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Prodrugs: bridging pharmacodynamic/pharmacokinetic gapsBernard Testa

In this mini review, prodrugs are discussed with a focus on their

pharmaceutical, pharmacokinetic, and pharmacodynamic

objectives, as well as on the resulting therapeutic benefits.

Carrier-linked prodrugs remain the most extensively

investigated and receive due attention here with recent

successes highlighted. A clear trend is apparent in modern

prodrug research, namely the increased attention given to the

knowledge-based design of bioprecursors, namely prodrugs

devoid of a detachable promoiety. In most cases, such

prodrugs are activated by in situ reduction, hence their

designation as bioreductive prodrugs. This is a particularly

active field in the design of more selective, small-molecule

antitumor agents. New antimicrobial agents are also in the

pipeline. In addition, biooxidative bioprecursors offer a

promising strategy in specific cases, as illustrated by the

successful antiaggregating agent clopidogrel.

Address

Pharmacy Department, University Hospital Centre, CHUV/BH-04, 46

Rue du Bugnon, CH-1011 Lausanne, Switzerland

Corresponding author: Testa, Bernard ([email protected])

Current Opinion in Chemical Biology 2009, 13:338–344

This review comes from a themed issue on

Next-generation therapeutics

Edited by Karl–Heinz Altmann and Dario Neri

Available online 25th May 2009

1367-5931/$ – see front matter

# 2009 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2009.04.620

IntroductionWhat makes prodrugs different from other drugs is the

fact that they are devoid of intrinsic pharmacological

activity [1�,2��,3–15]. Thus, the simplest and clearest

definition, in this writer’s view, is that given by Albert

[16], who coined the term. In modified form, the defi-

nition reads: ‘Prodrugs are chemicals with little or no

pharmacological activity, undergoing biotransformation

to a therapeutically active metabolite.’

Drug discovery projects are in danger of failure should

pharmacodynamic (PD) and pharmacokinetic (PK) pre-

requisites prove chemically incompatible that is imposs-

ible to combine in a single molecule. Because prodrugs

and their active metabolite have different chemical struc-

tures, they must differ in their biological behavior, a fact

that opens the door to dissociating PD and PK properties.


Why prodrugs?The prodrug concept has found a number of useful

applications in drug research and development. These

correspond to a number of objectives, as presented in

Figure 1. However, it should be clear that such a view is

too schematic, these objectives being often intertwined.

Thus, an improved solubility can greatly facilitate oral

absorption, while improving the chemical stability of an

active agent can allow tissue-selective delivery and even

lead to its in situ activation.

Pharmaceutical objectives

Pharmaceutical scientists are often faced with serious

formulation problems resulting from poor solubility,

insufficient chemical stability or poor organoleptic prop-

erties. While pharmaceutical technology can solve such

problems in favorable cases (e.g. by improving the solu-

bility of cyclosporine), success is not guaranteed and may

be time-consuming to achieve.

An example of chemical interest is that of isotaxel (1 in

Figure 2), a prodrug of the potent antitumor agent pacli-

taxel (2 in Figure 2) [17,18]. Paclitaxel has a very poor

water solubility (about 0.25 mg/L) which necessitates

coinjection of a detergent. Taking advantage of the

well-known mechanism of intramolecular nucleophilic

transacylation, an analog of paclitaxel was prepared hav-

ing the benzoyl group located on the a-hydroxy group

rather than on the amido group. What is an amido func-

tion in paclitaxel thus became a basic amino group in

isotaxel, and solubility was greatly improved by protona-

tion (water solubility of 1 HCl is 0.45 g/L). O,N-Migration

of the benzoyl moiety to the active paclitaxel was fast

under physiological conditions of pH and temperature,

but much slower under slightly acidic, pharmaceutically

compatible conditions.

Note that increasing solubility is not only a pharmaceu-

tical but also a PK objective. Indeed, and as made explicit

by the biopharmaceutics classification scheme (BCS) [19],

solubility is one of the main factors influencing oral

absorption.

Pharmacokinetic objectives

PK objectives are currently the most important ones in

prodrug research. Foremost among these is firstly, a need

to improve oral bioavailability, be it by improving the oral

absorption of the drug and/or by decreasing its presyste-

mic metabolism. Other objectives are secondly, to

improve absorption by parenteral (nonenteral, e.g. dermal

and ocular) routes; thirdly, to lengthen the duration of

action of the drug by slow metabolic release; and finally to

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http://dx.doi.org/10.1016/j.cbpa.2009.04.620

Bridging pharmacodynamic/pharmacokinetic gaps Testa 339

Figure 1

A schematic classification of objectives in prodrug research. Note that

many of these objectives are in fact intertwined (modified from [1�]).

achieve the organ/tissue-selective delivery of an active

agent. Some of these objectives are exemplified below

with clinically or potentially useful prodrugs.

Achieving improved oral absorption by a prodrug strategy

is a frequent rationale in marketed prodrugs, as aptly

illustrated by neuraminidase inhibitors of therapeutic

value against type A and B influenza in humans [20].

Target-oriented rational design has led to highly hydro-

philic agents which are not absorbed orally, as exempli-

fied by the zwitterion zanamivir which is administered in

aerosol form. Another active agent is Ro-64-0802, which

also shows very high in vitro inhibitory efficacy toward the

enzyme but low oral bioavailability, also being a (too)

hydrophilic zwitterion by virtue of basic amino and acidic

carboxylate groups. To circumvent this problem, the

active agent was derivatized to its ethyl ester known as

oseltamivir (3 in Figure 2). This prodrug is well absorbed

orally and undergoes rapid enzymatic hydrolysis to pro-

duce high and sustained plasma levels of the active agent

(structure marked in red).

Another PK objective is the organ-selective or tissue-

selective delivery of a given drug, also known as the

search for the ‘magic bullet’. Targeting a prodrug to

enzymes highly expressed in the target organ or tissue is

a useful strategy for selective delivery. Given the high

concentration of cytochromes P450 (CYPs) in the liver,

it comes as no surprise that CYP-activated prodrugs

should have been designed with a view of selectively

delivering antihepatitis virus agents to the liver. A good

example is provided by pradefovir (4 in Figure 2), a

prodrug currently in clinical trials as the cis-(2R,4S)-

isomer shown here [21]. When examined in rats, this 1-

aryl-1,3-propanyl prodrug showed a 12-fold improve-


ment in the liver/kidney ratio of adefovir (structure

marked in red) over adefovir dipivoxil, the prodrug in

current use.

Targeting prodrugs to enzymes overexpressed in cancer

tissues is a topic of major interest and promise in drug

discovery [22]. A clinically significant example is that of

capecitabine (5 in Figure 2), a multistep, orally active

prodrug of 5-fluorouracil (structure in red) [23]. Capeci-

tabine is well absorbed orally and undergoes three acti-

vation steps resulting in high tumor concentrations of the

active drug. It is first hydrolyzed by liver carboxylesterase,

the resulting metabolite being a carbamic acid which

spontaneously decarboxylates to 50-deoxy-5-fluorocyti-

dine. The enzyme cytidine deaminase present in the

liver and tumors then transforms 50-deoxy-5-fluorocyti-

dine into 50-deoxy-5-fluorouridine. Transformation into

5-FU is catalyzed by thymidine phosphorylase and occurs

selectively in tumor cells.

Capecitabine is of great interest in the context of this

review as it affords an impressive gain in therapeutic

benefit compared to 5-FU due to its oral bioavailability

and a relatively selective activation in and delivery to

tumors. Nevertheless, most of the current attention in

antitumor prodrug design is focused on reductive biopre-

cursors, namely prodrugs reduced in situ to a cytotoxic

metabolite.

Pharmacodynamic objectives

PD objectives can be understood as being synonymous

with decreasing systemic toxicity. Two major cases are

illustrated here, namely the masking of a reactive agent to

improve its therapeutic index, and the in situ activation of

a chemotherapeutic agent.

The masking of a reactive agent to improve its thera-

peutic index is aptly exemplified by the successful anti-

aggregating agent clopidogrel (6 in Figure 3). This

compound, whose molecular mechanism of action was

poorly understood for years, is now known to be a prodrug

[24,25,26�]. It is of interest among prodrugs in that its

major metabolic route in humans (about 80% of a dose) is

one of hydrolysis which leads to the inactive acid. In

contrast, much of the remaining dose is activated in a

three-step sequence. First, CYP3A oxidizes clopidogrel to

2-oxo-clopidogrel (7 with the corresponding substituents

R and R0). This is followed by a CYP-catalyzed sulfox-

idation to the cyclic sulfoxide intermediate. The latter

hydrolyzes spontaneously to a highly reactive sulfenic

acid metabolite (8 with the corresponding substituents R

and R0) which forms a covalent S–S bridge with a thiol

group in platelet ADP receptors, thereby causing their

long-lasting inactivation. The sulfenic intermediate can

also be reduced by glutathione, leading to the correspond-

ing thiol which for some years was thought to be the active

metabolite.


340 Next-generation therapeutics

Figure 2

Some prodrugs discussed in the text. An illustrative prodrug developed for improved water solubility is namely isotaxel (1), a promising prodrug of

paclitaxel (2). Examples of carrier-linker prodrugs (active drug shown in red) are the anti-influenza virus agent oseltamivir (3), the antihepatitis virus

agents pradefovir (4), and capecitabine (5), a well-known pro-prodrug of 5-fluorouracil.

It is interesting to compare the activation of clopidogrel

with that of prasugrel (9 in Figure 3), a more recent and

smartly designed analog [27–29]. In contrast to the for-

mer, the latter is activated by carboxylesterases 1 and 2 in

humans. Indeed, C(2) in prasugrel carries an acetyl ester

function whose hydrolysis yields the corresponding

hydroxy-thiophene which tautomerizes to the thiolactone

(7 with the corresponding substituents R and R0), a close

analog of 2-oxoclopidogrel. Sulfoxidation followed by

hydrolytic ring opening yields the (yet to be discovered)

active sulfenic acid metabolite (8 with the corresponding

substituents R and R0).

In situ activation to toxic agents is a part of the well-known

mechanism of action of the antibacterial and antiparasitic

nitroarenes such as metronidazol. This strategy is finding

a recent and promising revival with the discovery of


potent bioreductive prodrugs. These are designed to

be activated by a metabolic reaction of reduction in target

tissues or organisms with a low oxygen concentration.

Such a state of hypoxia may favor reduction reactions, but

another factor accounting for the shift toward reduction is

the hypoxia-increased expression of genes coding for

reductive enzymes. A recent and promising illustration

of bioreductive antimicrobial prodrugs is provided by the

antitubercular nitroimidazooxazine PA-824 (10 in

Figure 3) now in clinical trials [30�]. There is evidence

to indicate that the active species in antimicrobial nitroar-

enes is their nitrenium cation, a strong and highly reactive

electrophile [31].

The case of antitumor bioprecursors is best illustrated

with tirapazamine (11 in Figure 3), since this much

studied agent and its analogs have revealed inspiring



Figure 3

A comparison of the activation pathways of the antiaggregant prodrugs clopidogrel (6) and prasugrel (9). The former is activated by two sequential

CYP-catalyzed reactions, first to the thiolactone 7, then to its sulfoxide (not shown), followed by spontaneous hydrolytic ring opening to the reactive

sulfenic acid (8). The latter antagonizes platelet ADP receptors via a covalent S–S bridge. The first activation step of prasugrel (9) is by

carboxylesterase-catalyzed deacetylation, followed by the same subsequent steps as clopidogrel. The figure also shows the structure of the

antitubercular nitroimidazooxazine PA-824 (10) whose activation is by nitroreduction, and the activation of the hypoxia-selective antitumor agent

tirapazamine (11) to the radical 12.

information on their molecular mechanism of action

[32–34]. Tirapazamine is inactivated by two-electron

reduction steps catalyzed by quinone reductase, but

activated to a cytotoxic radical (12 in Figure 3) by a

one-electron reduction catalyzed by NADPH-cyto-

chrome P450 reductase and multiple reductases in the

nucleus. The strong redox potential of this benzotriazi-

nyl radical allows it to abstract a hydrogen radical from

DNA, thereby causing single-strand and double-strand

breaks.

How prodrugs?Given the stated objectives, how can medicinal chemists

design clinically useful prodrugs? As implicit above, two

main classes of prodrugs exist, namely the carrier-linked

prodrugs and the bioprecursors [3].


In carrier-linked prodrugs, the active agent (the drug) is

linked to a carrier (also known as a promoiety). In many

cases, carrier-linked prodrugs are esters activated by

enzymatic hydrolysis, although nonenzymatic hydrolysis

may contribute [11]. The variety of ester prodrugs in the

literature is immense and has served to mask carboxylate

groups (e.g. oseltamivir in Figure 2), phosphate groups

(e.g. pradefovir Figure 2), acidic tetrazolate groups, or

alcoholic or phenolic functions. Examples of promoieties

used to derivatize such active agents include simple or

functionalized alkyl groups, aryl groups, simple or com-

plex carboxylic acids, amino acids, and fractionable moi-

eties (e.g. carbonic acid esters).

Amides form another major group of carrier-linked pro-

drugs, the active agent usually being an amine. Here,



Figure 4

Two schematic situations in a research project. The two large ovals symbolize the undefined chemical space theoretically accessible in a given

research project. PD space symbolizes the ensemble of all molecules within this chemical space having the required pharmacodynamic profile. PK

space symbolizes the ensemble of all molecules within this chemical space having the required pharmacokinetic behavior. Case A represents the

favorable situation where there is partial overlap of the PD and PK chemical spaces, allowing lead optimization to evolve toward candidates which

combine good PD and PK properties. Case B represents the difficult situation where there is incompatibility between the structural conditions for the

required PD and PK properties, and where a traditional optimization strategy must fail. Here, a prodrug strategy may be the only alternative to bridge

the gap and save the project. The sooner a given research project is found to belong to Case B, the more successful a prodrug strategy may be

(modified from [9]).

useful promoieties include carboxylic acids, amino acids,

and carbonic acids to yield carbamates. Mannich bases

and even imines have also been prepared as potential

prodrugs of amines. Extensive compilations of promoi-

eties have been published [5,6,11].

In contrast to carrier-linked prodrugs, bioprecursors do

not contain a promoiety yet may be activated by hydration

(e.g. lactones such as some statins), oxidation (e.g. prasu-

grel in Figure 3), or reduction (e.g. PA-824 and tirapaza-

mine in Figure 3). This is currently a highly active field in

the search for more selective chemotherapeutic agents

(antibacterial, antiparasitic, or anticancer prodrugs) acti-

vated by reductases [35–41]. Such prodrugs include

nitroarenes, quinones, amidoximes, platinum(IV) com-

plexes, and N-oxides.

ConclusionThe gain in therapeutic benefit provided by prodrugs

relative to the active agent is a question that knows no

general answer. Depending on both the drug and its

prodrug, the therapeutic gain may be negligible, modest,

marked, or even significant. Nevertheless, a trend is

apparent from innumerable data in the literature and


when comparing marketed drugs and candidates in

R&D. In the case of marketed drugs endowed with useful

qualities but displaying some unwanted property ( post hocdesign), the therapeutic gain is usually modest yet real,

but it will be negligible when the drug defect is tolerable

or barely improved by transformation to a prodrug. In

contrast, it will be marked or significant when much

improved targeting is achieved.

In the case of difficult candidates showing excellent

target properties but suffering from some severe or

intractable physicochemical and/or PK drawbacks (e.g.

high hydrophilicity restricting bioavailability), ad hocdesign may lead to a marked or even significant benefit.

Here indeed, a prodrug form may prove necessary, and its

design should be integrated into the iterative process of

lead optimization. In other words, a prodrug approach is

most fruitful when optimization of a traditional lead

candidate fails because the structural conditions for PD

activity (i.e. the pharmacophore) are incompatible with

the preset pharmaceutical, PK or PD properties. In such

cases, the gap between activity and other drug-like prop-

erties may be of such a nature that only a prodrug strategy

can bridge it (Figure 4).



Conflicts of interestNone.

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