Complexation and Protein Binding

 Chapter -4 

COMPLEXATION AND PROTEIN BINDING

OBJECTIVES

• The term complexation is used to characterize the covalent or non-covalent interactions between two or more compounds capable of independent existence. The ligand, a molecule, interacts with substrate the molecule to form a complex. Drug molecules can form complexes with other small molecules or with macromolecules. Once complexation occurs, the solubility, stability, partitioning, energy absorption and emission, and conductance of the drugs are changed. Drug complexation, therefore, can lead to beneficial properties such as enhanced aqueous solubility and stability. Complexation can also be useful in the optimization of delivery systems and affect the distribution in the body after systemic administration due to protein binding. The drug-protein binding in this unit is covered in depth in the later part. Contrary, complexation can also lead to poor solubility or decreased absorption of drugs in the body. For certain drugs, complexation with certain hydrophilic compounds can enhance excretion. Overall, complexes can alter the pharmacologic activity of drugs.

 After studying the contents of the chapter, students are expected to:

• Understand the significance of complexation in pharmaceutical products. 

•  Understand the fundamental forces that are related to the formation of drug complexes. 

• Differentiate between different complexation types and understand the mechanism of complex formation. 

• Relate the formation of complexes with improvements in the physicochemical properties and bioavailability of drugs. 

• Identify the significance of protein-ligand interactions in drug action. 

• Understand properties of plasma proteins and its mechanism of interactions with drugs. 

• Understand the techniques of in vitro analysis and factors affecting complexation and protein binding.  

 INTRODUCTION 

• Complexes or co-ordination compounds result from a donor–acceptor mechanism or Lewis acid–base reaction between two or more different chemical components. The term complexation is used to characterize the covalent or non-covalent interactions between two or more compounds capable of independent existence. The ligand, a molecule, interacts with substrate, the molecule, to form a complex. Drug molecules can form complexes with other small molecules or with macromolecules. Once complexation occurs, the solubility, stability, partitioning, energy absorption and emission, and conductance of the drugs are changed. Drug complexation, therefore, can lead to beneficial properties such as enhanced aqueous solubility and stability. Complexation can also be useful in the optimization of delivery systems and affect the distribution of drug in the body after systemic administration due to protein binding. Contrary, complexation can lead to poor solubility or decreased absorption of drugs in the body. For certain drugs, complexation with certain hydrophilic compounds can enhance excretion. Overall, complexes can alter the pharmacologic activity of drugs.

• Complexes can be divided broadly into two classes depending on whether the acceptor component is a metal ion or an organic molecule; these are classified according to one possible arrangement. Another class, the inclusion/occlusion compounds, involves the entrapment of one compound in the molecular framework of another. Intermolecular forces involved in the formation of complexes are the van der Waals forces of dispersion, dipolar, and induced dipolar types. Hydrogen bonding provides a significant force in some molecular complexes, and co-ordinate covalence is important in metal complexes. Many drugs bind to plasma proteins which has significant influence on duration of drug action. Some drugs in body exist only in a bound form and proper distribution of such drugs into extra vascular part is governed by the process of dissociation of drugs from the complex. The fraction of drug that can be in free form can vary but may be as low as 1%. The other fraction remains in associated form as a complex with the protein. The free form of drug is pharmacologically active and is responsible for action on body. Thus, the protein binding features of the drug plays significant role in its therapeutic actions. 

CLASSIFICATION OF COMPLEXATION  

Based upon type of interaction, ligand-substrate complexes are classified as follows.

(I) Metal ion or co-ordination complexes:

• Inorganic type 
• Chelates 
• Olefin type 
• Aromatic type 

1. Pi (π) complexes 
2. Sigma (σ) complexes 
3. Sandwich compounds 

(II) Organic molecular complexes: 

• Quinhydrone type
• Picric acid type 
• Caffeine and other drug complexes 
• Polymer type 

(III) Inclusion or occlusion compounds:

• Channel lattice type 
• Layer type 
• Clathrates 
• Monomolecular type 
• Macromolecular type

(I) Metal Ion or Co-ordination Complexes:

• A satisfactory understanding of metal ion complexation is based upon a familiarity with atomic structure and molecular forces, and electronic structure as well as hybridization. The co-ordination complex or metal complex is a structure made-up of a central metal atom or ion (cation) surrounded by a number of negatively charged ions or neutral molecules possessing lone pairs. The ions surrounding the metal are known as ligands. The number of bonds formed between the metal ion and ligand is called as co-ordination number. 

Inorganic Complexes:

• Ligands are generally bound to a metal ion by a covalent bond and hence called to be coordinated to the ion. The interaction between metal ion and the ligand is known as a Lewis acid-base reaction wherein the ligand (base) donates a pair of electrons (to the metal ion, an acid) to form the co-ordinate covalent bond. For example, the ammonia molecules in hexamine cobalt (III) chloride, as the compound [Co (NH3)6] 3+ ⋅ Cl3 is called as the ligands and are said to be coordinated to the cobalt ion. The co-ordination number of the cobalt ion, or number of ammonia groups coordinated to the metal ions, is six. Other complex ions belonging to the inorganic group include [Ag (NH3)2] +, [Fe (CN)6] 4- , and [Cr (H2O)6] 3+.

Chelates: 

• A substance containing two or more donor groups may combine with a metal to form a special type of complex known as a chelate. Some of the bonds in a chelate may be ionic or of the primary covalent type, whereas others are co-ordinate covalent links. When the ligand provides one group for attachment to the central ion, the chelate is called monodentate. For example, pilocarpine behaves as a monodentate ligand toward Co (II), Ni (II), and Zn (II) to form chelates of pseudo tetrahedral geometry. 

• Chelation holds stringent steric requirements on both metal and ligands. Ions such as Cu (II) and Ni (II), which form square planar complexes, and Fe (III) and Co (III), which form octahedral complexes and can exist in either of two geometric forms. Because of this isomerism, only cis-coordinated ligands (ligands adjacent on a molecule) are readily replaced by reaction with a chelating agent. Vitamin B12 and the hemoproteins are incapable of reacting with chelating agents because their metal is already co-ordinated in such a way that only the trans-co-ordination positions of the metal are available for complexation. In contrast, the metal ion in certain enzymes, such as alcohol dehydrogenase, which contains zinc, can undergo chelation, suggesting that the metal is bound in such a way as to leave two cis-positions available for chelation. 

Applications of chelation: 

• Chlorophyll and hemoglobin, two extremely important compounds, are naturally occurring chelates involved in the life processes of plants and animals. Albumin is the main carrier of various metal ions and small molecules in the blood serum. The amino-terminal portion of human serum albumin binds to Cu (II) and Ni (II) with higher affinity than that of dog serum albumin. This fact partly explains why humans are less susceptible to copper poisoning than are dogs. The binding of copper to serum albumin is important because this metal is possibly involved in several pathologic conditions. The synthetic chelating agent ethylene diamine tetra acetic acid (EDTA) has been used to tie-up or sequester iron and copper ions so that they cannot catalyze the oxidative degradation of ascorbic acid in fruit juices and in drug preparations. In the process of sequestration, the chelating agent and metal ion form a water-soluble compound. EDTA is widely used to sequester and remove calcium ions from hard water. 

Olefin Type:

• Olefins belong to a family of organic compounds called hydrocarbons. They consist of different molecular combinations of the two elements, carbon and hydrogen. Another name for an olefin is an alkene. Alkenes contain one or more double bonds between the carbon atoms of the molecule. Olefins form different compounds based on their structure. Some have short chains with only two, three or four carbons, such as ethylene. Others form long chains or closed ring structures. Some have a combination of both. Alkenes are insoluble and exist in all three states of matter. Some short chain alkenes are gases at room temperature and pressure. More complicated structures exist as liquids and solids. 

• Olefin ligands are common in Organo transition metal chemistry. The first Organo transition metal complex, Zeise's salt (K[PtCl3(C2H4] ·H2O) was an olefin complex. The bonding of an olefin to a transition metal can activate the ligand to electrophilic or nucleophilic attack depending on the nature and charge of the metal center. For example, if there is a high formal charge on the metal center then the olefin is subject to attack by nucleophiles at the face opposite the metal (giving trans addition). Likewise, electron rich metal centers in low oxidation states are activated for attack by electrophiles at the C-C bond. 

Aromatic Type: 

Pi (π) complexes:

• The example of Pi complex is interaction of local anesthetic bupivacaine and its structural analogs such as 2,6-dimethylaniline, and N-methyl-2, 6-dimethylacetanilide, and cocaine, with several electron deficient aromatic moieties. In solution, the anesthetic, its analogs and cocaine are electron donors and form π-π charge transfer complexes with strong aromatic acceptors. The concentrations of free bupivacaine, its analogs and of cocaine are reduced from solution via binding to aromatic-functionalized silica

The rapid binding of bupivacaine 

1. Its analogs 2, 6-dimethylaniline 
2. 6-dimethylacetanilde 
3.  respectively, and of cocaine 
4. by the acceptor molecules.

•  The structures 1, 2, 3 and 4 show that the molecules are lipophilic in nature, a characteristic common to toxic molecules. 1, 2 and 3 include a benzene ring with two methyl and a nitrogen electron-donating groups, making this portion of the molecules π-electron rich, and hence strong π-donors. The aromatic ring of cocaine, 4, also has weak π-donor capability when complexed with a strong π-acceptor. The selective removal of excess bupivacaine and cocaine from solution is charge transfer complex formation of the π-π type through aromatic-aromatic interaction, based on the assumption that dinitro benzoyl groups possessing less π-electron density would not only bind with the more π-electron rich bupivacaine and cocaine but would also reduce their toxic effects. The LD50 of bupivacaine is 7.8 mg/kg subcutaneously. The effectiveness of this approach is based on the fact that only free, unbound molecules in the blood possess toxicity and that they lose toxicity once bound to or conjugated with another moiety. 

Sigma (σ) complexes:

• A uranium ion is a cyclohexadienyl cation that appears as a reactive intermediate in electrophilic aromatic substitution. This complex is also called a Wheland intermediate or a sigma complex or σ-complex. The smallest uranium ion is the benzene ion (C6H + 7), which is protonated benzene.

• Methylene uranium ion stabilization by metal complexation is another example of σ-complex. In the reaction sequence the R-Pd (II)-Br starting complex is stabilized by tetra methyl ethylene diamine (TMEDA) which is converted by 1,2-Bis(triphenylphosphine) ethane (DPPE) to metal complex. Electrophilic attack of methyl triflate forms methylene uranium ion with positive charge located in aromatic para position and with the methylene group at 6° out of the plane of the ring. Reaction first with water and then with triethylamine hydrolyzes the ether group.

Sandwich compounds:

• A sandwich compound is a metal bound by haptic covalent bonds to two arene ligands. The arenes have the formula CnHn, substituted derivatives (for example Cn (CH3) n) and heterocyclic derivatives (for example BCnHn+1). Because the metal is usually situated between the two rings, it is said to be "sandwiched". Special classes of sandwich complexes are the metallocene's. Metallocene's including just one facially bound planar organic ligand instead of two gives rise to a still larger family of half-sandwich compounds. The most famous example is probably methylcyclopentadienyl manganese tricarbonyl. Compounds such as the cyclopentadienyl iron Di carbonyl dimmer and cyclopentadienyl molybdenum tricarbonyl dimer can be considered a special case of half-sandwiches, except that they are bimetallic. 

Organic Molecular Complexes:

• An organic molecular complex consists of constituents held together. The forces involved are of donor and acceptor type or by hydrogen bonds. There is a difference between complexation and the formation of organic compounds. For example, dimethyl aniline and 2,4,6-trinitroanisole react at low temperature to give a molecular complex. The dotted line in the complex, Fig. 4.5, indicates that the two molecules are held together by a weak secondary valence force. It is not to be considered as a clearly defined bond but rather as an overall

• attraction between the two aromatic molecules. The type of bonding existing in molecular complexes in which hydrogen bonding plays no part is not fully understood, but it may be considered for the present as involving an electron donor–acceptor mechanism corresponding to that in metal complexes but ordinarily much weaker. 

Drug Complexes:

• In the formation of drug complex degree of interaction depends upon certain effects. For example, the complexing of caffeine with several acidic drugs. The interaction between caffeine and sulfonamide or barbiturate is a dipole–dipole force or hydrogen bonding between the polarized carbonyl groups of caffeine and the hydrogen atom of the acid. The secondary interaction occurs between the non-polar parts of the molecules and the resultant complex is “squeezed out” of the aqueous phase due to the great internal pressure of water. 

• The complexes formed between esters and amines, phenols, ethers, and ketones have been attributed to the hydrogen bonding between a nucleophilic carbonyl oxygen and an active hydrogen. There are no activated hydrogens on caffeine; the hydrogen at the number 8 position is very weak (Ka = 1 × 10−14) and is not likely to enter complexation, Fig. 4.8. The complexation occurs due to dipole–dipole interaction between the nucleophilic carboxyl oxygen of benzocaine and the electrophilic nitrogen of caffeine. 

Polymer Complexes: 

• The polymers containing nucleophilic oxygens such as polyethylene glycols, polystyrene, carboxymethylcellulose and similar can form complexes with various drugs. The examples of this type include incompatibilities of carbowaxes, Pluronic's, and tweens with tannic acid, salicylic acid, and phenol. The interactions may occur in suspensions, emulsions, ointments, and suppositories and are manifested as a precipitate, flocculate, delayed biologic absorption, loss of preservative action, or other undesirable physical, chemical, and pharmacological effects. The interaction of povidone (PVP) with ionic and neutral aromatic compounds is affected by several factors that affect the binding to PVP of substituted benzoic acid and nicotine derivatives. Ionic strength has no influence but the binding increases in phosphate buffer solutions and decreases as the temperature is raised. Risperidone, a cross-linked insoluble PVP, can bind drugs owing to its dipolar character and porous structure. There exits an interaction of risperidone with acetaminophen, benzocaine, benzoic acid, caffeine, tannic acid, and papaverine hydrochloride. This interaction is mainly due to any phenolic groups on the drug. Hexyl resorcinol shows exceptionally strong binding. 

Inclusion Compounds : 

• The inclusion or occlusion compounds results from the architecture of molecules. One of the constituents of the complex is trapped in the open lattice or cage like crystal structure of the other to yield a stable arrangement. 

Channel Lattice Type : 

• The bile acids especially cholic acids form a complex of deoxycholic acid in combination with paraffin, organic acids, esters, ketones, and aromatic compounds and with solvents such as ether, alcohol, and dioxane. The crystals of deoxycholic acid are arranged to form a channel into which the complexing molecule can fit. Camphor has been partially resolved by complexation with deoxycholic acid, and dl-terpineol using digitonin, which occludes certain molecules in a manner like that of deoxycholic acid. Urea and thiourea also crystallize in a channel-like structure permitting enclosure of unbranched paraffin, alcohols, ketones, organic acids, and other compounds. The well-known starch–iodine solution is another example of channel-type complex consisting of iodine molecules entrapped within spirals of the glucose residues. Monostearin, an interfering substance in the assay of dienestrol, could be extracted easily from dermatologic creams by channel-type inclusion in urea. Urea inclusion might become a general approach for separation of long-chain compounds in assay methods. 

Layer Type :      

• Some other examples include clay montmorillonite, the principal constituent of bentonite, can trap hydrocarbons, alcohols, and glycols between the layers of their lattices. Graphite can also intercalate compounds between its layers.

Clathrates:

• The clathrates crystallize in the form of a cage like lattice in which the co-ordinating compound is entrapped. Chemical bonds are not involved in these complexes, and only the molecular size of the encaged component is of importance. The stability of a clathrate is due to the strength of the structure. The highly toxic agent hydroquinone (quinol) crystallizes in a cage like hydrogen-bonded structure. The holes have a diameter of 4.2°A and permit the entrapment of one small molecule to about every two quinol molecules. Small molecules such as methyl alcohol, CO2, and HCl may be trapped in these cages, but smaller molecules such as H2 and larger molecules such as ethanol cannot be accommodated. It is possible that clathrates may be used to resolve optical isomers and to bring about other processes of molecular separation. The warfarin sodium USP, is a clathrate of water, isopropylalcohol, and sodium warfarin in the form of a white crystalline powder. 

Monomolecular Inclusion Compounds: Cyclodextrins 

• Inclusion compounds are of channel - and cage-type (clathrate) and mono- and macro molecular type. Monomolecular inclusion compounds involve the entrapment of a single guest molecule in the cavity of one host molecule. Monomolecular host structures are represented by the cyclodextrins (CD). These compounds are cyclic oligosaccharides containing a minimum of six dextro-glucopyranose units attached by α-1,4 linkages produced by the action on starch of Bacillus macerans amylase. The natural α−, β−, and γ− cyclodextrins consist of six, seven, and eight units of glucose, respectively. 

• Cyclodextrins are cyclic oligomers of glucose that can form water-soluble inclusion complexes with small molecules and portions of large compounds. These complexes are biocompatible and do not elicit any immune responses and have low toxicities in animals and humans. Some examples of cyclodextrins used in therapeutics along with their method of preparation are listed in Table 4.2. 

• Cyclodextrins has wide pharmaceutical applications such as improvement in the bioavailability of drugs of specific interest and delivery of nucleic acids. The CD has ability to form inclusion compounds in aqueous solution due to the typical arrangement of the glucose units. The cyclodextrin structure forms a doughnut ring. The molecule exists as a 

APPLICATIONS OF COMPLEXATION

• Solubility enhancement : The aqueous solubility of retinoic acid (0.5 mg/L), a drug used topically in the treatment of acne, is increased to 160 mg/L by complexation with β-CD. Derivatives of the natural crystalline CD have been developed to improve aqueous solubility and to avoid toxicity. Partial methylation (alkylation) of some of the OH groups in CD reduces the intermolecular hydrogen bonding, leaving some OH groups free to interact with water, thus increasing the aqueous solubility of CD. A low degree of alkyl substitution is preferable. Derivatives with a high degree of substitution lower the surface tension of water, and this has been correlated with the hemolytic activity observed in some CD derivatives. Amorphous derivatives of β-CD and γ-CD are more effective as solubilizing agents for sex hormones than the parent cyclodextrins. The relatively low aqueous solubility of the CD is due to the formation of intramolecular hydrogen bonds between the hydroxyl groups, which prevent their interaction with water molecules.

• Bioavailability enhancement : Dissolution rate plays an important role in bioavailability of drugs, fast dissolution usually favours absorption. The dissolution rates of famotidine (used in the treatment of gastric and duodenal ulcers) and that of tolbutamide (oral antidiabetic drug) is increased by complexation with β-CD. The testosterone complex with amorphous hydroxypropyl β-CD allow an efficient transport of hormone into the circulation upon sublingual administration. This route avoids metabolism in the intestines and first-pass decomposition in the liver and thus improves bioavailability.

• Modifying reactivity : Cyclodextrins may increase or decrease the reactivity of the guest molecule depending on the nature of the reaction and the orientation of the molecule within the CD cavity. For example, α-cyclodextrin favours pH-dependent hydrolysis of indomethacin in aqueous solution, whereas β-CD inhibits it. The water solubility of β-CD (1.8 g/100 mL at 25°C) is insufficient to stabilize drugs at therapeutic doses. It is associated with nephrotoxicity when CD is administered by parenteral routes. 

• Modifying drug release : The hydrophobic forms of β-CD have been found useful as sustained-release drug carriers. The release rate of diltiazem (water-soluble calcium antagonist) was significantly decreased by complexation with ethylated β-CD. The release rate was controlled by mixing hydrophobic and hydrophilic derivatives of CD at several ratios. Ethylated β-CD has also been used to retard the delivery of isosorbide dinitrate, a vasodilator. 

• Taste masking : Cyclodextrins may improve the organoleptic characteristics of oral liquid formulations. The bitter taste of suspensions of femoxetine (antidepressant) is greatly suppressed by complexation of the drug with β-CD.

• Administration of therapeutic agents : Some therapeutic agents administered only as complexes due to physicochemical limitations. For example, iron complex with ferrous sulphate and carbonate and insulin complex with Zn and Vitamin-B12. These complexes reduce the GIT irritation, increase the absorption after oral administration and causes less irritation at the site of injection. 

• Use of ion exchange : Cholestyramine resin (quaternary ammonium anion exchange resin) is used to relief pruritus, the resin exchange chloride ion from bile result in increased elimination of bile through faeces. 

• In diagnosis : Technetium 90 (a radionuclide) is prepared in the form of citrate complex and this complex is used in diagnosis of kidney function and glomular filtration rate. Squibb (complex of a dye Azure A with carbacrylic cation exchange resin) is used for detection of achlorhydria due to carcinoma and pernicious anemia.

• Complexation as a therapeutic tool : Complexing agents are used for variety of uses. Many of them are related to chelation of metal ion. One of the important uses is preservation of blood. EDTA and citrates are used for in-vitro to prevent clotting. For example, anticoagulant acid citrate dextrose solution and anticoagulant sodium citrate solution. Citrates act by chelating calcium ion in blood as it depletes body calcium.

• Treatment of poisoning : Therapeutic procedure involves complexation to minimize poisoning. It is possible by two pathways. First by absorption of toxicants from GIT using complexing and adsorbing agent and second by inactivation of toxic material systemically and enhanced elimination of toxic substance through use of dialysis. In case of heavy metal poisoning the basic step involve in detoxification wherein inactivation of metal present in body is carried out through chelation (metal chelates) and the water-soluble constituents are readily eliminated from body via kidney. 

• Arsenic and mercury poisoning: The most effective agent is BAL (Dimercaprol). The arsenical dimercaprol is shown as: CH2SCHSAs-RCH2OH. Two sulfhydryl groups chelate with metal and a free OH group promotes water solubility. BAL is effective in treatment of poisoning from gold, bismuth, cadmium and polonium. 

• Lead poisoning: Treatment of choice for acute/chronic lead poisoning is i.v. administration of calcium or disodium complex of EDTA. This complex chelates ions which exhibit a higher affinity of EDTA than do the calcium. The route of administration of complex is important and is given only by slow i.v. drip in isotonic NaCl or Sterile 5% dextrose solution. Oral administration promotes absorption of lead from GIT and increase body levels of lead. 

• Radioactive materials: Poisoning with radioactive materials particularly with long biological half-life encounters problems that metal has toxic effect and body suffer from radiation damage. Uranium and plutonium exposure have been successfully treated with Canada. Plutonium get deposited and chelate in bone so, prompt treatment is necessary.

• Dialysis and complexation in poisoning: Removal of poisons from systemic circulation can be done by artificial kidney or by peritoneal dialysis. Dialyzing fluid is injected into peritoneal cavity continually and circulated into and out of the cavity. The toxic material diffuses through the wall of the blood vessel into the fluid present in the cavity. The efficiency of this procedure is improved by using principle of complexation. If the toxicant is complexed with some high molecular weight non-diffusible component, the rate of dialysis of the toxicant is increased and complexed toxicant is prevented from returning into the circulation. It is useful in humans and animals. In the treatment of intoxication due to salicylates and barbiturates serum albumin is commonly used. 

METHODS OF ANALYSIS

• A determination of the stoichiometric ratio of ligand to metal or donor to acceptor and a quantitative expression of the stability constant for complex formation are important in the study and application of co-ordination compounds. A limited number of the more important methods for obtaining these quantities are described below. 

Method of Continuous Variation : 

• The use of an additive property such as the spectrophotometric extinction coefficient such as dielectric constant or the square of the refractive index may also be used for the measurement of complexation. If the property for two species is sufficiently different and if no interaction occurs when the components are mixed, then the value of the property is the weighted mean of the values of the separate species in the mixture. This means that if the additive property, say dielectric constant, is plotted against the mole fraction from 0 to 1 for one of the components of a mixture where no complexation occurs, a linear relationship is observed.

• If solutions of two species A and B of equal molar concentration (and hence of a fixed total concentration of the species) are mixed and if a complex form between the two species, the value of the additive property will pass through a maximum (or minimum) as shown by the upper curv. For a constant total concentration of A and B, the complex is at its greatest concentration at a point where the species A and B are combined in the ratio in which they occur in the complex. The line therefore shows a break or a change in slope at the mole fraction corresponding to the complex. The change in slope occurs at a mole fraction of 0.5 indicating a complex of the 1:1 type. 

• When spectrophotometric absorbance is used as the physical property, the observed values obtained at various mole fractions when complexation occurs are usually subtracted from the corresponding values that would have been expected had no complex resulted. This difference, D, is when plotted against mole fraction, as shown in Fig. 4.11 the molar ratio of the complex is readily obtained from such a curve. 

• By means of a calculation involving the concentration and the property being measured, the stability constant of the complex formation can be determined by a method described by Bent and French. If the magnitude of the measured property, such as absorbance, is proportional only to the concentration of the complex MAn, the molar ratio of ligand A to metal M and the stability constant can be readily determined. The equation for complexation can be written as 

pH Titration Method : 

• This is most reliable method and used whenever the complexation is attended by a change in pH. The chelation of the cupric ion by glycine is represented as

• In the reaction of equation since two protons are formed (equation 4.4) the addition of glycine to a solution containing cupric ions should result in a decrease in pH. The potentiometric titration curves are obtained from the results of data obtained by adding a strong base to a solution of glycine and to another solution containing glycine and a copper salt. The pH against the equivalents of base added is plotted as shown. The curve for the metal–glycine mixture is well below that for the glycine alone, and the decrease in pH shows that complexation is occurring throughout most of the neutralization range. Similar results are obtained with other zwitterions and weak acids (or bases), such as N, N′-diacetyl ethylene diamine diacetic acid. 

Distribution Method : 

• The method of distributing a solute between two immiscible solvents can be used to determine the stability constant for certain complexes. The complexation of iodine by potassium iodide may be used as an example to illustrate the method. The equilibrium reaction in its simplest form is

• Additional steps also occur in polyiodide formation; for example, 2I− + 2I2 I 2− 6 may occur at higher concentrations, but it need not be considered here. Higuchi investigated the complexing action of caffeine, polyvinylpyrrolidone, and polyethylene glycols on many acidic drugs, using the partition or distribution method. According to a study, the reaction between caffeine and benzoic acid to form the benzoic acid–caffeine complex is

Solubility Method : 

• According to the solubility method, excess quantities of the drug are placed in well-stoppered containers, together with a solution of the complexing agent in various concentrations, and the bottles are agitated in a constant-temperature bath until equilibrium is attained. Aliquot portions of the supernatant liquid are removed and analyzed.

• The solubility method was used to investigate the complexation of p-amino benzoic acid (PABA) by caffeine. The results of the study are plotted as shown in Fig. 4.13. The point A at which the line crosses the vertical axis is the solubility of the drug in water. With the addition of caffeine, the solubility of PABA rises linearly owing to complexation. At point B, the solution is saturated with respect to the complex and to the drug itself. The complex continues to form and to precipitate from the saturated system as more caffeine is added. At point C, all the excess solid PABA has passed into solution and has been converted to the complex. Although the solid drug is exhausted and the solution is no longer saturated, some of the PABA remains uncompleted in solution, and it combines further with caffeine to form higher complexes such as (PABA-2 caffeine) as shown by the curve at the right of the diagram. 

Spectroscopy and Change Transfer Complexation Method: 

• Absorption spectroscopy in the visible and ultraviolet regions of the spectrum is commonly used to investigate electron donor–acceptor or charge transfer complexation. When iodine is analyzed in a non-complexing solvent such as CCl4, a curve is obtained with a single peak at about 520 nm. The solution is violet. A solution of iodine in benzene exhibits a maximum shift to 475 nm, and a new peak of considerably higher intensity for the charge shifted band appears at 300 nm. A solution of iodine in diethyl ether shows a still greater shift to lower wavelength and the appearance of a new maximum. These solutions are red to brown. These curves.

• In benzene and ether, iodine is the electron acceptor, and the organic solvent is the donor; in CCl4, no complex is formed. The shift toward the ultraviolet region becomes greater as the electron donor solvent becomes a stronger electron-releasing agent. These spectra arise from the transfer of an electron from the donor to the acceptor in close contact in the excited state of the complex. The more easily a donor such as benzene or diethyl ether releases its electron, as measured by its ionization potential, the stronger it is as a donor. Ionization potentials of a series of donors produce a straight line when plotted against the frequency maximum or charge transfer energies (1 nm = 18.63 call/mole) for solutions of iodine in the donor solvents. 

Other Methods: 

Many other methods are available for studying the complexation of metal and organic molecular complexes. They include NMR and infrared spectroscopy, polarography, circular dichroism, kinetics, X-ray diffraction, and electron diffraction. Several of these will be discussed briefly in this section. 

• 1H-NMR method: Complexation of caffeine with L-tryptophan in aqueous solution was investigated by using 1H-NMR spectroscopy. Caffeine interacts with L-tryptophan at a molar ratio of 1:1 by parallel stacking. Complexation is a result of polarization and π - π interactions of the aromatic rings. The tryptophan, which is presumed to be the binding site in serum albumin for certain drugs, can interact with caffeine even as free amino acid. However, caffeine does not interact with other aromatic amino acids such as L-valine or Leucine. 

• Circular dichroism: The coil–helix transition of Poly adenylic acid induced by the binding of the catecholamines norepinephrine and isoproterenol, using circular dichroism. Most mRNA molecules contain regions of Poly adenylic acid, which are thought to increase the stability of mRNA and to favor genetic code translation. The change of the circular dichroism spectrum of Poly adenylic acid was interpreted as being due to intercalative binding of catecholamines between the stacked adenine bases. Catecholamines may exert a control mechanism through induction of the coil-to-helix transition of Poly adenylic acid, which influences genetic code translation. 

• Infrared spectroscopy: The infrared spectroscopy was also used to investigate the hydrogen-bonded complexes involving polyfunctional bases such as proton donors. This is a very precise technique for determining the thermodynamic parameters involved in hydrogen-bond formation and for characterizing the interaction sites when the molecule has several groups available to form hydrogen-bonded. Caffeine forms hydrogen-bonded complexes with various proton donors: phenol, phenol derivatives, aliphatic alcohols, and water. From the infrared technique, the preferred hydrogen-bonding sites are the carbonyl functions of caffeine. Seventy percent of the complexes are formed at the C=O group at position 6 and 30% of the complexes at the C=O group at position 2 of caffeine. Conductometric and infrared methods has also been used to characterize 1:1 complex between uranyl acetate and tetracycline. 

PROTEIN BINDING 

A complete analysis of protein binding, including the multiple equilibria below. 

COMPLEXATION AND DRUG ACTION  

The depicts transfer of Drug (D), Complex (DC), and complexing agent (C) across biological membrane. With subsequent dissociation of complex after transfer.

• The rate of transfer of total drug on the right side of the membrane is a function of rate of transport of drug in its free and complex form. If transport rate of complex is more than drug, the diffusion will be aided by complex formation. If complexing agent is not diffusible rate of appearance of drug will be a function of transfer of free (uncompleted) drug. If the complex is not transported, diffusion is retarded by complexation. The mechanism by which complex formation can affect the passage compound include alteration of o/w partition coefficient, apparent solubility, effective size of drug, change in the charge of the drug and alteration in diffusion of drug. 

• One of the best examples of this class is the interference of calcium ions with the intestinal absorption of tetracycline. Earlier tetracycline preparations were made with calcium diphosphate to treat gastric irritation that was administered with milk, but it was observed that poor absorption was due to the formation of relative insoluble complex of tetracycline and calcium. Other examples wherein absorption was decreased due to formation of complex include oral administration of neomycin and kanamycin with bile salt. Complexing agent EDTA depress the absorption of strychnine alcohol and sulfanilamide in animals. EDTA is thought to be related to their interaction with metal ion in the G.I.T. On the contrary, enhanced drug absorption through complex formation was also observed in some cases. Thus, complex formation was proved to be an effective means of enhancing the absorption of poorly absorbed drug. For example, improvement in intestinal absorption of tetracycline's with the addition of citric acid, glucosamine or sodium hexametaphosphate or use of tetracycline phosphate complex. Other examples where drug absorption includes heparin whose absorption is increased in G.I.T. in the presence of EDTA or SLS or dioctyl sodium sulfosuccinate. The intestinal absorption of various quaternary ammonium compounds, organic acids and some neutral molecules such as mannitol and inulin is also found to be increased in presence of EDTA.

• Other examples of drugs where complex has significant effect on drug action is through enhancing solubility and drug stability. For example, adrenochrome Monos Emi carbazone is complexed with sodium salicylate. Adrenochrome (active) was found to be unstable in solution and semi carbazone has only limited solubility at the pH at which it is stable. However, the stable product can be prepared by the addition of sodium salicylate which complexes with adrenochrome, and thus increases its apparent solubility by about 10 folds. The injectable caffeine and sodium benzoate is used as stimulant and diuretic. The complexation of caffeine by sodium benzoate increases solubility of caffeine.

•  The problem of stabilization of the ingredient presents in the preparation against hydrolysis, oxidation etc. is another instance where complexation formation has been used. The interaction of labile functional groups of a drug with complexing agent may protect the drug from the attack of other species or the interaction may alter the usual electronic properties of the drug that result into either increase or decrease in stability. For example, local anesthetic esters have been stabilized against hydrolysis by complexation with caffeine. The half-life for procaine in the solution has been observed to increase from 26 h in the absence of caffeine to about 46 h in the presence of 2% caffeine and to about 71 h in the presence of 5% caffeine. The stabilization of certain compound can be done by incorporation within the crystal lattice of a solid or with in the voids formed by the arrangement of large polymeric molecules in solution.

CRYSTALLINE STRUCTURES OF COMPLEXES

• Complex or co-ordination compounds cover the range from quite simple inorganic salts to elaborate metal-organic hybrid materials and intricate bioactive metalloproteins. Their present uses and their potential applications are diverse due to their compositions, their molecular and crystal structures and their chemical and physical properties. Besides their use as chemical reactants, complex compounds are considered for extraction processes and as active agent in remedies and for drug delivery.

THERMODYNAMIC TREATMENT OF STABILITY CONSTANTS

• The thermodynamics of metal ion complex formation provides much significant information. In particular, it is useful in distinguishing between enthalpic and entropic effects. Enthalpic effects depend on bond strengths and entropic effects have to do with changes in the order/disorder of the solution as a whole. The chelate effect, below, is best explained in terms of thermodynamics. 

• All published stability constant values refer to the specific ionic medium used in their determination and different values are obtained with different conditions, as illustrated for the complex Cull (L = glycinate). Furthermore, stability constant values depend on the specific electrolyte used as the value of Γ is different for different electrolytes, even at the same ionic strength. There does not need to be any chemical interaction between the species in equilibrium and the background electrolyte, but such interactions might occur in particular cases. For example, phosphates form weak complexes with alkali metals, so, when determining stability constants involving phosphates, such as ATP, the background electrolyte used will be, for example, a tetraalkylammonium salt. Another example involves iron which forms weak complexes with halide and other anions, but not with perchlorate ions.