Complexation and Protein Binding: Introduction and Applications

By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
UNIT-IV
Complexation
and
Protein binding

Complexation and protein binding: Introduction, Classification of Complexation,
Applications, methods of analysis, protein binding, Complexation and drug action,
crystalline structures of complexes and thermodynamic treatment of stability constants.

“Complexation is the process of combining individual atom
groups, ions or molecules to create large ion or molecule, in which one
atom or ion is the central point of complex that form bonding with other
atoms, ions or molecules.”

 Complexes results from some type of interactions among different
chemical species.
 Intermolecular forces involved in the formation of complexes:
1. Van der Waals forces.
2. Hydrogen bonds
3. Coordinate covalence
4. Charge transfer.
5. Hydrophobic interaction.

 Complexes possess some properties which are different from those of
its components.
 Properties which confirm the formation of complexes.
1. Solubility
2. Light absorption
3. Conductunce
4. Partitioning behaviour
5. Chemical reactivities

Applications of Complexes
1. Physical state
By complexation Liquid substances can be converted to solid complex.
Hence Processing characteristics can be improved.
Example.
 Nitroglycerine can be transformed to its crystalline complex with
ß-cyclodextrin.
 The complex contain 15.6% Nitroglycerin and is explosion proof.

2. Volatility
 By complexation Volatility of substance can be reduce to stabilize
the system or to overcome unpleasant odour.
Example: Formation of Iodine as a complex with Polyvinyl-
pyrrolidone (PVP).
3. Solid state Stability
 Solid state Stability can be enhanced By complexation.
Example: ß-cyclodextrin complexes of Vitamin A & D are stable
form.

4. Chemical Stability
 Complex formation alter the chemical reactivity. Either inhibitory
or catalytic effects may be observed.
Example: Rate of hydrolysis of benzocaine can be reduce by
complexing it with caffeine.
5. Solubility
 Solubility of many Drugs can be enhanced by Complexation. Solid
complex is most commonly used pharmaceutically for solubility
enhancement.
Example: At low concentration, Caffeine enhances the solubility of p-
aminobenzoic acid (PABA)

6. Dissolution
 If solubility is enhanced, the dissolution rate should also increase.
Complexation is one of the possible method to achieve this
objective.
Example: The dissolution rate of phenobarbital is enhanced by using
cyclodextrin inclusion complexes.
7. Absorption and Bioavailability
 Complexation may alter Absorption & Bioavailability depends on
chemical nature of drugs.
Example: ß-cyclodextrin complexes of indomethacin, Barbiturates
have enhanced bioavailability.

8. Partition coefficient
 Partition coefficient of some substances can be alter by
complexation.
Example: Permanganate ions can be transferred in to benzene phase
from water by complexation with crown ether.
9. Reduce toxicity.
 Toxicity of drugs can be reduce by complexation.
Example: Cyclodextrins are effective in reducing ulcerogenic effect
of indomethacin & local tissue toxicity of Chlorpromazine.

10. Antidote for Metal poisoning
 Toxic metal ions such as arsenic, mercury, antimony etc. bind to –
SH group of various enzymes and interfere with their normal
functions.
Example:
1. Compound such as Dimercaprol (BAL, British Anti- Lewisite)
form water soluble complex with metal ions and eliminate them
rapidly form the body.
2. Beryllium Poisoning------- Salicylic acid
3. Lead Poisoning------------- EDTA

11. Antibacterial activity
 Antitubercular drug, PAS (P-amino salicylic acid) form a cupric
complex & Cupric chelate.
 Cupric chelate has shown greater in vivo Antitubercular activity in
mice than cupric complex.
 The chelate is about 30 times more fat soluble than the ionic
complex.

I. Metal Ion Complexes
a. Inorganic type
b. Chelates
c. Olefin type
d. Aromatic type
II. Organic Molecular Complexes
a. Quinhydrone type
b. Picric acid type
c. Caffeine and other drug complexes
d. Polymer type
III. Inclusion Compounds
a. Channel lattice type
b. Layer type
c. Clathrates
d. Monomolecular type
Classification of Complexes

I. Metal ion Complexes
 In this type, metal ion constitutes the central atom (substrate) and
interacts with a base (electron-pair donor, ligand).
 This type of interaction leads to formation of coordination bonds
between the species.
A. Inorganic Complexes
 complex consists of a metal ion (e.g. Co, Fe, Cu, Ni and Zn) linked
with one or more counter ions or molecules to form a complex.
 The ions or molecules (e.g. Cl, NH3, H2O, Br, I, CN, etc.) directly
bound with the metal are called ligands.

 The interaction between the metal and the ligand represents a Lewis
acid-base reaction
 The metal ion (Lewis acid) combines with a ligand (Lewis base) by
accepting a pair of electrons from the ligand to form the coordinate
covalent.
 The number of ligands bound to the
metal ion is defined as coordination
number.
 The coordination number of cobalt is
six, since it complexed with six NH3
groups.

 Compound (e.g. NH2) which has a
single pair of electrons for bonding
with the metal ion, is called
unidentate ligand.
 Ligands with two or three groups are
known as bidentate or tridentate
respectively.
 Ethylene Di-amine Tetra-Acetic acid
(EDTA) has six points for attachment
(two nitrogen and four oxygen donor
groups) and is called hexadentate.

 Coordination number usually determine the geometry of the
complex.

 Chelation is the formation of two or more coordinate bonds between
a Multidentate ligand (chelating agent) and a single central atom.
 The bonds in the chelate may be ionic, primary covalent, or
coordinate type.
Chelates

II. Organic Molecular Complexes
 Organic molecular complexes are formed as a result of
noncovalent interactions between a ligand and a substrate.
 The interactions can occur through Vander waals forces, charge
transfer, hydrogen bonding or hydrophobic effects.
 Many organic complexes are so weak that they cannot be separated
from their solutions as definite compounds, and they are often
difficult to detect by chemical and physical means.

Quinhydrone Complex
 This molecular complex is formed by mixing alcoholic solution of
equimolar quantities of benzoquinone with hydroquinone.
 Complex formation is due to overlapping of the 𝜋 - framework of the
electron deficient benzoquinone with the 𝜋- framework of the
electron-rich hydroquinone.

Picric Acid Complexes
 Picric acid (2,4,6-trinitrophenol), is a strong acid that forms complexes
with many weak bases.
Example:
Butesin picrate (local anaesthetic) which is a complex formed between
2 molecules of butyl p-aminobenzoate with 1 molecule of picric acid.

Caffeine Complexes
Caffeine forms complexes with a number of drugs by following
mechanism:
 Hydrogen bonding between the polarizable carbonyl group of
caffeine and the hydrogen atom of the acidic drugs such as p-
amino benzoic acid and gentisic acid.

 Dipole-dipole interaction between the electrophilic nitrogen of
caffeine and the carboxy oxygen of esters such as Benzocaine or
Procaine

Polymer Complexes
 Polymeric materials such as Eudragit, Chitosan, PEG, PVP & CMC
which are usually present in liquid, semisolid and solid dosage
forms, can form complexes with a large number of drugs.
 Such interactions can result in precipitation, flocculation,
Solubilization, alteration in bioavailability or other unwanted
physical, chemical, and pharmacological effects.
 Polymer–drug complexes however can also be used to modify
biopharmaceutical parameters of drugs.
Examples: Polymeric complex between Naltrexone and Eudragit
improves the dissolution rate of Naltrexone.
Application

Inclusion Complexes
 An inclusion compound is a complex in which one chemical
compound (the ‘host’) forms a cavity in which molecules of a
second compound (‘guest’) are entrapped.
 These complexes generally do not have any adhesive forces
working between their molecules and are therefore also known as
no-bond complexes.

Channel Lattice Type
 In this complex, the host component crystallizes to form channel-
like structure into which the guest molecule can fit.
 The guest molecule must possess a geometry that can be easily fit
into the channel-like structure.
 Dissolution of vitamin-E and Famotidine can be increased by
complexation with Urea (host molecule).
Application

 The well-known starch–iodine complex is a channel-type complex
consisting of iodine molecules entrapped within spirals of the glucose
residues

Layer Type
 Layer type complex (or intercalation compound) is a type of
inclusion compound in which the guest molecule is diffused between
the layers of carbon atom, to form alternate layers of guest and host
molecules.
E.g. Montomorillonite, (the principal constituent of bentonite) can trap
hydrocarbons, alcohols, and glycols between the layers of their lattices.

Clathrates
“The clathrates are compounds that crystallize in the form of a cage-
like lattice in which the coordinating compound is entrapped.”
E.g.
 One official drug, Warfarin Sodium, is in the form of
crystalline clathrate containing water and isopropyl alcohol.
 Hydroquinone crystallizes in a cage-like hydrogen-bonded
structure, in which small molecules such as methyl alcohol,
CO2,and HCl may be trapped in these cages.

 Size of the guest molecule is important for complex formation.

Monomolecular Inclusion Compounds: Cyclodextrins
 Monomolecular inclusion complex involves the entrapment of
guest molecules into the cage-like structure formed from a
single host molecule.
 Cyclodextrins are a family of compounds made up of sugar
molecules bound together in a ring (cyclic oligosaccharides)
 They consist of 6, 7, and 8 units of glucose referred to as 𝛼, 𝛽,
and 𝛾- cyclodextrins, respectively.

 Cyclodextrins have truncated cone structure with a hydrophobic
interior cavity because of the CH2 groups, and a hydrophilic
exterior due to the presence of hydroxyl group.

 Molecules of appropriate size and stereochemistry get entrapped in the
cyclodextrin cavity by hydrophobic interaction by squeezing out water
from the cavity.
 𝛽-CD and 𝜸-CD are the most useful for Pharmaceutical technology
owing to their larger cavity size.

 Cyclodextrins can enhance the solubility and bioavailability
of hydrophobic compounds due to the large number of hydroxyl
groups on the CDs.
 CDs are used as sustained release drug carriers.
Application

 In addition to improving the solubility of compounds,
complexation with cyclodextrin has been used to improve the
stability of many drugs by inclusion of the compound and
protecting certain functional groups from degradation.
 Complexation with Cyclodextrins has also been used to mask
the bitter taste of certain drugs such as Femoxetine.

Method of Continuous Variation
 The stoichiometry of a metal-ligand complexation reaction can
be determined by three methods:
(A) Job's method
(B) Mole ratio method
(C) Slope ratio method

Job's Method
 In Job’s method, a series of solution are prepared with variable ratios
of metal and ligand but with fixed total concentrations.
 An additive property that is proportional to the concentration of the
formed complex (e.g. absorbance) is measured and plotted against the
mole fraction from 0 to 1 for one of the components of a mixture
(e.g. Ligand).
 For a constant total concentration of A and B, the complex is at its
greatest concentration at a point where the species A and B are
combined in the ratio in which they occur in the complex.
 The line therefore shows a break or a change in slope at the mole
fraction corresponding to the complex.

 Here, the change in slope occurs at a mole fraction of 0.75
XL / XM = 0.75 / (1- 0.75) = 3
 This indicate a complex formation of the 3:1 type (ligand : metal).

Mole Ratio Method
 In the mole ratio method, a series of solutions are prepared with a
fixed amount of the metal and a variable amount of the ligand (or vice
versa).
 An additive property that is proportional to the concentration of the
formed complex (e.g. absorbance) is measured and plotted against the
mole ratio of the component with the variable amounts (e.g. Ligand).
 The formed complex is at its greatest concentration at a point where
the species A and M are combined in the ratio in which they occur in
the complex (indicated by a change in the slope at the mole ratio that
forms the complex).

 The change in slope (a) occurs at a mole ratio of 1 indicating a complex
of the 1:1 type, while the change in slope (b) occurs at a ratio of 2
indicating a complex of the 2:1 type.

Slope Ratio Method
 In the slope-ratio method two sets of solutions are prepared.
 The first set of solutions contains a large excess of metal and a variable
concentrations of ligand (all the ligand reacts in forming the metal–ligand
complex).
 The absorbance of the formed complex is plotted against the ligand
concentration and the slope of the line is determined.
 A second set of solutions is prepared with a large excess of ligand and a
variable concentration of metal (all the metal reacts in forming the metal–
ligand complex).
 The absorbance of the formed complex is plotted against the metal
concentration and the slope of the line is determined.
The stoichiometric ratio of metal to ligand can be calculated by formula
Stoichiometric ratio (L:M) = SlopeM / SlopeL

The slope of the first line (variable metal) is 1.56×10-3 and the slope of
the other line (variable ligand) is 5.3×10-4 What is the stoichiometric
ratio of this complex?
Stoichiometric ratio (L:M) = 1.56×10-3 / 5.3×10-4
= 3
Stoichiometric ratio (L:M) = 3:1 (L:M)
Example

pH Titration Method
 pH titration method can be used whenever the complexation is
accompanied by a change in pH.
E.g. The chelation of the cupric ion by glycine:
 Because 2 protons are formed in the reaction, the addition of glycine to
solution should result in a decrease in pH.

 Titration curves can be obtained by adding a strong base to a solution of
glycine alone and to another solution containing (glycine + copper salt)
and plotting the pH against the volume of base added.
 The curve for the metal-glycine
mixture is well below that for
the glycine alone.
 The difference in pH for a
given quantity of base added
indicates the occurrence of a
complex.

Solubility Method
 Solubility method is the most widely used method is the study the
inclusion complexation.
 According to the solubility method, excess quantities of the drug are
placed in well-stoppered containers, with a solution of the complexing
agent in various concentrations.
 The bottles are agitated in a constant temp. bath until equilibrium is
reached. Then, the supernatant liquid are removed and analyzed to
obtain the total drug concentration.

 The concentration of the drug is plotted against the concentration of
complexing agent to obtain a curve that can be used to calculate the
Stoichiometric ratio.
 An example to illustrate the solubility method is the p-Amino benzoic
acid (PABA) Complexation by caffeine

 Point A is the solubility of the drug in water. With the addition of
caffeine, the solubility of PABA rises linearly owing to complexation.
 At point B, the solution is saturated with the complex and with 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 PABA has passed into solution and has
been converted to the complex.
 Some of the PABA combines further with caffeine to form higher
complexes such as (PABA- 2 caffeine)

Distribution Method
 The method of distributing a solute between two immiscible
solvents can be used to determine the stability constant for
certain complexes.
 The solute distribution pattern changes depending on the nature
of complex.
 The distribution method has been used to study caffeine and
polymer complexes with a number of acidic drugs such as
benzoic acid, salicylic acid, and acetylsalicylic acid.

 The Complexation of iodine by potassium iodide is an example to
illustrate this Method.
I2 + K+ I- K+ I-
3
 The Equilibrium Stability constant,
K+ I-
3
[K+ I-][I2]
K =
 The K value of iodine-potassium iodide complex = 954

Protein Bindings
 A drug in the body can interact with several tissue components of
which the two major categories are;
1. Blood, and
2. Extravascular tissues.
 The interacting molecules are generally the macromolecules such as
proteins, DNA or adipose. The proteins are particularly responsible
for such an interaction.
“ The phenomenon of complex formation with proteins is
called as protein binding of drugs.”

 Proteins such as albumin and globulin are present in the blood.
Among these, the level of albumin is very high and plays a crucial
role in the drug protein binding.
 Alpha-acid glycoprotein (α-AGP) levels are very low in normal
healthy population. But, in the diseased state, such as myocardial
infarction, AGP levels increases.
 When drugs are present in blood, it is possible that these
macromolecules (proteins) bind drugs.
 Albumin binds both acidic and basic drugs. AGP binds most of the
basic drugs. Globulins bind drug to a little extent.

Protein binding may be divided into
1. Intracellular binding – where the drug is bound to a cell
protein which may be the drug receptor; if so, binding elicits a
pharmacological response. These receptors with which drug
interact to show response are called as primary receptors.
2. Extracellular binding – where the drug binds to an
extracellular protein but the binding does not usually elicit a
pharmacological response. These receptors are called secondary
or silent receptors.

 Binding of drugs to proteins is generally reversible which suggests
that it generally involves weak chemical bonds such as;
1. Hydrogen bonds
2. Hydrophobic bonds
3. Ionic bonds, or
4. van der Waal’s forces
 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;
For example,
covalent binding of Chloroform and Paracetamol metabolites to
liver results in hepatotoxicity.
Mechanisms of Protein-Drug Binding

The influence of binding on drug disposition and clinical response

Factors affecting protein-drug binding
1. Drug related factors
a. Physicochemical characteristics of the drug
b. Concentration of drug in the body
c. Affinity of a drug for a particular binding component
2. Protein/tissue related factors
a. Physicochemical characteristics of the protein or binding agent
b. Concentration of protein or binding component
c. Number of binding sites on the binding agent
3. Drug interactions
a. Competition between drugs for the binding site
b. Competition between the drug and normal body constituents
4. Patient related factors
a. Age
b. Intersubject variations
c. Disease states

Applications of protein binding
1. Drug distribution: Protein binding decreases the distribution of
drugs. The protein-bound drug is big in size, which cannot easily cross
cell membranes and therefore, restricts to the blood pool. Only free
(unbound) drug can pass through the cell membranes and contributes
to the tissue binding. The higher the protein binding, the lower the
tissue distribution.
2. Metabolism: Protein binding decreases the metabolism of drugs and
enhances the biological half-life. Only that fraction of drug which is
free can get metabolized.

3. Excretion: Protein binding decreases the renal excretion of drugs and
enhances the biological half-life. Only the free drug can get excreted
through glomerular filtration.
For example: tetracyclines are excreted mainly by glomerular
filtration. These drugs bind to proteins and results in decreased renal
excretion.
4. Drug action: Protein binding inactivates the drug, because sufficient
concentration of drug cannot be built up in the receptor site for action.
Example is naphthoquinones. Certain drugs though bind to
proteins, still retain the drug activity. Examples are penicillin and
sulfadiazine.

5. Sustained release: The complex of drug-protein in the blood acts
as a reservoir and continuously supply the free drug for its action.
Example is suramin-protein binding for anti-trypanosomal
action.
6. Carrier systems: Protein-drug complexes act as transport systems
to carry drugs to the site of action. This transport is extremely
important for drugs that exhibit low solubility in water portion of
the plasma.
For example, bis-hydroxycoumarin (anticoagulant) is bound
in the blood to the extent of about 98%. This drug might have
precipitated in the blood, if it is not complexed with protein.

Binding kinetics
𝑷 + 𝑫 ⇋ 𝑷𝑫
𝑲 =
𝑷𝑫
𝑷 𝑫
𝑲 𝑷 𝑫 = 𝑷𝑫
 The reversible binding of protein (P) and drug (D) molecule is
written as:
Where,
K = association constant, L/mole
[P] = concentration of free protein, mole/L
[D] = concentration of free drug, mole/L
[PD] = concentration of protein drug complex, mole/L
 The equilibrium stability constant, association constant, K, cab be
written as:

If the total protein concentration is the sum of unbound protein and the
protein present in the complex.
𝑷 𝒕 = 𝑷 + 𝑷𝑫
or 𝑷 = 𝑷 𝒕 − 𝑷𝑫
Substituting [P] in the above equation, we get
𝑷𝑫 = 𝑲 𝑷 + 𝑷𝑫 𝑫
𝑷𝑫 = 𝑲 𝑷 𝒕 − 𝑷𝑫 𝑫
𝑷𝑫 = 𝑲 𝑫 𝑷 𝒕 − 𝑲 𝑫 𝑷𝑫
𝑷𝑫 + 𝑲 𝑫 𝑷𝑫 = 𝑲 𝑫 𝑷 𝒕
𝑷𝑫 𝟏 + 𝑲 𝑫 = 𝑲 𝑫 𝑷 𝒕
𝑷𝑫
𝑷 𝒕
=
𝑲 𝑫
𝟏+𝑲 𝑫

𝑷𝑫
𝑷 𝒕
=
𝑲 𝑫
𝟏 + 𝑲 𝑫
Where [𝑷𝑫]/[𝑷 𝒕 ] represents the average number of molecules
bound per mole of protein Pt
Replacing [𝑷𝑫]/[𝑷 𝒕 ] by r, we get,
𝒓 =
𝑲 𝑫
𝟏 + 𝑲 𝑫
Suppose n number of independent binding sites, than
𝒓 = 𝒏
𝑲 𝑫
𝟏 + 𝑲 𝑫

Equation can be modified to get plot, known as Scatchard plot
𝐫 𝟏 + 𝐊 𝐃 = 𝐧𝐊 𝐃
𝐫 + 𝐫𝐊 𝐃 = 𝐧𝐊 𝐃
𝐫 = 𝐧𝐊 𝐃 − 𝐫𝐊 𝐃
𝐫 = 𝐃 𝐧𝐊 − 𝐫𝐊
r
D
= 𝒏𝑲 − 𝒓𝑲
In Scatchard plot r/[D] is plotted against r to give a straight line if only
one class of binding sites is present. However, if more than one class of
binding sites exist, the graph is not linear but exhibits a curvature.

Fig. shows a Scatchard plot for the binding of bis-hydroxycoumarin to
human serum albumin at 20 and 40° C.
The curvature in both the plots. At 20 and 40° C indicate the presence of
more than one type of binding sites.

Thermodynamic treatment of stability constants
 Many of the pharmaceutical processes such as Complexation, protein
binding, the dissociation of a weak electrolyte, or the distribution of a
drug between two immiscible phases are systems at equilibrium and
can be described in terms of changes of the Gibbs free energy (∆G).
 Consider a closed system at constant pressure and temperature, such as
the chemical reaction;
aA + bB ⇋ cC + dD …. (1)
 Because G is a state function, the free energy change of the reaction
going from reactants to products is;
∆G = ∆Gproducts− ∆Greactants …. (2)

 Equation (1) represents a closed system made up of several
components. Therefore. At constant T and P the total free energy
change of the products and reactants in equation (2) is given as the
sum of the chemical potential μ of each component times the
number of moles.
∆G = cμC+dμD − aμA+bμB …. (3)
 Further mathematical treatment of equation (3) for ideal reactants
and products give;
∆G = ∆G° + RT ln K …….(4)

 Because ∆G° is a constant at constant T and P, RT is also constant.
Hence ∆G = 0 and equation (4) becomes
0 = ∆G° + RT ln K
or
∆G° = − RT ln K
or
∆G° = − 2.303 RT log K …….(5)
 The standard free energy change of Complexation is related to the
overall stability constant, K, (or any of the formation constants) by
the relationship shown in equation (5)

 The standard enthalpy change, ∆H, can be obtained from the slope
of a plot of log K versus 1/T, following the expression
log K = −
∆H°
2.303R
1
T
+ constant
 When the values of K at two temperatures are known, the following
equation can be used:
log
𝑲 𝟐
𝑲 𝟏
=
∆H°
2.303R
T2 − T1
T1T2

 The standard entropy change, ∆S°, is obtained from the expression
∆G° = ∆𝑯°
− T ∆𝑺°
 ∆H°and ∆S° generally become more negative as the stability
constant for molecular complexation increases.
 As the binding between donor and acceptor becomes stronger,
∆H°would be expected to have a larger negative value.

Complexation and Protein Binding: Introduction and Applications

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Complexation and Protein Binding: Introduction and Applications