Physical Pharmacy

1. Presented by
(Dr) Kahnu Charan Panigrahi
Asst. Professor, Research Scholar,
Roland Institute of Pharmaceutical Sciences,
(Affiliated to BPUT)
Web of Science Researcher ID: AAK-3095-2020
Complexation
And Protein Binding

2. Complex compounds are defined as those molecules in
which most of the bonding structures can be described by
classical theories of valency between atoms, but one/more
of these bonds are some what anomalous.
Complexes or coordination compounds, according to the
classic definition, result from a donor–acceptor mechanism
or Lewis acid–base reaction between two or more different
chemical constituents.
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 coordinate covalence is important in metal complexes

3. COMPLEXES
Metal
complexes
Organic
molecular
complexes
Inclusion
compounds
1. Inorganic types
2. Chelates
3. Olefin type
4. Aromatic type
1. Drug-caffine
complex
2. Polymer type
3. Picric acid type
4. Quinhydrone
type
1. Channel type
2. Layer type
3. Clathrates
4. Mono
molecular type

4. I) Metal complexes:
METAL
(substrate)
Central atom
BASE
(ligand)
Electron pair donor
COMPLEX
formed by
co-ordination
bond
In this complex metal ion constitute the central atom and
interact with a base.

5. A) INORGANIC COMPLEXES:
Werner postulates:
1. There are 2 types of valency primary (ionic), secondary
(coordinate).
2. Same type of anion/ radical/ molecule may be held by any one /
both type of valency.
3. Every central atom has fixed number of non-ionic valences (co-
ordination number)
4. The co-ordination atoms occupy the first sphere/coordination
sphere, other atoms occupy second/ ionization sphere.
5. Neutral molecules/ions may satisfy non-ionic valency.
6. The non-ionic valences are directed to specific positions in space.
Ex: [Co Cl (NH3)5] Cl2
Substrate Coordination sphere
Ionization sphere

6. Ex: [Co Cl (NH3)5] Cl2
1. Compound ionize to form [Co Cl (NH3)5]+2 and 2Cl- .
2. Central chlorine do not precipitate with silver nitrate.
3. Substrate and ligand are bonded with coordination bond.
4. Coordination number is maximum number of atoms and groups
that combine with central atom in coordination sphere.
5. Co-ordination number for cobalt is 6.
Participate in
complexation
B) CHELATES:
These are group of metal ion complexes in which a substrate/ ligand
provides 2/more donor groups to combine with a metal ion. (In case
of inorganic complex only one donor group present)
Ligands-didentate, tridentate, polydentate.

7. Hexadentate -
Ethylenediaminetetraacetic acid
(EDTA)- Has a total of six points
(4:0 and 2: N) for attachment of
metal ions.
Sequestering:
This is a process in which the property of
metal is suppressed without removing it
from the solution.
Sequestering Agent:
This is a ligand which forms a stable
water soluble metal chelate
Ex: chlorophyll, hemoglobin.

8. Chelates applications:
1. INCREASING SOLUBILITY:
Fruit juices and drugs (ascorbic acid) + Fe/Cu oxidative
degradation.
Add EDTA + Fe/Cu stable Chelate
2. PURIFICATION OF HARD WATER:
Hard water (Ca+2) + EDTA EDTA-Ca+2 (ppt)
3. DURG ANALYSIS:
Procainamide + cupric ions (1:1) at pH 4-4.5 Coloured complex
detect by Colourimetry.
4. ANTI-COAGULANT:
Blood (Ca+2) + EDTA/Citrates/Oxalates prevent thrombin
formation no clotting.

9. C,D: OLEFIN AND AROMATIC TYPE:
a. These involves Lewis acid-base reactions
b. These type of complexes can be used as catalysts in the
manufacturing of bulk drugs, intermediates and in drug
analysis.

10. II. ORGANIC MOLECULAR COMPLEXES:
1. Interaction between 2 organic molecules  Complex
temperature change  molecular compound.
2. These complexes have (H)bonds/ weak vander wall forces/
dipole-induced dipole interactions.
3. Energy of attraction is 3K.Cal/mole
4. Bond distance is 3A0

11. MECHANISM:
1. Donar-Acceptor type:-
Bonds between uncharged species is formed and stabilized by
dipole-dipole interactions.
EX: N-Dimethyl aniline + 2,4,6-Trinitro anisole.
2. Charge transfer
complexes:-
Complex is stabilized by resonance.
Ex: Benzene + Trinitro benzene.

12. A) DRUG & CAFFINE COMPLEX:
Acidic drugs (benzocaine, procaine) + Caffeine Complexes
Mechanism:
1. dipole-dipole forces/ hydrogen bonding between acid (H) atom
and caffeine carboxyl group.
2. Interaction of non-polar parts
Ex: Caffeine + Benzocaine.

13. DRUG & CAFFINE COMPLEX APPLICATIONS:
1. These complexes can improve / extend absorption and
bioavailability of drug.
2. These complexes can enhance/ inhibit solubility and dissolution
rate of drug.
3. Caffeine+ gentisic acid complexes mask bitter taste of caffeine.
B) POLYMER COMPLEXES:
Polymers with nucleophilic oxygen (PEG/CMC)
+Drugs(tannic acid/salicylic acid/phenols) Complexes.
Disadvantages:
1. Incompatibilities in suspension, emulsion, ointments.
2. Complexes + Container drug loss
3. Complexes + preservatives decrease preservative action.

14. C) PICRIC ACID COMPLEXES:
Picric acid (strong acid) + strong base Salt.
Picric acid (strong acid) + weak base Complexes.
Ex: BUTESIN PICRATE
Picric acid (antiseptic) + Butesin (anesthetic)
(Ratio- 2:1)
1% ointment used for burns and abrasions.

15. D) QUINHYDRONE COMPLEXES:
Alcoholic solutions of equimolar quantities of Hydroquinone and
Benzoquinone form Quinhydrone complexes (green crystals)
Mechanism:
1. Overlapping of π electrons of molecules
2. (H) bonding for stabilizing complex.
Applications:
Used as electrode in pH determination.
Hydroquinone
Benzoquinone

16. 3.INCLUSION COMPLEXES/OCCLUSION COMPOUNDS:
One compound is trapped in lattice/cage like structure of other
compound. Interaction are due to suitable molecular structure.
A) CHANNEL LATTICE TYPE:
• In case of starch-iodine solution iodine molecule are trapped within
spiral of glucose molecule.
• In case of Urea-methyl α-lipolate is a needle shaped hexagonal
channel complex in which urea act as host.
Host (tubular channel)- Deoxycholic acid, urea, thiourea, amylose
Guest (long unbranched straight chain compounds)- paraffin, esters,
acids, ethanol.
Applications:
• Seperation of isomers:
Dextro, levo-terpineol are separated using Digitonin.
• In analysis of dermatological creams, long chain compounds
interfere and removed by complexation with urea.

17. B) LAYER TYPES:
• They form monomolecular layer of guest and host.
• Host (Layers With Gaps)- clays, bentonite
• Guest (entrapped in gaps)- hydrocarbons, alcohols, glycols.
• Use: Due to their large surface area they are used as
catalysts.

18. C)CLATHRATES: (cage like structure):
• During crystallization some compounds (host) form cage like
structures in which coordinating compound (guest) is entrapped.
• Ex: warfarin sodium (water + isopropyl alcohol)
• Ex. Hydroquinone form cage with hydrogen bonds and hole have
diameter of 4.2A0.This can entrap methanol, carbon dioxide,
hydrochloric acid.
CAGE
Application:
• Synthetic metal-alumino silicate are known to be used as
molecular sieves.
• These are use to store volatile substances and toxic substances.

19. D) MONO MOLECULAR INCLUSION COMPLEX:
Single guest molecule entrapped by single host molecule.(generally
cyclodextrins)
Cyclodextrins:
Cyclodextrins are cyclic oligo sacchirides containing minimum of 6 D-
(+) gluco pyranose units attached by α-1,4 linkages.
Cyclodextrins Cavity diameter (Ao) Glucopyranose units
α 5 6
β 6 7
γ 8 8
Hydrophilic
entrance
Hydrophobic
interior

20. MONO MOLECULAR INCLUSION COMPLEX APPLICATIONS:
1. Enhanced solubility:
Retonic acid (solubility= 0.5mg/ml)
Retonic acid + β-CD (solubility= 160 mg/ml)
2. Enhanced dissolution:
Famotidine/ Tolbtamide + β-CD
3. Enhanced stability: Asprin/Ephedrine/Testosterone +
β-CD (no reaction with other functional groups)
4. Sustained release:
Ethylated β-CD + Diltiazem

21. Method of analysis:
Estimation of 2 parameters
1. Stoichiometric ratio of Ligand: Metal / Donar : Acceptor
2. Stability Constant of complex.
Methods:
1. Method of continuous variation.
2. Distribution method
3. Solubility method
4. pH titration method.

22. General procedure:
Equation for complexation
M + n A MAn
Stability constant
Applying Log on both sides
Log [MAn] = log K + log [M] + n log [A]
[M] = Conc. of Metal ion uncomplexed
[A] = Conc. of ligand uncomplexed
[MAn] = Conc. of complex
n = number of mole

23. 1. Method of continuous variation
1. Dielectric constant
2. Refractive index
3. Spectrophotometric
extinction coefficient
Physical
properties
Characteristics
of species.
A
+B
No
complexation
Complexatio
n
Physical
properties
are additive
values
Physical
properties
values different

24. 1. Due to
complexation
physical properties
result may be
maximum or
minimum.
2. At maximum/
minimum point note
concentration of
individual species.
3. Calculate
stoichiometric
ratio of species.

25. 2. Distribution method:
• Distribution of solute between two immiscible liquids is
expressed by Partition / Distribution coefficient.
• Partition coefficient / Distribution changes due to
complexation.
• By conducting 2 experiments stability constant is
estimated.
• Example: Iodine complex with Potassium iodide.
I2 + 𝐾+𝐼− = 𝐾+𝐼−3

27. 3. Solubility method:
• When mixture form complexes solubility may increase/ decrease.
• Experiments are conducted to estimate donor – acceptor ratio and
equilibrium constant.
• Example: PABA-caffeine complex
Experiment:
1. Caffeine (Complexing agent) taken in different concentrations in a
series of flask.
2. Excess PABA is added to all flask with agitation
3. Solution are filtered and analyzed for drug content.
4. At zero conc. of caffeine the first point indicates solubility of drug
PABA in water.
5. With addition of caffeine the solubility of PABA is increased up to
second point. At this point the solution is saturated with respect to
complex and drug itself.
6. On further addition complex precipitate up to third point. Atthis
point all excess PABA converted to complex.
7. On further addition of caffeine higher complex are formed.

29. 4. pH titration method:
This method is suitable if complexation produces change in pH.
Example: Chelation of cupric ions by glycine molecule
Chelation of calcium ion by EDTA
The reaction represented as
𝐶𝑢2+
+ 2𝑁𝐻3+
𝐶𝐻2𝐶𝑂𝑂−
= 𝐶𝑢 (𝑁𝐻2 𝐶𝐻2𝐶𝑂𝑂)2 + 2𝐻+
Experiment:
1. Glycine solution (75 ml) titrated with NaoH, pH is recorded.
2.Complex solution of Glycine solution (75 ml) and Cu+2 titrated
with NaoH, pH is recorded.
3.Complexation releases protons and pH decreases. Hence metal-
glycine complex curve below the glycine curve
4.The horizontal distance between the curve between the curve
gives amount of alkali cosumed.
5.Quantity of alkali = Concentration of ligand bound at that pH.
n= 𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑙𝑖𝑔𝑎𝑛𝑑 𝑏𝑜𝑢𝑛𝑑
𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑛𝑐, 𝑜𝑓 𝑚𝑒𝑡𝑎𝑙 𝑖𝑜𝑛

30. Stability constant represented as β
log β = 2 X p [A] (at n=1)
p [A] = pKa- pH- log ( [HA]initial - [NaoH] )
Where p [A] is related to Conc. of ligand bound (glycine)
pKa = Dissociation constant of ligand, glycine
[HA]initial = Conc. of glycine at initial stage
[NaoH] = Horizontal distance expressed in mole/litre

31. COMPLEXATION –Applications in pharmacy
•Physical state
•Volatility
•Solid state Stability
•Chemical stability
•Solubility
•Dissolution
•Partition coefficient
•Absorption & bioavailability
•Reduced toxicity
•Antidote in metal poisoning
•Drug action through metal
poisoning
•Antibacterial activity

32. 1. Physical state:
Liquid substance  Solid complex  improve process
characteristics.
Ex: Nitroglycerine (Explosive) + β-CD  Explosion proof
Complex
2. Volatility:
Substances Complex
(volatile / unpleasant odour)
 Reduce volatility
& Mask odour
3. Solid state stability
Vitamin-A,D + β-CD  Chemically stable solid complex.

33. 4. Chemical stability
Complexation  Reduce Reactivity, Improve stability.
Ex: Caffeine + Benzocaine Complex  Prevent
benzocaine hydrolysis.
5. Solubility:
PABA (insoluble) + Caffine  Complex improves
solubility of PABA
6. Dissolution:
Phenobarbitol (insoluble) + β-CD  Complex improves
Solubility & Dissolution.

34. 7. Partition Coefficient:
(Water + Benzene) + Permanganate ions  Partition in to
W
A
TER.
(Water + Benzene) + Permanganate ions + Crown ether 
Partition in to Benzene.
8. Absorption & bioavailability
β-CD + Barbiturates  Complex  Improves
Bioavailability
Tetracyclines + Ca+2 / Mg+2  Insoluble metal Complex
 Reduced Absorption & Bioavailability
9. Reduced Toxicity:
β-CD + Indomethacin  Reduce ulcerogenic effect
β-CD + Chlorpramazine  Reduce local tissue toxicity.

35. 10. Antidote in metal poisoning:
Arsenic, Mercury (Toxic metal ions) + (-SH) groups of
enzymes  Effect normal functioning.
Dimercaprol + Arsenic,  Complex  Easily eliminated
Mercury from body.
11. Drug action through metal poisoning:
8-Hydroxy Quinoline + Iron  Complex  Enter malarial
parasite  Accumulation of metal  Anti-Malarial action.
12. Antibacterial activity:
PAS + Cupric ions  Cupric Complex + Chelates.
(anti-Tubercular drug)
Chelates  30 times more fat soluble  Penetrate in to
cells  High anti-Tubercular action.

36. PROTEIN DRUG COMPLEXATION AND DRUG
ACTION
• The phenomenon of complex formation of drugs with
proteins is called protein binding.
• A protein bound drug is neither metabolized nor excreted
hence it is pharmacologically inactive. Binding of drugs to
proteins is generally of reversible & irreversible.
• Reversible binding generally involves weak chemical bond
such as: Hydrogen bonds, Hydrophobic bonds, Ionic bonds
and Van der waal's forces.
• While Irreversible drug binding, though rare, arises as a
result of covalent binding and is often result carcinogenicity
or tissue toxicity of the drug.
Protein + drug ⇌Protein-drug complex
Protein binding may be divided into:
1. Intracellular binding.
2. Extracellular binding.

38. BINDING OF DRUG TO BLOOD COMPONENTS
The order of binding of drugs:
albumin> α1-acid glycoprotein> lipoproteins> globulins

39. Binding of drug to Human serum albumin:
• The Molecular weight of albumin is 65,000 — 69,000.Albumin
is distributed in the plasma and in the extracellular fluids of
skin , muscle ,and various tissues.
• Elimination half life of albumin is 17-18 days . Albumin
concentration is 3.5-5.5% (w/v) or 4.5 mg/dl.
• Many weak acidic drugs bind to albumin by electrostatic and
hydrophobic bonds.
• There are four site of attachment of drug.
I. Warfarine & Azapropazone Site
II. Diazepam Site
III. Digoxine Site
IV. Tamoxifen Site

40. SITE l: To this specific site a large population of drugs bind like Non-
Steroidal AntiInflammatory Drugs mainly phenylbutazone,
indomethacin, many sulfonamides e.g.; sulfamethoxine,
sulfamethizole, and even many anti-epileptic drugs like phenytoin
etc. This site is also called as Warfarin binding site or as
Azapropazone binding site.
SITE Il: This is actually said to be Diazepam binding site.
Benzodiazepines, medium chain fatty acids, ibuprofen, ketoprofen,
etc. bind extensively at this site. This is due to structural changes the
following drugs have high and specific affinity for this site.
SITE Ill: This site is called as Digitoxin binding site
SITE IV: This is referred as Tamoxifen binding site.

41. Binding of drug to α1-acid glycoprotein
They ate also called as orosomucoid. The molecular weight of α1-
acid glycoprotein is 44,000. They are bound by Hydrophobic bonds
E.g. : Basic Drugs such as Imipramine , Amytriptyline , Lidocaine ,
nortriptyline, Propranolol, Quinidine and disopyramide
Binding of drug to Lipoprotines:
They are bound by hydrophobic bond. The molecular weight of
lipoprotein is 2-3 lakhs to 34 lakhs. Bound drug dissolve in lipid
core. Example acidic drug (diclofenac), Neutral (cyclosporin) and
Basic drug (chlorpromazine). They are classified as
• Chylomicrons
• Very low density lipoprotine
• Low density lipoprotine(more in human)
• High density lipoprotine

43. Binding Of Drugs To Blood cells
Red Blood Cells (RBC's) are the major blood cells which rates
about 40% of total blood. The red blood corpuscles constitute
95% of the total blood cells concentration in the body. Major
portion of red blood cells to which drugs can bind are:
i) Hemoglobin: The weight & structural is similar to that of
HSA but the concentration is much higher than of albumins in
blood. Examples of drugs that bind are phenytoin,
pentobarbital etc.
ii) Carbonic Anhydrase Inhibitors: Carbonic anhydrase
inhibitors mainly bind to the site like chlorthaizine.
iii) Red Blood cell membrane: Basic drugs like imipramine are
known to bind to RBC membrane.

44. BINDING OF DRUG TO EXTRAVASCULAR TISSUE PROTEIN
•
• Importance: 1. It increases apparent volume of distribution of drug.
2. localization of a drug at a specific site in body.
• Factor affecting: lipophilicity, structural feature of drug, perfusion
rate, pH differences.
Binding order: Liver › Kidney › Lung ›Muscles
Tissue Binding of
1.Liver Irreversible binding of Epoxides of
Halogenated Hydrocarbon & Paracetamol.
2.Lungs Basic drugs: Imipramine, Chlorpromazine,
&AntiHistaminics.

45. Tissue Binding of
3.Kidney Metallothionin protein binds to Heavy
metals & results in Renal accumulation
and toxicity.
4.Skin Chloroquine& Phenothiazine bind
to Melanin.
5.Eye Chloroquine & Phenothiazine also
binds to Eye Melanin & results in
Retinopathy.
6.Hairs Arsenicals, Chloroquine, &
Phenothiazine.
7.Bones Tetracycline(yellow discoloration of
teeth), Lead(replaces Ca & cause
brittleness)
8.Fats Lipophilic drugs
(thiopental), Pesticides
(DDT)
9.NucleicAcid Chloroquine & Quinacrine.

46. FACTORS AFFECTING PROTEIN DRUG BINDING
1. Drug-related factors
a. Physicochemical characteristics of the drug:-
Protein binding is directly related to the lopophilicity of drug. An increase
in lipophilicity increases the extent of binding.
b. Concentration of drug in the body:-
Alteration in the concentration of drug substance as well as the protein
molecules or surfaces subsequently brings alteration in the protein
binding process.
c. Affinity of a drug for a particular bindingcomponent:-
This factor entirely depends upon the degree of attraction or affinity the
protein molecule or tissues have towards drug moieties. For Digoxin has
more affinity for cardiac muscles proteins as compared to that of
proteins of skeletal muscles or those in the plasma like HSA.

47. 2. Protein/ tissue related factors:
a. Physicochemical characteristics of protein or binding agent:
•. Lipoproteins & adipose tissue tend to bind lipophilic drug by
dissolving them in their lipid core.
•. The physiological pH determines the presence of active anionic &
cationic groups on the albumin to bind a variety of drug.
b. Concentration of protein or binding component:
•. Among the plasma protein , binding predominantly occurs with
albumin, as it is present in high concentration in comparision to
other plasma protein.
•. The amount of several proteins and tissue components available for
binding, changes during disease state.

48. 3. Drug interactions
a. Competition between drugs for the binding sites :-
D2
D1+P D2+P
D1: Displaced drug. D2: Displacer drug.
e.g. Administration of phenylbutazone to a patient on Warfarin therapy
results in Hemorrhagic reaction.
b. Competition between drug & normal body constituents:-
The free fatty acids are known to interact with a no. of drugs that binds
primarily to HSA. the free fatty acid level increase in physiological, pathological
condition.

49. c. Allosteric changes in protein molecule:-
• The process involves alteration of the protein structure by the drug
or it’s metabolite thereby modifying its binding capacity.
e.g. aspirin acetylates lysine fraction of albumin thereby modifying its
capacity to bind NSAIDs like phenylbutazone.
4. Patient-related factors
Age:
1.Neonates: Low albumin content: More free drug.
2.Young infants: High dose of Digoxin due to large renal
clearance.
3.Elderly:Low albumin: So more free drug.
Intersubject variability: Due to genetics & environmental factors.

50. Disease states:-
Disease Influence on plasma
protein
Influence on protein drug
binding
Renal failure ↓ Albumin content ↓ binding of acidic drugs;
neutral and basic drugs are
un affected
Hepatic failure ↓ Albumin synthesis ↓ binding of acidic drugs;
and binding of basic drugs is
normal
Inflamatory states i.e,truama
surgery etc… ↑AAG levels
↑ binding of basic drugs;
neutral and acidic drugs are
un affected

51. Kinetics of Protein Binding:

52. A scatchard plot is useful when high conc. of free drug is there.

55. SIGNIFICANCE OF PROTEIN/TISSUE BINDING OF DRUG
a. Absorption-
• As we know the conventional dosage form follow first order kinetics.
So when there is more protein binding then it disturbs the absorption
equilibrium.
b. Distribution-
• A protein bound drug in particular does not cross the BBB, the
placental barrier, the glomerulus.
• Thus protein binding increases the distribution of drugs.
c. Metabolism-
• Protein binding decreases the metabolism of drugs & enhances the
biological half life.
• Only unbound fraction get metabolized.
• e.g. Phenylbutazone & Sulfonamide.

56. d. Elimination
•
•
•
Only the unbound drug is capable of being eliminated.
Protein binding prevent the entry of drug to the metabolizing organ
(liver ) & to glomerulus filtration.
e.g. Tetracycline is eliminated mainly by glomerular filtration.
e. Systemic solubility of drug
• Lipoprotein act as vehicle for hydrophobic drugs like steroids, heparin
etc.
f. Drug action-
• Protein binding inactivates the drugs because sufficient concentration of
drug can not be build up in the receptor site for action.
• e.g. Naphthoquinone

57. g. Sustain release-
• The complex of drug protein in the blood act as a reservoir &
continuously supply the free drug.
• e.g. Suramin sodium-protein binding for antitrypanosomal action.
h. Diagnosis-
• The chlorine atom of chloroquine replaced with radiolabeled I-
131 can be used to visualize-melanomas of eye & disorders of
thyroid gland.

58. THERMODYNAMIC TREATMENT OF STABILITY CONSTANTS:
• The standard free energy change of complexation is related to the
overall stability constant K (or any of the formation constants) by
the relationship.
ΠGo = -2.303RT log K
• The standard enthalpy change ΠH may be obtained from the slope
of a plot of log K versus l/T, following the expression:
log K = ΠH / 2.303R T + constant
• When the values of K at two temperatures are known, the
following equation may be 'used:
log(K2/K1 ) = - ΠH/2.303R(T2-T1/T1-T2)
• The standard entropy change is :
ΠGo = ΠH – T ΠS
• Andrews and Keefer demonstrated that ΠH andΠS generally
become more negative as the stability constant for molecular
complexation increases.
• As the binding between donor and acceptor becomes stronger, ΠH
would be expected to have a larger negative value.

59. Thank you