Complexation and protein binding

n n Chapter n n n n n n

6

Complexation and Protein Binding

Complexation is one of the several ways to enhance favourably the physicochemical properties of pharmaceutical compounds. Complexation may broadly be defined as covalent or noncovalent interactions between two or more species capable of independent existence.

Although the classification of complexes is somewhat arbitrary, the differentiation is usually based on the types of interactions and species involved (e.g. coordination complexes, organic molecular complexes and inclusion complexes). Drugs can form complexes with other small molecules, with other drugs or excipients and with macromolecules such as proteins. Once complexation occurs, the physical and chemical properties of the complexing species are altered. These properties include stability, solubility, partitioning and conductance of the drug.

The applications of complexation in pharmacy are enumerated as follows: Solubility/ Dissolution: Many examples of solubility enhancement by complexation have been reported. For example, complexation of theophylline with ethylenediamine to form aminotheophylline enhances solubility and dissolution.

Stability: Solid-state stability and chemical stability can be improved by complexation. For example, the rate of hydrolysis of benzocaine can be reduced by complexing it with caffeine and the volatility of iodine can be reduced by complexing it with PVP. Bioavailability: Complexing drugs with cyclodextrins results in complexes that exhibit higher ocular, oral and transdermal bioavailability compared to free drug.

Antidotes: Therapeutically chelating agents are used as antidotes in heavy metal poisoning.

For example, CaNa2EDTA is used in cases of lead poisoning, dimercaprol in cases of mercury and arsenic poisoning, deferoxamine mesylate in cases of iron poisoning and salicylic acid in cases of beryllium poisoning.

Therapeutics: Some complexes possess pharmacological actions and are thus used as drugs.

For example, 1. Cisplatin and carboplatin are platinum (II) complexes that are used as anticancer agents.

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2. Povidone–iodine is a water-soluble complex of PVP with iodine and is used as an effective topical antiseptic and germicidal.

3. EDTA is used in the treatment of urinary calauli, calciferous corneal deposits and hypocalcaemia.

4. 8-hydroxyl quinoline forms complex with iron, resulting in better penetration through the cell membranes of the malaria parasite and better antimalarial activity.

5. Cupric chelate of p-amino salicylic acid possesses better antitubercular activity.

Titrations: Complexometric titrations are valuable to assay drugs containing metal ions such as magnesium trisilicate, calcium gluconate and calcium lactate.

Dosage form design: Complexation of drugs with excipients and polymers results in the development of novel drug delivery systems and sustained drug release devices.

As bioconstituents: 1. Haemoglobin and myoglobin are iron complexes that are essential for transport of oxygen in the blood and tissues.

2. Cytochrome c is a naturally occurring chelate involved in photosynthesis and respiratory systems.

3. Copper ion is present in haemocyanin, superoxide dismutase and cytochrome oxidase.

4. Cobalt is present in complexed form in vitamin B12.

5. Zinc is an important constituent of insulin.

COMPLEXES

Coordination complexes

Organic molecular complexes

Inclusion or occlusion complexes

1. Based on entrapment of guest into host 2. No-bond formations

1. Based on addition mechanism 2. Noncovalent interactions

1. Based on the donor– acceptor mechanism 2. Covalent interactions

Inorganic type

Quinhydrone type

Clathrate

Chelates

Picric acid type

Channel lattice

Olefin type

Caffeine complex

Layer type

Aromatic type

Polymeric type

Monomolecular

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Complexation and Protein Binding n 155

CLASSIFICATION OF COMPLEXES Although classification based on a rigid set of conventions is difficult, complexes are generally classified according to the type of complex that is formed.

Coordination complexes A coordination complex consists of a transition-metal ion (central atom) linked or coordinated with one or more counter ions or molecules to form an electrically neutral complex. The ions or molecules (Cl, NH3, H2O, etc.) directly bound with the central atom are called coordinated groups or ligands. The interaction between the transition-metal ion and the ligand often resembles a Lewis acid–base reaction in which the transition-metal ion (Lewis acid) combines with a ligand (Lewis base) by accepting a pair of electrons from the ligand to form the coordinate covalent or electrostatic bond. For example, Co3+ + 6 (:NH3)

–

[Co(NH3)6]3+Cl3

Hexaminecobalt (III) chloride

1. Cobalt ion (Co3+) interacts with ammonia (:NH3) to form hexaminecobalt (III) chloride coordinate complex.

2. Cobalt ion (Co3+) is the central metal ion or Lewis acid having an incomplete electron shell.

3. Ammonia (:NH3) donates a pair of electron to central metal ion and is called as ligand or Lewis base.

4. The bonding between metal or ligand is either electrostatic or covalent.

5. In solution this complex ionizes to form [Co(NH3)6]3+ and 3Cl– ions.

6. The number of ligands bound to the transition-metal ion is defined as coordination number.

The coordination number of cobalt is six, since six ammonia groups are complexed with the central cobalt ion.

Compound such as ammonia, which has a single pair of electrons for bonding with the central metal ion, is called as unidentate ligand. Ligands such as ethylenediammine with two basic groups are known as bidentate. A molecule with three donor groups is called tridentate.

Hard ligands are electronegative with Ethylenediaminetetraacetic acid (EDTA) has six electrostatic interactions, such as F ions points (two nitrogen and four oxygen donor and H2O molecules. groups) for attachment to the metal ion and is Soft ligands are polarizable covalent called hexadentate. If the same metal ion binds bonds, such as I –, Br –, and Cl –. with two or more sites on a multidentate ligand, the complex is called a chelate (Table 6.1).

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Table 6.1 Description and example of different types of ligands Ligand type

Example

Monodentate

I-, Br-, Cl-, OH-, H2O, NH3

Bidentate

Ethylenediammine H2C — CH2 H2N

Tridentate

NH2

Diethylenetriamine CH2CH2 H2N

Tetradentate

CH2CH2 NH

Triethylenetetramine CH2CH2 H2N

Hexadentate

NH

CH2CH2 NH

CH2CH2 NH

NH

Ethylenediaminetetraacetate O

O

–

O — CCH2

CH2C — O

–

–

O — CCH2

CH2C — O

–

O

O

NCH2CH2N

Several theories such as crystal field theory, molecular orbital theory and valence bond theory have been postulated to describe coordinate complexes. Crystal field theory focuses on the electrostatic interaction between ligands and the central metal ion. The molecular orbital theory shows how electrons are oriented to form covalent bonds in coordinate complexes, whereas the valence bond theory explains the nature of hybridization and the geometry of the molecule. Coordination complexes are classified as inorganic complexes, chelates, olefin complexes and aromatic complexes based on the nature of ligand.

Inorganic Complexes Transition-metals such as cobalt, iron, copper, nickel and zinc use their 3d, 4s and 4p orbitals in forming hybrids. These hybrids results in different geometries often found for the complexes of the transition metal ions. Inorganic ligands such as H2O, NH3, Cl –, Br –, I – and CN – donate a pair of electrons that enters one of the unfilled orbitals on the metal ion to form an inorganic coordination complex.

n

Complexation and Protein Binding n 157

Examples 1. [Co(NH3)6]3+ Cobalt (atomic number 27) has the electronic structure 1s22s22p63s23p63d9. When it forms a Co3+ ion, it loses the 3d electrons to leave 1s22s22p63s23p63d6. The ground state electronic configuration is 3d ↑↓

↑

↑

4s ↑

4p

↑

In complexation, the electron in a half-filled orbital shifts to other orbitals to create vacant orbitals, which are filled by electron pairs donated by a ligand, thus resulting in complex formation. ↑↓ ↑↓

↑↓ d2sp3 octahedral

2. [Cu(NH3)4]2+ Copper (atomic number 30) has the electronic structure 1s22s22p63s23p63d104s1.

When it forms a Cu2+ ion, it loses the 4s electron and one of the 3d electrons to leave 1s22s22p63s23p63d9. The ground state electronic configuration is 3d ↑↓

↑↓

↑↓

4s ↑↓

4p

↑

In complexation, the electron in a half-filled d orbital shifts to the p orbital (stable state) to create vacant orbitals that are filled by electron pairs donated by the ligand, thus resulting in complex formation. The complex thus formed is called as inner sphere complex since the ligand lies below a partially filled orbital. ↑↓ ↑↓

↑↓ ↑↓

↑

dsp2 square planer 3. [Fe(CN)6]3+ Iron (atomic number 26) has the electronic structure 1s22s22p63s23p63d64s2. When it forms an Fe3+ ion, it loses the 4s electrons and one of the 3d electrons to leave 1s22s22p63s23p63d5. The ground state electronic configuration is 3d ↑

↑

↑

4s ↑

↑

4p

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In complexation, the electron in a half-filled orbital shifts to other orbitals to create vacant orbitals that are filled by electron pairs donated by the ligand, thus resulting in complex formation. The complex is called as outer sphere complexes since the ligand lies above a partially filled orbital. ↑↓ ↑↓

↑

d2sp3

Chelates Chelation is the formation of two or more separate coordinate bonds between a multidentate ligand and a single central atom. Usually the ligands are organic compounds, and are called chelators or chelating agents. In the process of sequestration, the chelating agent and metal ion form a water-soluble complex. The bonds in the chelate may be ionic or primary covalent type or coordinate type. Chelation places stringent stearic requirements on both metals and ligands and only cis-coordinated ligands will be replaced with a chelating agent. For example, zinc ion in enzyme alcohol dehydrogenase has two cis-positions available for chelation and hence can undergo chelation.

The compound ethylenediaminetetraacetic acid, on deprotonation, yields the hexadentate tetra-anion ligand EDTA, which forms remarkably stable complexes by simultaneously bonding through the two nitrogens and four oxygens, one each from the four acetate groups (Fig. 6.1).

Importance of chelates: 1. Haemoglobin and myoglobin are iron complexes that are essential for the transport of oxygen in the blood and tissues.

2. Cytochrome c is a naturally occurring chelate involved in photosynthesis and respiratory systems.

O O–

O O–

N M N

O– O–

O

O

Figure 6.1 Structure representing binding of metal ion to hexadentate EDTA.

n

Complexation and Protein Binding n 159 3. Albumin is the main carrier of metal ions in the blood plasma.

4. EDTA has been used to sequester calcium ions from hard water.

5. EDTA has been used to sequester iron and copper ions and it prevents oxidative degradation of creams, lotions and of ascorbic acid in fruit juices and in drug preparations.

6. EDTA is used to remove colour impurities from antibiotic preparations.

7. Therapeutically chelating agents are used as antidotes in heavy metal poisoning. For example, CaNa2EDTA is used in cases of lead poisoning, dimercaprol in cases of mercury and arsenic poisoning, deferoxamine mesylate in cases of iron poisoning and salicylic acid in cases of beryllium poisoning.

8. Sequestering agents are used in the treatment of urinary calauli, calciferous corneal deposits and hypercalcaemia.

9. EDTA may be used as an in vitro anticoagulant.

10. Chelate of p-amino salicylic acid possesses antitubercular activity, whereas chelates of 8-hydroxyl quinoline have antibacterial action.

11. Chelation can be applied to an assay of drugs such as magnesium trisilicate, calcium gluconate and calcium lactate.

Olefin Complexes Olefin complexes are formed by the interaction of aqueous solutions of metal ions (platinum, iron, palladium, mercury, silver) with olefin such as ethylene. These complexes are further classified as (1) monoolefins, (2) conjugated diolefins (e.g. butadiene) and (3) nonconjugated or chelating diolefins (e.g. cyclo-1,s-octadiene).

1. Complexes are usually water soluble.

2. Bonding in olefin complex is a sigma-type donation from the C=C π orbital with concomitant π-backbonding into an empty π* orbital on the ethylene (see Fig. 6.2).

3. Stability of the olefin complex depends on electronic and stearic factors.

s bond:

 backbond: C

M

C M

C Empty d-orbital

Filled ethylene -orbital

C filled d-orbital

Empty ethylene *-orbital

Figure 6.2 Sigma donation and π-backbonding in olefin complex

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Example Silver–olefin complex (Fig. 6.3) C

+ C

C

+ C

C

C

Ag+

Ag

Ag

Figure 6.3 Silver–olefin complex.

4. Used as stationary phase in the gas–liquid chromatographic (GLC) analysis of hydrocarbon mixtures, which are otherwise difficult to separate.

5. Resolution of optically active olefins such as trans-cyclo-octene.

Aromatic Complexes Aromatic complexes are formed by the interaction of metal ions as acceptors with aromatic molecules such as benzene, toluene and xylene as donors.

1. Stability of the complex depends on the basic strength of the aromatic hydrocarbon and increases with the increase in the basic strength of the aromatic hydrocarbon.

2. If the complex is formed by a π-bond between metal ions and the aromatic molecule, the complex is called π-bond complex.

Example: Complex of toluene with HCl (Fig. 6.4) CH3

CH3 + HCI =

HCI

Figure 6.4 Complex of toluene with HCl.

If the complex is formed by a sigma-bond between a metal ion and a carbon of the aromatic ring, the complex is called sigma-bond complex.

Example: Complex of toluene with catalyst couple HCl.AlCl3 (Fig. 6.5) H

H

H—C—H

H—C H+ AICI4–

+

H

H

H

H

Figure 6.5 Complex of toluene with catalyst couple HCl.AlCl3.

n

Complexation and Protein Binding n 161 1. If the complex is formed by a delocalized covalent bond between the d-orbital of a transition metal and a molecular orbital of the aromatic ring, the complex is called sandwich compounds

Example: Ferrocene or bisdicyclopentadienyl iron II complex (Fig. 6.6)

Fe

Figure 6.6 Structure of ferrocene or bisdicyclopentadienyl iron II complex.

ORGANIC MOLECULAR COMPLEXES Organic molecular complexes are formed as a result of noncovalent interactions between a ligand and a substrate. The interactions can occur through electrostatic forces, charge transfer, hydrogen bonding or hydrophobic effects. The attraction, which acts as a stabilizing force for the molecular complex, is created by an electronic transition into an excited electronic state, and is best characterized as a weak electron resonance.

Charge-Transfer Complexes A charge-transfer complex is an association of two or more molecules in which a fraction of electronic charge is transferred between the molecular entities. The molecule from which the charge is transferred is called the electron donor and the receiving species is called the electron acceptor.

1. Attraction in charge-transfer complexes is weaker than that in covalent forces.

2. Characterized by intense colour because the excitation energy of the resonance occurs in the visible region of the electromagnetic spectrum.

3. Usually these complexes are formed by sharing of π-electrons.

Example Complex between benzene and trinitro benzene (Fig. 6.7, 1:1) (polar nitro groups of trinitro benzene induce a dipole in the readily polarizable benzene molecule, resulting in electrostatic interaction)

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n n Physical Pharmacy NO2 δ–

δ+ O2N NO2

Figure 6.7 Complex between benzene and trinitro benzene.

Quinhydrone Complex This molecular complex is formed by mixing alcoholic solutions of equimolar quantities of benzoquinone with hydroquinone (Fig. 6.8).

1. Complex formation is due to overlapping of the π-framework of the electron-deficient benzoquinone with the π-framework of the electron-rich hydroquinone .

2. Complex appears as green crystals.

3. Used as an electrode to determine pH. O

OH

+ 2H+ + 2e OH

O

Hydroquinone

Benzoquinone

Figure 6.8 Quinhydrone complex.

Picric Acid Complexes Picric acid (2,4,6-trinitrophenol), being a strong acid, forms complexes with many weak bases such as polynuclear aromatic compounds.

1. Stability depends on the number of electron-attracting groups on the nitro group and the ring complexity.

Example Complex between two molecules of butyl p-aminobenzoate with one molecule of picric acid to give butesin picrate (local anaesthetic) (see Fig. 6.9).

n

Complexation and Protein Binding n 163 COOC4H9

O2N HO

NH2

NO2

O2N 2

Butesin picrate is used as a 1% ointment for burns and painful skin conditions since it has the anaesthetic property of butesin and the antiseptic property of picric acid.

Figure 6.9 Butesin picrate complex.

Hydrogen Bonded Complexes In this type, the complex is formed due to the attraction of the positive hydrogen atoms of one molecule towards the negative oxygen atoms of a second molecule. Hydrogen bonds are relatively weak bonds with about 10% of the strength of an ordinary covalent bond.

1. It is an example of dipole–dipole interaction.

2. Complex formation occurs if intermolecular hydrogen bonding is present.

Caffeine complexes Caffeine (Fig. 6.10) forms complexes with a number of drugs owing to the following factors: 1. Hydrogen bonding between the polarizable carbonyl group of caffeine and the hydrogen atom of the acidic drugs such as p-amino benzoic acid and gentisic acid.

2. Dipole–dipole interactions between the electrophilic nitrogen of caffeine and the carboxy oxygen of drugs such as benzocaine tetracaine or procaine. δ–

O

N

N CH

δ+ N N H 3C

O δ–

CH3

Figure 6.10 Structure of caffeine.

3. Caffeine drug complexes can enhance or inhibit the solubility, mask the bitter taste of drugs and improve the stability of drugs.

Polymer Complexes Polymeric materials such as eudragit, chitosan, polyethylene glycols, polyvinylpyrrolidone and sodium carboxymethyl cellulose, which are usually present in liquid, semisolid and solid

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dosage forms, can form complexes with a large number of drugs. Such interactions can result in precipitation, flocculation, solubilization, alteration in bioavailability or other unwanted physical, chemical and pharmacological effects.

1. Polymeric complex between naltrexone and eudragit improves the dissolution rate of naltrexone.

2. Intermolecular H-bonds between pectin and amoxicillin trihydrate to form polymer complex increase the therapeutic activity of the complexed drug.

3. Complexation of chitosan with sodium alginate makes them applicable for the design of more precisely controlled drug delivery systems.

4. Povidone–iodine is a stable complex of polyvinylpyrrolidone and iodine, which possess superior antibacterial activity.

INCLUSION COMPOUND (OR NO BOND COMPLEXES) An inclusion compound is a complex in which one chemical compound (the ‘host’) forms a cavity in which molecules of a second compound (‘guest’) are entrapped (see Fig. 6.11).

These complexes generally do not have any adhesive forces working between their molecules and are therefore also known as no-bond complexes.

Guest Host

Figure 6.11 Representation of an inclusion complex.

Clathrates Clathrates are inclusion compounds in which a molecule of a ‘guest’ compound gets entrapped within the cagelike structure formed by the association of several molecules of a ‘host’ compound (Fig. 6.12). The guest compound may be a solid, liquid or a gas and may be released from the complex by heating, dissolving or grinding the clathrate.

n

Complexation and Protein Binding n 165 HO

OH

Hydroquinone X H2N

NH2

urea (X=O), thiourea (X=S)

Perhydrotriphenylene OH

HO

COOH

Deoxycholic acid O O

O

O

O O

18-crown-6

Figure 6.12 Diagrammatic representation of clathrate and host molecules.

1. It is prepared by crystalling the host from a solution containing the guest compound.

2. Size of the guest molecule is important for complex formation.

3. If the size is too small, the guest molecule will escape from the cagelike structure of the host and if the size is too big, it will not be accommo­dated inside the cage.

Example 1. Entrapment of Krypton-85, methyl alcohol, HCl and CO2 in hydroquinone cage 2. Warfarin sodium (anticoagulant drug) is a clathrate of water, isopropyl alcohol and sodium warfarin in the form of a white crystalline solid

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Channel Lattice Complexes In this complex, the host component crystallizes to form a channellike structure into which the guest molecule can fit (see Fig. 6.13). The guest molecule must possess a geometry that can easily fit into the channellike structure.

1. Guest molecules are usually long, unbranched straight-chain compounds because the channels are springlike spirals.

2. Deoxycholic acid can take up organic acids, esters, ketones and aromatic compounds into its channellike structure.

3. Digitonin–cholesterol complex is an example of the cholic acid-type complex.

Figure 6.13 Channel lattice complex.

4. Channel lattice complexes provide a means of separation of petroleum products and optical isomers.

5. Vitamin A palmitate can be complexed with urea, which prevents its oxidation.

6. Dissolution of vitamin E and famotidine can be improved by complexation with urea.

Intercalation Compound or Layer-type Complexes Intercalation compound or layer-type complexes is a type of inclusion compounds in which the intercalate or guest molecule is diffused between the layers of carbon atom, hexagonally oriented to form alternate layers of guest and host molecules.

1. Montmorillonite, the principal constituent of bentonite clay, can entrap a number of hydrocarbons, alcohols and glycols between the layers of its lattices.

2. Graphite can also intercalate a number of compounds between its layers.

n

Complexation and Protein Binding n 167

Monomolecular Inclusion Compounds Monomolecular inclusion complex involves the entrapment of guest molecules into the cagelike structure formed form a single host molecule.

Example: Cyclodextrins 1. Represent a monomolecular host structure into which a number of guest molecules can get entrapped (see Fig. 6.14) 2. Possess cyclic oligosaccharides containing 6, 7 and 8 units of glucose referred to as α, β and γ cyclodextrins, respectively 3. Show doughnut ring or truncated cone structure 4. Interior of the cavity is relatively hydrophobic because of the CH2 groups, whereas the exterior is hydrophilic due to the presence of hydroxyl groups 5. β- and γ-cyclodextrins are more useful because of their larger diameters 6. Molecules of appropriate size and stereochemistry get entrapped in the cyclodextrin cavity by hydrophobic interaction by squeezing out water from the cavity Table 6.2 Classification of cyclodextrins Cyclodextrin type

Glucose units

Internal diameter

Aqueous solubility

USP name

α-cyclodextrin

6

4.7–5.3 Å

14.5 g/100 mL

Alfadex

β-cyclodextrin

7

6.0–6.5 Å

1.85 g/100 mL

Betadex

γ-cyclodextrin

8

7.5–8.3 Å

23.2 g/100 mL

Gammadex

Commercial formulations of CDs Piroxicam/β-CD tablet Cephalosporin/β-CD tablet Nimesulide/β-CD tablet Chlordiazepoxide/β-CD tablet Omeprazole/β-CD capsule Benexate/β-CD capsule Chloramphenicol/Me-β-CD eye drop Diclofenac/HP3-β-CD eye drop Cisapride/HP3-β-CD suppository Iodine/β-CD gargle PGE2/β-CD sublingual tablet Nitroglycerin/β-CD sublingual tablet

Modified cyclodextrins (CD) 1. Methyl, dimethyl and trimethyl CDs 2. Ethyl CDs 3. 2-hydroxyethyl CD 4. 3-hydroxypropyl CD 5. Carboxy methyl-, carboxy ethyl CD 6. Sulphoethyl ether CDs

1. Derivatives of natural cyclodextrins have been developed to improve the aqueous solubility and to avoid nephrotoxicity 2. Amorphous derivatives of β- and γ-cyclodextrins are more effective solubilizing agents 3. Hydrophobic β-cyclodextrins have been used to produce sustained release products

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n n Physical Pharmacy HOH2C O

O

O HOH2C

O

HO

HO

CH2OH

HO

OH

HOH2C O

O

O HO

OH

O HO

OH

HO

OH O

OH O

HO OH O

HO

CH2OH

O

CH2OH O

O

CH2OH

(a)

(b)

Figure 6.14 Chemical structure of cyclodextrin

4. Complexation with cyclodextrins has also been used to mask the bitter taste of certain drugs such as famoxetine 5. Cyclodextrin complexation has been found to stabilize and solubilize aspirin, ephedrine, sulphonamides, tetracyclines, morphine, benzocaine, reserpine, testosterone and retinoic acid.

Macromolecular Inclusion Compounds Macromolecular inclusion compounds or molecular sieves include synthetic zeolites, dextrans, silica and related substances. The atoms in these compounds are arranged in three dimensions to provide cages and channels and the guest molecules are entrapped within. Synthetic zeolites may be made to possess a definite pore size to separate molecules of different dimensions, and hence the name, molecular sieves. Synthetic metal-alumino silicates have been used to store gaseous, volatile and toxic materials; to dry gases; and to separate gaseous mixtures.

METHODS OF ANALYSIS Complexes are analysed for stoichiometric ratio of ligand to metal or donor to acceptor and for determining a quantitative expression for the stability constant for complex formation.

The equation for stability constant is written as follows:

n

Complexation and Protein Binding n 169 K= [DC] / [D][C] where [DC] is the concentration of the drug-complex (= total drug in solution – amount of uncomplexed drug), [D] the solubility of uncomplexed drug and [C] the concentration of uncomplexed complexing agent (= total complexing agent – amount of complexing agent in the drug complex [DC].

Job’s Method of Continuous Variation Job’s method of continuous variation is a simple method to determine the stoichiometric ratio of a complex based on measuring change in properties such as absorbance, mass of the precipitate, dielectric constant or square of the refractive index that are proportional to complex formation. The method is based on the assumption that the maximum change in these properties will occur at a stoichiometric ratio since the solution at that point will contain the highest concentration of the complex. In this method, the total molar concentration of the reacting species (metal and a ligand) is held constant, but their mole fraction is varied as shown in Table 6.3. A property such as absorbance of each solution at wavelength of maximum absorbance (λmax) is determined. The absorbance increases as the concentration of the reactant (metal ion) increases from zero because of the increase in the amount of complex.

A maximum value of absorbance is obtained for the solution, in which complex formation is maximum, i.e. at stoichiometric ratio. Further additions of metal ion give solutions containing insufficient ligand to complex with all the metal; hence, the absorbance due to the complex decreases. Thus, the maximum change observed in a plot of absorbance and mole fractions corresponds to the stoichiometric ratio of the two species (see Fig. 6.15). On the other hand, when no complex is formed, a linear relationship is obtained.

Table 6.3 Molar concentration of reacting species and their respective absorbance values Volume of metal ion (mL)

Volume of ligand (mL)

Absorbance (nm)

1

9

0.22

2

8

0.3

3

7

0.39

4

6

0.48

5

5

0.58

6

4

0.54

7

3

0.5

8

2

0.46

9

1

0.41

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This method can also be used to determine the stability constant for a complex, since the deviation of the experimentally determined curve from the extrapolated lines arises from dissociation of the complex. However, a similar deviation can be caused if Beer's law is not followed. Therefore, several conditions must be met in order for Job's method to be applicable: 1. The system must conform to Beer’s law.

2. One complex must predominate under the conditions of the experiment.

3. The total concentration of the two binding partners must be constant.

4. pH and ionic strength must be constant.

0.8

Absorbance (nm)

0.7 0.6 0.5 0.4 0.3

No complex formation

0.2

Stoichiometric ratio

0.1 0

1

2

3

4 5 6 Volume of metal ion (mL)

7

8

9

Figure 6.15 Plot of absorbance and molar concentration of metal ion.

pH Titration Method The formation of metal complexes often depends, to a great extent, on the pH of the solution because there is a competition between the metal ion and the proton for the ligand as they both bind to the same atoms of the ligand. Owing to this, the pH titration method is considered the most reliable method for studying complexation and can be used when complexation is accompanied by a change in pH. Method: Let us consider the complex formation between cupric ion and glycine: Cu2+ + 2NH3 + CH2COO– ⇔ Cu (NH2CH2COO)2 + 2H+ Since two protons are formed in the reaction, the addition of cupric ion to glycine solution should result in a decrease in pH. As a first step, the glycine solution is titrated with sodium hydroxide solution (standard basic solution) and a titration curve is obtained by plotting the pH versus the volume of the sodium hydroxide added. Then, the titration curve is obtained by adding sodium hydroxide to the solution containing glycine and copper salt. The titration curves are shown in Figure 6.16.

The curve for the copper–glycine complex is well below that for glycine alone and the decrease in pH indicates the occurrence of complexation throughout most of the neutralization range.

n

Complexation and Protein Binding n 171 Glycine

pH Glycine + Copper complex

Sodium hydroxide (ml)

Figure 6.16 Titration curve of glycine and copper–glycine complex.

The horizontal distances represented by the lines in Figure 6.16 between the titration curve of glycine alone and the titration curve of copper–glycine complex indicate the amount of sodium hydroxide used. This concentration of sodium hydroxide is equal to the concentration of ligand bound at any value of pH. The concentration of ligand bound divided by the total concentration of metal ion gives the value of n. Therefore, n = Total [Ligand] bound/Total [Metal ion] The concentration of the free glycine [G]free at any pH is considered as the difference between the total concentration of glycine [G] and the concentration of sodium hydroxide added.

[G]free = Ka ([G] – [NaOH])/[H3O+] or p[A] = pKa – pH – log ([G] – [NaOH]) The values of n and p[A] at various pH values are plotted to obtain the formation curve and the stability constants for the complexation can be obtained by treating the results mathematically.

Phase-Distribution Method For some complexes, the partitioning of solute between two immiscible solvents can be used to determine the stability constant. The complexation of iodine by potassium iodide (KI) may be used as an example to explain this method. Complexation between iodine and potassium iodide occurs and the following equilibrium reaction takes place: I2 + I– = I3– Method: First, the iodine is distributed between an immiscible system containing water (w) and carbon disulphide (o). The distribution coefficient in this system is given by K = [Iodine]o/[Iodine]w After analysis of iodine in both solvents, K is found to be 50.

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n n Physical Pharmacy

Now iodine is distributed between an immiscible system containing aqueous KI solution (30 M) and carbon disulphide. Let 250 M be the concentration of iodine in carbon disulphide and 25 M in an aqueous KI solution.

The distribution law expresses only the concentration of free iodine (i.e. only free iodine will partition), whereas chemical analysis yields the total concentration of iodine.

The results are summarized as follows: Total [Iodine] (free + complexed) in the aqueous KI solution is = 25 M Total [KI] (free + complexed) in the aqueous KI solution is = 30 M Distribution coefficient K = [Iodine]o/[Iodine]w = 50 The [Iodine]free in the aqueous KI solution is = [Iodine]o/50 = 250/ 50 = 5 M [Iodine]complexed = [Iodine] – [Iodine]free = 25 – 5 = 20 M Furthermore, iodine and KI combine in equimolar concentrations to form complex.

Therefore, [KI]complexed = [Iodine]complexed = 20 M Thus, [KI]free = [KI] – [KI]complexed = 10 M Now, K = [complex]/ [Iodine]free . [KI]free = 25/ 5 × 10 = 0.5 The distribution method has been used to study caffeine and polymer complexes with a number of acidic drugs such as benzoic acid, salicylic acid and acetyl salicylic acid.

Solubility Method The most widely used approach to study inclusion complexation is the phase solubility method described by Higuchi and Connors, which examines the effect of a complexing agent on the drug being solubilized.

Method: In this method, the increasing concentrations of a complexing agent in aqueous vehicle are taken in well-stoppered containers. An excess quantity of drug is then added to each of the containers. The containers are then placed in a constant temperature bath and agitated till equilibrium is attained. After equilibrium, an aliquot portion of the supernatant liquid is removed and analysed for drug concentration. The solubility of the drug is plotted against the molar concentration of the complexing agent as shown in Figure 6.17. Phase solubility diagrams are categorized into A and B types. A-type curves indicate the formation of soluble inclusion complexes and are subdivided into AL (linear increases of drug solubility as a function of CD concentration), AP (positively deviating isotherms) and AN (negatively deviating isotherms) subtypes. HP-β-CD usually produces soluble complexes and thus gives A-type systems. The B-type curve suggests the formation of inclusion complexes with poor solubility. A BS-type response denotes complexes of limited solubility and a BI curve indicates insoluble complexes. β-CD often gives rise to B-type curves due to their poor water solubility.

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Complexation and Protein Binding n 173 AP

St

AL AN [Dissolved guest] A SO SC

B BS BI

[Cyclodextrin]

Figure 6.17 Phase solubility diagram.

1. The solubility of the drug increases as the CD is added due to the formation of a soluble complex (So to A).

2. At point A, the solution is saturated with respect to both the drug and the complex.

3. With continuous addition of the CD, the complex continues to form and precipitates from the already saturated solution.

4. Point B indicates that all the excess drug has been used up for complexation.

5. On further addition of CD, one or more secondary complexes are formed.

The stoichiometry of the interaction can be determined from the phase solubility diagram (see Fig. 6.17).

Amount of drug entering the complex = Total drug – drug at point A or B Amount of CD in complex = CD at point B – CD at point A Ratio = moles of drug in complex/ moles of CD in complex If the ratio is 1, the complexing reaction can be written as D + C = DC and the stability constant can be written as K = [DC] / [D][C]

Protein Binding: Small Molecule-Macromolecule Complexes The interaction between macromolecules such as proteins and small molecules such as drugs is the most widely studied phenomenon. Protein–ligand interaction is important in drug

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binding to receptor, enzyme–substrate interaction in catalysis, antigen–antibody recognition and the binding between drugs and proteins in plasma.

Significance of Protein Binding Plasma proteins such as albumin, globulin and α1-acid glycoprotein or lipoproteins present in the body have been known to bind with a large number of drugs. This protein binding alters the biological properties of the drug molecule as the free drug concentration is reduced.

The bound drug inherits diffusional and other transport characteristics of the protein. The following are some of the properties of plasma protein binding: 1. Influences the way in which a drug distributes into tissues in the body 2. Limits the amount of free drug available to access sites of action in the cell 3. Reduces or completely eliminates the pharmacological activity of the drug since bound drug is not available for binding to the receptor site 4. Retards the excretion of a drug and increases its accumulation 5. Prolongs the duration of action of a drug 6. Co-administration of a different drug that Majority of ligands bind with albumin by also binds to plasma proteins may cause hydrophobic interactions because of the displacement of the (first) bound drug, immense flexibility in protein molecule resulting in significant toxicity as the free that allows for conformation change to drug interacts with the receptor to produce accommodate the different shapes of pharmacological response ligands.

7. The protein–drug complex itself has biological activity

Binding Equilibria Many of the interactions between the drug (D) and proteins (P) occur in a reversible manner according to the following equilibrium: Protein [P] + Drug [D] ↔ Protein – drug complex [PD] where [P], [D] and [PD] are the molar concentrations of unbound protein, unbound drug and protein–drug complex, respectively.

Applying the law of mass action, the expression becomes K = [PD]/[P][D] or [PD] = K [P] [D] The association constant describes a measure of the affinity between the protein and the drug.

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Complexation and Protein Binding n 175 Now consider that total protein concentration in the body is [PT]. The total protein concentration is the sum of the unbound protein and the protein present in the complex and therefore, [PT] = [P] + [PD] or [P] = [PT] – [PD] Substituting for [P] in the above equation, we get [PD] = K [D] ([PT] – [PD]) [PD] = K [D] [PT] – K [D] [PD] [PD] + K [D] [PD] = K [D] [PT] [PD] (1+K [D]) = K [D] [PT] Dividing both sides by [PT], we get [PD]/[PT] = K[D]/1 + K[D] Let r = [PD]/[PT], which relates to the concentration of bound drug to the total protein concentration. r = K [D]/1 + K[D] The above equation describes the simplest situation, where one mole of drug binds with one mole of protein to form a 1:1 complex. However, if the number of binding sites (n) is greater than one, the equation is modified as follows: r = n K[D]/1 + K[D] The plot of r versus [D] will yield a hyperbolic curve (see Fig. 6.18).

However, the equation is converted to a straight-line form to determine the magnitude of association constant (K) and the number of binding sites (n).

n

r

[D]

Figure 6.18 representation of hyperbolic curve.

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Slope = 1/nK

1/r

Intercept = 1/n

1/[D]

Figure 6.19 Representation of double reciprocal plot.

1. Using the double reciprocal method: 1/r = 1+ K[D]/n K[D] 1/r = 1/n K[D] + 1/n The plot of 1/r versus 1/[D] will yield a straight line with slope equal to 1/nK and intercept 1/n (see Fig. 6.19).

2. Using the Scatchard method, we get r = n K[D]/1+ K[D] r (1+ K[D]) = n K[D] r + r K[D] = n K[D] Dividing both sides by [D] gives r/[D] = n K[D]/[D] – r K[D]/[D] r/[D] = nK – rK

Intercept = nK

r/D

Slope = K

r

Figure 6.20 representation of Scatchard plot.

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Complexation and Protein Binding n 177 The plot of r/[D] versus r will yield a straight line with slope equal to – K and intercept nK (see Fig. 6.20).

Protein–drug binding studies have shown that even if there is more than one binding site for the drug, the binding affinity (K) is the same for all drug molecules. However, in some cases, drugs that have multiple binding sites have more than one binding association constant as represented in Figure 6.21.

Slope = – K1 r/[D] Slope = – K2

r

Figure 6.21 representation of Scatchard plot for drugs with multiple binding association constants.

Methods for Determining Protein Binding Equilibrium dialysis method Equilibrium dialysis is used to determine the extent of binding of a drug to plasma proteins (see Fig. 6.22). A semipermeable membrane (cellophane) separates a protein-containing compartment from a protein-free compartment. The semipermeable membrane is permeable to small drug molecules but does not allow the proteins to pass through. The proteincontaining compartment is immersed in a drug solution (protein-free compartment). The system is allowed to equilibrate at 37°C under slight agitation. The drug present in each compartment is quantified. The extent of binding is reported as a fraction unbound (Fu) value, which is calculated as follows: Fu = 1 – {(PC – PF)/PC} PC = drug concentration in protein-containing compartment PF = drug concentration in protein-free compartment 1. The same drug concentration in PC and PF indicates the absence of protein binding.

2. Higher drug concentration in PC compared to PF indicates that protein binding has occurred because both bound and unbound drugs are present in PC.

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n n Physical Pharmacy Before equilibrium PC

PF

Semipermeable membrane Protein Drug

After equilibrium PC

PF

PC

Drug–protein binding absent

PF

Drug–protein binding

Figure 6.22 Pictorial representation of the process of equilibrium dialysis.

Dynamic dialysis This is a kinetic method for studying the protein binding of drugs and is based on the fact that the rate of disappearance of drug from a dialysis cell is proportional to the concentration of the unbound drug. The apparatus consists of a temperature-controlled (jacketed) beaker into which a buffer solution is placed. A cellophane dialysis bag containing a solution of drug and protein is suspended in the buffer solution. Both the solutions are stirred and the samples are periodically removed from outside the dialysis bag and analysed. The benefit of this method is that it can be readily applied for studying the competitive inhibition of protein binding. The dialysis process follows the rate law: – d[DT]/ dt = k[DT] where [DT] is the total drug concentration, [Df] the concentration of free or unbound drug, – d[DT]/dt the rate of loss of drug from the bag and k the first order rate constant representative of the diffusion process

n

Complexation and Protein Binding n 179 The concentration of unbound drug [Df] in the bag can be calculated using the above equation if k and d[Dt]/dt are known. The rate constant k is obtained from the slope of a semilogarithmic plot of [Dt] versus time when the experiment is conducted in the absence of the protein.

UItracentrifugation Ultracentrifugation is a method by which compounds with different molecular weights are separated with the aid of centrifugal force. Ultracentrifuges can spin the mixtures at speeds in excess of 50,000 rpm. Separation of bound drug and unbound drug occurs depending on the relative settling of compounds with different molecular weights. The main advantages of the ultracentrifuge over other methods are the economy of time and small sample requirement.

In recent years, a number of spectroscopic methods, including ultraviolet, fluorescence spectroscopy, optical rotator dispersion, circular dichroism, nuclear magnetic resonance and electron spin resonance, have become very popular methods of analysing protein–drug interaction.

THERMODYNAMIC TREATMENT OF STABILITY CONSTANTS The relationship between the standard free energy change of complexation and the overall stability constant K can be given by the following equation: ΔG = – 2.303 RT log K The standard enthalpy change ΔH may be obtained from the slope of a plot of log K versus 1/T, following the expression: log K = – ΔH/2.303R 1/T + constant When the values of K at two temperatures are known, the following equation may be used: log (K2/K1) = – ΔH/2.303 R T2 – T1/T1T2 The standard entropy change may be obtained from the following expression: ΔG = ΔH – T ΔS As the stability constant for molecular complexation increases, ΔH and ΔS generally become more negative. As the binding between the donor and the acceptor becomes stronger, ΔH becomes more negative. Since the specificity of interacting sites or structural restraint also becomes negative, ΔS also becomes more negative. However, the extent of change in ΔH is large enough to overcome the unfavourable entropy change resulting in negative ΔG value and hence complexation.

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Questions 1. Give proper justification for the following: a. CaNa2EDTA is used as antidote in case of lead poisoning. b. Complex [Fe(CN)6]3+ is called as outer sphere complex. c. Complex [Cu(NH3)4]2+ is called as inner sphere complex. d. Caffeine can form complexes with acidic as well as basic drugs. e. Cyclodextrins form monomolecular inclusion complexes with drugs.

2. Write short notes on the following: a. Chelating agents b. Polymer complexes c. Inclusion complexes d. Molecular sieves e. Ultracentrifugation 3. Classify organic molecular complexes with suitable examples of each type.

4. Define stability constant and stoichiometric ratio of complex. Enumerate the methods to determine stability constant, and describe any one method in detail.

5. Deduce the equation and plot for drug–protein-binding equilibria.