COMPLEXATION & PROTEIN BINDING.pptx

PHYSICAL PHARMACEUTICS I.
B.PHARM III - SEMESTER.

1. COMPLEXATION AND PROTEIN BINDING BALASUNDARESAN M

2. Contents Introduction Classification of Complexation Applications Methods of analysis Protein binding Complexation and drug action Crystalline structures of complexes and thermodynamic treatment of stability constants.

3. Introduction A complex may simply be defined as a species formed by the association of two or more interactant molecules or Ions. Such an association takes place because one of the interactants called the substrate accepts a pair of electrons donated by the other interactant called the ligand . As a result a bond is formed between the two interactants (substrate and ligand) and the compound thus formed is called the complex. The complex formation may be represented by the following chemical equation. mS + nL ↔ S m L n Where, m= no. of substrate molecules n= no. of ligands S=substrate L=ligand S m L n =c o m p l ex

4. Types of Complexes Complexes may be divided broadly into two classes depending on whether the acceptor component (Substrate) is a metal ion or an organic molecule a. Metal-ion complexes In t h i s o ne i n t e r ac t a n t i s m e t a l i on ( sub s t r a t e) and oth e r interactant is inorganic or organic molecule /ion (Ligand). The bond formed as a result of transfer of lone pair of electrons is called coordinate bond . Th e m e t al- i on c oo r d i n a t e co m pl e xe s c on s i s t of met a l i on (substrate) bonded to a electron pair donor (ligand) .

5. Types of Complexes.. b. Organic Molecular complexes I n t h e se c o m p l e x e s , bo th t h e i n t e r ac t a n ts a r e o r g a n i c molecules . dono r - acce p t o r t yp e o f i n t e r ac ti o n s e x ist in A s ob s e rv e d in i no r g an i c - m e t a l c o m p l e x e s , e l ec t ro n o r g a n ic molecular complexes. The individual species are bound together by weak forces ( Van der Waals forces, dipole-induced dipole interactions) and hydrogen bonding .

6. Coordination number of the complex Th e no. of bonds be t w een t he me t a l i on and t he l i ga nd is called coordination number of the complex. Thus , coor d i na t i on nu m b er of Cu ( NH 3 ) 2 2 + and Cu ( NH 3 ) 4 2 + a r e 2 and 4 respectively. 2+ Cu(NH 3 ) 2 2 Cu(NH 3 ) 4

7. Unidentate Vs. Multidentate Ligand Ammonia has a single basic group capable of bonding to a metal ion and hence it is unidentate ligand . Ethylene diamine has two accessible basic binding sites and it is called bidentate ligand. Ethylene diamine tetraacetate is a hexadentate . If a metal ion binds with a multidentate ligand it usually forms with the ligand a cyclic complex called chelate . If the ligand forms a stable, water soluble metal chelate , it is called a sequestering agent.

8. Classification of Complexes Metal complexes Inorganic types Chelates Olefin type Aromatic types Organic molecular complexes Drug and caffeine complexes Polymer types Picric acid types Quinhydrone types Inclusion complexes Channel types Layer types Clatharates Monomolecular types

9. 1. Metal Complexes In this one interactant is metal ion (substrate) and other interactant is inorganic or organic molecule (Ligand) . The bond formed as a result of transfer of lone pair of electrons is called coordinate bond . Classification of Metalcomplex Inorganic types Chelates Olefin type Aromatic types

10. 1.A. Inorganic type Complexes Werner postulates A metal ion complex have two types of valences Primary valence Secondary valence Primary valences is ionisable and satisfy by negative ions Secondary valences is non-ionisable . They are satisfied by neutral or negative molecules . The secondary valance is equal to the coordination number. Same type of anion or molecule may be held by individually or both types of valences The nonionic valences are directed towards definite positions in space. Primary valence=2 Secondary valence=6 Coordination Sphere

11. The concept of hybridization can be extended to understand the formation of complexes Ligands donates lone pair of electrons to form complex and these electron pair is accepted by metal ion. In metals, all the orbitals such s , p and d are to be considered to account complexation Ground state electronic configuration of cobalt The electrons in half-filled orbitals shifts to other orbitals in order to fill them as electron pairs . This creates completely vacant orbitals in the metal electronic configuration. Now ligands (NH 3 and Cl ) donates lone pair of electrons to those vacant orbitals of the metal ion. This results in the formation of complexes .

12. 1.B. Chelates Chelates are a group of metal ion complexes in which ligand provides two or more donor groups (lone pair of elcetrons) to combine with a metal ion. Ammonia has a single basic group capable of bonding to a metal ion and hence it is unidentate ligand . Ethylene diamine has two accessible basic binding sites and it is called bidentate ligand. Ethylene diamine tetraacetate is a hexadentate . If a metal ion binds with a multidentate ligand it usually forms with the ligand a cyclic complex called chelate . If the ligand forms a stable, water soluble metal chelate , it is called a sequestering agent. Applications: Increase in stability, purification of hard water analysis of drugs Metal (M)

13. 2. Organic Molecular Complex In these complexes, both the interactants are organic molecules . The individual species are bound together by weaker forces (Van der Waals forces, dipole-induced dipole interactions) or hydrogen bonding. Classification of organic molecular complex Drug and caffeine complexes Polymer types Picric acid types Quinhydrone types

14. A number of acidic drugs are known to form complexes with caffeine . Drugs such as benzocaine, procaine and tetracaine form complexes with caffeine. Mechanism: Dipole-dipole force or hydrogen bonding between the polarized carbonyl groups (C=O) of caffeine and the hydrogen atoms of the acid . 2.A. Drug and caffeine complexes

15. 2.B. Polymer Complexes Many pharmaceutical additives such as polyethylene glycols (PEGs), carboxymethyl cellulose (CMC) contain nucleophilic oxygen . These can form complexes with various drugs. These types of complexes produce incompatibilities with the dosage forms which may delay absorption of drugs and produce physical, chemical and pharmacological effects. E.g. Polymers: carbowaxes, pluronics etc. Drugs: tannic acid, salicylic acid, phenols etc. C arboxyl m e t h y l c el l u l o s e ( C M C ) + A m phe t a m i ne absorbed complex = p oor l y Nucleophilic: tendency to donate electrons

16. 2.C. Picric acid Complexes Picric acid, being a strong acid , forms organic molecular complexes with weak bases , whereas it combines with strong bases (butesin) to yield salts . Ex: The antiseptic activity of picirc acid is combined with anesthetic activity of butesin.

17. 2.D. Quinhydrone Complexes T h e m o l e c u l a r c o m p l e x o f t h i s t y p e i s o b t a i n e d b y m i x i n g a l c o h ol i c solutions of equimolar quantities of hydroquinone and benzoquinone . This complex settles as green crytals.

18. 3. Inclusion Complexes Inclusion complexes are also called occlusion (closure) compounds in which one of the components is trapped in the open lattice or cage like crystal structure of other. Here, the interaction is not due chemical reactivity , but because of the favorable molecular architecture . Classifications of Inclusion complex Channel types Layer types Clathrates Monomolecular types

19. 3.A. Channel lattice type In t h is on e m o l ec u le ( ho s t) is tr a pp e d w it h in t h e s p ir a l o r c a g e li k e structure of others (guest). Ex: Starch-Iodin e, Urea-Methyl α-lipoate . Host: Urea, thiourea etc., Guest: Methyl α-lipoate, paraffins, acids, ethyl alcohol etc., Applications These are used for separation of optical isomers. Ex: Terpineol (guest) has been solved by use of digitoin (Host). In dermatological creams , long chain compounds (guest) interfere in the assay methods. Such ingredients are removed by using channel type complexation with urea.

20. 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. 3.B. Layer type H o st Host Guest Catalysis: acceleration of chemical reaction

21. 3.C. Clathrates During crystallization, certain substances form a cage-like lattice in which the coordinating compound is entrapped . Ex: Hydroquine molecules crystallize in to the cage like structures with hydrogen bonding. The holes permit the entrapment of small molecules such as methyl alcohol, Hcl, CO 2 H y droquine molecules Applications These materials are used to store gaseous, volatile and toxic substance by the mechanism of Clathrates

22. 3.D. Monomolecular type 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 . Th e i n te r i o r of t he ca v i t y i s re l at i v e l y hydrop h obic, w her e as t he entrance of the cavity is hydrophilic in nature. Applications Enhanced solubility Enhanced dissolution Enhanced stability (Protects the drug) Sustain release (Retard the drug release)

23. Method of Analysis of complexes Methods Continuous variation method Distribution method Solubility method pH variation method

24. 1. Continuous variation method Physical c ons t ant, p r op e rt ie s su c h as d ie l e c t ric re f ra ct iv e i ndex e t c . , a re characteristics of particular species. When no complexation b/w species (A and B) the value is linear (_ _ _ _). In case of c o m pl e xa t i on no li n e a r t he pheno m enon obs erv e d. Th i s is indication of complexation (1:1).

25. 2. Distribution method The distribution behavior of a solute b/w two immiscible liquids is expressed as distribution coefficient or partition coefficient. When the solute forms complex with added substance the distribution pattern changes depending on the nature of complex (water soluble or insoluble) Ex: I + K + I - 2 Iodine+Pottasium iodide K + I - 3   3 The equilibrium constant K + I - [I 2 ] [ K + I - ] K= Distribution coefficient of I 2 b/w carbon disulfide and water= 625 Distribution coefficient of K + I - Complex b/w carbon disulfide and water= 954 3 Iodine-Pottasium iodide complex

26. 3. Solubility method Caffeine-Diff conc. PABA- Excess conc. Agitated in constant temp bath The samples are filtered and analyzed for drug content At zero concentration of caffeine , The point A on y axis indicates the solubility of PABA in water. With addition of caffeine, the solubility of PABA is increased up to B. At point B , the solution is saturated with complex B. On further addition of caffeine, the complex continuous to form and starts precipitates upon the point C. At point C , Only small fraction of free PABA is available in solution. On further addition of caffeine, the free PABA combines with caffeine and forms higher complexes. When the components in a mixture produce a complex , the solubility of one of the components may be enhanced or inhibited . The change in solubility profile is taken as a criterion to decide the complexation behavior. Experimental

27. 4. pH Titration Method The pH titration method is used for the analysis of complexes provided such an interaction produces a change in the pH of the mixture. Ex: Chelation of cupric ions by glycine molecules Cu 2+ +2NH 3 + CH 2 COO -  Cu(NH 2 CH 2 COO) 2 +2H + Cupric ions+Glycine Since 2 H + is released on complexation results in decrease in pH Experimental A known quantity of glycine is titrated against NaoH Similarly, complex solution is titrated against NaoH Titration with NaoH permits the estimation of Conc. Of ligand (~2H + ) bound to metal ion The horizontal distance b/w two curves gives the amount of alkali (NaoH) consumed in the reaction. The quantity of NoaH is exactly equal to the concentration of ligand bound .

28. Applications Physical state: It is possible to convert a liquid to a solid complex and thus improve its processing characteristics. For example, nitroglycerin is transformed to its crystalline inclusion complex with β- cyclodextrin ; the complex contains 15.6% nitroglycerine and is explosion-proof . Volatility: When it is desirable to reduce substrate volatility in order can to stabilize a system or to overcome an unpleasant odour, complex can offer the advantage. For example., in the formulation, iodine is complexed with polyvinylpyrrolidone (PVP) Solid state stability: The solid state stability of drugs can he enhanced by complexation. For example, beta cyclodextrin complexes of vitamin A and D are stabilised chemically . Chemical stability: Complex formation will alter chemical reactivity . Either inhibitory or catalytic effects may be observed. For example, the rate of hydrolysis of benzocaine is reduced by complexing it with caffeine. Solubility: Many examples of solubility enhancement by complexation halves been reported. For example, at low concentrations, caffeine enhances the solubility of p- aminobenzoic acid (PABA). Dissolution: If solubility is enhanced , the dissolution rate should also increase and complexation is one possible method. The dissolution rate of phenobarbital is enhanced by using β-cyclodextrin inclusion complexes. Partition coefficients: This will be enhanced by complexation Ex: I 2 and KI complex

29. Applications.. Absorption and bioavailability: The absorption and bioavailability of tetracyclines was reduced when coadministered with divalent cations such as calcium, magnesium and aluminum, on account of formation of insoluble metal complexes. On the other hand, beta- cyclodextrin complexes of indomethacin and barbiturates have enhanced the drug bioavailability . Reduced toxicity: Cyclodextrins are effective in reducing the ulcerogenic effect of indomethacin and local tissue toxicity of chlorpromazine. Antidote for metal poisoning: Toxic metal ions such arsenic, mercury, antimony etc., bind to -SH groups of various enzymes and interfere with their normal function. Compounds such as dimercaprol (BAL. British Anti-Lewisite) form water soluble complexes with these metal ions and eliminate them rapidly from the body. Other examples are: Beryllium poisoning: Salicylic acid. Lead poisoning: EDTA Antibacterial activity: Antitubercular drug, PAS (p-aminosalicylic acid) is known to form a cupric complex and also a chelate. Cupric chelate has shown greater in vivo antitubercular activity in mice than cupric complex .

30. Complexation and Drug action/activity Write two applications of complexation each in formulation and drug action A: Formulation: Physical state, volatility, solid state stability, Drug action: Absorption and bioavailability, reduced toxicity, antibacterial activity. How does complexation influences the drug action? Explain with the help of two examples A:Once complexation occurs, the physical and chemical properties of the complexing species are altered. These properties include solubility, stability, partitioning, energy absorption and emission, and conductance of the drug. Complexes can alter the pharmacologic activity of the agent by inhibiting interactions with receptors. Examples: Absorption and bioavailability, reduced toxicity, antibacterial activity.

31. PROTEIN BINDING Protein Binding refers to complex formation between small molecules (Drug molecules) and blood proteins such as albumins and globulins . Of all the constituents of blood that might take part in the formation of complexes with drugs, the most important and most studied is the protein serum albumin . The concentration of this normal human serum albumin (HSA) is remarkably high in the blood.

32. Binding is a function of the affinity of the protein molecule for the drug molecules and also the concentration of drugs and proteins . The interaction between protein (P) and drug (D) for a simple case of 1:1 protein drug complex can be represented as follows: P + D PD Applying the law of mass action, the expression is K=[PD]/[P] [D] or, [PD] = K[P] [D] Where, K= Association Constant [P]= Concentration of unbound drug [D]= Concentration of bound drug [PD]= Concentration of protein drug complex

33. Methods of determining Protein Binding Methods: Equilibrium dialysis, Ultra filtration and Electrophoresis a. Equilibrium dialysis In this method, a protein solution is enclosed within a membrane (semi-permeable membrane such as cellophane) which is permeable to small drug molecules and not to macro-molecule, the protein molecules. Drug is placed in outer vessel. As the dialysis proceeds the drug molecule penetrate through the membrane and enters bag, as result protein binding takes place. Albumin, being a macromolecule cannot pass the membrane. Samples from inside the membrane are withdrawn and analysed. If binding occurs the drug concentration in the bag should be grater than its concentration out side

34. Methods of determining Protein Binding b. Dynamic dialysis method The dynamic dialysis method is based on the rate of disappearance of the drug from a dialysis bag which is proportional to the concentration of unbound drug. The dialysis process follows the rate law d [ Dt ]  k [ Df ] dt Where, [Dt]= conc. of total drug. Mol/L [Df]= conc. of free (unbound) drug in the bag, Mol/L k= First order rate constant or permeability constant

35. Equilibrium Vs. Dynamic dialysis Semi permeable membranes (cellophane): Permeable to small drug molecules and not to macro-molecule , the protein molecules

36. Significance of Protein Binding Plasma protein bound drugs are unable to penetrate biological membranes . Hence they cannot be excreted or biotransformed . Only free form of the drug is pharmacologically active. Binding between plasma protein and drug is a reversible interaction . The bound drug acts as a reservoir which releases the drug in the free form to replace the drug which is removed from the body by excretion or biotransformation . This effect prolongs the duration of action of the drug. Drugs may exhibit competitive binding due to differences in the affinities of the drugs for the binding sites in the protein molecule. Such competitive binding may be advantageous or disadvantageous . For example, displacement of antibiotics by other drugs provides better effect due to the release of free antibiotic for action . But displacement of protein bound anticoagulants may cause excessive bleeding. Drugs may interact with other tissues also similar to drug protein binding . For example, very lipid soluble drugs like thiopentone accumulate in the body fat, and tetracyclines in teeth. The concentration of a drug in the plasma is often a measure of drug distribution in the body since the drug distributed in various organs of the body show a dynamic equilibrium. If a drug protein binding is very high , the concentration of free form of the drug may not be sufficient to elicit pharmacological activity . This can be remedied by giving sufficiently more amount of drug to provide the required concentration for activity. Protein binding may lead to increase in solubility of certain drugs eg. discoumarol.

37. Complex as drugs a. Cisplatin This is a coordination compound that has broad application in human cancer chemotherapy. It has divalent platinum bound to two potentially leaving groups, the chlorides. Two NH3 groups are bound irreversibly and in firm coordinate covalent bond, trans- position to the chlorides.

38. Complex as drugs… (b) Povidone-iodine: Polyvinylpyrrolidone (PVP) is a water soluble- polymer and forms a water-soluble complex with iodine. Since, the complex is soluble in water, drug can be removed easily from the site of application. Povidone-Iodine is a safe and effective anti-bacterial and germicidal agent. It is available as soap for the hand wash of healthcare personal, surgical hand scrub and skin preparation. It is also used post-operatively for wound cleansing and protection.

39. Questions Essay (10 M) What is protein binding and explain the significance of protein binding. Write about the method of analysis of complexation Short notes (5 M) Write two applications of complexation each in formulation and drug action. How does complexation influences the drug action? Explain with the help of two examples D e f i n e c o m p l e x c o m p o u n d s . W r it e a bo u t t h e v a r i o u s t y p e s o f m e t al complexes. Explain about the werner theory of complexation. W r i t e a b o u t v a r io u s t h e t y p e s o f o r g a n i c m o l ec u l a r a n d i n c l u s i o n complexes.