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Complexation And Protein Binding:- PDF


             BP302T. PHYSICAL PHARMACEUTICS-I (Theory)

 Complexation is the process of complex formation that is the process of characterization
   the covalent or non-covalent interactions between two or more compounds.
 The ligand is a molecule that interacts with another molecule, the Drug, to form a
   complex. Drug molecules can form complexes with other small molecules or with
   macromolecules such as proteins.
 A coordination complex is the product of a Lewis acid-base reaction in which neutral
   molecules or anions (called ligands) bond to a central metal atom (or ion) by coordinate
   covalent bonds
 Simple ligands include water, ammonia and chloride ions.
 Once complexation occurs, the physical and chemical properties of the complexing
   species altered are;
      Solubility,
      Stability,
      Partition co-efficient,
      Energy absorption,
      Energy emission.
      Conductance of the drug.
 Forces involved in complex formation:
      Covalent bond.
      Co-ordinate covalent bond.
      Van der Waals force of dispersion.
      Dipole-Dipole interaction.
      Hydrogen bond.
 Beneficial effects of complexation:
      Drug complexation, therefore, can lead to beneficial properties such as enhanced
       aqueous solubility (e.g., theophylline complexation with ethylenediamine to form
       aminophylline) and stability (e.g., inclusion complexes of labile drugs with

      Complexation also can aid in the optimization of delivery systems (e.g., ion-exchange
       resins) and affect the distribution in the body after systemic administration as a result
       of protein binding.
      In some instances, complexation also can lead to poor solubility or decreased
       absorption of drugs in the body.
      For some drugs, complexation with certain hydrophilic compounds can enhance
 Metal ion complexes
   Metal ion includes the central atom as Drug and it interacts with a base (Electron-pair
   donor, ligand), forming co-ordination bonds between the species.
      Inorganic type
      Chelates
      Olefin type
      Aromatic type
       o Pi (π) complexes
       o Sigma (σ) complexes
       o “Sandwich” compounds
 Organic molecular complexes
      Quinhydrone type
      Picric acid type
      Caffeine and other drug complexes
      Polymer type
 Non-Bonded or Inclusion/ occlusion compounds
      Channel lattice type
      Layer type
      Clathrates
      Monomolecular type
      Macromoleular type
Metal ion complexes:
 Metal ion includes the central atom as Drug and it interacts with a base (Electron-pair
   donor, ligand), forming co-ordination bonds between the species.

Inorganic type –
 In inorganic metal complexes, the ligand provides only one site for binding with metal.
 The ammonia molecules in hexamminecobalt (III) chloride, as the compound
   [Co(NH3)6]3+ Cl3- , are known as the ligands and are said to be coordinated to the
   cobalt ion. The coordination number of the cobalt ion, or number of ammonia groups
   coordinated to the metal ions, is six. Other complex ions belonging to the inorganic group
   include [Ag(NH3)2]+, [Fe(CN)6]4-, and [Cr(H2O)6]3+.
 Each ligand donates a pair of electrons to form a coordinate covalent link between itself
   and the central ion having an incomplete electron shell.

 Hybridization plays an important part in coordination compounds in which sufficient
   bonding orbitals are not ordinarily available in the metal ion.
Chelates -
 The chelates are a group of metal ion complexes in which a substance (Ligands) provides
   two or more donor groups to combine with a metal ion.
 Some of the bonds in a chelate may be ionic or of the primary covalent type, whereas
   others are coordinate covalent links.
 When the ligand provides one group for attachment to the central ion, the chelate is
   called monodentate.
 Pilocarpine behaves as a monodentate ligand toward Co(II), Ni(II), and Zn(II) to form
   chelates of pseudotetrahedral geometry.

   Fig 1. Structure of EDTA.
Olefin type -
 The aqueous solution of certain metal ions like Pt, Fe, Pd, Hg and Ag can absorb olefins
   such as ethylene to yield water soluble complexes.
 These are uses as catalyst in the manufacture of bulk drugs and analysis of drugs.
Aromatic type -
 Pi (π) complexes – Aromatic bases (Benzene, toluene and Xylene) form pi-bond
   complexes with metal ions like Ag by Lewis acid-base reactions.

 Sigma (σ) complexes – sigma bond complexes involve in the formation of a sigma-bond
   between ion and a carbon of aromatic ring.
 “Sandwich” compounds – The are relatively stable complexes involving in the
   delocalized covalent bond between the d-orbital of transition metal and a molecular orbit
   of the aromatic ring.
Organic molecular complexes:
 Many organic complexes are so weak that they cannot be separated from their solutions
   as definite compounds.
 The energy of attraction between the constituents is probably less than 5 kcal/mole for
   most organic complexes.
 Because the bond distance between the components of the complex is usually greater than
   3 Å, a covalent link is not involved.
 An organic coordination compound or molecular complex consists of constituents held
   together by weak forces of the donor–acceptor type or by hydrogen bonds.
 Donor Acceptor type – In this the bond is between uncharged species but lacks charge
   transfer. The dipole-dipole interaction and London dispersion forces (Dotted lines) make
   the complex stable. Example - The compounds dimethylaniline and 2,4,6-trinitroanisole
   react in the cold to give a molecular complex.

 The charge transfer Complexes - In this one molecule polarizes the other, resulting in a
   type of ionic interaction or charge transfer, and these molecular complexes are often
   referred to as charge transfer complexes. The resonance makes the complex more stable.
   The intermolecular bonding is quite higher compared to donor-acceptor type complexes.
   For example, the polar nitro groups of trinitrobenzene induce a dipole in the readily.
Caffeine and other drug complexes -
 Drugs such as benzocaine, procaine and tetracaine form complexes with caffeine.
 A number of acidic drugs are known to form complexes with caffeine.

Fig 2. Structure of caffeine and Benzocaine.
Quinhydrone type –
 The molecular complex of this type is obtained by mixing alcoholic solutions of
   equimolar quantities of hydroquinone and benzoquinone.

Fig 3. The complexes of hydroquinone and benzoquinone.
Polymers Type –
 Many pharmaceutical additives such as polyethylene glycols (PEGs), carboxymethyl
   cellulose (CMC) contain nucleophilic oxygen. These can form complexes with various
 E.g. Polymers: carbowaxes, pluronics etc. Drugs: tannic acid, salicylic acid, phenols etc.
 Carboxy methyl cellulose + Amphetamine – Poorly absorbed drugs.
Picric acid types –
 Picric acid, being a strong acid, forms organic molecular complexes with weak bases,
   whereas it combines with strong bases (anesthetic activity of butesin) to yield salts.
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.
Channel types –
 Channels are formed by crystallization of the host molecules, the guest component is
   usually limited to long, unbranched straight chain compounds.
Layer types –
 Compounds such as clays, montomorillorite (constituent of bentonite), can entrap
   hydrocarbons, alcohols and glycols.

 They form alternate monomolecular (monoatomic) layers of guest and host.
 Their uses are currently quite limited; however these may be useful for catalysis on
   account of a larger surface area.
Clathrates -
 It is available as white crystalline powder, during crystallization, certain substances form
   a cage-like lattice in which the coordinating compound is entrapped.
Monomolecular types –
 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 interior of the cavity is relatively hydrophobic, whereas the entrance of the cavity is
   hydrophilic in nature.
Applications of Complexation:
 Physical state:
      Complexation process improves processing characteristics by converting liquid to
       soild complex. β-cyclodextrine complexes with nitroglyerine.
 Volatility:
      Complexation process reduces Drug volatility for following benefits;
       o Stabilise system.
       o Overcome unpleasant odour (I2 complexes with Poly Vinyl Pyrollidone, PVP).
 Solid state stability:
      Complexation process enhances solid state stability of drugs.
      β-cyclodextrine complexes with Vitamin A and D.
 Chemical stability:
      Complex formation inhibit chemical reactivity (Mostly inhibit).
      The hydrolysis of Benzocaine is decreased by complexing with Caffeine.
 Solubility:
      Complexation process enhances solubility of drug.
      Caffeine enhances solubility of PABA (Para Amino Benzoic Acid) by complex
 Dissolution:
      Complexation process enhances dissolution of drug.
      β-cyclodextrine increases the dissolution of Phenobarbitone by inclusion complex.

 Partition co-efficient:
      Complexation process enhances the partition coefficient of certain drugs.
      Permanganate ion with benzene.
 Absorption and Bioavailability:
      Complexation process reduces the absorption of Tetracycline by complexing with
       cations like Ca+2, Mg+2 and Al+3.
      Complexation process enhances the aborption of Indomethacine and Barbiturates by
       complexing with β-cyclodextrine.
 Reduced toxicity:
      β-cyclodextrine reduces ulcerogenic effects of Indomethacine.
      β-cyclodextrine reduces local tissue toxicity of Chlorpromazine.
 Antidote for metal poisoning:
      BAL (British Anti Lewisite) reduces toxicity of heavy metals by complexing with As,
       Hg and Sb.
 Drug actin through Metal Poisoning:
      8-Hydroxy quinoline complexes with Fe exhibit greataer antimalaria activity.
 Antitubercular activity:
      PAS (Para Amino Salysylic acid) complexes with Cupric ion exhibit greater
       Antitubercular activity.
 Development of Novel Drug delivery system:
      The Comlexation of drug with polymers used in the formulation of sustained drug
       delivery device.
 Assay of Drugs:
      The complexometric titrations are used to assay of the drug containing the metal ion.
 As therapeutic Tools:
      Both CITRATES and EDTA are used as preservation of blood as anti-coagulant.
 As Diagnostic agent:
      Ta90 complexes with citrate are used for diagnosis of Kidney and measurement of
       Glomerular Filtration Rate.
Methods of analysis Complexation:
Job’s Method of Continuous Variation:
 As per the Job, the species possess several characteristics that are;
      Dielectric constant.

      Refractive Index.
      Spectrophotometric extinction coefficient.
 Principle - When there is no complexation between the species, the value of property is
   additive. On complexation these properties changes but additive rule do not hold good.
   The change in the characteristics proves that the complexation has been taken place.
 Let’s take two species A and B whose individual dielectric constant in solid form and
   Absorbance in solution form were measured. Then two species in both forms were mixed.
   The dielectric constant and absorbance were determined.
 The individual values are subtracted with mixed additive values and result was found out.
 If result is zero then no complexation and if result is not zero then there is complextaion.
pH Titration Method:
 Principle - This method is applicable for that complex that produces the changes in pH on
   interaction. The significant change in pH will determine that complexation has been taken
 Let us take 75 ml of glycine solution and it is titrated with strong alkali NaOH solution.
   The pH was recorded. A graph was drawn between pH and volume of NaOH added.
 In another test, complex solution of glycine and copper salt is titrated. The change in pH
   with increments of NaOH solution also recorded. A graph was drawn between pH and
   volume of NaOH added.
 The two plots are compared and it is seen that the plot of glycine with copper is well
   below that of the pure glycine, which indicated that complexation is obtained throughout
   the titration range.
Distribution Method:
 The method of distributing a solute between two immiscible solvents can be used to
   determine the stability constant for certain complexes.
 The distribution behavior of a solute between two immiscible liquids is expressed by
   distribution or partition co-efficient.
 Principle - When a solute complexes with an added substance, the solute distribution
   pattern changes depending on the nature of the complex.
 The complexation of iodine by potassium iodide.
    I2 + K+I- ---------- K+ I3-
 The Equilibrium stability constant,
   K = [K+ I3-]/ [I2] [K+I-]

 The distribution coefficient of iodine between disulfide and water is 625.
 The K value of Iodine-Potassium iodide complex is 954.
 This change in distribution coefficient proves that the complexation has taken place.
Solubility Method:
 Principle - When the component in a mixture produce a complex, the solubility of one of
   the components may be increased or decreased. The change in solubility is a sign of
 The experimental data can be used to analyse complexes in terms of donor-acceptor ratio
   and equilibrium stability constant.
 Example – PABA and Caffeine and Paracetamol – Caffeine.
Spectroscopy Method:
 The study of donor acceptor (D-A) or charge transfer complexation is generally
   undertaken with absorption spectroscopy in the visible and UV regions of the spectrum.
  D+A ======== DA
 Where, D and A represents electron donor and acceptor, k1 and k2 are interaction rate
 K = k1/ k2 = Equilibrium or stability constant for complexation.
 The absorbance A of the charge transfer band is measured at a definite wavelength and
   the constant K is obtained from the Benesi-Hildebrand equation.
 A0/A = (1/ε) + (1/Kε) (1/D0)
 Where, A0 and D0 are the initial concentration of acceptor and donor species in mole/litre.
   Ε is the molar absorptivity of the charge-transfer complex at its particular wavelength and
   K is the stability constant in litre/mole.
 A plot of A0/A versus 1/D0 results in straight line with a slope of 1/Kε and an intercept of
 The spectrometric method used to investigate the interaction of nucleic acid bases with
   catechol, epinephrine and isoproterenol.
Protein binding:
 The interacting molecules are generally the macromolecules such as protein, DNA or
   adipose. The proteins are particularly responsible for such an interaction.
 The phenomenon of complex formation of drug with protein is called as protein binding
   of drug.

 As a protein bound drug is neither metabolized nor excreted hence it is pharmacologically
   inactive due to its pharmacokinetic and Pharmacodynamics inertness.
 Protein + drug ⇌ Protein-drug complex.
 Protein binding may be divided into - Intracellular binding. 2. Extracellular binding.
Mechanisms of protein drug binding:
 Binding of drugs to proteins is generally of reversible and irreversible.
 Reversible generally involves weak chemical bond such as: 1. Hydrogen bonds 2.
   Hydrophobic bonds 3. Ionic bonds 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.
 Absorption - As we know the conventional dosage form follow first order kinetics. So
   when there is more protein binding then it disturbs the absorption equilibrium.
 Distribution - A protein bound drug in particular does not cross the BBB, the placental
   barrier, the glomerulus. Thus protein binding decreases the distribution of drugs.
 Metabolism - Protein binding decreases the metabolism of drugs and enhances the
   biological half life. Only unbound fractions get metabolized. • e.g. Phenylbutazone and
 Elimination – Only the unbound drug is capable of being eliminated. Protein binding
   prevent the entry of drug to the metabolizing organ (liver) and to glomerulus filtration. •
   e.g. Tetracycline is eliminated mainly by glomerular filtration.
 Systemic solubility of drug – Lipoprotein act as vehicle for hydrophobic drugs like
   steroids, heparin, oil soluble vitamin.
 Drug action - Protein binding inactivates the drugs because sufficient concentration of
   drug cannot be build up in the receptor site for action. • e.g. Naphthoquinone.
 Sustain release – The complex of drug protein in the blood act as a reservoir and
   continuously supply the free drug. e.g. Suramin sodium-protein binding for
   antitrypanosomal action.
 Diagnosis – The chlorine atom of chloroquine replaced with radiolabeled I- 131 can be
   used to visualize-melanomas of eye and disorders of thyroid gland.
Factors affecting protein binding:
 Drug related Factor.
       o Physicochemical properties of drug Increase in lipophilicity increases the drug
           binding with the protein.

       o Total concentration of drug – Alternation in drug and protein concentration alter
           the drug protein binding.
 Protein related Factors.
       o Physicochemical properties of protein – Lipoprotein bind with lipophilic drugs.
       o Quantity of protein – Disease state affect the concentration of protein in blood.
       o Number of binding sites – Albumin has more no of binding sites.
 Affinity and Magnitude of association constant.
 Drug Interaction.
          Displacement reaction
          Composition of drugs and normal body constituents.
          Allosteric changes in protein molecules.
 Patient related factors.
      Age – Noenata have low albumin content, thus less drug binding.
      Disease state – Disease sate alter the drug binding.
Binding of drug to blood plasma proteins –
 The binding of drugs to plasma proteins is reversible.
 The extent or order of binding of drug to plasma proteins is: Albumin › ὰ1-Acid
   glycoprotein › Lipoproteins › Globulins.
 Binding of drug to human serum Albumin –
      It is the most abundant plasma protein (59 %).
      Having M.W. of 65,000 with large drug binding capacity.
      Both endogenous compounds such as fatty acid, bilirubin as well as drug bind to
      Four different sites on HSA for drug binding.
      Site I: warfarin and azapropazone binding site.
      Site II: diazepam binding site.
      Site III: digitoxin binding site.
      Site IV: tamoxifen binding site.
 Binding of drug to α1-Acid glycoprotein –
      It is called as orosomucoid. It has a M.W. 44,000.
      Its plasma conc. range of 0.04 to 0.1 g %.
      It binds to no. of basic drugs like imipramine, lidocaine, propranolol, and quinidine.

 Binding of drug to Lipoproteins –
      Binding by Hydrophobic Bonds, Non-competative.
      Mol wt: 2-34 Lacks dalton.
      Lipid core composed of: Inside: triglyceride & cholesteryl esters. Outside:
       Apoprotein. e.g. Acidic: Diclofenac. Neutral: Cyclosporin A. Basic: Chlorpromazine.
      Its types are LDL, HDL, VLDLand Chylomicrons.
 Binding of drug to Globulins –
      α1 Globulin (Transcortine /Corticosteroid Binding globulin) - Steroidal drugs,
       Thyroxin & Cyanocobalamine (Vit B12).
      α2 Globulin (Ceruloplasmine) - Vitamin A, D, E, K.
      β1 Globulin (Transferin) - Ferrous ions.
      β2 Globulin – Carotinoids.
      γ Globulin – Antigens.
Kinetics of Protein Binding:
 An equation relating reaction velocity to Drug concentration (Mol/L) for a system where
   a Drug D binds reversibly to an Protein P of to form an Protein-Drug complex .
 This system can be represented schematically as follows:
   P + DF ======= PD
 Applying the law of mass action, the equilibrium or association constant (K) is;
   K = [PD]/ [P] [DF]
 The [PD], [P] and [D] are the concentration of protein-drug complex, protein and drug in
   K[P][ DF] = [PD]
 Free protein concentration can obtain as;
   [PT] = + [PD]
   [P] = [PT] _ [PD]
 [PT] is the total protein.
 Substituting the [P] in last equation, K[P][ DF] = [PD]
   K ([PT] – [PD]) [DF] = [PD]
 Where, DF is the free drug.
   K [PT] [DF] – K [PD] [DF] = [PD]
   K [PT] [DF] = [PD] + K [PD] [DF]
   K [PT] [DF] = [PD] (1+ K [DF])

   [PD] = (K [PT] [DF])/ (1+ K [DF])
   [PD]/ [PT] = K [DF]/ 1+ K [DF]
 Let R be expressed as moles of drug bound [PD] per mole of total protein [PT]
   R = [PD]/ [PT] = K [DF]/ 1+ K [DF]
 If V is the number of independent binding sites available then R,
   R = V (K [DF]/ 1+ K [DF])
   1/R = 1/VK[DF] + 1/V
 The graph is plotted between 1/R versus 1/[DF], called Klotz reciprocal plot, gives a
   straight line whose slope is 1/VK and intercept is V.

Fig 1. Klotz reciprocal plot.
R + R K [DF] = V K [DF]
R/[DF] = VK - RK
Complexation and drug action:
 Protein binding inactivates the drugs because sufficient concentration of drug cannot be
   build up in the receptor site for action. • e.g. Naphthoquinone.
 Only free drug participate in drug action.
 Complexation can alter the pharmacological action of drug by interfering interaction with
 The action of drug to remove the toxic effect of metal ion from the human bodies is
   through the complexation reaction.

 It has been seen that in some instance complexation can also lead to poor solubility or
   decreased absorption of drug in the body, which decreases the bioavailability of drug in
   the blood. Thus the drug action gets altered.
 Drug complex with hydrophilic drug also enhance the drug elimination, thus helps in drug
   action termination and reduction in drug toxic action.
 Examples :
      Tetracycline and Calcium – Poor absorbed complex.
      Polar drug and complexing agent – Well absorbed lipid soluble complex.
      Carboxy methyl cellulose and amphetamine – Poor absorbed complex.
      PVP and I2 – Better absorption.
Thermodynamic treatment of stability constants Complexes:
 The relationship between the standard free energy change of complexation and the over
   all stability constant K is related as;
   ΔG = -2.303RT Log K
 The Standard Enthalpy Change ΔH may be obtained from the slope of a plot of Log K
   Versus 1/T, thus the equation will be;
   Log K = - (ΔH/2.303R) × (1/T) + Constant
 When the value of K at two temperatures are known, the following equation can be
   written as;
   Log (K2/K1) = - (ΔH/2.303R) × (T2-T1/T1T2)
 The Standard entropy change may be obtained from the expression;
   ΔG = ΔH - T ΔS
 As the stability constant for molecular complexation increases, ΔH and ΔS becomes more
 As binding between the donor and receptor becomes stronger, ΔH becomes more
 Since the specificity of interacting sites becomes negative, ΔS also become more
 But the extent of change in ΔH is large enough to overcome the unfavourable entropy
   change resulting in negative ΔG value and hence complexation.

References –
1. Subrahmanyam CVS. Physical Pharmaceutics-I. 1ST Edition. New Delhi: Vallabh
   Pralashan; 2019. pp. 450-484.
2. Agarwal SP, Khanna R. Physical Pharmacy. 2nd Edition. New Delhi: CBS Publishers
   & Distributors Pvt. Ltd; 2009. pp. 191-220.

Submitted by:
Dr. Bhabani Shankar Nayak
M. Pharm (Pharmaceutics), Ph.D.,
Assoc. Prof, Institute of Pharmacy& Technology
Salipur, Cuttack,Odisha – 754202, India.
Email ID- [email protected],
Tel: 09938860284