[D]

Where, [P] = Cone, of free protein [0] = Cone. of free drug [PO] = Cone. of protein-drug-complex Ka = Association rate constant The association constant is measure of affinity between drug and protein. If total protein concentration is suppose to be [PT]. Therefore

(eq 7.15)

N Physical

146

[PT]

[P] = [PT] ~ [PD]

Or [PT] is sum of unbound protein BY substituting

= [P] + [PD](eq

Pharmaceutics-I

7.16) (eq 7.17)

and protein present in complex.

[P] in equation 7.15

[PD] [PD]

= Ka

[D] ([PT] - [PDD

[D] [PT] - Ka [D] [PD]

(eq 7.19)

= [PD] + Ka [D] [PD] [D] [PT] = [PD] ( 1+ Ka [DD

(eq 7.20)

Ka [D] [PT] Ka

(eq 7.18)

(eq 7.21)

Ka [D] [PT] [PD] = ---1+ Ka [D] (eq 7.22) Dividing both side by [PT], we obtain [PD]

. Ka [DJ.

--=

[PT]

1+ Ka [D] (eq 7.23)

To study the behaviour of drugs, a determinable

ratio r is defined as follows:

moles of drug bound r=

total moles of protein (eq 7.24) r=

[PD] [PT]

[PD] [PT] and from above equation

(eq 7.25)

Ka[D] 1+ Ka [D] (eq 7.26)

Compiexation and Protein Binding

147

Ka [D] r=

1 Ka [D]

(eq 7.27)

This equation describes the simplest situation, in which 1 mole of drug bind to 1 mole of protein in 1: 1 complex. If there are 'N' identical independent binding sites per protein molecule, then Ka [D] r=

1 Ka [D]

(eq 7.28)

If there are more than one type of binding site and the drug binds independently on each binding site with its own association constant, then equation (eq. 7.28) expands to the following:

r=--+--+l+Kl[D)

l+~[D1

(eq 7.29) The value of association constant, Ka and the number of binding sites N can be obtained by plotting the above equation in four different ways 1. Direct plot 2. Scatchard plot 3. Klotz plot 4. Hitchcock plot

7.9.1 Direct plot A direct plot of "r" Vs [D] can be used to find out the number of binding sites on protein 'N' (plateau value). Association constant (Ka) is obtained by finding drug concentration required to saturate the half of the total binding sites available (i.e NI2).

i --------~---+~

PLATEAU

[D]

Figure 7.18: Direct plot

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Physical Pharmaceutics-/

7.9.2 Scatchard plot: r =

N Ka [D]

(eq 7.30)

1+ Ka[D]

By rearrange the equation into linear form r + r Ka [D] = N Ka [D]

(eq 7.31)

r = N Ka [D] - r Ka [D]

(eq 7.32)

_r_=NKa

- rKa

[D]

A plot of [~]

vs r yields a straight

(eq 7.33)

line with X & Y intercepts equal to 'N' & ' NKa ' & the

slope is equal to Ka.

r/[O]

N --

•• r

Figure 7.19: Scatchard plot

7.9.3 Klotz plot Also known as Double reciprocal plot ( line weaver -burk plot ). By Reciprocating the equation 1 1 1 - = +r Nka[D] N A plot of ~ vs ~ r

. 1 mtercept -. N

(eq 7.34)

yields a double reciprocal plot. It is straight line with slope _1_ and YNka

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149

lIr

i IIN --.~

lID

Figure 7.20: Klotz plot

7.9.4 Hitchcock plot It is obtained by rearranging the equation as Nka[D] --"----"-=1+ r

(eq 7.35)

Ka[D]

Dividing both sides by N ka gives

[D]

-=--+r NKa A plot of

[D] yields a straight line with slope ~ and intercept _1_ rVs [D] N Nka

[D]/r

i IINKa --.~

[D]

Figure 7.21: Hitchcock plot

(eq 7.36)

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Physical Pharmaceutics-I

7.10 METHODS FOR MEASURING THE UNBOUND DRUG CONCENTRATION There are various methods used for determining the unbound drug concentrations in plasma, serum, or diluted tissue homogenate Equilibrium dialysis and ultrafiltration are the two most commonly used methods. Equilibrium dialysis is considered to be the standard method for protein binding measurements. Ultrafiltration can be adopted as the initial method for conducting protein binding studies, because it is less time-consuming and involves simpler sample preparation.

7.10.1 Equilibrium Dialysis In this method, serum albumin is kept in Visking Cellulose tube or bag. The tube is closed tightly and placed in vessel containing drug in different concentrations. The equilibrium dialysis chambers are separated by a semipermeable membrane, which allows only low-molecular-weight ligands, such as drug molecules, to transport between the two chambers. When dialysis continues, drug molecule enter into bag through membrane permeation. But albumin cannot pass through membrane to outer vessel. With timing, more amount of drug get permeated and concentrated in bag. If binding occur, drug concentration in bag containing protein will be higher than its concentration in outer vessel. Temperature sensor Sample withdrawl

tube

\ -+--t-t--

Visking cellulose bag Drug solution

Jacketed

beaker

Magnetic stirrer

Figure 7.22: Apparatus for equilibrium dialysis method

The ratio of unbound and total drug concentrations in plasma can be estimated after equilibrium dialysis.

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151

7.10.2 Dynamic Dialysis: The Apparatus consist of 400 ml jacketed beaker in which 200 ml buffer solution is kept. A dialysis bag having solution of drug and protein is suspended in buffer solution. Solution is stirred. Periodically sample is removed from dialysis bag and analyzed. This method is based on rate of disappearance of drug from dialysis bag is proportional to concentration of unbound drug. d[Dt) =k[Dd dt

(eq 7.37) where [Dll is concentration of total drug [Dd is concentration of free drug in bag k is first order rate constant or apparent permeability rate constant. The concentration of unbound drug [Dd can be calculated by above equation if d[Dll / dt and k are known. Rate constant k obtained from slope of serniloarithmic plot of [Dtl vs time, when experiment is conducted in absence of protein Temperature sensor Sample withdrawl tube

\ 1--+--+-+--

Dialysis bag 200ml, pH buffer

Jacketed beaker

Magnetic stirrer

Thermostat magnetic stirrer

Figure 7.23: Apparatus for Dynamic Dialysis

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Physical Pharmaceutics-I

7.10.3 Ultrafiltration Ultrafiltration is based on physical separation of free drug molecules from drug bound to plasma proteins by centrifugation. Concentration of a drug in an ultrafiltrate is an unbound drug concentration at the particular plasma drug concentration examined.

Advantages of ultrafiltration method over equilibrium dialysis. For ultrafiltration, Small fraction of sample for drug assay is required. Ultrafiltration takes about 30 min, which is significantly faster than equilibrium dialysis. Clean up procedure of the ultrafiltration device is easy.

7.11 THERMODYNAMIC

TREATMENT OF STABILITY CONSTANTS

The stability constants of the metal complexes are related to thermodynamic properties such as free energy charge (~G), enthalpy (~H) and entropy change (~S). These values can be computed by usual equations: ~G

= -2.303 RT log K

(eq 7.38)

The standard enthalpy change (~H) obtained from slope of plot of log K vs 1fT Log K = - ~H !2.303RT + constant

(eq 7.39)

When value of K at two temperatures are known. Then K2 and KJ are the stability constants at the absolute temperatures T2 and T, respectively. Log K2! Kl

= - 6.H / 2.303R (TrT

T, T2)

(eq 7.40)

Then standard entropy change is (eq 7.41)

6.H and ~S become negative, If stability constant of complexes increase. As binding between

donor and acceptor is stronger then ~

also become negative. When specificity of interacting

site become negative, then ~S also become negative.

Compiexation and Protein Binding

153

REVIEW QUESTIONS SUBJECTIVE PART VERY SHORT ANSWER QUESTIONS 1.

Defme Complexation Answer- It is the association between two or more molecules to form a non bonded entity with a well-defined stoichiometry

Define Ligands Answer- The ligand is a molecule that interacts with another molecule ( the substrate) by co-ordinate bonds and form a complex.

What do you mean by Chelatation? Answer- It is the process of formation of two or more separate coordinate bonds between a polydentate ligand and a single central atom.

What are Inclusion complexes? Answer- These are the compounds in which one of the components is trapped in the open lattice or cage like crystal structure of the other.

What is Protein Binding? Answer- This is the phenomenon of complex formation of drugs with proteins

SHORT ANSWER QUESTIONS 1.

How complexes are formed? Answer- Complexes are formed because of the donar acceptor mechanism. Donor is the neutral molecule or ion of non metallic substance that can donate the lone pair of electrons. Acceptor is the metallic ion or sometimes it might a neutral atom.

How protein binding affect distribution

of drugs?

Answer- Protein binding decrease distribution of drug because protein bound drug does not cross Blood Brain Barrier. placental barrier etc. 3.

Write AdYHntages of ultraDltratJon metbod oyer elJuDibrium dialysis method. Answer. During Ultrafiltration. Small fraction of sample for drug assay is required. Ultrafiltration takes about 30 min. which is significantly faster than equilibrium dialysis. Clean up procedure of the ultrafiltration device is easy.

Dissolution rate of ajmaline is enhanced by complexation with PVP. Why? Answer- This is due to the aromatic ring of ajmaline and the amide groups of PVP to yield a dipole dipole induced complex

How EDT A improve stability of ascorbic acid in Pharmaceutical

preparations?

Answer. When EDT A is added to drug preparations containing ascorbic acid. it celates trace metals. As a result oxidative degradation can be prevented. So stability of ascorbic acid is improved.

154

PhysicaJ Pharmaceutics-J

LONG ANSWER QUESTIONS 1.

Define complexation. What are types of complexes? Write detail about inclusion complexes. Refer article 7.2 Discuss Several methods used for estimation of complexes Refer article 7.4

Define protein binding. Explain kinetics of protein binding. Refer article 7.9

Write note on a. What is Equilibrium Dialysis method? Refer article 7.10.1 b. What is Klotz plot? Explain with labelled diagram Refer article 7.9.3

What are the significance of protein binding? Explain Methods for measuring the unbound drug concentration. Refer article 7.7, 7.10

Write note on a. Thermodynamic Treatment Of Stability Constants. Refer article 7.11 b. Application of complexation in pharmacy Refer article 7.3

Discuss the factors affecting protein drug binding. Refer article 7.8

OBJECTIVE PART MULTIPLE CHOICE QUESTIONS 1.

Ethylenediaminetetraacetic

a. Unidentate b. Bidentate c. Tetradentate d. Hexadentate Which of the following is unidentate ligand

a. Ammonia b. Oxalate ion c. EDTA d. Ethylene diarnine Which of the following organic solvent is used to form complex or IodIne a. b. c. d.

Toluene Aniline Hexane Cyclohexane

acid (EDTA) is

type or ligand

Complexation and Protein Binding

155

Which of the foUowing is not classification of Organic molecular complexes a. Quinhydrone type b. Caffeien complex c. Acetic acid type d. Polymeric complex

Ligands with multiple binding sites are caUed a. Unidentate b. Bidentate c. Polydentate d. Hexadentate

The value of association constant, Ka and the number of binding sites N can be obtained by a. Direct plot b. Scatchard plot c. Klotz plot d. All of the above

Which of the foUowing is application of complexation in pharmacy a. Complex formation is used in various types of poisoning b. Complexation is used in solubilisation c. Complexation is used for Stability of product d. AD of the above

Which of the foUowing drug bind to a. Steroids b. Ferrous ion c. Carotenoid d. Vitamin D

Which of the foUowing methods is! are used to measure unbound drug concentration a. dynamic dialysis b. Equilibrium dialysis c. Ultrafiltration d. All of the above

u,- globulin

10. EDTA i a. Ethylene diamine tetra acetic acid b. Ethylene diamine tri acetic acid c. Ethyl dicarboxylique tri acetic acid d. Ethyl dibutyl tri acetic acid 11. The process of formation of two or more separate coordinate bonds between a polydentate ligand and a single central atom a. Chelatation b. Complexation c. Protein binding d. Association

ANSWERS Id

lOa

lla