Preclinical Drug Discovery in Pharmacology.

1. SUBMITTED BY :
Sourin De
M PHARM PARMACOLOGY 2nd SEM.

2. Prodrugs are inactive compounds which are metabolized
either chemically or enzymatically in a controlled or
predictable manner to the parent active drug inside the
body.
Prodrugs can enhance the therapeutic efficacy and/or
reduce the adverse effect via different mechanisms
including increased solubility, improved permeability and
bioavailability , prolonged half life and tissue target
delivery.

3. The release of active drug only occurs at its site of action ,
thereby:
oReduce toxic side effects due to high plasma concentration
or
oReduce non-specific updates by other body tissues.
Site specific drug delivery is achieved by:
oSite directed drug delivery
oSite directed bioactivation

4. Site directed drug delivery
Anticonvulsant activity of Pragabide
oGABA is too polar to cross BBB.
oLipophilic analog of GABA crosses the BBB and releases
GABA inside the brain .
oThus shows anti convulsant activity
o
PRAGABIDE
GABA

5. Increased lipid solubility may enhance uptake in other
tissue with a resultant increase in toxicity.
These problems may be overcome by utilizing a
dihydropyridine-pyridine-pyridinium salt type redox
system.
The drug [D] which is aimed to be delivered to the brain ,
is coupled to a quaternary carrier QC [ ie. N-methyl
nicotinic acid ].
QC is reduced to neutral, lipophilic dihydro form [D-DHC]
D + QC D-QC reduced to D-DHC[prodrug]
D-DHC oxidation D-QC hydrolysis QC + D

6. After administration , this compound is distributed
throughout the body including the brain.
D-DHC is then enzymatically oxidized back to the original
D-QC .
The ionic form is trapped in the brain and undergo slow
enzymatic cleavage releasing the active agent.
Only small amount of free drug is released in the blood.
Eg : dopamine , phenytoin , penicillin

7. Beta-lactam antibiotics are used in the treatment of
bacterial meningitis.
oThey are hydrophilic , enter the brain very slowly.
oDihydropyridine-penicillin prodrug deliver beta-lactam
antibiotics in higher concentration into the brain.

8. Site specific bio-activation
Designing a prodrug that require activation by an enzyme
found predominantly at the desired site.
Eg : acyclovir , antiviral agent
o Acyclovir thymidine kinase acyclovir monophosphate
oAcyclovir monophosphate cellular kinase Acyclovir di
and
triphosphate
oTriester formation takes place much greater extend in herpes
–infected cells, than uninfected cells.

9. Acyclovir Acyclovir monophosphate
Acyclovir triphosphate
Virus thymidine kinase
Cellular
kinase

10. pH dependent bio-activation
Omeprazole : antiulcer drug
Omeprazole prodrug is converted to their active
sulphonamide in the acidic condition of parietal cell.
H+
OMEPRAZOLE
[inactive]
sulfonamide intermediate
[active]
Disulphide complex

11. It can be achieved by making a long chain aliphatic ester
because these ester hydrolysis slowly.
Fluphenazine antipsychotic drug
oFluphenazine has shorter duration of action[6-8 hrs]
oFluphenazine enanthate and decanoate [prodrug] has
duration of action about a month.

12. R
Fluphenazine
Fluphenazine
decanoate[prodrug]
R = H
R = CO[CH2]8CH3

13. Vasopressin used for the treatment of bleeding
varicose veins in the lower end of the esophagus .
oVasoconstrictor action of drug stops bleeding .
oBut short duration of action
oIncreasing dose leads to toxic side effect
oGlypressin [gly-gly-gly-lys-vacopressin] prodrug of
vasopressin
oMinimize possibility of unwanted side effects caused by
high blood pressure

14. The rationale for prodrug design is to achieve favourable
physicochemical characteristics [ chemical stability,
solubility, taste or odor ], biopharmaceutical properties
[oral absorption, first-pass metabolism, permeability
across biological membranes such as blood brain barrier ,
reduced toxicity] or pharmacodynamic properties [ reduced
pain or irritation].

15. Improving formulation and administration.
Enhancing permeability and absorption.
Changing the distribution profile.
Protecting from rapid metabolism and excretion.
Overcoming toxicity problems.
Managing life cycle.

16. Dissolution rate limiting step of absorption.
Prodrugs are an alternative way to increase the aqueous
solubility of parent drug molecule by improving
dissolution rate via attached ionizable or polar neutral
groups such as phosphates, amino acids, or sugar moiety.
Phosphate esters are widely used to increase the aqueous
solubility of orally and parenterally administered drug.

17. Prednisolone sodium phosphate is an example of phosphate
prodrug.
oIt is highly water soluble [ 30 times greater than prednisolone]
oThe phosphate prodrug moiety is linked directly to a free
hydroxyl group on prednisolone.
oThe orally administered prodrug rapidly hydrolyzed by gut
epithelial alkaline phosphatases to their respective parent drug.
Phosphatase
Prednisolone sodium
phosphate[prodrug]
Prednisolone

18. Amino acid esters or amides are also commonly used
ionizable groups which can be introduced into the hydroxyl,
thiol, amine or carboxylic acid functionalities of parent
drugs.
A variety of esterases , amidases, peptidases can bioconvert
their prodrug into active counterparts.
Sugar moiety glucose , galactose, or glucuronic acid
Enzymes glucosidases, galactosidases, or glucuronidase

19. A hydrophilic hydroxyl, thiol, carboxyl, phosphate or
amine group on parent drug can be converted to more
lipophilic alkyl or aryl esters .
oThese prodrugs are readily converted to their active
species by ubiquitous esterase.
Oseltamivir is an orally active ethyl ester prodrug .
oSelective inhibitor of viral neuraminidase glycoprotein .
oUsed in the treatment of influenza type A and type B .

20. oAfter absorption , oseltamivir undergoes rapid bioconversion to
its parent drug mostly by the action of carboxylesterase.
oBioavailability of oseltamivir is 80%comparing to free
carboxylate which is 5%.
Oseltamivir [prodrug]
Carboxylesterase

21. Tazarotene is an example of marketed ethyl ester prodrug
with enhanced transdermal drug delivery.
oTazarotene is used for psoriasis and acne treatment and
causes less skin irritation than the parent drug
tazarotenic acid .
Dipivefrin improve ocular absorption .
oDipivefrin is dipivalyl diester of epinephrine.
oDipivefrin penetrates the human cornea 17 times more
rapidly than epinephrine.
oLower the intraocular pressure.

22. A great many prodrug have increased efficacy and safety
profile because of their targeting properties.
Anti-Parkinson agent L-DOPA .
oDopamine[hydrophilic] not able to cross BBB .
oAmino acid prodrug of dopamine , L-DOPA enable the
uptake and accumulation in the brain.

23. Dopa
decarboxylase
Levodopa [prodrug] Dopamine
o L type amino transporter 1 for the uptake of L DOPA
o After uptake LDOPA is bioactivated by aromatic L amino
acid decarboxylase to hydrophilic dopamine.

24. Rapid metabolic break down of the drug can be protected
by prodrug.
This is carried out by masking the metabolically labile but
pharmacologically essential functional groups of the drug.
Terbutaline is a bronchodilator and beta 2 agonist, used
in the treatment of asthma.
oTerbutaline undergo rapid pre systemic metabolism.
oIt is prevented by converting its phenol group to bis
dimethyl carbamate [bambuterol]

25. Bambuterol [prodrug] is slowly bioactivated to Terbutaline
predominantly by non specific butyrylcholine esterase
outside the lungs.
As a result of slower release and prolonged action , once
daily administration of bambuterol provides relief of
asthma.
butyrylcholinesterase
Bambuterol [prodrug] Terbutaline

26. Kidneys are the principle organ of excretion.
It efficiently eliminate water soluble substance from the
body.
Therefore use of lipophilic promoieties to decrease the
solubility is one way to prolong the duration of action of
very water soluble drug.

27. Reduced toxicity can be achieved by targeting drug to
desired cells and tissues via site selective drug delivery .
Therefore antibody directed enzyme prodrug therapy and
gene-directed enzyme prodrug therapy and virus directed
enzyme prodrug therapy are widely used , especially with
high toxic compounds.

28.  Prodrug can offer a valuable opportunity to improve the life
cycle management of parent drug.
Fosamprenavir is water soluble prodrug.
oThis phosphate ester of the HIV protease inhibitor amprenavir
was originally designed to improve solubility and oral
bioavailability.
oAt the same time, this prodrug will continue the life cycle of
amprenavir.
oBecause the fosamprenavir patent will last until 2017, whereas
patent on amprenavir will expire on 2013

29. Prodrug research encounter various challenges.
Many enzymes involved in the prodrug activation are
subjected to interindividual variabilities in their activities.
Intrinsic factor : polymorphism in the gene coding the
enzymes.
Extrinsic factor :interaction caused by other drugs or
xenobiotics.
Both these factors may cause insufficient or excessive
conversion of prodrug into their active form.

30. Esterase catalyzed hydrolysis of prodrug
The majority of carboxylesterases are found in 2 isozyme
families : CES1 and CES2.
In human CES1 is highly expressed in the liver .
CES2 is highly expressed throughout the intestine and
kidney.
The difference in both localization of CES1 and CES2 I
humans and their substrate specificities can take into
account in prodrug design.
CES1 prefer the hydrolysis of esters with large acyl and
small alcohol groups.

31. CES2 hydrolyses esters of smaller acyl and larger alcohol
groups.
CES1 can hydrolysis , Temocapril [small alcohol group and
large acyl group] with higher efficiency than CES2.
Temocapril [prodrug]
CES1
Temocaprilat

32. Prodrug bioactivation by cytochrome P450
enzymes
The P450 enzymes are super family enzymes accounting
for up to 75% of all metabolism of drugs, including
prodrugs.
Genetic polymorphism of prodrug activating P450s
contribute substantially to the variability in prodrug
activation .
Thus affect the efficacy and safety of drugs using this
bioactivation pathway.
Several drug-metabolizing P450 genes are known to be
polymorphic including CYP2A4, CYP2B6 etc…

33. In particular , the CYP2D6 gene is highly polymorphic
with 75 different major alleles currently known.
Some of these alleles are associated with reduced enzyme
activity or even the absence of enzyme function.
Anti cancer drugs [cyclophosphamide, ifosfamide and
trofosfamide] are prodrugs .
oBioconversion of these cyclic phosphate drugs to the
corresponding cytotoxic species is initiated by P450
enzymes.

35. AIM : targeting hypoxia condition of solid tumor by
hypoxia activated prodrug [HAP]
INTRODUCTION
Hypoxia is an important characteristics of solid
malignancies.
Hypoxia is closely related to tumor prognosis and
therapeutic resistance.
HAP
Hypoxia activated prodrug are bioreductive prodrug that
are activated in the hypoxia condition .
It is a site specific drug delivery targeting the hypoxia
region of solid tumor.
This will reduce toxicity to normal tissues.

36. Tirapazamine
1st activated prodrug reported in 1986.
In hypoxic condition , Tirapazamine undergo 1 electron
reduction by the enzyme CYP450 and form free radicals

37. Chromosomal aberration and DNA double strand break
Preclinical and clinical effect
As of 2006, Tirapazamine is undergoing phase 3 clinical
trial .
In preclinical studies , Tirapazamine effectively inhibited
tumor in hypoxia cells.
But Tirapazamine has only limited effect on clinical trial ,
The affinity of Tirapazamine for hypoxia tissue is much
lower in vivo compared to in vitro.
Clinical trial also report significant toxicity like
ototoxicity, muscle cramping , nausea, vomiting etc…

38. Prodrug from serendipity to rational design : Kristiina M.
Huttunen, Hannu Raunio and Jarkko Rautio
:pharmacological review :63:750-771, 2011
Drug design : Dr. V.M Kulkarni and Dr. K.G. Bothara
K ,.Ilango vol 1 : text book of medicinal chemistry