2.

Complexation and Protein Binding

2.1. Definitions:

• A complex is a species formed by the reversible or

irreversible association of two or more interacting
molecules or ions.

• Complexes have been usually referred to as

coordination compounds.

1
 2.1. Definitions:
• Once complexation occurs, the physical and chemical
properties of the complexing species are altered (solubility,
stability, partitioning, energy absorption, and emission and
conductance)

• Complex formation usually alters the physical and chemical

properties of the drug. For examples: (1) chelates of
tetracycline with calcium are less water soluble and are poorly
absorbed, (2) theophylline complexed with ethylenediamine to
form aminophylline is more water soluble and is used for
parenteral and rectal administration, (3) cyclodextrins are used
to form complexes with many drugs to increase their water
solubility.

2.1. Definitions:

• Complexes, according to the classic definition, result from a

donor-acceptor mechanism or Lewis acid-base reaction
between two or more different chemical constituents.

• A Lewis acid is a molecule or ion that accepts an electron pair

to form a covalent bond. The acceptor, or constituent that
accepts a share in the pair of electrons, is frequently a metallic
ion, although it can be a neutral ion.

• A Lewis base is a molecule that provides a pair of unshared

electrons by which the base coordinates with the acid. Any
nonmetallic atom or ion, whether free or contained in a neutral
molecule or in an ionic compound, that can donate an electron
pair may serve as the donor.

2
 2.2 Classification of Complexes

• Intermolecular forces involved in the formation of complexes

are
 Van der Waals forces of dispersion
 Dipolar and induced dipolar types
 Hydrogen bonding: provide a significant force in some
molecular complexes
 Coordinate covalence is important in metal complexes.
 Charge transfer
 Hydrophobic interaction

2.2 Classification of Complexes

• Complexes may be divided broadly into two classes depending

on whether the acceptor component is a metallic ion or an
organic molecule; these are classified according to one
possible arrangement in the following table.

• A third class, the inclusion / occlusion compounds, involving

the entrapment of one compound in the molecular
framework of another is also included in the table.

3
 2.2 Classification of Complexes

1. Metal Ion Complexes:

a. Inorganic Type
b. Chelates
2. Organic molecular Complexes
3. Inclusion/occlusion Complexes
a. channel lattice type
b. layer type
c. clathrates
d. monomolecular inclusion compounds

2.3. Metal Complexes:

a. Inorganic Complexes:

• Examples of electron donors include :NH3, H2O:, CN:- and Cl:- can act
as ligands.

• Metal ions are electron acceptors so that are capable of binding

such ligands. Examples include Co3+ , Ni2+, Ag+, Fe2+ and Cr3+.

• Hybridization plays an important part in coordination compounds in

which sufficient bonding orbitals are not ordinarily available in
metal ions.

• The type of the hybridization in the complex affects its molecular

structure or shape (refer to the textbook; Table 10-2).

4
 2.3. Metal Complexes:
a. Inorganic Complexes:

2.3. Metal Complexes:

a. Inorganic Complexes:

• Example:
[Co(NH3)6]3+3Cl-

– The ammonia molecules in the hexamineocobalt III chloride is

called the ligand.
– The ligands are coordinated to the cobalt ion.
– The coordination number of the cobalt ion is the number of
ammonia molecules coordinated to the ion (6).
– Each ligand donates a pair of electrons to form a coordinate
covalent link between itself and the central ion.

Co3+ + 6:NH3 = [Co(NH3)6]3+

5
 2.3. Metal Complexes:
a. Inorganic Complexes:

– Co and Co3+ has the following ground state electronic configuration:

Co: 1s22s22p63s23p63d74s24p04d0
Co3+: 1s22s22p63s23p63d64s04p04d0

3d 4s 4p

– Hybridization produces 6 orbits (d2sp3) with no electrons which can form a

covalent coordinate bond with an electron pair donor (NH3). The
Co(NH3)63+ has an octahedral structure.

.. .. .. .. .. ..
3d d2sp3

2.3. Metal Complexes:

a. Inorganic Complexes:

• In case of [Ni(CN)4]-2 the ground state electronic configuration of Ni and Ni+2

is:
Ni: 1s22s22p63s23p63d84s24p04d0
Ni2+: 1s22s22p63s23p63d84s04p04d0
3d 4s 4p

• Hybridization produces 4 orbits of the type dsp2 that can be filled with
electron pairs donated by the ligand. The dsp2 hybridization produces a
square planar structure.

.. .. .. ..
3d dsp2 4p

6
 2.3. Metal Complexes:
b. Chelates:

– When a ligand provides one group for attachment to the central

ion, then its called monodentate.
– Molecules with two or three groups are called bidentate and
tridentate respectively (multidentate or polydentate).
– If a metal ion binds to two or more sites on a multidentate
ligand, a cyclic complex is formed; this cyclic complex is known as
a chelate.
– Chelates are complexes that typically involve a ring-like structure
formed by the interaction between a partial ring of atom and a
metal.
– In chelates, ligands are usually organic molecules, known as
chelating agents, chelators, chelants or sequestering agents.

7
 2.3. Metal Complexes:
b. Chelates:

• Some of the bonds in a chelate may be ionic or of

the primary covalent type, while others are
coordinate covalent links.

• The formation of chelate complexes is controlled

by stringent steric requirements on both the
metal ion and the ligand.

2.3. Metal Complexes:

b. Chelates:

• Many biologically important molecules (e.g.

hemoglobin, insulin, cyanocobalamine,
chlorophyll) are chelates.

• Other biological chelates include albumin, the

most common plasma protein which acts as a
carrier of various metal ions (Cu2+ and Ni2+) and
small molecules in the blood.

8
 2.3. Metal Complexes:
b. Chelates:

• In chlorophyll the central ion is magnesium, and the large organic

molecule is a porphyrin. The porphyrin contains four nitrogen atoms
that form bonds to magnesium in a square planar arrangement.

2.3. Metal Complexes:

b. Chelates:

• Hemoglobin also contains a porphyrin chelating agent bonded

to an iron II ion.

9
 2.3. Metal Complexes:
b. Chelates:
Vitamin B12, Cyanocobalamin

2.3. Metal Complexes:

b. Chelates:

• EDTA is a synthetic chelating agent used to sequester ions

(iron and copper) that catalyzes oxidative degradation
reactions in drug preparation.
• EDTA is also widely used to sequester and remove calcium
ions from hard water.

10
 2.3. Metal Complexes:
b. Chelates:

• Ethylenediamine tetraacetic acid (EDTA) has six points for

attachment to the metal ion and accordingly is hexadentate.

EDTA Gadolinium Complex

(atomic number 64)

2.3. Metal Complexes:

• The chelating properties of procainamide (Sodium channel

blocker, Class IA antiarrhythmic) has been used as an assay for its
content in pharmaceutical preparations. Complex formation with
Cu2+ results in a colored compound that can be measured by
visible spectrophotometry. Thus calorimetric methods to assay
procainamide in injectable solutions is based on the formation of
a 1:1 complex of procainamide with cupric ion at pH 4 to 4.5.

11
 2.3. Metal Complexes:
b. Chelates:

• Tetracycline antibiotics are capable of acting as chelating agents

and binding a variety of polyvalent metal ions (Fe2+, Mg2+, Al3+,
Bi3+ ).

• The complexation results in changes in both the drugs’ and the

metal ions’ physical and chemical properties.

• The complexation between tetracycline antibiotics and metal

ions either in food (cabbage) or in pharmaceutical preparations
(iron containing supplements) has been found to reduce both
the solubility and bioavailability of the antibiotics.

2.3. Metal Complexes:

b. Chelates:

• Tetracyclines are contraindicated in pediatric patients since

they are prone to tetracycline complexation of calcium in
teeth and bones resulting in teeth discoloration and bone
growth problems.

http://www.medicinescomplete.com/mc/rem/2012/images/c33-fig-33-4.png

12
 2.4. Organic Molecular Complexes:

• An organic coordination compound or molecular complex is made of

constituents held together by weak forces of:
 Hydrogen bonds. Or
 The donor-acceptor type: in these complexes, the electron donor-
acceptor mechanisms involve basically van der Wall’s forces or charge
transfer (i.e. not covalent or coordinate bonds as in metal ion
complexes)).

• In charge transfer complexes, one molecule polarizes the other

resulting in a type of ionic interaction. London forces, dipole-induced
dipole and dipole-dipole interactions play a role in the stability of the
complex.

2.4. Organic Molecular Complexes:

• For example, the polar nitro groups of trinitrobenzene induce

a dipole in the readily polarizable benzene molecule and the
electrostatic interaction that results leads to complex
formation.

13
 2.4. Organic Molecular Complexes:

• A factor of importance in the formation of molecular

complexes is the steric requirement. If the approach and close
association of the donor and acceptor molecules are hindered
by steric factors, the complex is not likely to form.

• Many organic complexes are so weak that they can not be

separated from their solutions as definite compounds and
they are often difficult to detect by chemical and physical
means.

2.4. Organic Molecular Complexes:

• Many drug complexes are of this type, of special importance is

the complexation of esters.

• Many important drugs are esters, they can form complexes

through their carboxyl oxygen which usually have a partial
negative charge and is strongly nucleophilic or basic and
capable of bonding with an active electrophilic hydrogen or
any other electrophilic center of the other molecule.

14
 2.4. Organic Molecular Complexes:

The complexation between caffeine and benzocaine can occur as a result of

dipole-dipole interaction between the nucleophilic carboxyl oxygen of
benzocaine and the electrophile nitrogen of caffeine.

2.4. Organic Molecular Complexes:

• The incompatibility of certain polymers used in suspensions,

emulsions, ointments and suppositories and certain drugs
may be due to the formation of organic molecular complexes.
The incompatibility may be manifested as a precipitate,
flocculate, delayed biological absorption, loss of preservative
action, or other undesirable physical, chemical, and
pharmacologic effect.

15
 2.5. Inclusion Compounds:

• This class of complexes differ from the previously discussed

classes in that they are mainly the result of the architecture of
the molecules rather than their chemical affinity.

• In this class of complexes, one of the constituents of the

complex is trapped in the open lattice or cage like structure of
the other to yield a stable arrangement.

• Some times they are referred to as occlusion compounds.

16
 2.5.1. Channel Lattice Type:

• In this type of inclusion compounds, the crystals of the host

constituent are arranged to form a channel into which the
complexing molecule can fit.

• A very common example of such complexes is the one formed

by starch and iodine where iodine molecules are trapped
within channels consisting of spirals of glucose residues of
starch.

2.5.1. Channel Lattice Type:

17
 2.5.1. Channel Lattice Type:

• Other materials capable of forming these channels include bile

acids, urea and thiourea.

 Such phenomena has been

used to resolve mixtures of
optical isomers because of
the high stereo specificity of
the complexation.

2.5.2. Layer Type:

• Clays such as montmorillonite ([(Mg0.33Al1.67)Si4O10(OH)2]Na0.33),

principal component of bentonite have the ability to arrange
themselves in layers.

• Many molecules can be trapped within these layers to form

inclusion complexes (e.g. HCs, alcohols and glycols).

• Minimal stereo-specificity in comparison to channel type inclusion

complexes.

18
19
 2.5.3. Clathrates:

• The term Clathrates refers to a group of compounds

which crystallizes in the form of a cage-like lattice in
which the coordinating compounds are trapped.

Cage-like structure of clathrates

2.5.3. Clathrates:

• Chemical bonds are not involved in these complexes and only the
molecular size of the encaged component is of importance.

• For example, hydroquinone crystallizes in a cagelike hydrogen-bonded

structure. The holes permit the entrapment of one small molecule to
every two hydroquinone molecules. Small molecules such as
methanol, HCl and CO2 may be trapped, but smaller molecules as H2
and larger molecules as ethanol cannot be accommodated.

• Warfarin sodium USP is an example of a clathrate of water, isopropyl

alcohol and sodium warfarin in the form of a white crystalline powder.

20
 2.5.3. Clathrates:

• The stability of a clathrate may be related to the confinement

of a prisoner. The stability of a clathrate is due to the strength
of the structure, that is, to the high energy that must be
expanded to decompose the compound, just as a prisoner is
confined by the bars that prevent his escape.

2.5.4. Monomolecular inclusion Compounds:

• In this class of inclusion compounds, a single guest molecule is

entrapped in the cavity of one host molecule.

• A representative example of such compounds is cyclodextrins.

• Cyclodextrins are cyclic oligosaccharides containing a minimum

of six D (+) glucopyranose units attached by an -1,4 linkage.

• Cyclodextrins are produced from starch by the action of bacterial

amylase.

21
 2.5.4. Monomolecular inclusion Compounds:

• The naturally occurring -CD, -CD and -CD contain 6, 7

and 8 units of glucose respectively.

• The cyclodextrins form a ring and the molecule actually

exists as a truncated cone in which guest molecules can be
accommodated to form an inclusion complex.

• The size of the cavity increases by increasing the number

of glucose units, -CD being the smallest, -CD is not very
useful for pharmaceutical applications, -CD and -CD are
more useful owing to the large cavity.

2.5.4. Monomolecular inclusion Compounds:

22
 2.5.4. Monomolecular inclusion Compounds:

• The interior of the CD cavity is usually hydrophobic because of

the CH2 groups, while the exterior of the cavity is hydrophilic
because of the presence of the hydroxyl groups.

• Complexation with CD does not ordinarily involve the

formation of covalent bonds. Molecules of appropriate size
and stereochemistry can be included in the cyclodextrin cavity
by hydrophobic interaction.

2.5.4. Monomolecular inclusion Compounds:

• The aqueous solubility of many lipophilic drugs is

improved by complexation with CD and CD derivatives.

• The bioavailability of many of these drugs has been

improved as well.

• CD has been used to improve the organoleptic

characteristics (bitter taste) of oral liquid formulations.

• Hydrophobic CD derivatives has been used as sustained

release drug carriers. Ethylated -CD has been used to
reduce the release rate of the water soluble drug
diltiazem.

23
 2.6. Methods of Analysis:

• Different applications of complexes in pharmaceutical

sciences require a quantitative knowledge of the
complexation process and product.

– The Stoichiometric Ratio of ligand to metal or donor to

acceptor.
– The Stability Constant of the formed complex.

2.6.1. Method of Continuous Variation:

• An additive property such as the spectrophotometric extinction

coefficient or dielectric constant which changes with changing the
composition of a mixture can be used for the measurement of
complexation.

• If the property for two species is 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.

• A straight line will result from the plot of the additive property vs.
the composition of the mixture (mole fraction) if no complexation
occurs.

24
 Explanatory example

2.6.1. Method of Continuous Variation:

Complex
(Dielectric Constant)
Additive Property

Curve for no
Complex

100% Composition 100%

A B
Mole fraction

25
 2.6.1. Method of Continuous Variation:

• If complexation occurs then the value of the additive property

will decrease or increase depending on the nature of the
complex formed and will pass through either a minima or
maxima.

• The point of inflection represents the ratio of the two

components in the complex.

• For a constant total concentration of both species A and B, the

complex is at its greatest concentration at a point where the
species A and B are mixed at the same ratio in which they
occur in the complex.

2.6.1. Method of Continuous Variation:

A plot of an additive property against mole fraction of one of the species in which
complexation between the species has occurred. The dotted line is that expected if no
complex had formed

26
 2.6.1. Method of Continuous Variation:

A plot of absorbance difference against mole fraction showing the result of

complexation

2.6.1. Method of Continuous Variation:

• This method has been applied for the determination of the

stoichiometric ratio of complexation between Cu+2 (blue
solution) with thiobarbiturate (colorless solution) to produce a
green complex.

• In such cases the equation for complexation can be written as

follows:
M + nA  MAn

• The stability constant is:

K
MAn 
[ M ][ A]n

27
 2.6.1. Method of Continuous Variation:

• By taking the log of the previous relation we get:

log [MAn] = log K + log [M] + nlog [A]
Where
• [MAn] is the concentration of the complex
• [M] is the concentration of the uncomplexed metal ion
• [A] is the concentration of the uncomplexed ligand
• K is the stability constant
• n is the stoichiometric ratio of the metal – ligand in the complex
• We use the above equation to determine the stability
constant for complex formation K.

2.6.1. Method of Continuous Variation:

• The concentration of the metal ion is held constant and the

concentration of the ligand is varied, the corresponding
concentration of the complex [MAn] is obtained by
spectrophotometry.

• If log [MAn] is plotted against log [A] a straight line is obtained

with a slope of “n” or the coordination number and and
intercept of ([M] + log k).

• [M] is constant and so log k can be calculated.

28
 2.6.1. Method of Continuous Variation:

log [MAn]

Slope = n

log [M] + log k

log [A]

2.6.2. Distribution Method:

• Distribution of solute between two phases can be used to calculate

the stability constant of complexes.
• This depends on the fact that the distribution coefficient applies
only to the species common to both phases.
• The complexation of iodine by potassium iodide can be represented
by the following equilibrium:
I2+I-=I3-
• See problem 11-3 page 278 from the physical pharmacy book by
Martin (4th edition). If you have the fifth edition of the physical
pharmacy book by Martin, see problem 11-3 page 715. If you have
the sixth edition of the physical pharmacy book by Martin, see
problem 10-2.

29
 2.6.2. Distribution Method:
Example 10-2: Martin’s 6th ed.
Ko/w=625; [KI]=0.125 M; [I2]o= 0.1896 M; [I2]w, total= 0.02832 M

Ko/w=625= [I2]o/ [I2]w= 0.1896/ [I2]w…………… [I2]w,free= 0.000303 M

[I2]w, total= 0.02832 M = [I2]w,free + [I2]complexed= 0.000303 + [I2]complexed
……. [I2]complexed= 0.02802 M= [I-]complexed= [I3-]complexed
[I-]free= [I-]total – [I-]complexed= 0.125 – 0.02802= 0.09698M

K = [I3-]complexed/ [I2]w,free* [I-]free

K = 0.02802/ 0.000303*0.09698= 954

2.6.3. Solubility Method:

Solutions of the Excess solid

complexing agent (Drug) In
in various stoppered
concentrations containers

Bottles are agitated in a

constant temperature bath
until equilibrium is attained

Aliquot portions of the

supernatant liquid are
removed and analyzed

30
 2.6.3. Solubility Method:
Point B
Saturation
Point C
Molar Concentration of the Drug

All excess solid

converted to
complex

Point A
Solubility of
the Drug

Molar Concentration of the Ligand

Solubility profile of a drug in the presence of a complexing agent

2.6.3. Solubility Method:

• Point A in the previous graph represents the solubility of the drug in

water.
• As we add the ligand, the drug complexes with it and more solid drug
is withdrawn into solution to maintain the free drug concentration
constant, resulting in increased total drug concentration.
• Consequently the concentration increases to reach point B.
• At point B the system is saturated with respect to both the drug and
the complex.

31
 2.6.3. Solubility Method:

• In the plateau BC, the complex continues to form and precipitate,

however, the total concentration does not change because of the
presence of excess solid.

• At point C, all the excess solid has been exhausted and turned into
the complex.

• The decline in the total concentration is due to the formation of

higher complexes with lower solubility.

2.6.3. Solubility Method:

• To calculate the ratio of the constituents of the complex we

use the solubility profile.

• First we calculate the amount of the ligand entering the

complex throughout the plateau (B-C).

• The amount of the drug entering the complex during this

plateau is the solid drug that dissolves during this period
which is obtained by subtracting the amount dissolved at
point B from the total amount of solid added initially.

32
 2.6.3. Solubility Method:

• To calculate the stability constant we will use the AB segment.

– Choose any ligand concentration and then extrapolate to get

the corresponding drug concentration.
– The concentration of the complex at that specific ligand and
drug concentrations is obtained by subtracting the drug
concentration from the free drug saturation concentration A.
– The concentration of the free drug is A.
– The concentration of the free ligand is obtained by
subtracting the concentration of the complex from the
originally chosen ligand concentration.
•

Example 10-3 (Martin’s 6th ed.): PABA (drug) and caffeine (ligand)
Initial PABA concentration is 7.3*10-2 mol/L.
Solubility of PABA in water is 4.58*10-2 mol/L
At point B:
PABA conc.= 5.5*10-2 mol/L and caffeine conc.= 1.7*10-2 mol/L
At point C:
Caffeine conc.= 3.5*10-2 mol/L
Answer:
To calculate the stoichiometric ratio:
The concentration of caffeine in BC region is:
3.5*10-2 – 1.7*10-2 = 1.8*10-2 mol/L.
PABA conc. entered the complex within BC region is:
7.3*10-2 – 5.5*10-2 = 1.8*10-2 mol/L.
So the stoichiometric ratio is 1: 1

33
Example 10-3 (Martin’s 6th ed.): PABA (drug) and caffeine
(ligand)……..cont,
To calculate K
In AB region take a point, say (caffeine conc., PABA conc.) =
(1.0*10-2 mol/L, 5.31*10-2 mol/L )
Providing that solubility of PABA in water is 4.58*10-2 mol/L
Answer:
[PABA]complexed= 5.31*10-2 – 4.58*10-2 = 0.73*10-2 mol/L=
[caffeine]complexed=[complex]
[caffeine]free= 1*10-2 – 0.73*10-2 = 0.27*10-2 mol/L

K= (0.73*10-2 )/ (4.58*10-2 ) (0.27*10-2 )= 59

2.6.3. Solubility Method:

• The problems to be solved in relation to the solubility

method from the physical pharmacy book – 4th
edition are: 11-4, 11-5, 11-6 – all from page 279. If
you have the fifth edition, solve the following
problems in relation to solubility: 11-4, 11-5, 11-6 –
all from page 715.

• Example 11-3 page 265 in the fourth edition from the

physical pharmacy book. If you have the fifth edition,
see example 11-3 page 283.

34
 2.6.3. Solubility Method:

• If you have the sixth edition of the physical pharmacy

book by Martin, see Example 10-3 page 212 and
solve problem 10-3 in relation to the solubility
method.

2.7. Protein Binding:

• Protein-ligand interaction is important in drug binding to

receptor for pharmacologic activity, enzyme-substrate
interaction in catalysis and antigen-antibody recognition in
immunity, and the interaction of drugs with plasma proteins.

• Large drug complexes, such as drug-protein complexes, do not

cross the cell membrane easily. These complexes must
dissociate to free drug for absorption at absorption site, or to
permit transport across cell membrane or glomerular
filtration before the drug is excreted in the urine.

35
 2.7. Protein Binding:

• The binding of drugs to proteins in the body can affect their

actions by:

– Affecting the drug distribution throughout the body.

– Affecting the activity of the drug by reducing amount of

free drug available to bind the receptor site.

– Retard the excretion of the drug and increase its half life.

2.7. Protein Binding:

• Since proteins are molecules composed of different types of

amino acids, the interactions between proteins and small
molecules can occur through one or more than one of the
followings:
– Hydrogen bonding
– Electrostatic interactions
– van der waals interaction
– Hydrophobic interactions

36
 Figure - Schematic representation of hydrophobic interaction

2.7. Protein Binding:

• The two most important parameters of protein binding are:

– Affinity of binding , expressed using the association
constant and is a measure of the strength of interaction
between the protein and drug molecule.
– Capacity referring to how extensive the drug-protein
binding is in terms of how many sites on the protein are
available for drug to bind.

37
 2.7.1. Binding Equilibria:

• The interaction between a group or free receptor P in a

protein and a drug molecule D is written as:

P+D PD

• The equilibrium constant can be written as:

[ PD ]
K
[ P][ D f ]
• In which K is the association complex, [P] is the concentration
of the protein in term of free binding sites, [Df] is the
concentration of the free drug and [PD] is the concentration
of the protein-drug complex.

2.7.1. Binding Equilibria:

• If the total protein concentration is [Pt], it can be

presented as:
[Pt] = [P] + [PD]
or
[P] = [Pt] - [PD]
• Substituting the expression for [P] into the equilibrium
expression gives:
[ PD ]
K
([ Pt ]  [ PD ])[ D f ]

[ PD]  K[ D f ]([ Pt ]  [ PD])

[ PD]  K[ D f ][ PD]  K[ D f ][ Pt ]

38
 2.7.1. Binding Equilibria:

• Rearrangement to get a ratio of moles of drug bound

[PD] per mole of total protein [Pt] gives:

[ PD ] K[D f ]
r 
[ Pt ] 1  K [ D f ]

• This equation assumes the presence of one binding

site, in case of the presence of v independent binding
sites, the equation becomes:

K[D f ]
r v
1  K[D f ]

2.7.1. Binding Equilibria:

Analysis of Plasma Protein Binding:

• Mathematical analysis of protein-drug interactions is

performed to evaluate the binding affinity or association
constant and the capacity as measured by the number of
binding sites.

• In vitro analysis of protein-drug interactions is usually studied

using a single protein solution (albumin) in conditions that
simulate the physiologic environment (aqueous buffer at 7.4,
ionic strength 0.16 and temperature of 37oC).

39
 2.7.1. Binding Equilibria:
Analysis of Plasma Protein Binding:

• The interactions between protein and drugs are described

according to the law of mass action:

D+P DP
• At equilibrium
Df + (Pt - Db) DP
R (relation between bound drug to total protein concentration)
as we have seen before is equal to
[ PD ] vK[ D f ]
r 
[ Pt ] 1  ( K [ D f ])

2.7.1. Binding Equilibria:

Analysis of Plasma Protein Binding:

• Cross multiplying of the previous equation gives:

vK[ D f ]  r (1  K [ D f ])
vK[ D f ]  r  rK[ D f ]
• Bringing r to the left and dividing both sides by [D f] gives

r vK[ D f ] rK[ D f ]
 
[D f ] [D f ] [D f ]
r
 vK  rK
[D f ]

40
 2.7.1. Binding Equilibria:
Analysis of Plasma Protein Binding:
• The previous equation is a straight line equation, we can plot
r/[Df] vs r which is the ratio of the bound drug to the total
protein concentration and what we get is a straight line with a
slope equal to –K and a y-intercept of vK.
• The plot of r/[Df] vs. r is known as the Scatchard plot.

Intercept=vK

Slope=-K
r / [Df]

2.7.1. Binding Equilibria:

Analysis of Plasma Protein Binding:

• Solve problems 11-15 (a) page 281 and 11-18 page

282 from the physical pharmacy book by Martin – 4th
edition. If you have the 5th edition of the book, solve
problems 11-15 (a) page 718 and 11-18 page 718.

41
 2.7.2. Determination of Protein Binding:

• Protein binding can be evaluated using many techniques

including equilibrium dialysis, ultrafiltration and dynamic
dialysis which depend on the selective diffusion of small
molecules through semipermeable membranes.

2.7.3. Equilibrium Dialysis:

• The protein (e.g. serum albumin or other protein under

investigation) at a specific concentration is placed in a
cellulose semipermeable tubing that is tied securely and
suspended in vessels containing the drug in various
concentrations.

• If binding occurs, the drug concentration in the sac containing

the protein is greater at equilibrium than the concentration of
the drug in the vessel outside the sac.

• Samples are collected and analyzed to obtain the

concentration of free and complexed drug.

42
 2.7.3. Equilibrium Dialysis:

Semipermeable membrane
allowing drug to diffuse in
while preventing protein
from diffusing out

Protein Solution

Drug (Free & Bound)

Drug (Free)

Drug Solution

2.7.3. Equilibrium Dialysis:

• The fraction bound of the drug (β) and the ratio of
bound drug to total protein (r) inside sac can be
calculated as follows:

[bound drug] [bound drug]

r 
[total protein] [total drug]
• See problem 11-16 page 281 from physical pharmacy by
Martin (4th edition). If you have the fifth edition, see problem
11-16 page 718.

43
 2.7.4. Ultrafiltration:

• In ultrafiltration, a hydraulic pressure is used to force the free drug

molecules to diffuse through a semipermeable membrane while
the protein and its bound drug trapped by the membrane.

• The ultrafiltrate is then analyzed for the free drug concentration.

• Similar to the equilibrium dialysis method, a potential error in the

ultrafiltration techniques may result from the drug binding to the
membrane.

• This method is usually less time consuming than the equilibrium

dialysis method. The choice between ultrafiltration and equilibrium
dialysis methods depends on the characteristics of the drug.

2.7.4. Ultrafiltration:

Hydraulic
pressure

Drug and
Ultrafiltrate protein
(Free drug) solution

44
 2.7.4. Ultrafiltration:
Example (10-5) (Martin’s 6th ed.):
[Dt]= 3.24*10-5 M; [Pt]= 1*10-4 M.
After equilibrium, the ultrafiltrate has absorbance, A, 0.559 at
540 nm. The molar absorptivity of the drug, ɛ, is 5.6 * 104
L/mol.cm
Calculate r and the percentage of drug bound.
Answer:
A=ɛbc……. A=ɛb[Df]… [Df]= 0.559/1 (5.6*104)= 0.99*10-5M
[Db]= [Dt] – [Df] = (3.24*10-5) – (0.99*10-5) = 2.25 *10-5 M

r = [Db]/ [Pt] = 2.25 * 10-5/ 1 * 10-4 = 0.225

% of bound drug is ([Db]/ [Dt])*100 = 69%

2.7.4. Ultrafiltration:

• Solve problem 11-19 page 282 from physical pharmacy by

Martin (4th edition). If you have the fifth edition of the
book, solve problem 11-19 page 718. If you have the sixth
edition of the book, solve problem 10-11.

• See example 11-5 page 270 from physical pharmacy by

Martin (4th edition). If you have the fifth edition, see
example 11-5 page 288. If you have the sixth edition, see
example 10-5 page 217.

45
 2.7.5. Dynamic Dialysis:

• This is a modified form of equilibrium dialysis where known

concentrations of protein and ligand are placed in the dialysis
tubing.

• Periodically, the outside buffer solution is replaced with fresh

buffer to maintain sink conditions.

• Samples of the solution outside the dialysis bags are collected

periodically and assayed for the drug content.

• Dynamic dialysis significantly accelerates the diffusion of free

ligand into the buffer solution and thus can shorten the
dialysis time

2.7.5. Dynamic Dialysis:

• The rate of disappearance of drug from a dialysis cell is

proportional to the concentration of the unbound drug.

• The dialysis process follows the rate law:

d [ Dt]
  K[ D f ]
dt
Where:
– [Dt] is the total drug concentration,
– [Df] is the concentration of free or unbound drug in the
dialysis bag
– -d[Dt]/dt is the rate of loss of drug from the dialysis bag

• The equation can be rewritten as follows:

ln [Dt] = ln[Df] - kt

46
 2.7.5. Dynamic Dialysis:

• In the presence of protein, the rate of loss of drug from the

dialysis bag is slowed in comparison with the rate in the
absence of protein.

• K in the equation is a rate constant associated with the

diffusion of free drug only and can be obtained from the slope
of the semilogarithmic plot of [Dt] vs time when the
experiment is conducted in the absence of protein.

2.7.5. Dynamic Dialysis:

1.00 Drug with

protein
Total drug within the

Dynamic dialysis
0.80
plot
bag Dt

0.60

0.40
Drug without
protein
0.20

Time

47
 2.7.5. Dynamic Dialysis:

1.00

Drug with
protein
Total drug with the

0.10
Dynamic dialysis
bag Dt

plot

0.01 (log transformation)

Drug without
0.001 protein

0.0001

Time

48