S SNS COLLEGE OF PHARMAC ©) eet ND HEALTH SCIEN UNIT-IV. COMPLEXATION AND PROTEIN BINDING: INTRODUCTION: V 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; ¥ Chemical (pH), instrumental (UV/IR spectra, conductance) Y Formulation (solubility, partitioning, stability, drug delivery by ion- exchange resins. Example: Delsym, Tussionex, Kayexalate, Renagel, Cholestyramine) ¥ Pharmacokinetic — permeability (bioavailability), protein binding (distribution) 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 benefi icial properties such as enhanced aqueous solubility (e.g,, theophylline complexation with cthylenediamine to form aminophylline) and stability (eg, inclusion complexes of labile drugs with cyclodextrins), Complexation also can aid in the optimization of delivery systems (c.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 excretion, CLASSIFICATION OF COMPLEXATION: COMPLEXES Organic Inclusion molecular esrncleed compounds 1. Inorganic types Pama Ty SU Ou mE ug 2; Layer type Ee ary eC 4, Aromatic type Ltn Creer MDa ay i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. aad te Cos bond: METAL rea (ric) rae eee Beco ard Inorganic complexes: Werner postulates: 1 There are 2 types of valences. primary (ionic), secondary (coordinate). type of anion/ radical/ molecule may be held by any one / both type of 'y central atom has fixed number of non-ionic valences (co- ordination tion atoms occupy the first sphere/coordination sphere, other second/ ionization sphere.3. Substrate and ligand are bonded with coordination bond. 4. Coordination number is maximum number of atoms and groups that combine with central atom in coordination sphere. 5. Co-ordination number for cobalt is 6, 4p @®OQ0QOO O OOO @WODEODOO O BSR 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. v Pilocarpine behaves as a monodentate ligand toward Co(II), Ni(II), and Zn(II) to form chelates of pseudotetrahedral geometry. > Hexadentate - ethylenediaminetetraacetic acid (EDTA)- Has a total of six points (4:0 and 2: N) for attachment of metal ions. HOOC=H,¢ eH,-CooH’ N= CHZCH, = | Hooc-H,c ‘cH COOH! _ EDTA | Figure: Structure of EDTA, Sequestering: This is a process in which the property of metal is suppressed without removing it from the solution. Sequestering Agent: This is a ligand which forms a stable water soluble metal chelateExample: chlorophyll, hemoglobin. Chelates applications: 1. INCREASING SOLUBILITY: Fruit juices and drugs (ascorbic acid) + Fe/Cu > oxidative degradation. Add EDTA + Fe/Cu ® stable Chelate 2. PURIFICATION OF HARD WATER: Hard water (Ca+2) + EDTA > EDTA-Cat2 (ppt) > filter > Pure water. 3. DURG ANALYSIS: Procainamide + cupric ions (1:1) at pH 4-4.5 ® Coloured complex > detect by Colourimetry. 4. ANTI-COAGULANT: Blood (Ca+2) + EDTA/Citrates/Oxalates > prevent thrombin formation > no clotting. 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 (x) complexes — Aromatic bases (Benzene, toluene and Xylene) form pi- bond complexes with metal ions like Ag by Lewis acid-base reactions. > Sigma (o) complexes — sigma bond complexes involve in the formation of a sigma-bond between ion and a carbon of aromatic ring. > Sandwich compounds — These 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: 8 are so weak that they cannot be separated from their > Many organic comple: solutions as definite compoundsv 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 A, a covalent link is not involved. Complex Molecular compound Reaction in cold temperature Reaction in hot temperature Weak attraction forces Strong electrostatic interactions Complexes cannot be separated from solutions. Compounds can be separated from solutions 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. On chy ) " Oa cn, Sf + u,co- no, ¢ ne HCO Nk / \ \ mo" en, ON NOx ON 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 resananes makes the complex more stable. The intermolecular bonding is quite higher compared to donor-acceptor type complexes. For ex: ‘ample, 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. > Anumber of acidic drugs are known to form complexes with caffeine. > Acidic drugs (benzocaine, procaine) + Caffeine =.» Complexes Mechanism: 1. Dipole-dipole forces/ hydrogen bonding between acid (H) atom and caffeine carboxyl group. 2. Interaction of non-polar parts. Example: Caffeine + Benzocaine. Figure: Structure of caffeine and Benzocaine. Applications: 1. These complexes can improve / extend absorption and bioavailability of drug. 2, These complexes can enhance! inhibit solubility and dissolution rate of drug. 3. Caffeine+ gentisic acid complexes mask bitter taste of caffeine. Quinhydrone type — > The molecular complex of this type is obtained by mixing alcoholic solutions of equimolar quantities of hydroquinone and benzoquinone (green crystals). Mechanism: 1, Overlapping of x electrons of molecules 2. (H) bonding for stabilizing complex. Applications: Used as electrode in pH determination.Hydroquinone no-,\)- OH (electron rich 1 Overlap cloud) ox | Yeo Benzoquinone deficient) Figure: 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 drugs. v Examples are: Polymers: carbowaxes, pluronics ete. © Drugs: tannic acid, salicylic acid, phenols ete. > Carboxy methyl cellulose + Amphetamine — Poorly absorbed drugs. Disadvantages: 1. Incompatibilities in suspension, emulsion, ointments. 2. Complexes + Container = drug loss 3. Complexes + preservatives === decrease preservative action. 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. > Picric acid (strong acid) + strong base =m Salt Picric acid (strong acid) + weak base =a Complexes. Example: BUTESIN PICRATE y Picric acid (antiseptic) + Butesin (anesthetic) - 1% ointment used for burns and abrasions0=§-0-C4H19 HO: | iby no~/ \-xo: a [~ sm 2 NOx Disadvantages: Y Picric acid + Carcinogenic Agents =m complex =p increase carcinogenic activity. Inclusion Complexes: > One compound is trapped in lattice/cage like structure of other compound. > Interaction are due to suitable molecular structure. > Prediction of complex formation is difficult. Channel types - > Channels are formed by crystallization of the host molecules, the guest component is usually limited to long, unbranched straight chain compounds. > Host (tubular channel)- Deoxycholic acid, urea, thiourea, amylose > Guest (long unbranched straight chain compounds)- paraffin, esters, acids, ethanol. > Example: Starch-iodine solution (starch-host) Urea-methyl a-lipolate (urea- host) Applications: Y Seperation of isomers; Dextro, levo-terpineol are separated using Digitoxin. ¥ In analysis of dermatological creams, long chain compounds interfere and removed by complexation with urea.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. Use: Y Due to their large surface area they are used as catalysts. Clathrates - > It is available as white crystalline powder, during crystallization, certain substances form a cage-like lattice in which the coordinating compound is entrapped. Example: warfarin sodium (water + isopropyl alcohol) » Hydroquinone form cage with hydrogen bonds and hole 4.2A9, have diameter of > This can entrap methanol, carbon dioxide, hydrochloric acid. Applications:1. Synthetic metalo- alumino silicates act as molecular sieves. 2. The pores store volatile gases and toxic substances. 3. The entrapped molecule can be removed by physical process. 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 cavity is hydrophilic in nature. pyranose units attached by a-1,4 linkages. Guest motecate hydrophobic Applications: ‘The interior of the cavity is relatively hydrophobic, whereas the entrance of Cyclodextrins are cyclic oligo sacchirides containing minimum of 6 D- glucoiced solubility: Retonic acid (solubility= 0.5mg/ml) Retonic acid + B-CD lubility= 160 mg/ml) - Enhanced dissolution: Famotidine/ Tolbtamide + B-CD 3. Enhanced stability: Asprin/Ephedrine/Testosterone + B-CD (no reaction with other functional groups) 4. Sustained release: Ethylated B-CD + Diltiazem Applications of Complexation: > Physical state: * Complexation process improves processing characteristics by converting liquid to soild complex. + Example: B-cyclodextrine complexes with nitroglyerine and forms crystalline inclusion complex which is explosion proof. > Volatility: + Complexation process reduces Drug volatility for following benefits; © To stabilise system. o Overcome the unpleasant odour © Example: in the formulation, 12 complexes with Poly Vinyl Pyrollidone, PVP. » Solid state stability: + Complexation process enhances solid tability of drugs. + Example: B-cyclodextrine complexes with Vitamin A and D are stabilised. > Chemical stability: ¢ Complex formation alter chemical reactivity (Mostly inhibit and sometim jalytic), + Example: The hydrolysis of Benzocaine is decreased by complexing with Caffeine. > Solubility: ¢ Complexation process enhances solubility of drug,a « Example: At low concentrations, caffeine enhances solubility of PABA (Para Amino Benzoic Acid) by complex formation. > Dissolution: * Complexation process enhances dissolution of drug which is possible by enhanced solubility. « Example: B-cyclodextrine increases the dissolution of Phenobarbitone by inclusion complex. > Partition co-efficient: * Complexation process enhances the partition coefficient of certain drugs. Example: Permanganate ion with benzene through ion pair mechanism. > Absorption and Bioavailability: Complexation process reduces the absorption and bioavailability of Tetracycline by complexing with cations like Ca", Mg” and AI. © Complexation process enhances the bioavailability of Indomethacine and Barbiturates by complexing with B-cyclodextrine. > Reduced toxicity: ¢ B-cyclodextrine reduces ulcerogenic effects of Indomethacine. + B-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 that is normally present in the body. + The complex penetrate cell membranesof the malarial parasite and thereby exhibit greater antimalaria activity. > Antitubercular activity: * PAS (Para Amino Salysylic acid) complexes with Cupri¢ 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: * Ta” complexes with citrate are used for diagnosis of Kidney and measurement of Glomerular Filtration Rate. Prepared by: Sridevi Murugesan Department of Pharmaceutics SNS College of Pharmacy and Health SciencesComplexes me Gonmed becuse 9 Hs door Arc epton mecloutom . lou is te norilial Molecule DA Love non-mitollr Bbubsterregr pst corm t Lorra Per 9 Bienen ccepenn is maine Ape torn dovsLé Adds BP302T. PHYSICAL PHARMACEUTICS-I (Theory) uM the nn wr ng be a neutered CADW UNIT-IV - COMPLEXATION AND PROTEIN BINDING: * INTRODUCTIOT is the process of complex formation that is the process of characterization > Complexati 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 st small molecules or with v complex. Drug molecules can form complexes with othe ‘macromolecules such as proteins. ‘A coordination complex is the product of a Lewis acid-base reaction in which neutral wond to a central metal atom (or ion) by coordinate vy molecules or anions (called ligands) be covalent bonds ° ‘Simple ligands include water, ammonia and chloride ions. Once complexation occurs, the physical and chemical properties of the complexing Dayperwnrl SRP juvotucl an species altered are; A : - © Solubility, The fi Mee aud abit ed puerlapS tou dangly meted Lo1n © Stal «Partition co-efficient, Orbe f Bee aeeception, AA 100 hiqewal (e wis base) wales Clechinr Pair. Energy emission. © Conductance of the drug. AA fle . Forces involved in complex formations sy mae, LO Ucceph a one F COveate 2 Lt |p iF ‘© Covalent bond '* Co-ordinate covalent bond. vv v © Van der Waals force of dispersion. ‘* Dipole-Dipole interaction. OnL alom ayes © Hydrogen bond. bot oven. ond Conus > Beneficial effects of complexation: Co- Ord jncte onsen athe Catlg of Drug complexation, therefore, can lead to beneficial properties such as enhanced aqueous solubility (e.g., theophylline complexation with ethylenediamine to form inclusion complexes of labile drugs with f & aminophylline) and. stability (e cyclodextrins).«Complexation also can aid in the optimization of delivery systems (¢.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 excretion. CLASSIFICATION OF COMPLEXATION: > 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 © Pi(x) complexes © Sigma (a) complexes © “Sandwich” compounds > Organic molecular complexes © Quinhydrone type © Pioric 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 providles only one site for binding with metal » The ammonia molecules in hexamminecobalt (Ill) chloride, as the compound {Co(NH3)6]3+ CI3-, 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(H20)6]3+. Sach ligand donates a pair of electrons to form a coordinate covalent link between itself and the central ion having an incomplete electron shell. CoS + 6NHy = (Co(NH)o]** 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. WOOe-HRe cH,= COOH) Ne cugcts-W | yaestacacks Hooc- CH= COOH fecal enras Es 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 (x) complexes — Aromatic bases (Benzene, toluene and Xylene) form pi-bond complexes with metal ions like Ag by Lewis acid-base reactions.i in ion of a sigma-bond > Sigma (6) complexes — sigma bond complexes involve in the formati ig between ion and a carbon of aromatic ring. ath in the > “Sandwich” compounds — The are relatively stable complexes involving ager 3 orbit delocalized covalent bond between the d-orbital of transition metal and a molecular 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 A, 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. ON ON _— cu, : a ony 7 « \< + ncot \-x0.- CV« +HCO- ( — cH, — : ( N Hy ON 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. example, the polar nitro groups of trinitrobenzene induce a dipole in the readily. and other drug complexes - +h as benzocaine, procaine and tetracaine form complexes with caffeine, of acidic drugs are known to form complexes with caffeine, ailCaffeine Benzccaine Fig 2. Structure of caffeine and Benzocaine, Quinhydrone ype — > The molecular complex of this type is obtained by mixing alcoholic solutions of equimolar quantities of hydroquinone and benzoquinone. Hydroquinone HO OH (electron rich) } Overlap (x cloud) ° O Benzoquinone (electron = deficient) 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 drugs. > Eg. Polymers: carbowaxes, pluronics etc. Drugs: tannic acid, salicylic acid, phenols ete. Carboxy methyl cellulose + Amphetamine — Poorly absorbed drugs. erie acid types — eric acid, being a strong acid, forms organic molecular complexes with weak bases, it combines with strong bases (anesthetic activity of butesin) to yield salts. are also called occlusion compounds in which one of the components is open lattice or cage like crystal structure of the other. d by crystallization of the host molecules, the guest component is snbranshed straight chain compounds.jest and host. P : or nic) layers of gu > They form alternate monomolecular (monoatomic) shit rust ese may be U > Their uses are currently quite limited; however th account of a larger surface area. Clathrates - i form owder, during crystallization, certain substances > Itis available as white crystalline p 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. f-eyclodextrine complexes with nitroglyerine > Volatility: © Complexation process reduces Drug volat ity for following benefits; © Stabilise system. © Overcome unpleasant odour (I2 complexes with Poly Vinyl Pyrollidone, PVP). > Solid state stability: * Complexation process enhances solid state stability of drugs © B-cyclodextrine complexes with Vitamin A and D. > Chemical stability: it) * The hydrolysis of Benzocaine is decreased by complexing with Caffeine. Solubility: * Complex formation inhibit chemical reactivity (Mostly inhil © Complexation process enhances solubility of drug, Caffeine enhances solubility of PABA (Para Amino Benzoie Acid) by complex formation. fution: plexation process enhances dissolution of drug, lodextrine 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’?, Mg"? and AI’ © Complexation process enhances the aborption of Indomethacine and Barbiturates by complexing with f-cyclodextrine, > Reduced toxicity: © B-cyclodextrine reduces ulcerogenic effects of Indomethacine. * B-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: Ta” complexes with citrate are used for diagnosis of Kidney and measurement of Glomerular Filtration Rate. of analysis Complexation: fethod of Continuous Variation: er the Job, the species possess several characteristics that are;© Refractive Index. * Spectrophotomettic extinction coefficient ; ue of property is Id good. > Principle - When there is no complexation between the species, the val additive, On complexation these properties changes but additive rule do not hol ‘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 place, > 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 geaph 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 immis ible 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. Ie - When a solute complexes with an added substance, the solute distribution sm changes depending on the nature of the complex. complexation of iodine by potassium iodide. ae K* ly librium stability constant, PV (h] [KT]> The distribution coefficient of iodine between disulfide and water is 625 > The K value of lodine-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 decteased, The change in solubility is a sign of complexation, > 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. ki D+A DA ky > Where, D and A represents electron donor and acceptor, ki and kz are interaction rate constants. > K=ki/ k= Equil } 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. > AdlA = (I/e) + (1/Ke) (1/Dp) rium or stability constant for complexation, > Where, Aoand Do are the initial concentration of acceptor and donor species in mole/litre. Eis the molar absorptivity of the charge-transfer complex at its particular wavelength and K is the stability constant in litre/mole. > A plot of Ao/A versus 1/Do results in straight line with a slope of I/Ke and an intercept of We. The spectrometric method used to investigate the interaction of nucleic acid bases with hol, epinephrine and isoproterenol. binding: ing molecules are generally the macromolecules such as protein, DNA or proteins are particularly responsible for such an interaction. ‘of complex formation of drug with protein is called as protein binding> AS aprotein 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: be 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. Tonic bonds 4. Van der Waal’s forces, > Irreversible drug binding, though rare, arises as a result of covalent binding and is often a sason for the carcinogenicity or tissue toxicity of the drug, > Absorption - As we know the conventional dosage form follow first order kineties. So When there is more protein binding then it disturbs the absorption equilibrium. Distribution ~ A protein bound drug in patticular does not eross 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, Sulfonamide, + 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. ©.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. + ¢.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, .rs affecting protein binding: ig related Factor. Physicochemical properties of drug Increase in lipophilicity increases the drug binding with the protein. 10y Won UU - Ue ty fas I cl | a bloc vlan Ce ae "chia SAP od Pong _ Cty jug laimatory t/¢ 9 ROY rrabys) aMlal b[¢ © Total concentration of drug - Alternation in drug and protein concentration alter the drug protein binding, > Protein related Factors, © Physicochemical properties of protein ~ Lipoprotein bind with lipophilic drugs. © Quantity of protein — Disease state affect the concentration of protein in blood i © Number of binding sites ~ Albumin has more no of binding sites. TAO Yaa Hag im Band > Affinity and Magnitude of association constant. bo Why ze > 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 > &l-Acid glycoprotein Lipoproteins » Globulins. > Binding of drug to human serum Albumin ~ ‘© Itis 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 HA. CHY Men Seu abbrarey, ‘© Four different sites on HSA for drug binding. ao ‘ educa tel like NSAUDS Site I: warfarin and azapropazone binding site, — Site I: diazepam binding site, — an Xyeky MI ulcig Sager , Site I: digitoxin binding site. ~ N@ carb doub'ure Site IV; tamoxifen binding ste, Cancer b/& ling of drug to a1-Acid glycoprotein — , called as orosomucoid. It has a M.W, 44,000. 1a cone. range of 0.04 t0 0.1 g %. Aono, of basic drugs lke imipramine, lidocaine, propranolol, and quinidine, ‘ aijaa @ bounded fuel Play > Binding of drug to Lipoproteins ~ bond Se * Binding by Hydrophobic Bonds, Non-competative © Mol wt: 2-34 Lacks dalton, / cl sters. Outside: © Lipid core composed of: Inside: triglyceride & cholesteryl “ “ i asic: Cl mazine. Apoprotein, e.g, Acidic: Diclofenac. Neutral: Cyclosporin A. Basic: Chlorpro © Its types are LDL, HDL, VLDLand Chylomicrons. Binding of drug to Globulins — v © al Globulin (Transcortine /Corticosteroid Binding globulin) - Steroidal drugs, Thyroxin & Cyanocobalamine (Vit B12). * a2 Globulin (Ceruloplasmine) - Vitamin A, D, E, K. * BI Globulin (Transferin) - Ferrous ions. * B2 Globulin ~ Carotinoids. * yGlobulin— 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+ Dp == D > Applying the law of mass action, K=[PD)/ [P] [Dr] > The [PD], [P] and [D] are the concentration of protein-drug complex, protein and drug in Mol/L. K[PI[ Dr] = [PD] > Free protein concentration can obtain as; +[PD] Pr] _ [PD] > [Pr] is the total protein, the equilibrium or association constant (K) is; > Substituting the (P] in last equation, K[P][ Dy] = [PD] K ([Pr] - [PD}) [Dr] = [PD] > Where, Dris the free drug, K [Pr] [Dr] —K [PD] [Dr] = [PD] K [Pr] [Dr] = [PD] + K [PD] [Dr] K [Pr] [Dr] = [PD] (1+ K [Dr])[PD] = (K [Px] [Ds])/ (1+ K [De]) [PDJ/ [Px] = K [De]/ 1+ K [De] > Let R be expressed as moles of drug bound [PD] per mole of total protein [Pr] R=[PDJ/ [Pr] = K [DrJ/ 1+ K [Dr] > IfVis the number of independent binding sites available then R, R=V(K [Dr]/ 1+ K [De]) /R= 1/VK[Dg] + 1/V > The graph is plotted between 1/R versus 1/[De], called Klotz reciprocal plot, gives 4 straight line whose slope is 1/VK and intercept is V. 1/DF Fig 1. Klotz reciprocal plot. R+RK [Dr]=VK [Dr] R/[Dr] = 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 receptor. The action of drug to remove the toxic effect of metal ion from the human bodies is through the complexation reaction.AL Yy Shability complexes Nes ces brucliny g bet'™ Donon £ acceptar is Brruqa thor Any algo becowmo —LWO. > Ithas 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 help in drug action termination and reduction in drug toxic a > Examples : ion, + Tetracycline and Calcium — Poor absorbed complex. * Polar drug and complexing agent — Well absorbed lipid soluble complex. * Carboxy methyl cellulose and amphetami —Poor absorbed complex. * PVP and I~ 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; AG = -2.303RT Log K © ‘The Standard Enthalpy Change AH may be obtained from the slope of a plot of Log K ‘Versus 1/T, thus the equation will be; Log K = - (AH/2.303R) x (1/T) + Constant > When the value of K at two temperatures are known, the following equation can be written as; Log (K2/K1) = - (AH/2.303R) * (T2-TVTiT2) > The Standard entropy change may be obtained from the expression; AG=AH-TAS > As the stability constant for molecular complexation increases, AH and AS becomes more negative, > As nding between the donor and receptor becomes stronger, AH becomes more negative. > Since the specificity of interacting sites becomes negative, AS also become more negative. > But the extent of change in AH is large enough to overcome the unfavourable entropy change resulting in negative AG value and hence complexation. Tha Stability cans} g medal complexes ooo to thenrodynumre Rasp. titer Pres er0eg y (Mr GW eubupy (as) :References — 1. Subrahmanyam CVS, Physical Pharmaceuties-1, 15" Edition. New Delhi: Vallabh Pralashan; 2019. pp. 450-484. 2, Agarwal SP, Khanna R. Physical Pharmacy. 2" 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 Pharm: Salipur, Cuttack, Odisha ~ 754202. India. Tel: 09938860284