complexation and Protein bindings

This PDF file content is about complexation and Protein bindings as per the PCI syllabus for B.Pharm Second year for the subject Physical Pharmaceutics

1. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
UNIT-IV
Complexation
and
Protein binding

2. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Complexationandproteinbinding:Introduction,ClassificationofComplexation,
Applications,methodsofanalysis,proteinbinding,Complexationanddrugaction,
crystallinestructuresofcomplexesandthermodynamictreatmentofstabilityconstants.

3. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
“Complexationistheprocessofcombiningindividualatom
groups,ionsormoleculestocreatelargeionormolecule,inwhichone
atomorionisthecentralpointofcomplexthatformbondingwithother
atoms,ionsormolecules.”

4. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Complexesresultsfromsometypeofinteractionsamongdifferent
chemicalspecies.
Intermolecularforcesinvolvedintheformationofcomplexes:
1.VanderWaalsforces.
2.Hydrogenbonds
3.Coordinatecovalence
4.Chargetransfer.
5.Hydrophobicinteraction.

5. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Complexespossesssomepropertieswhicharedifferentfromthoseof
itscomponents.
Propertieswhichconfirmtheformationofcomplexes.
1.Solubility
2.Lightabsorption
3.Conductunce
4.Partitioningbehaviour
5.Chemicalreactivities

6. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Applications of Complexes
1. Physical state
BycomplexationLiquidsubstancescanbeconvertedtosolidcomplex.
HenceProcessingcharacteristicscanbeimproved.
Example.
Nitroglycerinecanbetransformedtoitscrystallinecomplexwith
ß-cyclodextrin.
Thecomplexcontain15.6%Nitroglycerinandisexplosionproof.

7. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
2. Volatility
BycomplexationVolatilityofsubstancecanbereducetostabilize
thesystemortoovercomeunpleasantodour.
Example:FormationofIodineasacomplexwithPolyvinyl-
pyrrolidone(PVP).
3. Solid state Stability
SolidstateStabilitycanbeenhancedBycomplexation.
Example:ß-cyclodextrincomplexesofVitaminA&Darestable
form.

8. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
4. Chemical Stability
Complexformationalterthechemicalreactivity.Eitherinhibitory
orcatalyticeffectsmaybeobserved.
Example:Rateofhydrolysisofbenzocainecanbereduceby
complexingitwithcaffeine.
5. Solubility
SolubilityofmanyDrugscanbeenhancedbyComplexation.Solid
complexismostcommonlyusedpharmaceuticallyforsolubility
enhancement.
Example:Atlowconcentration,Caffeineenhancesthesolubilityofp-
aminobenzoicacid(PABA)

9. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
6. Dissolution
Ifsolubilityisenhanced,thedissolutionrateshouldalsoincrease.
Complexationisoneofthepossiblemethodtoachievethis
objective.
Example:Thedissolutionrateofphenobarbitalisenhancedbyusing
cyclodextrininclusioncomplexes.
7. Absorption and Bioavailability
ComplexationmayalterAbsorption&Bioavailabilitydependson
chemicalnatureofdrugs.
Example:ß-cyclodextrincomplexesofindomethacin,Barbiturates
haveenhancedbioavailability.

10. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
8. Partition coefficient
Partitioncoefficientofsomesubstancescanbealterby
complexation.
Example:Permanganateionscanbetransferredintobenzenephase
fromwaterbycomplexationwithcrownether.
9. Reduce toxicity.
Toxicityofdrugscanbereducebycomplexation.
Example:Cyclodextrinsareeffectiveinreducingulcerogeniceffect
ofindomethacin&localtissuetoxicityofChlorpromazine.

11. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
10. Antidote for Metal poisoning
Toxicmetalionssuchasarsenic,mercury,antimonyetc.bindto–
SHgroupofvariousenzymesandinterferewiththeirnormal
functions.
Example:
1.CompoundsuchasDimercaprol(BAL,BritishAnti-Lewisite)
formwatersolublecomplexwithmetalionsandeliminatethem
rapidlyformthebody.
2.BerylliumPoisoning-------Salicylicacid
3.LeadPoisoning-------------EDTA

12. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
11. Antibacterial activity
Antituberculardrug,PAS(P-aminosalicylicacid)formacupric
complex&Cupricchelate.
CupricchelatehasshowngreaterinvivoAntitubercularactivityin
micethancupriccomplex.
Thechelateisabout30timesmorefatsolublethantheionic
complex.

13. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
I.Metal Ion Complexes
a.Inorganic type
b.Chelates
c.Olefin type
d.Aromatic type
II.Organic Molecular Complexes
a.Quinhydronetype
b.Picric acid type
c.Caffeine and other drug complexes
d.Polymer type
III.Inclusion Compounds
a.Channel lattice type
b.Layer type
c.Clathrates
d.Monomolecular type
Classification of Complexes

14. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
I.Metal ion Complexes
Inthistype,metalionconstitutesthecentralatom(substrate)and
interactswithabase(electron-pairdonor,ligand).
Thistypeofinteractionleadstoformationofcoordinationbonds
betweenthespecies.
A.Inorganic Complexes
complexconsistsofametalion(e.g.Co,Fe,Cu,NiandZn)linked
withoneormorecounterionsormoleculestoformacomplex.
Theionsormolecules(e.g.Cl,NH
3,H
2O,Br,I,CN,etc.)directly
boundwiththemetalarecalledligands.

15. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
TheinteractionbetweenthemetalandtheligandrepresentsaLewis
acid-basereaction
Themetalion(Lewisacid)combineswithaligand(Lewisbase)by
acceptingapairofelectronsfromtheligandtoformthecoordinate
covalent.
Thenumberofligandsboundtothe
metalionisdefinedascoordination
number.
Thecoordinationnumberofcobaltis
six,sinceitcomplexedwithsixNH
3
groups.

16. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Compound(e.g.NH
2)whichhasa
singlepairofelectronsforbonding
withthemetalion,iscalled
unidentateligand.
Ligandswithtwoorthreegroupsare
knownasbidentateortridentate
respectively.
EthyleneDi-amineTetra-Aceticacid
(EDTA)hassixpointsforattachment
(twonitrogenandfouroxygendonor
groups)andiscalledhexadentate.

17. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Coordinationnumberusuallydeterminethegeometryofthe
complex.

18. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Chelationistheformationoftwoormorecoordinatebondsbetween
aMultidentateligand(chelatingagent)andasinglecentralatom.
Thebondsinthechelatemaybeionic,primarycovalent,or
coordinatetype.
Chelates

19. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
II. Organic Molecular Complexes
Organicmolecularcomplexesareformedasaresultof
noncovalentinteractionsbetweenaligandandasubstrate.
TheinteractionscanoccurthroughVanderwaalsforces,charge
transfer,hydrogenbondingorhydrophobiceffects.
Manyorganiccomplexesaresoweakthattheycannotbeseparated
fromtheirsolutionsasdefinitecompounds,andtheyareoften
difficulttodetectbychemicalandphysicalmeans.

20. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Quinhydrone Complex
Thismolecularcomplexisformedbymixingalcoholicsolutionof
equimolarquantitiesofbenzoquinonewithhydroquinone.
Complexformationisduetooverlappingofthe??????-frameworkofthe
electrondeficientbenzoquinonewiththe??????-frameworkofthe
electron-richhydroquinone.

21. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Picric Acid Complexes
Picricacid(2,4,6-trinitrophenol),isastrongacidthatformscomplexes
withmanyweakbases.
Example:
Butesinpicrate(localanaesthetic)whichisacomplexformedbetween
2moleculesofbutylp-aminobenzoatewith1moleculeofpicricacid.

22. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Caffeine Complexes
Caffeineformscomplexeswithanumberofdrugsbyfollowing
mechanism:
Hydrogenbondingbetweenthepolarizablecarbonylgroupof
caffeineandthehydrogenatomoftheacidicdrugssuchasp-
aminobenzoicacidandgentisicacid.

23. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Dipole-dipoleinteractionbetweentheelectrophilicnitrogenof
caffeineandthecarboxyoxygenofesterssuchasBenzocaineor
Procaine

24. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Polymer Complexes
PolymericmaterialssuchasEudragit,Chitosan,PEG,PVP&CMC
whichareusuallypresentinliquid,semisolidandsoliddosage
forms,canformcomplexeswithalargenumberofdrugs.
Suchinteractionscanresultinprecipitation,flocculation,
Solubilization,alterationinbioavailabilityorotherunwanted
physical,chemical,andpharmacologicaleffects.
Polymer–drugcomplexeshowevercanalsobeusedtomodify
biopharmaceuticalparametersofdrugs.
Examples:PolymericcomplexbetweenNaltrexoneandEudragit
improvesthedissolutionrateofNaltrexone.
Application

25. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Inclusion Complexes
Aninclusioncompoundisacomplexinwhichonechemical
compound(the‘host’)formsacavityinwhichmoleculesofa
secondcompound(‘guest’)areentrapped.
Thesecomplexesgenerallydonothaveanyadhesiveforces
workingbetweentheirmoleculesandarethereforealsoknownas
no-bondcomplexes.

26. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Channel Lattice Type
Inthiscomplex,thehostcomponentcrystallizestoformchannel-
likestructureintowhichtheguestmoleculecanfit.
Theguestmoleculemustpossessageometrythatcanbeeasilyfit
intothechannel-likestructure.
Dissolutionofvitamin-EandFamotidinecanbeincreasedby
complexationwithUrea(hostmolecule).
Application

27. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Thewell-knownstarch–iodinecomplexisachannel-typecomplex
consistingofiodinemoleculesentrappedwithinspiralsoftheglucose
residues

28. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Layer Type
Layertypecomplex(orintercalationcompound)isatypeof
inclusioncompoundinwhichtheguestmoleculeisdiffusedbetween
thelayersofcarbonatom,toformalternatelayersofguestandhost
molecules.
E.g.Montomorillonite,(theprincipalconstituentofbentonite)cantrap
hydrocarbons,alcohols,andglycolsbetweenthelayersoftheirlattices.

29. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Clathrates
“Theclathratesarecompoundsthatcrystallizeintheformofacage-
likelatticeinwhichthecoordinatingcompoundisentrapped.”
E.g.
Oneofficialdrug,WarfarinSodium,isintheformof
crystallineclathratecontainingwaterandisopropylalcohol.
Hydroquinonecrystallizesinacage-likehydrogen-bonded
structure,inwhichsmallmoleculessuchasmethylalcohol,
CO
2,andHClmaybetrappedinthesecages.

30. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Sizeoftheguestmoleculeisimportantforcomplexformation.

31. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Monomolecular Inclusion Compounds: Cyclodextrins
Monomolecularinclusioncomplexinvolvestheentrapmentof
guestmoleculesintothecage-likestructureformedfroma
singlehostmolecule.
Cyclodextrinsareafamilyofcompoundsmadeupofsugar
moleculesboundtogetherinaring(cyclicoligosaccharides)
Theyconsistof6,7,and8unitsofglucosereferredtoas�,�,
and�-cyclodextrins,respectively.

32. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa

33. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Cyclodextrinshavetruncatedconestructurewithahydrophobic
interiorcavitybecauseoftheCH
2groups,andahydrophilic
exteriorduetothepresenceofhydroxylgroup.

34. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Moleculesofappropriatesizeandstereochemistrygetentrappedinthe
cyclodextrincavitybyhydrophobicinteractionbysqueezingoutwater
fromthecavity.
�-CDand??????-CDarethemostusefulforPharmaceuticaltechnology
owingtotheirlargercavitysize.

35. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Cyclodextrinscanenhancethesolubilityandbioavailability
ofhydrophobiccompoundsduetothelargenumberofhydroxyl
groupsontheCDs.
CDsareusedassustainedreleasedrugcarriers.
Application

36. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Inadditiontoimprovingthesolubilityofcompounds,
complexationwithcyclodextrinhasbeenusedtoimprovethe
stabilityofmanydrugsbyinclusionofthecompoundand
protectingcertainfunctionalgroupsfromdegradation.
ComplexationwithCyclodextrinshasalsobeenusedtomask
thebittertasteofcertaindrugssuchasFemoxetine.

37. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa

38. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Method of Continuous Variation
Thestoichiometryofametal-ligandcomplexationreactioncan
bedeterminedbythreemethods:
(A)Job'smethod
(B)Moleratiomethod
(C)Sloperatiomethod

39. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Job's Method
InJob’smethod,aseriesofsolutionarepreparedwithvariableratios
ofmetalandligandbutwithfixedtotalconcentrations.
Anadditivepropertythatisproportionaltotheconcentrationofthe
formedcomplex(e.g.absorbance)ismeasuredandplottedagainstthe
molefractionfrom0to1foroneofthecomponentsofamixture
(e.g.Ligand).
ForaconstanttotalconcentrationofAandB,thecomplexisatits
greatestconcentrationatapointwherethespeciesAandBare
combinedintheratioinwhichtheyoccurinthecomplex.
Thelinethereforeshowsabreakorachangeinslopeatthemole
fractioncorrespondingtothecomplex.

40. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Here,thechangeinslopeoccursatamolefractionof0.75
X
L/X
M=0.75/(1-0.75)=3
Thisindicateacomplexformationofthe3:1type(ligand:metal).

41. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Mole Ratio Method
Inthemoleratiomethod,aseriesofsolutionsarepreparedwitha
fixedamountofthemetalandavariableamountoftheligand(orvice
versa).
Anadditivepropertythatisproportionaltotheconcentrationofthe
formedcomplex(e.g.absorbance)ismeasuredandplottedagainstthe
moleratioofthecomponentwiththevariableamounts(e.g.Ligand).
Theformedcomplexisatitsgreatestconcentrationatapointwhere
thespeciesAandMarecombinedintheratioinwhichtheyoccurin
thecomplex(indicatedbyachangeintheslopeatthemoleratiothat
formsthecomplex).

42. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Thechangeinslope(a)occursatamoleratioof1indicatingacomplex
ofthe1:1type,whilethechangeinslope(b)occursataratioof2
indicatingacomplexofthe2:1type.

43. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Slope Ratio Method
Intheslope-ratiomethodtwosetsofsolutionsareprepared.
Thefirstsetofsolutionscontainsalargeexcessofmetalandavariable
concentrationsofligand(alltheligandreactsinformingthemetal–ligand
complex).
Theabsorbanceoftheformedcomplexisplottedagainsttheligand
concentrationandtheslopeofthelineisdetermined.
Asecondsetofsolutionsispreparedwithalargeexcessofligandanda
variableconcentrationofmetal(allthemetalreactsinformingthemetal–
ligandcomplex).
Theabsorbanceoftheformedcomplexisplottedagainstthemetal
concentrationandtheslopeofthelineisdetermined.
Thestoichiometricratioofmetaltoligandcanbecalculatedbyformula
Stoichiometricratio(L:M)=Slope
M/Slope
L

44. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Theslopeofthefirstline(variablemetal)is1.56×10
-3
andtheslopeof
theotherline(variableligand)is5.3×10
-4
Whatisthestoichiometric
ratioofthiscomplex?
Stoichiometric ratio (L:M) = 1.56×10
-3
/ 5.3×10
-4
= 3
Stoichiometric ratio (L:M) = 3:1 (L:M)
Example

45. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
pHTitrationMethod
pHtitrationmethodcanbeusedwheneverthecomplexationis
accompaniedbyachangeinpH.
E.g.Thechelationofthecupricionbyglycine:
Because2protonsareformedinthereaction,theadditionofglycineto
solutionshouldresultinadecreaseinpH.

46. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Titrationcurvescanbeobtainedbyaddingastrongbasetoasolutionof
glycinealoneandtoanothersolutioncontaining(glycine+coppersalt)
andplottingthepHagainstthevolumeofbaseadded.
Thecurveforthemetal-glycine
mixtureiswellbelowthatfor
theglycinealone.
ThedifferenceinpHfora
givenquantityofbaseadded
indicatestheoccurrenceofa
complex.

47. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Solubility Method
Solubilitymethodisthemostwidelyusedmethodisthestudythe
inclusioncomplexation.
Accordingtothesolubilitymethod,excessquantitiesofthedrugare
placedinwell-stopperedcontainers,withasolutionofthecomplexing
agentinvariousconcentrations.
Thebottlesareagitatedinaconstanttemp.bathuntilequilibriumis
reached.Then,thesupernatantliquidareremovedandanalyzedto
obtainthetotaldrugconcentration.

48. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Theconcentrationofthedrugisplottedagainsttheconcentrationof
complexingagenttoobtainacurvethatcanbeusedtocalculatethe
Stoichiometricratio.
Anexampletoillustratethesolubilitymethodisthep-Aminobenzoic
acid(PABA)Complexationbycaffeine

49. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
PointAisthesolubilityofthedruginwater.Withtheadditionof
caffeine,thesolubilityofPABAriseslinearlyowingtocomplexation.
AtpointB,thesolutionissaturatedwiththecomplexandwiththedrug
itself.
Thecomplexcontinuestoformandtoprecipitatefromthesaturated
systemasmorecaffeineisadded.
AtpointC,alltheexcesssolidPABAhaspassedintosolutionandhas
beenconvertedtothecomplex.
SomeofthePABAcombinesfurtherwithcaffeinetoformhigher
complexessuchas(PABA-2caffeine)

50. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
DistributionMethod
Themethodofdistributingasolutebetweentwoimmiscible
solventscanbeusedtodeterminethestabilityconstantfor
certaincomplexes.
Thesolutedistributionpatternchangesdependingonthenature
ofcomplex.
Thedistributionmethodhasbeenusedtostudycaffeineand
polymercomplexeswithanumberofacidicdrugssuchas
benzoicacid,salicylicacid,andacetylsalicylicacid.

51. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
TheComplexationofiodinebypotassiumiodideisanexampleto
illustratethisMethod.
I
2+K
+
I
-
K
+
I
-
3
TheEquilibriumStabilityconstant,
K
+
I
-
3
[K
+
I
-
][I
2]
K=
TheKvalueofiodine-potassiumiodidecomplex=954

52. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Protein Bindings
Adruginthebodycaninteractwithseveraltissuecomponentsof
whichthetwomajorcategoriesare;
1.Blood,and
2.Extravasculartissues.
Theinteractingmoleculesaregenerallythemacromoleculessuchas
proteins,DNAoradipose.Theproteinsareparticularlyresponsible
forsuchaninteraction.
“Thephenomenonofcomplexformationwithproteinsis
calledasproteinbindingofdrugs.”

53. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Proteinssuchasalbuminandglobulinarepresentintheblood.
Amongthese,thelevelofalbuminisveryhighandplaysacrucial
roleinthedrugproteinbinding.
Alpha-acidglycoprotein(α-AGP)levelsareverylowinnormal
healthypopulation.But,inthediseasedstate,suchasmyocardial
infarction,AGPlevelsincreases.
Whendrugsarepresentinblood,itispossiblethatthese
macromolecules(proteins)binddrugs.
Albuminbindsbothacidicandbasicdrugs.AGPbindsmostofthe
basicdrugs.Globulinsbinddrugtoalittleextent.

54. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Proteinbindingmaybedividedinto
1.Intracellularbinding–wherethedrugisboundtoacell
proteinwhichmaybethedrugreceptor;ifso,bindingelicitsa
pharmacologicalresponse.Thesereceptorswithwhichdrug
interacttoshowresponsearecalledasprimaryreceptors.
2.Extracellularbinding–wherethedrugbindstoan
extracellularproteinbutthebindingdoesnotusuallyelicita
pharmacologicalresponse.Thesereceptorsarecalledsecondary
orsilentreceptors.

55. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Bindingofdrugstoproteinsisgenerallyreversiblewhichsuggests
thatitgenerallyinvolvesweakchemicalbondssuchas;
1.Hydrogenbonds
2.Hydrophobicbonds
3.Ionicbonds,or
4.vanderWaal’sforces
Irreversibledrugbinding,thoughrare,arisesasaresultofcovalent
bindingandisoftenareasonforthecarcinogenicityortissue
toxicityofthedrug;
Forexample,
covalentbindingofChloroformandParacetamolmetabolitesto
liverresultsinhepatotoxicity.
MechanismsofProtein-DrugBinding

56. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
The influence of binding on drug disposition and clinical response

57. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Factors affecting protein-drug binding
1.Drug related factors
a.Physicochemicalcharacteristicsofthedrug
b.Concentrationofdruginthebody
c.Affinityofadrugforaparticularbindingcomponent
2.Protein/tissuerelatedfactors
a.Physicochemicalcharacteristicsoftheproteinorbindingagent
b.Concentrationofproteinorbindingcomponent
c.Numberofbindingsitesonthebindingagent
3.Druginteractions
a.Competitionbetweendrugsforthebindingsite
b.Competitionbetweenthedrugandnormalbodyconstituents
4.Patientrelatedfactors
a.Age
b.Intersubjectvariations
c.Diseasestates

58. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Applications of protein binding
1.Drugdistribution:Proteinbindingdecreasesthedistributionof
drugs.Theprotein-bounddrugisbiginsize,whichcannoteasilycross
cellmembranesandtherefore,restrictstothebloodpool.Onlyfree
(unbound)drugcanpassthroughthecellmembranesandcontributes
tothetissuebinding.Thehighertheproteinbinding,thelowerthe
tissuedistribution.
2.Metabolism:Proteinbindingdecreasesthemetabolismofdrugsand
enhancesthebiologicalhalf-life.Onlythatfractionofdrugwhichis
freecangetmetabolized.

59. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
3.Excretion:Proteinbindingdecreasestherenalexcretionofdrugsand
enhancesthebiologicalhalf-life.Onlythefreedrugcangetexcreted
throughglomerularfiltration.
Forexample:tetracyclinesareexcretedmainlybyglomerular
filtration.Thesedrugsbindtoproteinsandresultsindecreasedrenal
excretion.
4.Drugaction:Proteinbindinginactivatesthedrug,becausesufficient
concentrationofdrugcannotbebuiltupinthereceptorsiteforaction.
Exampleisnaphthoquinones.Certaindrugsthoughbindto
proteins,stillretainthedrugactivity.Examplesarepenicillinand
sulfadiazine.

60. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
5.Sustainedrelease:Thecomplexofdrug-proteininthebloodacts
asareservoirandcontinuouslysupplythefreedrugforitsaction.
Exampleissuramin-proteinbindingforanti-trypanosomal
action.
6.Carriersystems:Protein-drugcomplexesactastransportsystems
tocarrydrugstothesiteofaction.Thistransportisextremely
importantfordrugsthatexhibitlowsolubilityinwaterportionof
theplasma.
Forexample,bis-hydroxycoumarin(anticoagulant)isbound
inthebloodtotheextentofabout98%.Thisdrugmighthave
precipitatedintheblood,ifitisnotcomplexedwithprotein.

61. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Binding kinetics
??????+??????⇋????????????
??????=
????????????
????????????
??????????????????=????????????
Thereversiblebindingofprotein(P)anddrug(D)moleculeis
writtenas:
Where,
K=associationconstant,L/mole
[P]=concentrationoffreeprotein,mole/L
[D]=concentrationoffreedrug,mole/L
[PD]=concentrationofproteindrugcomplex,mole/L
Theequilibriumstabilityconstant,associationconstant,K,cabbe
writtenas:

62. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
If the total protein concentration is the sum of unbound protein and the
protein present in the complex.
??????
??????=??????+????????????
or??????=??????
??????−????????????
Substituting [P] in the above equation, we get
????????????=????????????+??????????????????
????????????=????????????
??????−??????????????????
????????????=??????????????????
??????−????????????????????????
????????????+????????????????????????=??????????????????
??????
????????????�+????????????=??????????????????
??????
????????????
??????
??????
=
????????????
�+????????????

63. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
????????????
??????
??????
=
????????????
�+????????????
Where [????????????]/[??????
??????]represents the average number of molecules
bound per mole of protein P
t
Replacing [????????????]/[??????
??????]by r, we get,
??????=
????????????
�+????????????
Suppose nnumber of independent binding sites, than
??????=??????
????????????
�+????????????

64. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Equation can be modified to get plot, known as Scatchard plot
??????�+????????????=??????????????????
??????+??????????????????=??????????????????
??????=??????????????????−??????????????????
??????=??????????????????−????????????
r
D
=????????????−????????????
InScatchardplotr/[D]isplottedagainstrtogiveastraightlineifonly
oneclassofbindingsitesispresent.However,ifmorethanoneclassof
bindingsitesexist,thegraphisnotlinearbutexhibitsacurvature.

65. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Fig.showsaScatchardplotforthebindingofbis-hydroxycoumarinto
humanserumalbuminat20and40°C.
Thecurvatureinboththeplots.At20and40°Cindicatethepresenceof
morethanonetypeofbindingsites.

66. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Thermodynamic treatment of stability constants
ManyofthepharmaceuticalprocessessuchasComplexation,protein
binding,thedissociationofaweakelectrolyte,orthedistributionofa
drugbetweentwoimmisciblephasesaresystemsatequilibriumand
canbedescribedintermsofchangesoftheGibbsfreeenergy(∆G).
Consider a closed system at constant pressure and temperature, such as
the chemical reaction;
aA+bB⇋cC+dD…. (1)
Because Gis a state function, the free energy change of the reaction
going from reactants to products is;
∆G= ∆G
products
− ∆G
reactants
…. (2)

67. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Equation(1)representsaclosedsystemmadeupofseveral
components.Therefore.AtconstantTandPthetotalfreeenergy
changeoftheproductsandreactantsinequation(2)isgivenasthe
sumofthechemicalpotentialμofeachcomponenttimesthe
numberofmoles.
∆G=cμ
C
+dμ
D
−aμ
A
+bμ
B
…. (3)
Further mathematical treatment of equation (3) for ideal reactants
and products give;
∆G=∆G
°
+RTlnK …….(4)

68. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Because∆G
°
isaconstantatconstantTandP,RTisalsoconstant.
Hence∆G=0andequation(4)becomes
0=∆G
°
+RTlnK
or
∆G
°
=−RTlnK
or
∆G
°
=−2.303RTlogK …….(5)
ThestandardfreeenergychangeofComplexationisrelatedtothe
overallstabilityconstant,K,(oranyoftheformationconstants)by
therelationshipshowninequation(5)

69. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
The standard enthalpy change, ∆H, can be obtained from the slope
of a plot of log Kversus 1/T, following the expression
logK=−
∆H
°
2.303R
1
T
+constant
When the values of K at two temperatures are known, the following
equation can be used:
log
??????
�
??????
�
=
∆H
°
2.303R
T
2
−T
1
T
1
T
2

70. By: Khalifa M Asif Y Asst. Professor Pharmaceutics Dept. AACOP Akkalkuwa
Thestandardentropychange,∆S°,isobtainedfromtheexpression
∆G
°
=∆??????
°
−T∆??????
°
∆H°and∆S°generallybecomemorenegativeasthestability
constantformolecularcomplexationincreases.
Asthebindingbetweendonorandacceptorbecomesstronger,
∆H°wouldbeexpectedtohavealargernegativevalue.