Complexation and protein binding

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Complexation Protein Binding
Physical Pharmacy
Unit II
By
Abhijit Debnath Asst. Professor Faculty of
Pharmaceutical Sciences PDM University
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About Abhijit
Asst. Professor Faculty of Pharmaceutical
Sciences PDM University

• Worked at Belco Pharma as Asst- Manufacturing
  Chemist.
• M.Pharma from Pt. B D Sharma University of Health
  Sciences, Rohtak 2016
• B.Pharma form Tripura University, Agartala (1st
  Div) 2014
• DOEACE O- Level from NIELT, Agartala ( Grade
  A) 2011
• Other Courses-

Biochemistry IIT Kharagpur (NPTEL) 2016
Membrane Biogenesis, Porosomes, Health Jawaharlal Nehru University (GIAN) 2016
Bio-Assays Bio-Efficacy in new Drug Discovery Biotechnology Research Jawaharlal Nehru University (GIAN) 2016
Introduction to research IIT Madras (NPTEL) 2016
Health Research Fundamentals National Institute of Epidemiology and ICMR (NPTEL) 2016
Functional Genomics IIT Kanpur (NPTEL) 2017
Biomedical Nanotechnology IIT Roorkee (NPTEL) 2017
Introduction to Data Analytics IIT Madras (NPTEL) 2017
Twitter/Instagram/Youtube TheAbhijitDN
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COMPLEXATION PROTEIN BINDING
What's on your mind?

• Complex

Cheleates
Precipitation
Drug ? Protein Drug Protein Complex
Metal complexes
Metal complexes
Applications

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Outline of the Talk
? 1. Definition Applications of Complexation
in Pharmacy ? 2. Classification of Complexes ?
3. Methods of Analysis ? 4. Significance of
Protein Binding ? 5. Kinetics of Protein
Binding ? 6. Methods for Determining Protein
Binding
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• 1. Definition Applications of Complexation in
  Pharmacy

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• Protein Binding This is the phenomenon of
  complex formation of drugs with proteins.

1.1 Definitions

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

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

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

Eg In complex ion Cu(NH3)4 2 4 ammonia ligand
are attached to the central metal ion Cu. Hence
the coordination number of Cu2 ion in above
complex ion is 4.

• Precipitation It is the Process of forming
  insoluble solid mass from the reaction of ions
  present in a solution.

• Agglutination It is the Process of forming a
  solid mass from particles present in a solution.

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1.2 Steps involved in drug Complexation
Different steps involved in drug Complexation are
1. The filled ligand orbital overlaps the empty
metal ion orbital.
2. 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.
5. Such a bond, in which one atom in the bond
contributes both electrons, is called
a coordinate covalent bond.
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1.3 Applications 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 CaNa2EDTA 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
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1.3 Applications of Complexation in Pharmacy
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.
8. As therapeutic tool Both EDTA CITRATES are
used in Preservation of blood. They are used as
anticoagulant.
9. Assay of drug Complexometric titration are
used to assay of drug containing metal ion such
as magnesium trisilicate
10. Development of novel drug delivery system
Complexation 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.
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Outline of the Talk
? 1. Definition Applications of Complexation in
Pharmacy ? 2. Classification of Complexes ? 3.
Methods of Analysis ? 4. Significance of Protein
Binding ? 5. Kinetics of Protein Binding ? 6.
Methods for Determining Protein Binding
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• 2. Classification of Complexes

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21-Feb-16
2. Classification of Complexes
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21-Feb-16
The ligand provides only one site for binding
with metal!
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21-Feb-16
Chelatation is the process of formation of two or
more separate coordinate bonds between a
polydentate ligand and a single central atom.
Eg citric acid, Tartrates, EDTA
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21-Feb-16

• 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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21-Feb-16

• 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 pi
  bond complex.
• If complex is formed by sigma bond between metal
  ion and aromatic molecule, then it is called
  sigma- bond complex.

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• They are formed when the alcohol solution of
  benzoquinone and alcoholic solution of the
  hydroquinone is mixed in equimolar concentration.

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• A number of acidic drugs are known to form
  complexes with caffeine. Drugs such as
  benzocaine, procaine and tetracaine form
  complexes with caffeine.
• 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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• Picric acid being a strong acid, forms organic
  molecular complexes with weakbases.
• The complex is indication of magnitude of
  carcinogenic activity.

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• 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.

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

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• The guest molecule is entrapped in the layers.
  Compounds such as claymontmorillonite, 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.

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• It's a cage like complex in which the
  coordinating compound is entrapped.
• For example Cage like structure formed through
  hydrogen bonding of hydroquinone molecules.
• Small molecules in cage get entrapped and form
  clatharates.

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• They are the inclusion compounds. Monomolecular
  inclusion compounds involve the entrapment of a
  single guest molecule in the cavity of one host
  molecule.
• The entrance of the cavity is hydrophilic in
  nature and the interior of the cavity is
  relatively hydrophobic.

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Outline of the Talk
? 1. Definition Applications of Complexation in
Pharmacy ? 2. Classification of Complexes ? 3.
Methods of Analysis ? 4. Significance of Protein
Binding ? 5. Kinetics of Protein Binding ? 6.
Methods for Determining Protein Binding
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• 3. Methods of Analysis

pH Titration method
Method of continuous variation
Distribution method
Solubility method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis
The analysis of complexes involves the estimation
of two parameters.
1. The stoichiometric ratio of ligand-to-metal or
donor to-acceptor.
2. Stability constant of the complex.
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3. Methods of Analysis
pH Titration method
Distribution method
Method of continuous variation
Solubility method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis
pH Titration method
Method of continuous variation
Solubility method
Distribution method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis

• This method is used when the complexation is
  achieve by change in pH. This method
  isconsidered as most reliable method for
  studying complexation.
• Eg. chelation of cupric ion by glycine.

pH Titration method
Method of continuous variation
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.
Solubility method
Distribution method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis
pH Titration method

• The physical properties, such as dielectric
  constant, refractive index and spectrophotometric
  extinction coefficient, are characteristics of a
  particular species.
• When there is no complexation between these
  species (A and B), the value of property is
  additive.
• This particular behaviour is represented in
  figure as dotted line. In case of complexation,
  these properties change, i.e. additive phenomena
  do not hold good.

Method of continuous variation
Solubility method
Distribution method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis

• 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. The results of
  above experiments is plotted as molar conc.of
  PABA Vs. molar conc. of caffeine.

pH Titration method
Method of continuous variation
Solubility method
Distribution method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis

• The distribution behaviour of a solute between
  two immiscible liquids is expressed by
  distribution coefficient or partition
  coefficient.
• When a solute complexes with an added substance,
  the solute distribution pattern changes depending
  on the nature of a complex.
• A set of such experiments provides data for the
  estimation of complex equilibrium stability.

pH Titration method
Method of continuous variation
Solubility method
Distribution method
Spectroscopy and Charge Transfer complexation
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Spectroscopy for complexation UV Spectroscopy
3. Methods of Analysis
pH Titration method
Method of continuous variation
Solubility method

• 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.

Distribution method
Spectroscopy and Charge Transfer complexation
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3. Methods of Analysis
pH Titration method
Method of continuous variation
Solubility method
Distribution method
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Outline of the Talk
? 1. Definition Applications of Complexation
in Pharmacy ? 2. Classification of Complexes ?
3. Methods of Analysis ? 4. Significance of
Protein Binding ? 5. Kinetics of Protein
Binding ? 6. Methods for Determining Protein
Binding
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• 4. Significance of Protein Binding

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4. Significance of Protein Binding
a. Absorption Protein binding disturb Absorption
equilibrium.
b. Distribution Protein binding decrease
distribution of drug because protein bound drug
does not cross Blood Brain Barrier, placental
barrier etc.
c. Metabolism Protein binding decreases the
metabolism of drugs enhances the biological
half life
d. 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
e. Drug action Protein binding inactivates the
drugs because sufficient concentration of drug
can not be build up in the receptor site for
action
f. In diagnosis The chlorine atom of chloroquine
replaced with radiolabeled I- 131. It can be used
to visualize-melanomas of eye disorders of
thyroid gland.
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Outline of the Talk
? 1. Definition Applications of Complexation
in Pharmacy ? 2. Classification of Complexes ?
3. Methods of Analysis ? 4. Significance of
Protein Binding ? 5. Kinetics of Protein
Binding ? 6. Methods for Determining Protein
Binding
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• 5. Kinetics of Protein Binding

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5. Kinetics of Protein Binding
The value of association constant, Ka and the
number of binding sites N can be obtained by
plotting the above equation in four different
ways1. Direct plot2. Scatchard plot3. Klotz
plot4. Hitchcock plot
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Outline of the Talk
? 1. Definition Applications of Complexation
in Pharmacy ? 2. Classification of Complexes ?
3. Methods of Analysis ? 4. Significance of
Protein Binding ? 5. Kinetics of Protein
Binding ? 6. Methods for Determining Protein
Binding
43

• 6. Methods for Determining Protein Binding

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6. Methods for Determining Protein Binding

• The most commonly available methods are used to
  determine Protein Bindings are

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Outline of the Talk
? 1. Definition Applications of Complexation
in Pharmacy ? 2. Classification of Complexes ?
3. Methods of Analysis ? 4. Significance of
Protein Binding ? 5. Kinetics of Protein
Binding ? 6. Methods for Determining Protein
Binding
46
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