Physical Pharmaceutics I Unit-III PDF

BP302T: Physical Pharmaceutics I Unit-III Topic Complexation and Protein Binding  Contents Complexation and protein binding: Introduction, Classification of Complexation, Applications, methods of analysis, protein binding, Complexation an...

BP302T: Physical Pharmaceutics I Unit-III Topic Complexation and Protein Binding  Contents 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 a process where complexes or coordination compounds are formed which involves association or interaction of 2 chemical species. Association of two or more species capable of individual existence. Complexes are formed because of the donar acceptor mechanism. Donor (Ligand) is the neutral molecule or ion of non metallic substance that can donate the lone pair of electrons. Acceptor (Substrate) is the metallic ion or sometimes it might a neutral atom.  As a result a bond is formed between the tow interactants (L&S) and the compound thus formed is called as complex. mS + nL ------ SmLn S – Substrate L – Ligand SL - Complex Following intermolecular forces are involved in the formation of complexes. 1. Van der Waals forces 2. Dipolar or induced dipolar type 3. Hydrogen bonding 4. Covalent or co-ordinate bonds  Applications of Complexation Physical state Volatility Stability of drugs Solubility Dissolution Absorption and Bioavailability Antidote for metal poisoning Antibacterial activity  Classification of complexes Inorganic types Metal Complexes Chelates Olefin and aromatic types Drug & Caffeine complexes Organic Molecular Polymer types Complexes Picric acid types Quinhydrone types Channel type Layer type Inclusion Complexes Clathrates Monomolecular types  Metal-ion complexes In this type metal ion constitutes central atom and interacts with ligands. a) Inorganic type: The central atom / acceptor is metal ion. Coordination number is maximum number of atoms or groups that can combine in coordination sphere with the central atom. b) Chelates: Chelates are group of metal ion complexes in which a substance (ligand) provides 2 or more donor groups to combine with metal ion or central atom. Example is EDTA –hexadentate ligand Applications of Chelates in Pharmacy 1. Purification of water EDTA combines with Calcium and Magnesium ions present in hard water and settles as precipitate. 2. Improves stability of drug 3. Analysis for drug molecule 4. Anticoagulants for blood. c) Olefin and Aromatic type: These types of Complexes are used as catalyst in the manufacturing of bulk drugs, intermediates and in the analysis of drugs. i) Olefin complex They are formed by interaction of aqueous solutions of metal ions (platinum, mercury, silver) with olefin such as ethylene. Eg: Ag-olefin complex. ii) Aromatic complex They are formed by interaction of metal ions as acceptors with aromatic molecules such as benzene, toluene and xylene. Eg: Complex of toluene with HCl.  ORGANIC MOLECULAR COMPLEXES Organic molecules are involved in complexation Weak forces - dipole-dipole forces and hydrogen bonding involved. Experimental conditions should be constant as any alteration in it molecular compounds are formed instead of molecular complexes. Eg: Iodine with Tolnaftate enhances antifungal activity Difference between Molecular complex and Molecular compound SL. MOLECULAR MOLECULAR No COMPLEX COMPOUND 1. It is formed when reaction Formed at elevated or high takes place in cold condition temperature 2. Weak forces are involved Strong electrostatic bonds are involved 3. Complexes cannot be They can be separated from separated from solutions solutions a) Quinhydrone complex The molecular complex formed by mixing alcoholic solutions of Benzoquinone and Hydroquinone in the ratio 1:1. Green crystals of Hydroquinone are obtained. Applications: used in manufacturing Quinhydrone electrode for pH determination. b) Picric acid Complex Picric acid is strong acid. If reacted with strong base molecular compounds are formed If reacted with weak base, molecular complex is formed. Eg: Butesin picrate as 1 % ointment for burns and paingul skin abrasions. c) Drug Caffeine complex Many acidic drugs forms complexes with Caffeine by dipole dipole interactions. Eg: Caffeine and Benzocaine Eg 2: Caffeine and Gentisic acid complex in chewable tablets. d) Polymer Complex Polymers are pharmaceutical additives which have nucleophilic oxygen. Eg: PEG, CMC. This polymers form complexes with drug molecules like phenol, tannic acid and salicylic acid. Disadvantages: Loss of preservative action, delay in absorption and undesirable physical, chemical and pharmacological effects. Loss of therapeutic activity of the drug when stored in polymer containers.  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. Classifications of Inclusion complex 1. Channel types 2. Layer types 3. Clathrates 4. Monomolecular types 1) Channel types Molecule crystallises in the form of long chains forming hollow chain like structure and other molecules get entraped into the channel like structure. Eg: Urea crystallises to hollow structure and methyl-α-lipoate fits in urea molecule. Applications: separation of optical isomers and analysis of dermatological creams. 2) Layer types In layer type of complexes one layer gets sandwiched between 2 parallel layers of host molecule. GUEST HOST Eg: Clays, montomorillorite entraps hydrocarbons, alcohols and glycols. Applications: used in the process of catalysis. 3) Clathrates Complexes formed are cage like structure wherein one molecule undergoes crystallisation and other molecule is involved in entrapment. Eg: Hydroquinone molecule forms a small hole within it and the guest molecule undergoes rearrangement and entraps within it. Applications: Storage of gases, toxic compounds or volatile substances. 4) 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.  APPLICATION OF COMPLEXATION Physical state : liquid to solid-nitroglycerine inclusion complex with beta Cyclodextrin----15% nitroglycerine containing complex Reduction in Volatility : Reduce volatility and odor. Ex.PVP iodine complex. Solid stability : stability of drug enhanced. Ex. β cyclodextrin with vit. A and D Chemical stability : Alter the chemical reaction by (inhibitory or catalytic) Benzocaine- caffine complex Solubility : solubility enhanced. Ex.low conc of Caffine enhance the solubility of PABA Dissolution : Increase solubility….dissolution also increased. Ex. phenobarbital inclusion complex with β cyclodextrin Absorption and bioavailability : Tetracycline complex with cal.,mag., Alu.----- in soluble complex β cyclodextrin complex with indomethacin ,barbiturates---- more soluble complex. Reduced toxicity : cyclodextrin reduce ulcerogenic effect of indomethacin and local tissue toxicity of chlorpromazine. Antidote for metal poisoning: toxic metal ions- arsenic,mercury,antioney  In diagnosis: Technetium 90 (a radionuclide) is prepared in the form of citrate complex this complex is used in diagnosis of kidney function & GFR. Squibb (complex of a dye Azure A with carbacrylic cation exchange resin): used for detection of achlorhydria due to condition such as carcinoma, pernicious anaemia.  METHOD OF ANALYSIS OF COMPLEXES Compound A + Compound B New compound AB (Complex AB) Properties of Cpd. A and Cpd. B is changed while forming Complex AB. 1. pH 2. Solubility 3. Distribution 4. Absorption 5. Dielectric constant The analysis of a Complex involves the estimation of two parameters 1. Stoichiometric ratio of ligand to metal or donor to acceptor. 2. Stability constant of the complex. Equation for complexation is written as D+C DC stability constant (K) = [ DC] / [ D] [C] Keeping the conc of metal ion or drug constant, the conc of ligand may be varied. The corresponding changes in the conc of DC can be estimated by suitable analytical method. Determination of the stoichiometric ratio of ligand to the metal or donor to the acceptor and a quantitative expression of the stability constant for complex formation are important in the study of complexes. Several methods of estimation of complexes have been developed as follows: 1. Job’s continuous variation method 2. pH Titration method 3. Distribution method 4. Solubility method 5. Spectroscopy method 1. Job’s continuous variation method This process uses the estimation of certain additive properties of the complex like dielectric constant, spectrophotometric absorbance, Refractive Index etc. 2. pH Titration method 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 place. Stability constant: Log β = 2 Χ p [A] P [A] = pKa - pH – log ([HA] initial – [NaOH]) 3. Distribution Method When a solute complexes with an added substance, the solute distribution pattern changes depending on the nature of the complex. 4. Solubility Method When the components 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 complexation. 5. Spectroscopy method K = k1/ k-1 = 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) Other Method NMR Infrared spectroscopy polarography circular dichroism kinetics X-ray diffraction electron diffraction  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. Types: 1. Reversible binding- involves weak chemical bonds such as H-bonds, hydrophobic bonds, ionic bonds, van der waal’s forces. 2. Irreversible binding- arises as a result of covalent bonding & is often a reason for carcinogenecity or toxicity of drug. Proteins which can be bounded by drugs: 1. Human serum albumin 2. Alpha 1 acid glycoprotein 3. Lipoproteins 4. Blood cells 1) Human serum albumin: Molecular weight: 65000- 69000 Synthesized in liver. Conc of albumin in extracellular fluid is about 60% Elimination half life: 17-18 days Conc: 3.5- 5.5 % (w/w) Possess 4 binding sites. 2) Alpha 1 acid glycoprotein (orosomucoid): Molecular weight: 44000 Bound by hydrophobic bonds. E.g. Basic drugs such as imipramine, amitriptyline, lidocaine, nortryptyline, Quinidine, disopyramide, etc bound to this. 3) Lipoproteins: Molecular weight: 2-3 lakhs to 34 lakhs. Bound drug dissolve in lipid core. E.g. Acidic drugs ( diclofenac), neutral (cyclosporine), basic drug (chlorpromazine). 4) Blood cells: 1. Hemoglobin 2. Carbonic anhydrase inhibitors 3. 3. Red blood cells membrane Order of binding: albumin> Alpha 1 acid glycoprotein>lipoproteins> globulins  Significance/ effects of protein binding: 1. Absorption- protein binding with drugs decreases free drug conc & disturbs abs equilibrium. 2. Decrease in Distribution of drugs 3. Decrease metabolism by preventing entry of drug to metabolizing organs & enhances biological half life. 4. Only unbound drug is capable of being eliminated 5. Diagnosis of diseases or disorders by using radio active substance 6. Site specific drug delivery of hydrophilic moieties.  Factors affecting protein (A) Drug factors: 1. Physicochemical properties of drug: 2. Conc of drugs 3. Affinity of drug for binding sites: (B) Protein related factors: 1. Physicochemical properties of protein 2. 2. Conc of protein 3. 3. Number of binding sites on the protein (C) Drug interactions: 1. Displacement reactions 2. Competition between drugs and normal body constituents 3. Allosteric changes in protein molecules (D) Patient related factors: 1. Age 2. Inter subject variations 3. Disease states  Kinetics of protein binding If ‘P‘ represent protein & ‘D’ represent the drug then applying law of mass action to reversible protein binding, P+ D PD The association constant (Ka) = [PD] / [P] [D] or [PD] = Ka [P] [D] The ‘Pt’ is the total conc of protein present, unbound and bound. Pt = [PD]+ [P] To study the behavior of drugs, a determinable ratio ‘r‘ is as follows. r = Moles of drug bound / total moles of protein r = [PD] / Pt r = Ka [D] / Ka [D] + 1 The above equation holds when only one binding site on protein & protein-drug complex is 1:1 Complex. The value of association constant, Ka & no. of binding sites ’n‘ can be obtained by plotting. 1. Direct plot 2. Scatchard plot 3. Klotz plot 4. Hitchcock plot 1) Direct plot: A direct plot of ‘r‘ vs [D] can be used to find no of binding sites on protein. ‘Ka’ is obtained by finding drug conc required to saturate half of total binding sites available i.e. N/2. 2) Scatchard plot: r = Ka [D] / Ka [D] + 1 By rearranging the equation into linear form r/ [D] = N Ka – r Ka A plot of r/ [D] vs r yeilds straight line with X & Y intercepts equal to ‘N‘ & 'Nka’ the slope is equal to Ka, Klotz plot: By reciprocating the equation 1/ r = 1/ N Ka [D] + 1/ N A plot of 1/r & 1/ [D] yeilds a double reciprocal plot. Hitchcock plot: Obtained by rearranging the equation as N Ka [D] / r = 1+ Ka [D] Dividing both sides by N Ka [D] /r = 1/ Nka + [D] / N A plot of [D] / r vs [D] yeilds a straight line with slope 1/N & intercept 1/ NKa  Measurement of protein binding 1. Equilibrium dialysis 2. Dynamic dialysis 3. Ultrafiltration Equilibrium dialysis: Dialysis ( Gr. dia-through, lysis- loosening/splitting) “the separation of particles in a liquid on the basis of differences in their ability to pass through the membrane” Principle: two solutions are separated by a semipermeable membrane, allowing sufficient time to pass, the conc of diffusible substances will be equal on both sides of the membrane. Method: 1. Serum albumin is kept in visking cellulose tube/ bag, bag is closed tightly & placed in vessel containing drug. 2. Semipermeable membrane allow only low molecular weight ligands, such as drug molecules to transport between the chambers. 3. If binding occurs drug conc in bag containing protein will be higher than its conc in outer vessel. Dynamic dialysis: Apparatus consists of 400ml jacketed beaker in which 200ml buffer sol is kept. Dialysis bag having solution of drug & protein is suspended in buffer solution & Solution is stirred. Periodically sample is removed from dialysis bag & analyzed. Principle: rate of disappearance of drug from dialysis bag is proportional to conc. of unbound drug. d [Dt] / dt = K [ Df] Ultrafiltration: Based on physical separation of free drug molecules from drug bound to plasma proteins by centrifugation. Thermodynamic treatment of stability constants: The stability constant of metal complexes are related to thermodynamic properties such as free energy charge (∆G), enthalpy (∆H), & entropy change (∆S). These values can be computed by usual equation ∆G = - 2.303 RT log K The std enthalpy change (∆H) obtained from slope of plot of log K vs 1/T log K = - ∆H / 2.303 RT + constant Std entropy change is ∆G = ∆H – T ∆S If stability constant increase= ∆H & ∆S become negative, As binding between donor & acceptor is stronger then ∆H also become negative. 57