Prodrugs in medicinal chemistry and enzyme prodrug therapies

Raoul Walther, Jarkko Rautio, Alexander N. Zelikin

PII: S0169-409X(17)30097-2
DOI: doi:10.1016/j.addr.2017.06.013
Reference: ADR 13139

To appear in: Advanced Drug Delivery Reviews

Received date: 12 January 2017

Revised date: 27 June 2017
Accepted date: 29 June 2017

Please cite this article as: Raoul Walther, Jarkko Rautio, Alexander N. Zelikin, Prodrugs
in medicinal chemistry and enzyme prodrug therapies, Advanced Drug Delivery Reviews
(2017), doi:10.1016/j.addr.2017.06.013

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 ACCEPTED MANUSCRIPT

Prodrugs in medicinal chemistry and enzyme prodrug therapies

Raoul Walther,1 Jarkko Rautio,2* Alexander N. Zelikin1,3*

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Department of Chemistry, Aarhus University, Denmark

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2
School of Pharmacy, University of Eastern Finland, Kuopio, Finland

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3
iNano Interdisciplinary Nanoscience Centre, Aarhus University, Denmark

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Email : jarkko.rautio@uef.fi (JR), zelikin@chem.au.dk (ANZ)

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Abstract

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Prodrugs are cunning derivatives of therapeutic agents designed to improve the pharmacokinetics profile of
the drug. Within a prodrug, pharmacological activity of the drug is masked and is recovered within the
human body upon bioconversion of the prodrug, a process that is typically mediated by enzymes. This
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concept is highly successful and a significant fraction of marketed therapeutic formulations is based on
prodrugs. An advanced subset of prodrugs can be engineered such as to achieve site-specific bioconversion
of the prodrug – to comprise the highly advantageous “enzyme prodrug therapy”, EPT. Design of prodrugs
for EPT is similar to the prodrugs in general medicinal use in that the pharmacological activity of the drug is
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masked, but differs significantly in that site-specific bioconversion is a prime consideration, and the enzymes
typically used for EPT are non-mammalian and/or with low systemic abundance in the human body. This
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review focuses on the design of prodrugs for EPT in terms of the choice of an enzyme and the corresponding
prodrug for bioconversion. We also discuss the recent success of “self immolative linkers” which
significantly empower and diversify the prodrug design, and present methodologies for the design of
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prodrugs with extended blood residence time. The review aims to be of specific interest for medicinal
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chemists, biomedical engineers, and pharmaceutical scientists.

Keywords : prodrugs; enzyme prodrug therapy; drug delivery; self immolative linkers; enzyme
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Contents

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Introduction ....................................................................................................................................................... 3
General medicinal prodrugs............................................................................................................................... 8

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Esterases: a “universal” tool for drug recovery ............................................................................................. 8

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Prodrugs for phosphate and phosphonate groups .......................................................................................... 9
Phosphatase (Phosphoesterases) : bioconversion of phosphate prodrugs ................................................... 11

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Enzyme prodrug therapies ............................................................................................................................... 12
Cytosine deaminase ..................................................................................................................................... 12

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Phosphatases and the origin of EPT ............................................................................................................ 13
Viral thymidine kinase ................................................................................................................................ 15
β-Lactamase................................................................................................................................................. 16
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Nitroreductase.............................................................................................................................................. 17
Glucuronidase .............................................................................................................................................. 19
Other glycosidases ....................................................................................................................................... 20
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Self immolative linkers.................................................................................................................................... 21

Long circulating prodrugs ............................................................................................................................... 26
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Conclusions and Outlook ................................................................................................................................ 29

Acknowledgments ........................................................................................................................................... 29
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References ....................................................................................................................................................... 33
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Introduction
The traditional path to a successful drug on the market starts with identification of the disease and

the proper drug target, finding the lead and the pharmacophore, and optimizing the interaction of

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the drug lead molecule with the target. Successful to this point, the program of drug development

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may still fail due to poor pharmacokinetics (PK) of the molecule. Shortcomings in PK may be

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associated with each of the four constituting barriers, namely Absorption, Distribution, Metabolism,

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and Elimination (known as ADME). In many successful examples of drugs being brought to the

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market, pharmacological lead is significantly optimized to improve its PK and overcome such

barriers using prodrugs. By definition, prodrugs are derivatives or precursors of therapeutically

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active molecules, which undergo bioconversion into their active form inside the body, be it via

spontaneous processes (e.g. hydrolytic degradation) or through a biocatalytic mechanism. [1, 2]

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Application of a prodrug strategy in drug delivery typically seeks to aid the drug in overcoming a
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barrier, physical or metaphorical, to enhance the deliverable payload of the drug. Such barriers

include (but are not limited to) poor aqueous solubility – which can significantly limit utility of the
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drug in medicinal use; poor absorption from the gastro-intestinal tract into blood; poor rates of cell

entry; etc. Table 1 lists the “barriers” related to ADME as well as toxicity and provides examples of
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prodrugs, which were designed specifically with the view of overcoming the nominated problem.

Of the small molecule (non-biological) drugs that entered the market in the USA in 2015, 7 out of

32 molecules were prodrugs. One prominent example of prodrugs is Sofosbuvir, the active

ingredient in Harvoni, the second most selling drug in 2015. [3]

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Formulation,
development or Drug Prodrug strategy
Limitation Representative prodrug
ADMET
property

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Formulation and  Propofol  Insufficient solubility for aqueous  Fospropofol  Phosphonooxymethyl ester [4]

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administration dosage form
 Chloramphenicol  Unpleasant taste  Chloramphenicol palmitate  Palmitate ester [5]

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 Amprenavir  Insufficient dissolution rate in  Fosamprenavir  Phosphate ester [6]
gastrointestinal tract due to low

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aqueous solubility

Absorption
  Poor membrane permeability and low  

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Oseltamivir carboxylate Oseltamivir Ethyl ester [7]
 nucleotide monophosphates oral or topical (ocular) bioavailability  Sofosbuvir  aryloxy phosphoramidate [8]

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 travoprost carboxylate due to poor lipophilicity  travoprost  isopropyl ester [9]

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Distribution  Dopamine  Lack of site specificity (brain)  Levodopa  Carboxylation [10]
  Short duration of action  

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Metabolism and Terbutaline Bambuterol Bis-dimethylcarbamate [11]
excretion
 5-fluorouracil  Lack of site specificity  Capecitabine  5’-deoxy-5-fluorouridine and N4-

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Toxicity (pentyloxycarbonyl)cytidine [12]
 Phenytoin CE
 Irritation or pain after local
administration
 Fosphenytoin  Phosphonooxymethyl [13]

  Development of a prodrug with  

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Amprenavir Fosamprenavir Phosphate ester [14]
Life cycle
improved properties that may
management
represent a life cycle management
opportunity for an existing drug

Table 1: Examples of drugs, their respective shortcomings in terms of ADMET properties, and successful prodrugs that overcome the
nominated pharmacokinetic limitation of the drug.

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Prodrug bioconversion processes can arbitrarily

be grouped into two categories, namely i)

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prodrug degradation with resulting drug

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recovery, and ii) prodrug activation (Scheme 1).

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For the prodrug degradation class, a prodrug

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molecule represents a conjugate of the parent

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drug; bioconversion reaction involves removal

of a masking group (often termed “promoiety”);

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and the chemical complexity of the molecule Scheme 1. Schematic illustration of prodrug classes: prodrug
activation (e.g. conversion of 5-fluorocytosine to 5-
fluorouracil, phosphorylation of nucleoside analogues) or drug
decreases. Within the prodrug activation recovery (e.g. hydrolysis of esters, glucuronides, etc).
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category (also referred to as “bioprecursor” category [1]), the prodrug undergoes a point chemical
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modification (e.g. carbamine-to-carbonyl transformation [15]) whereby the chemical complexity of

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the molecule is relatively unchanged whereas therapeutic activity is markedly enhanced.

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Alternatively, prodrug activation involves a conjugation reaction (e.g. phosphorylation for

nucleoside analogues) such that chemical complexity of the molecule is increased. Here and below,
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these two classes of prodrug bioconversion are denoted as “drug recovery” and “prodrug

activation”, respectively.

Design strategy for a prodrug first of all depends on the structural features of the parent drug

molecule and specifically the availability of appropriate chemical functionalities that can be used to

mask pharmacodynamic activity of the drug through e.g. an attachment of the modifying group. The

second, equally important consideration pertains to the bioconversion mechanisms of drug release.

This process may be spontaneous, yet in the overall majority of cases, drug synthesis (via recovery

or activation) is carried out through an enzymatic process. Conventional prodrugs in medicinal

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chemistry are typically designed to achieve a quantitative recovery of the drug and this is the prime

objective. In these applications, there typically is little to no consideration of the drug distribution

within the body, and the enzyme to perform the bioconversion may be distributed throughout the

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body (e.g. esterases, phosphoesterases). In some instances, specific enzyme may be predominantly

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expressed within a particular organ such as in the liver and bioconversion of a prodrug is achieved

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predominantly therein (e.g. first step of capecitabine bioconversion performed by carboxylesterases

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[16] and HepDirect™ prodrugs for activation by cytochromes[17]). Design of these prodrugs for

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general medicinal use is reviewed rather well [1] [18], [19] [20]and the presentation below is

considered only briefly to provide a proper context, scientific as well as historical.

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The main focus of this review is the design of prodrugs for an advanced drug delivery opportunity
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termed Enzyme Prodrug Therapy (EPT). For this, a cunning sub-class of prodrugs is designed such
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that bioconversion is performed by a specifically nominated enzyme placed in a particular location

in the body. Through this, recovery or activation of the drug is achieved only at the location of the
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enzyme. In contrast to the general medicinal prodrugs, in the case of EPT, quantitative drug
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recovery is less important and the prime goal is to achieve a site-specific drug recovery.
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Localization of the enzyme at the desired site can be accomplished using a number of ways, with

varied degrees of success and progression from lab to clinic. Historically, antibody-directed

enzyme-prodrug therapy (ADEPT) marked the earliest broadly-recognized success of EPT. [21]

This approach to EPT is injection-based whereby the enzyme is conjugated to an antibody and the

latter facilitates anchorage of the enzyme at the site of action. Examples of ADEPT typically rely on

extracellular prodrug bioconversion through drug recovery mechanisms. Early successes were also

documented using encapsulated enzymes surgically placed at the site of resected tumour for post-

operative chemotherapy.[22, 23] This mode of EPT has recently seen a considerable revival of

interest.[24, 25] The mode of EPT with currently the highest number of ongoing clinical trials is

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that of gene-directed EPT (GDEPT), also known as “suicide gene therapy”.[26] In this case, the

enzyme for prodrug conversion is expressed by the cells upon transduction of the latter and this is

most successfully accomplished using viral vectors. GDEPT examples almost exclusively rely on

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intracellular activation of the administered prodrugs.

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The aim of this review is to provide a comparative analysis of the enzymes and corresponding

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prodrugs and to consider the diverse aspects that contribute to the utility of these enzyme-prodrug

pairs in biomedicine and EPT in particular. Such aspects include pharmacological and

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pharmacokinetics properties of the prodrugs and the change in these properties compared to the
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parent therapeutic, as well as the ease of prodrug synthesis (for an overview, see Table 2).

Presentation of prodrugs is organized by the choice of enzymes starting with esterases - the most
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important enzymes exploited in prodrug design in general medicinal chemistry, then presenting
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phosphoesterases, and finally presenting the enzymes used specifically for EPT. We also discuss the

utility of “self immolative linkers” (SIL), a relatively recent development in medicinal chemistry,
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which significantly broadens synthetic scope with regards to chemical functionalities amenable for
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prodrug design. Finally, we discuss the emerging methodologies to create pools of prodrugs with
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long blood residence time.

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General medicinal prodrugs

Esterases: a “universal” tool for drug recovery

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Bioconversion mechanism for approximately

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half of the marketed prodrugs relies on the

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hydrolysis of an ester bond by esterases (Scheme

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2). [27] Two equally important reasons for this

are i) abundance of hydroxyls and carboxylic

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acid functionalities in the marketed drugs, and ii) Scheme 2. Bioconversion of ester-based prodrugs into a
corresponding carboxylic acid and an alcohol, each of which
can serve as a promoiety or as a drug.
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esterases being ubiquitous such that metabolic

regeneration of drugs in the body is a facile process.[28]

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Carboxylic acid is a highly polar functionality, typically charged at the physiological pH values, and
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this often times diminishes oral absorption of the drug – in which case esters are employed to mask
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the carboxylate. Prime example where this design strategy is highly warranted is the synthesis of
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penicillin antibiotics, e.g. pivampicillin and bacampicillin. Another good example is oseltamivir

carboxylate, a selective inhibitor of neuramidase enzyme of the influenza viruses A and B. While
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this hydrophilic drug has the oral bioavailability of only 5% in humans, its more lipophilic ethyl

ester (Tamiflu) is rapidly and readily absorbed after oral administration, and at least 80% of an oral

dose reaches the systemic circulation as the active oseltamivir carboxylate.[29] After absorption,

oseltamivir undergoes bioconversion to oseltamivir carboxylate and ethanol mainly by

carboxylesterase-1 in the liver.

Similarly, hydroxyl-containing drugs can be acylated with aliphatic or aromatic carboxylic acids to

increase lipophilicity. Famciclovir is a diacetylated (and 6-deoxy) prodrug of an antiviral agent

penciclovir with oral bioavailability of 75% vs 4% for the parent penciclovir.[30] In general, it is

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Scheme 3. Bioconversion of sofosbuvir, an anti-HCV medication, into the corresponding nucleoside analogue and further
into the therapeutically active triphosphate.
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possible to synthesize esters with virtually any degree of lipophilicity and hydrophilicity. For

example, long-chain fatty acids to form decanoates, palmitates, cypionates, or valerates have
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resulted in several marketed sustained-release injectables of, for example, estrogens, neuroleptics,
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and steroids. [18] Hydroxyl-containing drugs can also be acylated with carboxylic acids contained
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within amino acids. Such prodrugs may become suitable substrates of endogenous oligopeptide

transporters (e.g. PePT1) to result in markedly improved oral absorption. Examples of such design

are valyl esters of acyclovir and ganciclovir with oral bioavailability increased by three- to tenfold

over the parent drug, respectively.[31, 32]

Prodrugs for phosphate and phosphonate groups

Phosphate (-OPO(OH)2) and phosphonate (-C-PO(OH)2) groups have recently emerged among the

most important sites for modification during prodrug design, specifically due to the high demand of

antiviral drugs, many of which are nucleoside analogues. [33] [34] [35] Nucleosides have to

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undergo triphosphorylation to become substrates for nucleic acid polymerases whereby the first

phosphorylation is often rate limiting. Design of drugs to contain the phosphate (or phosphonate) is

poised to overcome this limitation but this measure tremendously lowers the rate of drug cell entry

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and oral bioavailability. This is because these groups are charged within the physiological range of

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pH and exhibit minimal permeation through the lipid bilayer that makes up the membrane of

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mammalian cells. Prodrug design has been highly successful to mask the phosphate/phosphonate

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functionality, specifically through ester formation. Early focus was on acyloxymethyl esters such as

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used in the marketed drug adefovir dipivoxil, a pivaloyloxymethyl prodrug of adefovir. A related

strategy relies on the use of carbonate esters such as isopropyloxycarbonyloxymethyl derivatives as

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found in a clinically successful prodrug tenofovir disoproxil.[36] In both cases, ester hydrolysis

(and subsequent decarbonylation for tenofovir disoproxil) affords oxymethylene-protected

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phosphonates and these spontaneously release the antiviral drugs. Hostetler et al.[35] laid the basis
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for the highly promising alkoxyalkyl prodrugs of nucleoside analogues that afford drug recovery
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through the action of phospholipases. The latter prodrugs are hydrophobic and exhibit highly
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advantageous extended body residence times with opportunities for long-lasting antiviral

formulations (e.g. one-monthly drug administration). [35] The work by McGuigan et al. established
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the ProTide technology (prodrug for nucleotide) through a design of aryloxy phosphates and

phosphoramidates (Scheme 3),[37] [38] [39] a technology that inspired the development of highly

successful drugs that are on the market [40] and more undergo clinical trials. The best known

example of these is sofosbuvir, an anti-hepatitis C virus drug, that was second most grossing

therapeutic on the US market in 2015. Sofosbuvir is a pyrimidine nucleotide analogue that inhibits

non-structural protein 5B (NS5B) polymerase of hepatitis C virus and is, therefore, used for the

treatment of hepatitis C infection in adults as a once-daily dosage regimen.[41] Early discovery

studies focused on 2´-deoxy-2´-fluoro-2´-C-methyluridine that was inactive in the HCV replicon

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assay, but its triphosphate form showed to be a potent inhibitor of NS5B with a Ki of 0.42 μM.[8]

This triphosphate can be enzymatically formed in HCV replicon cells from the monophosphate

uridine derivative. However, the 2´-deoxy-2´-fluoro-2´-C-methyluridine failed to undergo the initial

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intracellular monophosphorylation step. To overcome this, and drawing inspiration from early work

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by McGuigan et al., [37] [39] phosphoramidate prodrug strategy was developed to allow the

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delivery of this highly hydrophilic monophosphate form in the body. The phosphoramidate prodrug

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was designed to undergo a cascade of enzymatic and chemical reactions which starts with an

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intracellular cleavage of the sterically most accessible ester functionality, predominantly by

cathepsin A.[42]. (Scheme 3). Worthy of note, bioconversion of sofosbuvir is naturally targeted to
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the liver, the organ mostly affected by the hepatitis C virus, through the first pass effect such that

the active drug is predominantly synthesised within this organ. [8]

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Phosphatase (Phosphoesterases) : bioconversion of phosphate prodrugs

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While the charged character of the phosphate

group poses a problem for drugs with

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intracellular targets, it can also be highly

Scheme 4. Bioconversion of the phosphate ester-based
beneficial in the design of prodrugs, specifically prodrugs to release the corresponding drug.

to increase the solubility of poorly soluble therapeutics.[43] After the administration, the phosphate

promoiety is cleaved by alkaline phosphatase (Scheme 4), a nonspecific esterase found in all tissues

throughout the entire body, including the liver, kidneys, and apical membrane of enterocytes. The

use of phosphate esters has resulted in several successfully marketed prodrugs such as

fosamprenavir.[43] Amprenavir is an anti-HIV agent and being a protease inhibitor (in contrast to

the above discussed nucleoside analogues) does not need to be phosphorylated to exert its

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therapeutic effect. In this case, a phosphate group is attached to its secondary alcohol group to

produce fosamprenavir – a prodrug with approximately 10 times higher aqueous solubility as a

calcium salt than the parent amprenavir.[44] Other examples of water-soluble phosphate prodrugs

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are fosphenytoin, fosaprepitant and ceftaroline fosamil. With fosphenytoin, a phosphate ester is

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attached to an acidic NH-group of phenytoin via an oxymethylene linker. Fosaprepitant employs a

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direct amide N-phosphate strategy, and ceftaroline fosamil is a phosphonate prodrug of a primary

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amine.

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Enzyme prodrug therapies

Cytosine deaminase
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Historically, development of EPT can be traced

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back to the 1980s and the first example of use of

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cytosine deaminase (CDase) to achieve a localized

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Scheme 5. Bioconversion of 5-fluorocytosine into 5-

conversion of 5-fluorocytosine (5FC) to a potent fluorouracil performed by cytosine deaminase .

anticancer agent, 5-fluorouracil (5FU). In humans,

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5FU has a highly variable absorption; coupled a rather narrow therapeutic window, it makes safe

dosing of 5FU challenging. Inspired by the use of 5FC as a drug against the CDase-expressing

fungi, Sakai et al [23] and Nishiyama et al [22] performed immobilization of CDase at the tumor

site, first by injection and subsequently through immobilization of the enzyme-containing reservoirs

enclosed within dialysis bags. Specific success of these reports was that quantification of 5FC and

5FU in the mice blood and at the site of tumor provided direct evidence for a higher concentration

of the anticancer drug at the tumor site compared to the systemic (blood) concentration. These

reports are surprisingly all but forgotten but should be recognized as those that paved the way to the

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development of all the arms of EPT. Specifically, 5FC/CDase is showing significant promise in

Phase II/III clinical trials.[15] [45] Expression of CDase now is engineered into the cancerous cells

through a retrovirus-assisted gene transfer making up the “gene directed enzyme prodrug therapy”,

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GDEPT (sometimes also referred to “suicide gene therapy”). [15] [45] The only obvious drawback

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of 5FC/CDase pair is that there is little if any substrate flexibility and 5FC is the only substrate that

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is used in pair with CDase. However, to our knowledge, GDEPT and specifically this

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prodrug/enzyme pair is currently the most successful and progressed to the highest level of clinical

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trials of all EPT and has a strong development partner aiming to commercialize this technology.
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Phosphatases and the origin of EPT

Second, and possibly independent origin of development of EPT relates to the phosphate prodrugs
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discussed above. While phosphoesterases are quite well distributed in the body, a member of this
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enzyme family, alkaline phosphatase, was in fact the first enzyme used in the antibody-directed
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enzyme prodrug therapy, ADEPT. The latter term was independently coined by Senter and
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Bagshawe [46, 47] to describe a two-step process whereby first, an antibody-enzyme conjugate is

administered to the patient such as to achieve a predominant association of the conjugate with a
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specific antigen, e.g. achieve targeting to the tumor cells. The second step is administration of the

prodrug, which is done after the clearance of the free (un-associated) antibody-enzyme conjugate

from the blood stream. In such a case, enzyme mediated prodrug activation is achieved only by the

antigen-bound enzyme which ensures a highly localized drug delivery.

Prime envisioned utility of ADEPT is in the anticancer treatment and the first drugs used in ADEPT

were anticancer drugs clinically approved to that point in time, namely etoposide (topoisomerase

inhibitor), mitomycin (DNA crosslinking agent), and doxorubicin (a DNA intercalator and

topoisomerase inhibitor).[46] These drugs served well as model compounds but were limited in

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their scope in that their activity focuses on fast dividing cells and leaves hypoxic and dormant cells

alive. To overcome this, in later studies, Bagshawe et al proposed to use nitrogen mustards as the

model drug candidates due to their broader killing potential regardless of the cell’s cell cycle.

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Early studies highlighted that prodrug stability under physiological conditions is a key determinant

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of success since instability gives rise to premature release of the toxic drug, promoting undesired

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side effects.[46] Also a highly important characteristic is the difference in toxicity for the prodrug

and the drug (defined as QIC50, the ratio of IC50 values for the prodrug and the drug). Naturally,

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prodrugs are best to exhibit minimal toxicity whereby the corresponding active therapeutic is sought

to be highly potent. In such a case, systemic distribution of the prodrug leads to minimal toxicity
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and side effects whereas site-specific activation of the prodrug elicits the desired therapeutic

effect.[48] From this perspective, phosphate derivatives of anticancer drugs are quite attractive in
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that these prodrugs exhibit greatly diminished cell entry and are therefore significantly less toxic
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than their parent drugs in vitro. Alkaline phosphatase/phosphate pair was an excellent system to
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show the efficacy of ADEPT due to the synthetic simplicity of the construction of the prodrugs
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(one-step condensation of drugs with phosphoryl chloride) as well as their inability to cross cell
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membranes. The limiting factor of this system is the high endogenous concentration of phosphatase

in healthy tissue and plasma, causing premature drug release and toxicity.[46]

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Viral thymidine kinase

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A highly promising strategy to EPT is to engineer

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prodrug bioconversion using enzymes of non-

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mammalian origin or those that have low
Scheme 6. Bioconversion of acyclovir into acyclovir
abundance in the human body. In such a case, non- phosphate performed by viral thymidine kinase.

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specific, systemic prodrug activation is suppressed to low levels and this enhances the site-specific
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nature of prodrug activation. This strategy was highly important for the development of ADEPT

and GDEPT. Examples of prodrugs used for this methodology are nucleoside analogues and
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specifically those that are good substrates for the viral thymidine kinase.[49] [26] The mechanism
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of antiviral activity of acyclovir and many other nucleoside analogue type drugs is that in their

triphosphorylated form, these drugs become substrates for nucleic acid polymerases. Once
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incorporated into the de novo synthesised chain of nucleic acid, these drugs become “chain
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terminators” by virtue of lacking the 3’-hydroxyl group for chain extension. Of the three steps of
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phosphorylation, the first step is the rate limiting. Utility of acyclovir as an antiviral drug is highly

facilitated by that the targeted pathogen, herpes simplex virus (HSV), uses its own, viral thymidine

kinase, which is brought into the cell upon viral infection. Viral thymidine kinase is markedly more

effective than the human analogue towards phosphorylation of acyclovir and this means that

acyclovir phosphorylation is significantly more effective within the virus-infected cells than in the

healthy ones. Correspondingly, concentration of acyclovir triphosphate in healthy cells during the

antiviral treatment is rather low and toxicity of antiviral treatment is also low. ADME

characteristics of acyclovir are challenging, specifically due to low aqueous solubility of this drug,

and a suit of successful prodrugs was developed to optimize the treatment against HSV, such as

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ester-type prodrug ganciclovir. [1] High level of activity of the viral thymidine kinase and the

existence of marketed (pro)drugs that require this enzyme for activation made this system a prime

candidate for the development of EPT and specifically the suicide gene therapy (GDEPT). [49] [26]

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Transduction of tumor cells can be achieved to instruct the cells to produce this kinase and upon

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administration of prodrugs of ganciclovir, toxicity of the corresponding triphosphate is specific to

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the transduced cells (with only a minor “bystander effect”, that is, toxicity to the non-transduced

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cells). GDEPT is currently undergoing clinical trials and we refer to current reviews on the subject

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for pharmacological details and clinical aspects of this EPT. [26]
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β-Lactamase
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Scheme 7.Drug recovery from the cephalosporin-based prodrugs as performed by β-lactamases with resulting liberation of the drug,
designated with LG for “leaving group”.
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Another class of non-mammalian enzymes with a high historical importance in the context of EPT

is bacterial lactamases. An aspect that contributed to the development of lactamase-based prodrug

therapy is that corresponding (pro)drugs, cephalosporins, are market-validated drugs with well-

established methods of their synthesis. ADEPT or EPT made great use of the old chemistry and

incorporated the cephalosporin platform into a prodrug strategy.[50] The synthesis of

cephalosporins and their analogues is well established and can be accomplished starting from a

marketed antibiotic drug, cephalotin (Scheme 7). This synthesis was accomplished for a variety of

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drugs, including doxorubicin[51], mitomycin[52], paclitaxel[53], and nitrogen-mustards.[54]

Incorporating the drug through either a carbamate or a carbonate bond to a cephalosporin was

shown to benefit the release kinetics.[51, 55] For a more in depths synthetic review about β-lactam

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prodrugs, the reader is referred to Jungheim et. al.’s review.[56] β-Lactamase specific prodrugs

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indicated that efficient release of drugs depends on the accessibility of the hydrolysis site for the

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enzyme and leaving group potential of the drug. The overall broad substrate acceptance of β-

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lactams enabled many research groups to develop various different prodrugs for ADEPT some of

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which showed good success.[52, 57-59] On the down side, toxicity ratio between drug and prodrug

(QIC50) is not as good as in the case of e.g. phosphate derivatives of drugs. Senter et al. showed that
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the toxicity ratio could be improved tremendously when incorporating a polar side group for R,

such as a sulfate- or carboxylic acid group.[52, 59]

D
TE

Nitroreductase
P
CE
AC

Scheme 8. Bioactivation of CB1954 into its active toxic form.

In the late 1960s,[60] various nitrogen-mustard analogues were synthesized and tested against

different cancer cell lines. Compound CB1954 received plenty of attention due to its impressive

toxicity profile in rat Walker 256 carcinoma cells documented both in vitro and in vivo.

Interestingly, this effect was not confirmed in human cancer cell lines. Analysis of this phenomenon

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revealed that mechanism of action of CB1954 involves an initial activation by a nitroreductase

enzyme into an extremely potent DNA-DNA double strand cross linking agent (Scheme 8). This

enzyme is far less active in healthy mammalian cells. [60] Delivering or expressing this protein at

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the nominated site of action would therefore lead to a site-specific prodrug activation, making

IP
nitroreductase a prime candidate for the use in EPT and specifically GDEPT.

R
SC
The scope of nitroreductase use in EPT became significantly broader with a realization that

nitrobenzyl alcohol linkers in their nitroreductase/prodrug pair effectively serve as a universal

NU
promoiety for diverse drugs such as doxorubicin, mitomycin C, and nitrogen mustards.[61] Key to

this platform is that reduction of the nitro group results in an oxime-containing molecule, which
MA
spontaneously undergoes 1,6-elimination and releases the attached drug molecule in its pristine

form (Scheme 8).The scope of this methodology[62] was expanded to nitrobenzyl-carbamate

D

containing prodrugs of enediynes[63], benzodiazepine, benzamide mustard[64], doxorubicin[65],

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campthothecin[66], nitrobenzyl-NO-releasing drugs[67], fluorescent turn-ON probes[68], and

P

oligonucleotides [69]. The attractive feature of nitrobenzyl alcohol-based prodrugs is the simplicity
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of their synthesis.[70] In general, a free-amine on the drug molecule is condensed with p-nitro-
AC

benzylchloroformate to yield the carbamate in high yields. The reaction requires the use of standard

solvents, such as dichloromethane, chloroform, THF, and DMF, and a mild base like triethylamine

or Hünigs base. The major drawback of these prodrugs is limited options to tune pharmacokinetics

of the prodrugs as well as modest value of QIC50.[61]

Scheme 9. Bio-activation of the nitrophenyl prodrugs by the nitroreductase with ensuing release of the drug molecule
(designated as “leaving group”, LG).

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Glucuronidase

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Glucuronide prodrugs (Scheme 10) and their

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corresponding enzyme, β-glucuronidase (β-Gluc),

R
appear to be the most suitable enzyme-prodrug

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Scheme 10. Bioconversion of the glucuronide-based prodrugs
pair for EPT. Glucuronides are natural metabolites into the corresponding drug molecule.

for many marketed drugs and their toxicity profile is well-documented.[71] Similarly to the

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phosphate-based prodrugs, glucuronides are highly polar and exhibit a minimal rate of cell

entry.[72] In stark contrast to phosphatases, β-Gluc is far less abundant in human body and is
MA
present in low concentrations intracellularly within lysosomes.[73] Largely due to this, toxicity

changes dramatically upon conversion of the prodrug to the parent therapeutic molecule.[74]
D

Bacterial copy of β-Gluc is markedly more active compared to the mammalian (human) enzyme.
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However, the use of endogenous β-Gluc can resolve the concerns regarding immunogenicity of
P

bacterial of fungal enzymes used in EPT.[75-78]

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Synthetically, glucuronide prodrugs and their precursors are more challenging to make as compared
AC

to the straightforward syntheses of e.g. phosphate or other glycoside prodrugs. One must

acknowledge the synthetic differences, such as reduced reactivity and increased tendency of side

reactions of glucuronic acid derivatives compared to its reduced counterpart glucose, the latter

being one of the most studied hexoses.[79-81]. Synthesis of glucuronides is a multi-step path with

several protection/deprotection steps as well as a glycosylation step. From our own experience and

based on Paulsen’s[82] and Stachulski’s[79, 80] seminal reviews, it seems fair to say that there are

no universal reaction conditions to synthesize a broad range of glucuronides and for each prodrug,

the synthesis is rather individual. Nevertheless, compared to the phosphate-, carboxypeptidase-, and

other enzyme-activated prodrugs, the major benefit of glucuronide prodrugs lies in the immensely

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decreased toxicity of the prodrug as compared to the released drug, with reported changes in IC50

being as high as 1000-fold.[83, 84]

T
IP
Other glycosidases

R
SC
Glycoside prodrugs relying on β-

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galactosidase/glucosidase (Scheme 11) have been

investigated heavily in the context of ADEPT,

MA
Scheme 11. Bioconversion of the glycoside-based prodrug
GDEPT as well as monotherapy.[85, 86] into the corresponding drug.

Similarly to β-Gluc, β-glycosidases reside exclusively within the cells providing stability of the
D

prodrugs within the blood stream. These enzymes are especially abundant within the liver, spleen,
TE

intestinal cells, and lymphocytes. Compared to glucuronides, glycosidic prodrugs based on glucose
P

and galactose have a significantly higher ability to enter mammalian cells. De Graaf et al. show in
CE

their cell uptake studies that the concentration of glucoside- and galactoside-prodrugs of

daunorubicin in the supernatant drops to around 75%, meaning a cellular uptake of roughly 25% of
AC

the prodrugs.[87] On the other hand, Haisma et al. show for glucuronide prodrugs of doxorubicin

and daunorubicin that there is no significant uptake.[88] Furthermore, glycoside prodrugs enable

selective tumor targeting due to the tumor’s overexpression of the insulin-independent glucose

transporters (GLUT-1). This overexpression can be tracked back to the faster proliferation, and

hence, higher energy demand of carcinogenic cells compared to healthy cells. This phenomenon

was first discovered and named by Warburg.[89-92] Tietze et al. did inspiring work in the field of

EPT with their work on glycosidase triggered prodrugs of duocarmycin derivatives.[93] Further

glycosylated prodrugs include doxorubicin[94-96], daunorubicin[87, 97], a mustard prodrug[98], 8-

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hydroxyquinoline and derivatives[99] benzodiazepins[100], NO-releasing prodrugs[101, 102], 5FU

prodrugs[103], and chloramphenicol[104].

T
R IP
Self immolative linkers

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Enzymatic conversion of prodrugs is critically dependent on accessibility of the scissile bond to the

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enzyme. This is easy to appreciate on an example of propofol (an anesthetic) and its two clinically
MA
tested prodrugs, a phosphate ester and a phosphonoxymethyl propofol Scheme 12 A. The two

prodrugs are similar in that in both cases drug release is initiated by the cleavage of a phosphoester

bond. However, the two molecules differ in that phosphonoxymethyl propofol has an additional
D
TE

oxymethylene spacer between the scissile phosphoester and the releasable drug. Bioconversion of

the phosphonoxymethylene prodrug proved to be markedly faster compared to the phosphate

P

prodrug – reflecting the steric hindrance effect exerted by two iso-propyl ortho-substituents (in the
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phosphate prodrug) which is overcome by placing the scissile bond to a position more accessible by
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the enzyme.[1] Another example underlining the importance of accessibility of the scissile bond is

Scheme 12. Illustrative examples of improving enzymatic hydrolysis of phosphate prodrugs.

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paclitaxel phosphate and paclitaxel-“trimethyl lock” (TML)-phosphate (Scheme 12B). Alkaline

phosphatase is unable to convert the phosphate prodrug into its pristine drug, whereas incorporation

of the TML spacer facilitates successful conversion.[105] These examples illustrate the concept and

T
the marketed use of the “self immolative linkers”, SIL (Scheme 13). Such linker is installed

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between the trigger and the deliverable drug and is designed such as to degrade spontaneously and

R
fast, and produce byproducts with acceptable safety profile. For diverse enzymes (glucuronidase,

SC
peptidases, esterases, etc) this concept has documented greatly enhanced rates of bioconversion of

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prodrugs.[106] SILs are already found in prodrugs successfully marketed worldwide, including

pivampicillin (oxymethylene spacer) and antibody-drug conjugate brentuximab vedotin (p-

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aminobenzyl alcohol spacer, PABA).
D
P TE
CE

Scheme 13. Schematic illustration of the concept of “self immolative linkers” as spacers placed between the promoity and
the conjugated drug whereby enzymatic or chemical transformation occurs at the distal end of the SIL with ensuing
spontaneous elimination or degradation of the SIL and release of the pristine drug.
AC

Historically, development of SILs can be traced back to the original work by Cain, relying on

spontaneous cyclization of a phthalide derivative upon activation [107] and the development of

more bioavailable ampicillin derivatives, relying on the incorporation of oxymethylene spacer[108].

Katzenellenbogen et al. developed the 1,6-elimination based PABA linker and coined the term “self

immolative connector” (Scheme 14 A)[109]. The field of prodrug design attracts significant

research attention with very recent examples covering diverse, innovative applications of the SIL

methodology [110, 111] including orally administered, triggered drug release formulations. [112]

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SIL systems have now also gained prominence in other branches of biomedical engineering and

materials science with utility in sensor technologies, self-immolative polymers, amplification

systems etc.[113, 114] It is also important to appreciate that SIL is an enabling technology and

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allows to temporarily install additional chemical diversity into drug molecules for greater

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opportunities in prodrug design. Good case in point is the thiol-containing SIL linkers that allow

R
using the highly advantageous intracellular thiol-disulfide reshuffling for bioconversion of prodrugs

SC
– applied to the drug molecules that are devoid of thiol functionality. [115-117]

NU
PABA linker as introduced in the early work of Katzenellenbogen et al. [109] remains among the

most widely applied SIL. Specifically for the development of glucuronide prodrugs (in our opinion,
MA
the most promising prodrug for EPT), incorporation of PABA facilitates the synthesis of the

prodrugs meaning that solving every single glycosylation of an aglycon separately becomes
D

irrelevant.[118, 119] Successful examples of glucuronide prodrugs employing SIL include

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doxorubicin[88, 120-122], histone deacetylase inhibitor MS-275[123], MMAE[124],

P

paclitaxel[125-128], camptothecin and its analogues[129-132]. Key consideration for design of

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these (and other) prodrugs is the “quality” of the leaving group, which facilitates or impedes the
AC

drug release (for good and bad leaving groups, respectively). The ability of a drug molecule to be

released from an SIL-linker can be closely, but not exclusively, related to its pKa value: the lower

the pKa, the better is the leaving group ability.[113, 133] Thus, phenolic alcohols can be recovered

from the PABA-containing glucuronide prodrugs whereas aliphatic alcohols and amines cannot

[134-136] [130]. The introduction of carbonate/carbamate groups is a neat tool to transform amines

and alcohols into better leaving groups because carbonic and carbamic acids are good leaving

groups but are unstable and immediately fragment liberating CO2 and the amine/alcohol

respectively. The liberation of CO2 makes the reaction irreversible as well. The introduction of

cyclization based SIL linkers is another approach to efficiently release aliphatic alcohols and

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amines (Scheme 14 C-E)[137]. To our knowledge, the first synthesis of cyclization based diamine

SIL linker was reported by Saari et al. (Scheme 14 E).[138] Two SIL can be installed to act in

sequence, as was applied for delivery of aliphatic alcohol containing drugs such as paclitaxel

T
(Scheme 14 C).[125, 139] TML is a successful SIL-linker established by Borchardt et al. [140].

IP
Inspired by intramolecular lactonization, Borchard et al. synthesized various compounds of the

R
general structure depicted in Scheme 13D and investigated their potential to release amine-

SC
functionalities upon removal of the trigger unit.[141] Independently, Berglund et al. demonstrated

NU
the intramolecular lactonization of quinone analogues upon reduction to the corresponding

hydroquinone derivatives, and highlighted the potential use in targeted drug delivery, nitroreductase
MA
was mentioned as a possible enzyme pair.[142] Subsequent papers showed the effective release of

amine containing cargo with esterase-, and redox-sensitive trigger units, which also was shown to
D

work for the PABA linker.[143-145] Phosphorylase as trigger was also utilized to show the
TE

potential release of peptidic cargo and amine containing analogue of combretastatin A4.[146, 147]
P
CE
AC

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T
R IP
SC
NU
MA
D
P TE
CE

Scheme 14: General illustration of the self-immolation process for the different SIL linker types; PG =protecting group (e.g.
glucuronic acid); LG = leaving group, that is, a drug molecule; R = variable; X = O or NH.
AC

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Long circulating prodrugs

Prodrug designs presented above are elaborate and

successful but are not without shortcomings. Specifically

T
IP
for the prodrugs with relevance to EPT and in particular

R
for glucuronides, a significant limitation lies in that these

SC
small, water-soluble molecules have a short half-life.
Short body residence time for prodrugs means that only a
Scheme 15. General structure of the maleimide-
containing prodrugs of doxorubicin designed to release
the drug upon prodrug bioconversion mediated by
proteases (left); structure of 5-FU prodrug by He et al.

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(right). Such prodrugs undergo covalent ligation to
minor fraction of the drug is recovered from the albumin resulting in a long circulating prodrug.

administered prodrug. It also means that to maintain the concentration of the active drug within the
MA
therapeutic window for extended times, such prodrugs would necessitate frequent drug

administration or even infusion. Potential methods to overcome this may be found in the existing
D

toolbox of biomedical engineering and specifically, using technologies behind therapeutic agents
TE

with extended blood residence time.

P
CE

Historically, highest success in creating long-circulating drug depots was achieved using tools of

nature and specifically albumin. [148-151] The latter is the most abundant protein in human plasma
AC

(with concentration around 50 g/L) and has a phenomenal blood residence time (half-life of 19

days). This is achieved through biological mechanisms of albumin recycling, specifically through

recognition with the neonatal FcRn receptor within acidified cellular sub-compartments upon

lysosome-endosomal trafficking and ensuing exocytosis of receptor-bound albumin. [148] Albumin

is used on the market with highest success to ensure extended circulation life-time of biological

drugs (insulin, glucagon-like peptides, Factor IX), for which purpose drug molecules are bound to

albumin through either covalent or non-covalent interactions.[152-154]

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R IP
SC
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Scheme 16. General structure of albumin-responsive glucuronide prodrugs; R = H in case of Doxorubicin; R = Me in case of
MMAE.
MA
In the field of anti-cancer drug delivery, albumin was used as a carrier for the conjugated drugs

(doxorubicin, MMAE, MMAF) whereby drug release was mediated by the lysosomal proteases.
D

Overall prodrug design followed the development path applied successfully to the antibody-drug
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conjugates [155] and made use of the valine-citrulline amino acid pair as an enzyme degradable

trigger for drug release and PABA to optimize release kinetics.[134] In the recent advanced designs,
P

prodrugs were engineered such as to undergo covalent ligation to albumin in vivo upon prodrug
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infusion – thus eliminating the necessity for albumin handling, sample purification etc. This
AC

approach is based on maleimide-containing prodrugs that react with the cysteine thiol of the

albumin. Kratz et al. are pioneers in this field and have synthesized various doxorubicin prodrugs

containing a maleimide moiety and a peptide linker (Scheme 15), which is cleaved specifically by

various overexpressed enzymes within the tumor, such as prostate specific antigen [156-158],

urokinase-type-plasminogen activator [159], and matrix metalloproteinase 2.[160] Other examples

designed on similar guidelines include prodrugs for doxorubicin [161, 162] and methotrexate.[163]

He et al. have synthesized an albumin-binding 5FU prodrug relying on ubiquitous esterases for

enzymatic release. Their targeting strategy is based on the passive accumulation of albumin in

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tumors through the EPR effect. They show enhanced tumor activity of the prodrug compared to the

pristine drug 5-FU (Scheme 15).[164]

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In an example most relevant to EPT, Legigan et al. developed albumin-carried, β-Gluc-responsive

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prodrug of doxorubicin (Scheme 16). Glucuronides have particularly short plasma half-lives and

R
would benefit greatly if successfully engineered to exhibit extended blood residence times. This was

SC
achieved using albumin as a carrier and an SIL linker – importance of which in this case extended

NU
beyond considerations of efficient drug release. The SIL linker contained a side functionality to

achieve covalent ligation of the prodrug to albumin. In vitro, the authors showed nearly identical
MA
IC50 values for the prodrug upon activation compared to the parent drug, doxorubicin. In vivo, the

prodrug showed superior activity compared to that of HMR-1826 (a known glucuronide prodrug of
D

doxorubicin unable to bind to albumin) and similar activity compared to doxorubicin, without
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showing any sign of weight loss and toxicity.[165] Most recently, the same group has published
P

results on the identical prodrug system, swapping doxorubicin with the highly potent MMAE. The
CE

group showed impressive results with their in vivo studies on various different model cancer types,

including mouth epidermal carcinoma, triple-negative breast cancer (TNBC), and pancreatic cancer.
AC

In all cases, the prodrug exhibited superior anti-cancer activity than the pristine drug, MMAE.

Moreover, the prodrug was well tolerated and in case of TNBC, 50% of mice showed complete

remission after day 70, and in case of mice with pancreatic cancer xenografts 33% of mice showed

complete remission. Worthy of note, prodrug conversion was achieved with no external tools of

EPT, that is, without targeting the expression of β-Gluc and relying only on the over-expression of

this enzyme within tumors. [166]

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Conclusions and Outlook

This review aimed to present the enzymes used to achieve bioconversion of prodrugs as well as the

T
considerations related to the synthesis and utility of prodrugs specific to these enzymes

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(summarized in Table 2), with a particular emphasis on enzyme-prodrug therapies. Current state of

R
the art in prodrug design already presents this field as highly successful, so many of marketed

SC
therapeutics being prodrugs. In contrast, from advent several decades ago, EPT has revealed minor

success in transition from lab to clinic and to our knowledge, there is no product on the market

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making use of this technology. We believe that knowledgeable, cunning design of prodrugs is key
MA
to the overall translational success of EPT. Specifically, development of long-lasting prodrugs is

particularly important for EPT in that this methodology relies on the external administration of
D

prodrugs, short blood residence times of the prodrugs limiting success of EPT. One documented
TE

successful way to overcome this is the association of prodrugs with albumin to ensure extended

plasma residence times of the prodrugs, that is – development of long-circulating prodrug depots.
P

Further efforts are now needed to engineer long-circulating prodrug design into an overall
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methodology of EPT. From a different perspective, in our view, recent surge of interest into “self
AC

immolative linkers” is highly important and fruitful and provides enabling methodologies and

chemical diversity to the conjugation techniques. SILs simplify prodrug synthesis through making it

modular and are powerful to optimize drug release specificity and kinetics. It would take much

more than just successful prodrugs to make EPT a clinical success, yet we believe that cunning

design of prodrugs with the understanding of hurdles that are specific to EPT is key to the overall

success of this methodology.

Acknowledgments

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This work was financially supported by the Lundbeck Foundation (grants R108-2012-10354 and

R164-2013-15291), the European Research Council Consolidator grant (ANZ, ERC-2013-CoG

617336 BTVI) and the Academy of Finland (#308329).

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R IP
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D
P TE
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Table 2. Comparative analysis of prodrugs with regards to their pharmacodynamics and pharmacokinetic properties. QIC50 is a measure of fold-change in toxicity between the prodrug
and the corresponding drug (QIC50 = IC50 prodrug / (IC50 free drug or prodrug in presence of the enzyme). Values of QIC50 are typically determined in vitro and therefore do not take into
account the biodistribution of the enzyme in vivo, and thus do not reflect the prodrug safety observed in vivo. Abbreviations: antibody-directed enzyme prodrug therapy (ADEPT), gene-
directed enzyme prodrug therapy (GDEPT), bacteria-directed enzyme prodrug therapy (BDEPT), substrate-mediated enzyme prodrug therapy (SMEPT).

T
IP
Prodrug Type Intra- or extra- cellular Administration
(corresponding A : activation; activation (I or E, QIC50 (O : oral ; P : Application and selected publications

CR
enzyme) R : release respectively) parenteral)

US
General prodrug design (improve oral
Esters (Esterases) R I, E n/a O
bioavailability)

N
P

MA
Phosphoesters ~100 ( O: if General prodrug design [1]
(Phosphoesterase, R E
[167, 168] further ADEPT [167, 168]
phospholipase)
masked)

D
TE
Nucleoside
a) General prodrug design [1];
analogues A I n/a O
b) GDEPT [49] [26]
(kinase)

P
Nitroreductase
A I, E
CE20-100
10-75
[61]
[63] P
ADEPT[61]
BDEPT [169]
substrates
AC
5-200 [65] GDEPT [63, 65]

<5 [50]
Lactams
R E 10-22 [52] P
(Lactamase) ADEPT[50, 52, 53, 56]
<20 [53]

ADEPT [125]
glucuronides >100-1000
R E P SMEPT [24]
(glucuronidase) [83, 84, 131, 165, 166]
Monotherapy [124, 131, 165, 166]

Glycosides >1000-10000 ADEPT[48, 83, 85, 94, 170]

R I P
(glycosidase) [48, 83, 85, 170] monotherapy [171]

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IP
CR
N US
MA
D
PTE
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