Strategies in the designing of prodrugs, taking into account the antiviral and anticancer compounds

Accepted Manuscript Strategies in the designing of prodrugs, taking into account the antiviral and anticancer compounds Monika A. Lesniewska-Kowiel, Izabela Muszalska PII:

S0223-5234(17)30076-4

DOI:

10.1016/j.ejmech.2017.02.011

Reference:

EJMECH 9207

To appear in:

European Journal of Medicinal Chemistry

Received Date: 9 November 2016 Revised Date:

13 January 2017

Accepted Date: 5 February 2017

Please cite this article as: M.A. Lesniewska-Kowiel, I. Muszalska, Strategies in the designing of prodrugs, taking into account the antiviral and anticancer compounds, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Strategies in the designing of prodrugs, taking into account the antiviral and anticancer

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compounds

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Monika A. Lesniewska-Kowiel, Izabela Muszalska*

5 Poznan University of Medical Sciences, Faculty of Pharmacy, Department of Pharmaceutical

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Chemistry, Grunwaldzka Str. 6, 60-780 Poznań, Poland

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8 9 ABSTRACT

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Prodrugs are a wide group of substances of low or no pharmacological activity. The search for

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prodrugs is aimed at obtaining drugs characterized by better pharmacokinetic properties,

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pharmaceutical availability and selective activity of the active substance. Prodrug strategies

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involve chemical modifications and syntheses of new structures as well as the establishment

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of systems that deliver active substances for therapeutic aims that is prodrug-based treatments.

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The paper describes decisive factors in prodrug designing, such as enzymes participating in

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their activation, concepts of chemical modifications in the group of antiviral drugs and new

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anticancer treatments based on prodrugs (ADEPT, GDEPT, LEAPT). Prodrugs are seen as a

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possibility to design medicines which are selective for their therapeutic aim, for example a

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tumorous cell or a microorganism. Such an approach is possible thanks to the knowledge on:

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pathogenesis of diseases at molecular level, metabolism of healthy and affected cells as well

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as metabolism of microorganisms (bacteria, fungi, protozoa, etc.). Many drugs which have

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been used for years are still studied in relation to their metabolism and their molecular

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mechanism of operation, providing new knowledge on active substances. Many of them meet

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the criteria of being a prodrug. The paper indicates methods of discovering new structures or

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modifications of known structures and their synthesis as well as new therapeutic strategies

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using prodrugs, which are expected to be successful and to broaden the knowledge on what is

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happening to the drug in the body, in addition to providing a molecular explanation of

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xenobiotics activity.

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Keywords: Prodrugs, enzymes, chemical modifications, ADEPT, GDEPT

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*Corresponding author. E-mail address: [email protected] 1

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1. Introduction

2 Prodrugs are substances of a low or non-existent biological activity. They become active

4

by releasing active substances through chemical or enzymatic transformations occurring in the

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body [1-4]. The release of the active substance from the prodrug occurs before, during or after

6

its absorption, sometimes even after it reaches the target [1, 2]. The term “prodrug” was used

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for the first time by Adrien Albert in 1958 [5]. However, the idea of prodrugs was created a

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long time before that. Probably the first deliberately designed prodrug was methenamine,

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which was introduced to pharmacies in 1899 [6, 7]. The first substance which complied with

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the criteria of being a prodrug was acetanilide. It has been used since 1867 as an anti-

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inflammatory drug. However, it was the discovery that its activity is caused by acetaminophen

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created as a result of aromatic ring hydroxylation that resulted in acetanilide being classified

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as a prodrug [8].

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A constantly growing interest in obtaining and applying prodrugs has been observed

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since 1960s. It has been estimated that around 10% of the medicines worldwide available are

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prodrugs, while in 2008 they constituted 1/3 of all registered drugs with a small molecular

17

mass [9].

The aim of designing prodrugs is to optimize the properties of compounds which have the

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required pharmacological effect, but cause problems in further development of the drug.

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There are three basic aims related to obtaining prodrugs, which frequently overlap each other:

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pharmaceutical – decreasing inconveniences in the form of the drug by improving its solubility, chemical stability, organoleptic properties (taste, smell) and decreasing the

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irritation and pain it causes after it is administered locally •

pharmacokinetic – improvement of ADME properties (absorption, distribution,

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metabolism, excretion) involving, among other things, better absorption (in case of oral

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use, but also other forms of administration), limitation of the drug metabolism until it

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reaches its target, increasing the selectiveness of carrying the drug to its effector site,

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modification of the method of crossing the blood-brain barrier and improving the drug life

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time

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•

pharmacodynamical – decreasing its toxicity, improving the therapeutic index, creating

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drugs with two active substances (the co-drugs strategy) [1, 8, 10].

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The majority of the prodrugs currently used are the so-called carrier-linked prodrugs (Fig.

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1a). These are compounds obtained as a result of a simple modification to the functional

34

group of an active substance made by creating ester, amid, carbonate, carbamate, oxime, 2

ACCEPTED MANUSCRIPT phosphate, N-Mannich base, imine or PEG (polyethylene glycol) conjugate [8, 11]. In the

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body, the prodrug is subjected to transformation by removal of the carrier which realizes the

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active substance. It is necessary to choose an appropriate carrier, which will secure the active

4

substance, last during drug storage and administration, and once it releases the active

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compound, the carrier should be subject to biodegradation, be decomposed into non-active

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metabolites and quickly excreted from the body. An ideal carrier should be non-expensive,

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easy to obtain and have no immunogenic properties [1, 8, 12, 13].

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Carriers linked prodrugs can be divided into bipartite ones, where the carrier is directly

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attached to the active substance, and tripartite drugs, where the carrier is linked to the drug via

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linker. In addition, there are also co-drugs (mutual drugs) created by the combination of two

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active substances, acting as carriers to one another [8, 11]. The combination of L-DOPA and

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entacapone in a form of carbamate is an example of a co-drug. It increases the efficiency of

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delivering dopamine to the brain. Another example is the L-ascorbic and retinoic acid ester,

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thanks to which the skin absorption of both drugs is increased [14]. Another type of

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substances which require activation in the body is bio precursors (Fig. 1b). Those compounds

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do not include a carrier, and their structure is different than that of an active substance. Due to

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that, activation of bioprecursors is not based on a simple removal of the functional group, but

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rather on transformation into another compound, usually via oxidation or reduction. As a

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result of the reaction, a biologically active substance is created or it is further transformed into

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an active metabolite [8, 11, 15]. Bioprecursors include, among other things, dexpanthenol,

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sulindac and nabumetone [2, 8, 16].

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The knowledge on the biotransformation processes has contributed to the discovery of

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new medicines. Many drugs are transformed into active metabolites inside the body. They

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frequently have a better safety profile than their parent substance and they can become drugs

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by themselves. The best example of such a case if acetaminophen, which is a metabolite of

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phenacetin. In comparison to the original substance, it shows better painkilling properties and

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it does not cause methaemoglobinaemia or haemolytic anaemia [15, 17].

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What is significant for the effectiveness of both prodrugs and their bio precursors is the

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speed of biotransformation into an active substance after the drug reaches the effector site

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(speed constant kbio). It has to be quicker than the speed of elimination of the unchanged

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prodrug (speed constant kel1) and the speed of elimination of the active substance (speed

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constant kel2). Only then can the biologically active substance obtain a higher concentration

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than the threshold (Fig. 1).

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ACCEPTED MANUSCRIPT An alternative method of obtaining prodrugs is the intramolecular chemical approach in

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which the designing is made on the basis of calculations with the use of molecular orbital

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(MO) methods, molecular mechanics (MM) and correlation between values obtained as a

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result of an experience and those obtained from calculations. In this method, no enzyme takes

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part in the conversion of the prodrug into the parent substance. Interconversion of the prodrug

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is controlled only at the stage of limiting the speed of intramolecular reaction [11].

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2. Discussion

9 2.1. Enzymes activating prodrugs

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In accordance with the definition, a prodrug is a non-active medicine. Because of that, its

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activation in the body is of a key important when obtaining a desired pharmacological effect.

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This activation may be due to their chemical susceptibility to degradation leading to the

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formation of a pharmacologically active substance (e.g. influence of pH) or enzymatic

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metabolism. The majority of prodrugs are subject to enzymatic activation, usually with the

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participation of hydrolases or P450 cytochrome enzymes [1, 18]. It is necessary to remember

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that the activation of prodrugs is subject to individual differences. The causes of such

19

differences result from genetic polymorphism, the influence of medications and xenobiotics

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taken at the same time, age, sex and co-existing diseases [19]. The aforementioned factors

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impede the ability to foresee the degree and speed of the prodrug conversion into its active

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form. There are many interspecific differences in enzymatic activation which makes it

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impossible to foresee what will happen to the prodrug in the human body based on animal

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observations [1].

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When designing a prodrug, it is necessary to ensure that the structural modification

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introduced not only has a beneficial influence on the active substance’s pharmacokinetic and

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pharmacodynamical parameters but also makes it possible for the prodrug to be activated by

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an appropriate enzyme. Usually, in cases of systematically active parent drugs, attempts are

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made so that the prodrug is a substrate of generally available hydrolases which accept various

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substances, such as peptidase, phosphatase and especially, esterase.

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2.1.1. Esterase and paraoxonase

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ACCEPTED MANUSCRIPT Many prodrugs have an ester group in their structure. These include, among others,

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penicillin (bacampicillin, pivampicillin), cephalosporin (cefuroxime acetyl, cefatamet

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pivoxil), macrolides (erythromycin cyclic carbonate), vitamins (retinol acetate, α-tocopherol

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acetate), β-adrenolytic (timolol benzoate) and β-adrenergic (ibuterol, bambuterol) drugs. The

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removal of an ester bond is usually done by the participation of esterases which generally

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occur in the body, such as carboxylesterase (CES), acetylcholinesterase (AChE),

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butyrylcholinesterase (BChE), paraoxonase (PON) and arylesterase. The decay of an ester

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bond is also possible to obtain by oxidation catalysed by P450 cytochrome enzymes [18].

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Carboxylesterase, acetylcholinesterase and butyrylcholinesterase belong to α/β-

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hydrolases. They have a similar construction and activity mechanism. As serine must be

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present in order for them to become active, they are sometimes referred to as serine esterases.

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Reactions catalysed by them use the so-called catalytic triad composed of amino acid residues

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of serine, glutamic acid and histidine [20, 21]. Those enzymes are independent from any

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cofactors, such as nonorganic ions, but they are inhibited by organophosphates. It is estimated

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that 50% of currently used prodrugs are metabolised by this group of enzymes [8].

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Carboxylesterases play a significant role in the metabolism of xenobiotics and

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endogenous substances (for example palmitoyl-CoA). They show a wide substrate specificity

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and, apart from carboxyl acid esters, they also hydrolyse, for example, thioesters. In addition,

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they show activity of aryl esterases, acetyl esterases, amidases and lipases, and they take part

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in the transesterification processes [18]. As carboxylesterases are located in the whole body, it

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is thought that the probability of their saturation and the possibility of interaction with

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substrates with other drugs are low [1, 18]. The majority of carboxylesterases belong to one of

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the two families: CES1 or CES2. In people, the level of CES1 group enzymes present is very

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high in the liver. However, they are also present in other tissues, with the exception of

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intestines. The expression of CES2 carboxylesterases is significantly lower and occurs mainly

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in kidneys and intestines [22]. Both groups are characterized by various substrates. CES1

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prefers hydrolysis of esters with a big acyl and small alcohol group, for example temocapril,

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while the CES2 group is more efficient in hydrolysis of esters with a small acyl group and a

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greater alcohol component, such as irinotecan [23]. Thanks to the aforementioned differences

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in the location and the substrate specificity, the use of carboxylesterases can be useful during

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prodrug designing, especially in the case of ester and amid prodrugs. The prodrugs activated

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by carboxylesterases include, among others, capecitabine [24], 2-ethyl carbonate of paclitaxel

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[25] and ester prodrugs of propranolol [26].

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term

cholinesterase

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both

acetylcholinesterase

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butyrylcholinesterase, often referred to as pseudocholinesterase. Those enzymes differ in their

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substrate specificity and some of their inhibitors. Acetylcholinesterase shows high catalytic

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activity and is one of the fastest enzymes. Its main substrate is acetylcholine, which

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decomposes in cholinergic and neuromuscular synapses. In addition, AChE is present in the

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bloodstream in the area of erythrocyte cell membranes. Its substrates include also chosen

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esters, amides and anilides. AChE also takes part in the activation of such prodrugs [18, 27] as

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propanol esters [26], acyclovir (ACV) [28] and dipivefrin hydrochloride [29]. Contrary to

9

carboxylesterases, this enzyme shows a high substrate specificity. It probably results from the

10

difference in the construction of the active enzyme site [21]. During the last few years, it has

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been suggested that acetylcholinesterase plays a significant role in many biological processes.

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Studies involve, for example, the relation between concentrations of AChE and paraoxonase

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in the serum and anxiety [30]. Other research suggests a relation between acetylcholinesterase

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and atherosclerosis [31].

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Butyrylcholinesterase shows high catalytic activity too, however, it is selective for

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butyrylcholine and propionyl choline. It has a wider substrate specificity than AChE. It is

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mainly produced by the liver and its highest concentration can be found in the plasma. BChE

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hydrolyses many esters and because of that, it is engaged in processes of xenobiotic

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detoxification, playing an important role in the metabolism of, for example, local anesthetics

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[32], succinylcholine [33], aspirin [34] and heroin [35]. In addition, it takes part in the

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activation of such prodrugs as propranolol esters [26], bambuterol [36], methylprednisolone

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acetate [37] and isosorbide diaspirinate [38].

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Paraoxonases belong to another important group of enzymes. They act via a different

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mechanism than the aforementioned enzymes as their activity depends on calcium ions. Three

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enzymes from this group, PON1, PON2 and PON3, catalyse the hydrolysis reaction of a wide

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spectrum of compounds, such as aromatic esters of carboxyl acids, lactones, cyclical

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carbonates, organic phosphates and phosphonates [18]. The PON1 enzyme is mainly

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produced by the liver and it is present in the blood and the liver’s microsomal fraction. PON3

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is present mainly in the liver and in the serum (in a lower amount), while PON2 seems to be

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located in all tissues, with the exception of the plasma. PON1 plays an important role in

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activation of a widely used antiplatelet drug – clopidogrel (Fig. 2). It is responsible for the

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second stage of this prodrug transformation into its active form. At the same time, the activity

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of PON1 is strongly dependent on its gene polymorphism, which has been found to have

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almost 200 varieties. That explains why there have been various levels of patients’ response to

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ACCEPTED MANUSCRIPT clopidrogrel treatment [39-41]. The remaining paraoxonases also show a significant level of

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polymorphism. Other prodrugs subject to activation under the influence of paraoxonases are:

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prulifloxacin [42, 43], simvastatin and lovastatin [42]. This group of enzymes shows

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antioxidant activity and thus it seems to play a significant role in detoxification and

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prevention of atherosclerosis and cardiovascular diseases [18]. The activity of paraoxonases is

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affected not only by genetic factors, but also by environmental ones. Some drugs (e.g.

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phenobarbital), alcohol and mercury compounds inhibit PON’s activity, while cigarette smoke

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increases it.

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Taking into account, the wide substrate specificity of enzymes it becomes obvious that

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esterases take part not just in the decomposition of substances of an ester structure. Blood

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serum albumins are present in the plasma and extracellular fluids. Their main role is to bind

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and transport numerous substrates in the blood. They also show catalytic activity and take part

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in prodrug activation [44]. Human serum albumin (HSA) can be useful in prodrug activation

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because it can convert an inactive prodrug to an active drug by hydrolysis with or without its

15

modification and with or without binding the prodrug to the protein. Serum albumin has

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esterase activity as a result of the presence Tyr411 or Lys199, and thioesterase activity due to

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the presence Cys34 [45]. Their activity is, however, much lower than that of typical esterase.

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Among the other activities of HSA are glucuronidase, phosphatase, amidase, isomerase and

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dehydration properties [44, 45]. For nicotine acid esters, the constants of the speed of

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hydrolysis of reactions catalysed by albumins were 4,000 – 900,000 times less than that of the

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reactions catalysed by carboxylesterases [44]. HSA may play a role in the in vivo conversion

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of sulfenamide prodrugs to their active form as shown by the example of the sulfenamide

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prodrugs of linezolid: N-(phenylthio)-linezolid and N-((2-ethoxycarbonyl)-ethylthio)-

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linezolid [46]. Apart from albumins, the esterase activity is also shown by: carboxypeptidase

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A [47], aldehyde dehydrogenase [48], carbonate anhydrases B and C [49], trypsin [50] and

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lipase [51]. This should be taken into consideration when designing prodrugs of an ester

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structure.

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2.1.2. P450 cytochrome enzymes

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The second method of activation of prodrugs in the body is based on the activity of P450

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cytochrome (CYP). It is estimated that the P450 cytochrome enzymes family takes part in

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75% of all enzymatic reactions during metabolism of medicines and prodrugs. There is a lot

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of evidence that genetic polymorphism of P450 cytochrome contributes to variability in the 7

ACCEPTED MANUSCRIPT activation of prodrugs, and thus to different efficiency and safety of using prodrugs activated

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in this way [1, 52, 53]. Isoenzymes which are characterized by polymorphism are: CYP2A6,

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CYP2B6, CYP2C9, CYP2C19, and especially CYP2D6 (75 alleles are present within its

4

area). Some of CYP2D6 alleles determine the decreased activity or even the loss of the

5

enzymatic function [54]. Prodrugs which are subject to activation with the participation of

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P450 cytochrome enzymes include, among others, losartan activated via CYP2C9 [51],

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lovastatin and simvastatin – CYP3A4 [56], clopidogrel – CYP1A2, CYP2B6, CYP2C9,

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CYP2C19, CYP3A4 (Fig. 2) [57-60], codeine and tramadol – CYP2D6 [61],

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cyclophosphamide – CYP2B6, CYP3A4, CYP2C19 [18, 62], tegafur – CYP2A6, CYP1A2, CYP2C8 [62] and tamoxifen – CYP2D6, CYP3A4 [18, 62, 63].

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2.1.3. Enzymes and prodrug designing

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When designing prodrugs, it is necessary to take into consideration the fact that the

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aforementioned difficulties exist when applying data obtained from studies on animals to the

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studies on the effects on humans. Rodents usually show a higher activity of plasma hydrolases

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than humans, while the hydrolases activity in the small intestine of dogs is significantly lower

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[64]. Carboxylesterases are present both in human and rat brains. However, only in the case of

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people they are included in the brain-blood barrier and play a role in limiting the permeation

20

of membrane cells by substances [18, 65]. In the majority of mammals, PON1 paraoxonase is

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present in all tissues, while in the case of humans, it can be found only in the blood and liver.

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The differences in enzymes’ tissue expression have to be taken into consideration as well as

23

the fact that esterases are frequently present in the body in various forms. AChE and BChE

24

cholinesterases take the following forms:

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type I – amphiphilic dimers

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type II – amphiphilic monomers and dimers

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type III – tetramers with hydrophobic residues

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type IV – forms with collagen-like residues and asymmetric forms

29

•

type V – tetrameric soluble forms [66, 67].

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Monomeric and tetrameric forms dominate in the brain, and their activity is related to the

31

Alzheimer’s syndrome [18]. Thirty types of carboxylesterases were distinguished in the

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human brain, while five were found in the liver. It has been observed that cyclic carbonates

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and glycocorticosteroids γ-lactones, which are quickly inactivated in the human plasma, have

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ACCEPTED MANUSCRIPT a limited systemic exposure and adverse side effects, show a satisfactory stability in the lung

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tissue [18, 68]. Apart from that, in the case of cancer, various expressions of, for example,

3

carboxylesterases between the area of the purse seines cancer tissue and adjacent healthy

4

tissues, have been observed [69]. The differences in the interspecific expression can be

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limited with the use of recombined enzymes or human tissue extracts, while the knowledge on

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the differences in the enzyme activity in cancers can be used to create selectively active

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anticancer drugs.

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In peptide prodrugs designing it is important to achieve the solid form of prodrug and the

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possibility to optimize the therapy i.e. the possibility of their application once a day or once a

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week. Such possibilities can give the synthesis of conjugates type of polyethylene glycol

11

(PEG)-linker-drug (peptide/protein). In this type of activation of prodrugs involving proteases

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and the linker sequence the therapeutic effect can be optimized for a given peptide by release

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a tracer and free peptide with half-life times from 1 to 42 h [70].

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However, there is an issue related to the use of prodrugs which relates to esterase

15

substrates caused by their hydrolysis happening too early. It usually occurs when the prodrugs

16

are absorbed in the enterocytes of the digestion system. If the active substance, which is

17

usually more polar and has more difficulty in penetrating biological membranes, is released in

18

the enterocytes, its ability to enter the bloodstream is limited. This can result in a lower

19

bioavailability of some prodrugs, for example, those from the group of cephalosporins or

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inhibitors of the enzyme converting angiotensin [71].

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2.2. Prodrugs in antiviral therapy

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2.2.1. Phosphates (phosphonates) as nucleoside prodrugs

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Synthetic nucleosides are an essential part of both chemotherapy and treatment for

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diseases induced by a viral infection. However, in the majority of cases, nucleosides

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themselves are inactive and require transformation to bioactive nucleotides – triphosphates of

29

nucleosides which bind natural oligonucleotides (RNA, DNA) leading the inhibition of

30

uncontrolled cell proliferation or virus replication. Monophosphorylation is a stage limiting

31

the creation of an active triphosphate, hence the concept of creating prodrugs which contain

32

phosphate groups in their structure. Enzymatically and chemically stable phosphonate

33

analogues, which imitate monophosphates of nucleosides and allow elimination of

34

preliminary enzymatic phosphorylation, can potentially be more effective antivirals. A 9

ACCEPTED MANUSCRIPT limitation in designing such structures intended for oral administration is the necessity of

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blocking the possibility of deproteinization of an active phosphonate group in a physiological

3

pH environment. Their ionizing capacity is the cause of new bioavailability as a result of

4

inability to overcome the mucous membrane barrier and mucosa in the gastrointestinal tract.

5

Meanwhile the possibility of oral administration is very important in the therapy of chronic

6

diseases. Thus, the development of the structure should be characterized by suitable stability

7

in an environment with the pH value of 2, 8.5-11 and in the serum. Hence, numerous

8

developments of both aliphatic (ANP) and cyclic (CNP) monophosphonates of nucleosides

9

[72, 73]. For aliphatic structure, the heterocyclic principle is connected to the lateral aliphatic

10

chain which contains the phosphonomethyl residue. The methylene bridge between the

11

phosphonate acid group and the rest of the molecule exclude dephosphorylation and ensures

12

enzymatic stability. The lack of a glycosidic bond, on the other hand, additionally increases

13

resistance to chemical and biological degradation. The elasticity of the acyclic chain allows

14

the adoption of appropriate conformation which makes it possible to bind the molecule with

15

active places of various target enzymes involved in DNA biosynthesis (DNA polymerase of

16

DNA viruses, reverse transcyptase of retroviruses) [73]. Esterification increases lipophilicity

17

of the molecule and promotes an increase in its bioavailability. Among numerous candidates

18

for drugs, adefovir dipivoxyl (reverse transcriptase inhibitor; HBV – Hepatitis B Virus, HSV

19

– Herpes Simplex Virus, HIV – Human Immunodeficiency Virus therapy) and tenofovir

20

disoproxyl fumaran (TDF, reverse transcriptase inhibitor; HIV, HBV therapy) (Fig. 3) [74,

21

75]. Attention should also be paid to tenofovir prodrugs (TFV) active towards HIV-1 and

22

HBV: hexadecyloxy-tenofovir and hexadecyloxypropyl cidofovir which are metabolised with

23

the participation of phospholipase C and isopropylalaninyl monoamidate-tenofovir, tenofovir

24

alfenamide (TAF) hydrolysed under the influence of cathepsin (CatA) (Fig. 3) [74-76]. TAF

25

is approx. 10 times more active towards HIV-1 than TDF, which allows the reduction of dose

26

to achieve therapeutic concentrations of TFV. The use of TDF has a disadvantageous effect

27

on the kidney function and bone mineralization. Such effects were not observed for TAF. As

28

TAF causes fewer adverse effects it was recommended for HIV combined therapy [76, 77].

29

However, it should be taken into account that pivaloyl acid and formaldehyde belong to

30

products of dipivoxyl adefovir metabolism, which may influence their toxicity (Fig. 4) [73].

31

The cidofovir molecule (in the form of acyclic and cyclic monophosphonate) was also

32

subjected to a N4-acylation reaction using the reactivity of a small amine group in position C4

33

of the cytosine ring (Fig. 3). It was shown that the N4-behenoyl derivative is 20-30 times more

34

active toward HCMV (Human Cytomegalovirus) than acyclic cidofovir monophosphonate.

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ACCEPTED MANUSCRIPT 1

This activity increased only 2-5 times towards HSV and VZV (Varicella Zoster Virus). While

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additional esterification of the phosphonate residue and formation of monopivaloyl weakens

3

antiviral activity [78]. Other most significant examples of phosphate formation as nucleoside prodrugs include

5

the so-called ProTides (phosphoramides, esters of amino acid bonded with nucleoside by an

6

N-P aryl phosphonate bond), SATE (S-acylthioethyl phosphates), HepDirect (cyclic 1-aryl-

7

1,3-propanyl phosphates) and cycloSal (salicylic phosphates) (Fig. 5) susceptible to

8

bioactivation under the influence of esterases or in the case of HepDirect with the

9

participation of CYP 450 enzymes [72, 73, 79-83].

RI PT

4

ProTides can be defined as a structure of isopropyl alaninyl of tenofovir-monoamide and

11

sofosbuvir (Fig. 3) which shows activity against HCV (Hepatitis C Virus). Sofosbuvir is

12

bioactivated under the influence of cathepsin A whose high expression takes place in

13

hepatocytes ensuring a proper activation of active metabolite in the liver [74]. The

14

introduction of the amid phosphate group as a modification of (-)-β-D-(2R, 4R)-1,3-dioxolan

15

adenosine nucleosides (Fig. 3) in the ProTides, caused an increase in the activity of anit-HIV-

16

1 and up to 300-times increase in anti-HBV as compared to these nucleosides. At the same

17

time, a considerable decrease in cytotoxicity is observed [84]. An intensive search for

18

compounds that are active towards the Ebola virus led to synthesis of adenosine analogue GS-

19

5734, prodrug from the monophosphoramides (ProTides) (Fig. 6). Tests of effectiveness of

20

the therapy conducted on primates encouraged scientists to undertake pharmacokinetic and

21

clinical tests [85].

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10

Another approach in designing prodrugs is an introduction of cyclic disulphide into the

23

SATE structure which would force bioactivity of such a structure with the participation of

24

cytosol glutathione (GSH) ensuring greater plasma stability as compared to thioesters (SATE)

25

or ProTides [72]. The introduction of the phosphoramide group, on the other hand, (Fig. 5)

26

into the structure of dideoxyadenosine, abacavir or acyclovir influences the increase of their

27

antiviral activity towards HIV-1 and -2, and in the case of stavudine, inhibition of cancer cell

28

activity was observed. Such connections are bioactivated with the participation of

29

carboxypeptdase Y [86].

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30

In the case of topical or percutaneous administration, a two-directional procedure is

31

advantageous: using DDAK (dodecyl ester of 6-(dimethylamino)hexanoic acid; (CH2)2N-

32

(CH2)5COOC12H25) which increases the penetration of the antivirally active substance 2-11

33

times e.g. acyclic phosphonate nucleosides of 2,6-diaminopurine derivatives, which allows

34

their percutaneous activation; or chemical modification with the formation of their esters e.g. 11

ACCEPTED MANUSCRIPT 1

hexadecyloxypropyl ((CH2)3OC16H33) the so-called lysolipid-like prodrugs, which protect

2

against systemic absorption and can be used in topical therapy [87]. The concept of developing nucleoside triphosphates (NTP) also seems interesting [83,

4

88]. For a thymidine analogue – 3’-deoxy-2’,3’-didehydrothymidine (d4T), highly lipophilic

5

acyl residues (Fig. 7) lead to an increase in mucous membrane permeability which causes an

6

intracellular increase in metabolite concentrations and antiviral activity as compared to the

7

primary nucleoside, which was shown in cellular extracts of human CD4+ lymphocytes T.

8

These derivatives are characterized by high selectivity and are high inhibitors of HIV-1 and

9

HIV-2 [88]. NTPs give hope for the acquisition of a range of new structures with antiviral activity targeted at inhibiting activity of HCV polymerase [83].

SC

10 11 12

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3

2.2.2. Prodrugs activated with the participation of oxidase and dipeptidyl peptidase

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6-deoxy-2-aminopurine derivatives (e.g. famciclovir), which are deprived of antiviral

15

activity, are characterized by better bioavailability and increased absorption after oral

16

administration, which promotes their synthesis as prodrugs activated with the participation of

17

xanthine oxidase or aldehyde oxidase. In this way, 6-deoxycyclopropavir (a potential prodrug)

18

undergoes enzymatic oxidation to cyclopropavir, which exhibits higher activity towards

19

HCMV than gancyclovir (Fig. 8) [89].

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The possibility of creating peptide conjugates for which good solubility in water

21

environment is an advantage as it increases pharmaceutical bioavailability after oral

22

administration. For acyclovir (ACV), a new approach is attempts at creating amid connections

23

with amino acids using reactivity of the amine group in position C5 ACV (Fig. 9). Such

24

potential prodrugs are bioactivated under the influence of dipeptidyl peptidase IV (DPP IV

25

which is also called CD26) as opposed to esters which hydrolyse to ACV under the influence

26

of esterases. A comparison of enzymatic stability (DPP IV) and activity towards HSV-1 and -

27

2, showed that the amid bond with the following tetrapeptide structure Val-Pro-Val-Pro-ACV

28

(Fig. 9) is bioactivated to ACV under the influence of DPP IV. While the ester bond with the

29

following tripepeptide structure Val-Pro-Val-ACV (Fig. 9) is hydrolysed to valacyclovir

30

(VACV) under the influence of DPP IV. Both structures are more stable in a phosphate buffer

31

solution (PBS) than VACV and their activity towards HSV-1 and -2 is 2-10 times higher than

32

ACV, which results from an increase in the availability of the primary compound (ACV) [90].

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2.2.3. Prodrugs designed for therapy of eye diseases 12

ACCEPTED MANUSCRIPT 1 For drugs used in the therapy of viral eye diseases, the possibility of producing

3

conjugates increasing the permeability through biological membranes and constituting a

4

substrate for membrane transporters is considered. Such a solution can increase the chances

5

for the achievement of therapeutic concentrations in eye structures after topical

6

administration. Transporters which take part in the transfer of molecules through biological

7

membranes include peptides/histidine, organic cation transporters, folic acid, amino acid

8

transporters and sodium-dependent multivitamin transporter (SMVT). SMVT occurs in

9

human retinal epithelial cells and takes part in vitamin transport, including biotin, which was

10

used in the development of a new class of prodrugs. For this reason, the usefulness of

11

structures which combine antivirally active nucleoside, e.g. ACV [91-93], gancyclovir (GCV)

12

[94] or cidofovir (cyclic monophosphate, cCDF) [95] with biotin or through a lipid chain of

13

various lengths which increases the lipophilicity of the molecule (Fig. 10). It was shown that

14

the length of the lipid connector determines the affinity to SMVT and, as a result, determines

15

the accumulation of the prodrug in the cell and can be an effective method of retinitis

16

treatment.

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17

19

2.2.4. Prodrugs as substrates of enzymes encoded by viruses

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For an increase in specificity or selectivity of the action of antiviral drugs, prodrugs are

21

being looked for which would constitute substrates for specific enzymes encoded by viruses.

22

Proteases belong to such enzymes. For example, HCMV encodes specific proteases splitting

23

bonds between alanine and serine. The results of research have shown that α-amino

24

substituted esters Ala-GCV can constitute a substrate for HCMV protease. The location of

25

alanine in position α is advantageous and it has a stabilizing effect, which was confirmed in

26

homogenate of Caco-2 cells, HLM (Human Liver Microsomes) and in human and rat plasma.

27

A promising candidate for a drug proved to be acyl monoester of glutamine-alanine dipeptide

28

of gancyclovir (Ac-L-Gln-L-Ala-GCV). Esters: tert-butoxycarbonyl, carbobenzyl and

29

benzyloxycarbonyl turned out to be much less susceptible to hydrolysis under the influence of

30

protease, which results from their hydrophobic properties [96].

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31 32

2.2.5. Other concepts of searching for and modification of the active compound structure

33

13

ACCEPTED MANUSCRIPT The sources of searching for prodrugs which are an improved form of the primary

2

compound are also disadvantageous biological properties determining adverse effects of their

3

action. Ribavirin (RBV), which is responsible for haemolysis and, as a result, anaemia

4

symptoms, is such an example. Ribavirin is recommended for the therapy of RSV infections

5

(Respiratory Syncytial Virus) and HCV, in therapy combined with PEGylated α-interferon

6

(PEG-αINF) and with protease inhibitors (boceprevir, telaprevir). Modifications of the

7

ribavarin molecule which limit its concentration in blood, amongst other things by reduced

8

penetration through membranes, weaken the haemolytic effect and involve the formation of:

9

3-carboxyamid derivative (taribavirin); L-enantiomer (levovirin); alkoxy alkyl phosphate

10

ester; conjugate with haemoglobin (Hb-RBV) and polymers: polyvinylpyrrolidone,

11

polyacrylic acid, polymethacrylic acid or poly-N-(2-hydroxypropyl)methacrylamide (PVP-

12

RBV, PAA-RBV, PMAA-RBV, PHPMA-RBV) (Fig. 11). Levovirin does not show antiviral

13

activity but immunomodulatory one which influences the anti-HCV effect comparable with

14

RBV [97, 98].

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1

In the group of HCV polymerase inhibitors used in the combined therapy, also balapiravir

16

should be included into potential prodrugs with an ester structure (Fig. 12), which is also

17

active towards the Dengue Virus [99-101]. In the case of infections with the Dengue Virus, an

18

elevated level of cytokines (TNF-α, IL-10, IFN-β) is observed, which inhibits the

19

phosphorylation process of the active form of balapiravir and reduces the effectiveness of the

20

therapy [100]. Compounds with activity against NS5B polymerase (HCV) which are being

21

designed in such large numbers now are usually large molecules with poor solubility

22

inhibiting the formulation of drugs for oral administration. To obtain structures with improved

23

solubility in SEDDS (Self-Emulsifying Drug Delivery Systems) based on lipids, the most

24

effective seems to be the strategy based on the modification of the carboxyl group of the

25

primary drug to the ester one in a reaction with glycolic acid amide [102]. In the group of

26

tricyclic indole derivatives (Fig. 12), it was shown that dimethylaminoethyl esters have the

27

strongest influence on their pharmacokinetic parameters after oral administration. They are

28

characterized by good absorption and effective transformation to the primary compound under

29

the influence of cholinesterase [103].

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15

30

Already at the stage of synthesis of new structures - dihydropirimidine derivatives of α,γ-

31

diketone butane acid (Fig. 12) – targeted at inhibiting HIV integrase activity, it was observed

32

that esterification of the carboxyl group with the formation isopropyl ester is important

33

antiviral activity and for keeping the cytotoxicity effect [104]. A similar observation was 14

ACCEPTED MANUSCRIPT 1

made in the case of the structure of the primary diketone butane acid built into the piridinone

2

structure (Fig. 12). Prodrugs, especially isopropyl esters considerably increased the anti-HIV

3

activity and they became quickly bioactivated to the primary compound in cells [105].

4 5

2.3. Anticancer therapies based on prodrugs

RI PT

6 A new approach using prodrugs are ADEPT therapies (Antibody-Directed Enzyme

8

Prodrug Therapy), GDEPT (Gene-Directed Enzyme Prodrug Therapy) and LEAPT (Lectin-

9

Directed Enzyme-Activated Prodrug Therapy) [15, 106]. Their aim is selective delivery of

10

cytotoxic compounds to cancer cells. The knowledge of the specifics of the tumor

11

microenvironment (e.g. pH, hypoxia) which influence the development of resistance can also

12

be used for delivery of active substances to the tumor.

14

2.3.1. Antibody-directed therapy

15

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7

The ADEPT method uses monoclonal antibodies or their fragments to transfer enzymes

17

capable of specific activation of non-toxic prodrugs to cytotoxic compounds (Fig. 13) [15,

18

106-109]. At the first stage, the enzyme-antibody conjugates (mAb-enzyme) which binds to

19

the specific antigen which is present only on the surface of cancer cells. After the time needed

20

to remove the conjugate that is not bound to the antigen, the prodrug is administered. Its

21

enzymatic activation occurs in the extracellular space of the extracellular tissue. In this way,

22

the cytotoxic effect also effects neighbouring cancer cells which do not have an antigen on the

23

surface (the so-called bystander effect). At the same time, the ability of the enzyme molecule

24

to activate many molecules of the prodrug results in much higher concentrations of the drug in

25

the cancerous tissue than in the healthy one [106-113]. The specification of the prodrug

26

administration time is very significant from the point of view of the patient's protection

27

against the activation of the prodrug in the plasma and next the development of systemic

28

toxicity [101]. Enzymes used the implementation of this concept should be characterized by

29

significant ability to activate as large a number of prodrug molecules as possible. Hence, the

30

use

31

carboxypeptidase A, nitroreductase, carboxypeptidase G2, organic phosphatase and other

32

[106, 107, 114-124]. Also the use of β-lactamase is a promising treatment strategy to enhance

33

the therapeutic effect and safety of cytotoxic agents. A conjugate (antibody-β-lactamase

34

fusion protein) is employed to precisely activate nontoxic cephalosporin prodrugs at the tumor

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16

of

the

following

enzymes

is

considered:

β-glucoronidase,

β-glucosydase,

15

ACCEPTED MANUSCRIPT 1

site. A major obstacle to the clinical application is the low catalytic activity and high

2

immunogenicity of the wild-type enzymes. The way to evade it, is to create a conjugate with a

3

cyclic decapeptide (RGD4C) [125]. The modification of the ADEPT method is the ADAPT

4

system (Antibody-Directed Abzyme Prodrug Therapy) in which the enzyme is replaced with a

5

catalytic antibody [126]. Prodrug in the ADEPT method should be characterized by good solubility in water,

7

stability in the physiological pH and appropriate pharmacokinetic parameters. Moreover, it

8

must be a good substrate for the activating enzyme used and must be considerably less toxic

9

than the active substance [112]. Specific requirements must also be met by enzymes used for

10

the creation of conjugates in the ADEPT system. They should be characterized by high

11

catalytic activity at the place of operation, good stability and ability to activate a large number

12

of prodrug molecules [112].

SC

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6

Prodrugs used in the ADEPT therapy are usually derivatives of well-known, highly

14

active, anti-cancer drugs whose use is limited by considerable systemic toxicity and a narrow

15

therapeutic index. The ADEPT therapy, however, is not free from limitations. The most

16

significant of these include too low a number of cancer antigens to achieve a therapeutic

17

effect and high immunogenicity of conjugates preventing the repetition of the therapy. A

18

problem will also be extracellular activation of the prodrug, which requires the use of

19

substances capable of permeating through mucous membranes [107, 112, 113]. The majority

20

of ADEPT systems are at the stage of pre-clinical research, some are at the 1st phase of

21

clinical tests. A system formed by a conjugate of the scFv fragment of the MFE-23 antibody,

22

anti-CEA and bacterial carboxypeptidase G2 and 4{[di(2-iodoethyl)amino]phenyl}oxy-

23

carbonyl-L-glutaminate as a prodrug. This system, as opposed to the majority of the other

24

ones, is characterized by low immunogenicity and quick elimination of its unbound part from

25

the body [127, 128]. Moreover, ADEPT systems are constructed for doxorubicine prodrugs

26

(phosphate [114], phenoxy acetamide [115], glucuronide [116], combinations with

27

cephalosporin [117]), nitrogen mustard (combination with cephalosporin [118]), melphalan

28

(phenoxyacetamide [115]), methotrexate (α-alanin derivative [119]), 5-fluorouracil (5-

29

fluorocytosine [120]), amygdalin [122] and etoposide (phosphate [121]). In preclinical animal

30

models and clinical trials the effective prodrugs with tumor-selectivity (e.g. prodrugs of

31

paclitaxel, 5-fluoro-2’-deoxyuridine, doxorubicine, vinblastine) were designed for activation

32

by PSMA (prostate specific membrane antigen) and PSA (prostate specific antigen) [129].

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33

Numerous limitations connected with the ADEPT system result from the fact that the

34

delivery of a large amount of conjugate is limited in poorly vascular tumours. A low level of 16

ACCEPTED MANUSCRIPT the enzyme makes it difficult to produce appropriate quantities of an active drug (necessary

2

for the cytotoxic effect). Moreover, the binding of the conjugate to the cell surface is limited

3

by inhomogeneity of the antigen. Other drawbacks include immunogenicity of antibodies,

4

costs and difficulties in the development and cleaning of antibodies, availability for the

5

tumour of the enzyme-antibody conjugate and transformation of prodrugs in non-cancerous

6

tissues. The main problem related to the immunogenicity of the enzyme-antibody conjugate

7

can be bypassed by using humanized proteins with a simultaneous immunosuppresive

8

therapy. Another method is the use of recombinant DNA technology to produce a fusion

9

protein with specific characteristics and, to avoid additional antibody purification stages,

10

which reduce enzymatic activity or the bond of the antibody with the conjugate [106]. Protein

11

drugs must be closely supervised by the immune in the patient's body which forces the search

12

for methods of bioinformatics involving the development of algorithms for optimization

13

strategy deimmunization proteins [130].

15

2.3.2. Gene-directed therapy

16

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14

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1

Another example of therapy using prodrugs and guaranteeing selective killing of cancer

18

cells is GDEPT, which is also called the suicide gene therapy [123, 124]. In this method,

19

enzyme-encoding genes are introduced into cancer cells which are capable of activating the

20

subsequently delivered prodrug to cytotoxic substances (Fig. 14) [15, 106, 131-133]. Usually

21

enzymes of viral or bacterial enzymes are introduced which normally do not occur in

22

mammals or human enzymes which are absent from cancer cells or occur only at low

23

concentrations [8, 107]. The introduction of genes into cells in the GDEPT methods occurs by

24

using peptides, cation lipids or naked DNA. The use viral vectors (e.g. adenoviruses,

25

retroviruses, lentivirus) are characteristic of the VDEPT system (Virus-Directed Enzyme

26

Prodrug Therapy) [131-135]. The first clinical trials assessing the safety and the extent of

27

providing a high concentration of anti-cancer drug to the tumors involve the use of TG4023

28

for the treatment VDEPT. TG4023 is a modified vaccinia virus Ankara (MVA) expressing

29

cytosine deaminase and uracil phosphoribosyltransferase enzymes. It transforms the prodrug

30

flucytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5'-monophosphate

31

(5-FUMP), respectively [136]. Non-viral vectors are generally less effective than viral vectors

32

due to the short-term expression of the therapeutic gene. Naked DNA is used in clinical tests,

33

however, low cellular absorption and rapid clearance remain the main obstacle to the

34

effectiveness of such conjugates [134]. In both methods, the prodrug is activated only after it

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17

17

ACCEPTED MANUSCRIPT permeates through the mucous membrane inside the cell. Therefore, it is important that

2

physicochemical properties of the substance used as a prodrug made it possible to overcome

3

biological barriers. The ability of the active drug to permeate to surrounding cells (bystander

4

effect) and influence cancer cells at each stage of the cell cycle is beneficial for the

5

effectiveness of the therapy [15, 137]. The bystander effect can be a two-edged sword. While

6

these effects are beneficial for increasing the toxicity effect several times, they can also exert

7

toxic effects in healthy cells, which reduce the clinical usefulness of GDEPT. For example,

8

gancyclovir should be administered at low doses as at high doses it can have adverse effects

9

on non-target tissues, such as bone marrow cells [134]. Limitations related to GDEPT and

10

VDEPT involve a lack of efficient vectors that are selective towards cancer cells which carry

11

enzyme-encoding genes. A frequent problem is also an ineffective in vivo process of cell

12

transduction [133].

SC

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1

Prodrugs, which are often used in the GDEPT method, are nucleoside analogues and

14

alkalizing compounds [123, 124, 133]. In the therapy of multiforme glioblastoma and brain

15

tumours, the introduction of a gene encoding thymidine kinase of the HSV virus. In this way,

16

it is possible to transform the administered gancyclovir into active triphosphate, which after

17

being built into the DNA of the cell causes inhibition of nucleic acid synthesis and,

18

subsequently, death of the cell [106, 123, 138, 139]. In chemotherapy of cancer of the

19

gastrointestinal tract, breast, head and neck, 5-fluorouracyl (5-FU) is often used. The

20

replacement of 5-FU with 5-fluorocytosine (5-FC), preceded by the introduction of a cytosine

21

deaminase gene of the Escherichia coli bacteria into cancer cells reduces the systemic

22

cytotoxicity of the drug with a simultaneous increase in the concentration of the substance in

23

the affected tissue [123, 124, 140-143]. In addition, the effectiveness of introducing the rat

24

CYP2B1, CYP2C11 and CYP2C6 isoenzyme to selectively increase the exposure of these

25

cells to toxic metabolites of cyclophosphamide and phosphamide (mustard derivatives of

26

phosphoric acid) [15, 123, 144, 145]. Another approach is the use of horseradish peroxidase

27

in the GDEPT system, which activates the indole-3-acetic acid leading to the inhibition of

28

colony formation in mammal cells, showing increased effectiveness only in such a therapeutic

29

system at the same time [123, 124, 146]. A stronger cytotoxic effect towards cancer cells was

30

observed for a derivative of 5-fluoroindole-3-acetic acid [124].

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31

In the in vivo and in vitro studies demonstrated synergy of the combined MSC-based

32

gene therapy and significant therapeutic effect on experimental lung metastases induced by

33

human breast adenocarcinoma cells MDA-MB-231/EGFP cell line but the therapeutic effect

34

might be limited by the sensitivity of tumor cells [147, 148]. 18

ACCEPTED MANUSCRIPT 1

GPAT is a variation of GDEPT which uses the knowledge of differences in the

2

transcription between normal and cancer cells in the area of selective expression of the

3

prodrug-metabolizing enzyme. The so-called specific cancer transcriptive regulatory elements

4

(TRE) are placed above the gene of the enzyme leading to selective expression [106]. The co-expression of immunomodulators such as IL-2, IL-12, IFN-γ and TNF-α is used

6

to enhance the bystander effect. The dependence between the bystander effect and GDEPT

7

and immunity also results from immunogenicity of GDEPT enzymes. When enzymes are

8

immunogenic, the likelihood of toxicity outside the target cell is lower. In summary, the

9

toxicity of the bystander effect is not a problem in the majority of cases. However, one should

10

be careful, if immunomodulators are used to increase the immune response against cancer

11

antigens [134].

SC

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5

The gene therapy is a promising approach, however, it is still at an early stage of

13

development. One of the most important challenges is the influence on improving gene

14

delivery and, as a result, therapeutic effectiveness. The GDEPT therapeutic system offers both

15

possibilities and hopes for the future. However, clinical tests require that the safety of its use

16

and toxicity should be determined [123].

17

19

2.3.3. Lectin-directed therapy

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18

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12

The LEAPT therapy, on the other hand, is a two-part system which consists of selectively

21

provided glycosylated enzyme and a prodrug combined with a sugar group to cancer cells.

22

This method uses enzymes that occur naturally in a given species, e.g. α-rhamnosidase, which

23

is absent in mammals, which are synthetically glycosylated [15, 149, 150]. In this form, the

24

enzyme is bound with a receptor that is specific for carbohydrates and is provided to target

25

cells by means of endocytose. Next, a prodrug is administered with a connected sugar

26

fragment (e.g. rhamnose) which constitutes a substrate for the enzyme used. After the prodrug

27

reaches target cells, the sugar residue is detached and the active substance is released [150].

28

The effectiveness of the LEAPT therapy was shown for the combination of doxorubicine

29

with rhamnose used to decrease a tumour in a model of hepatocellular cancer (Hep2). It was

30

shown that the model used makes it possible to obtain a higher concentration of the drug at

31

the place of action [150]. Due to numerous carbohydrate-binding processes occurring in the

32

body, the possibility of using the aforementioned method also for other types of cells seems

33

likely. It is necessary to identify receptors of potential medical importance and the use of

34

carbohydrates specific for target cell receptors. It is potentially possible, amongst other things,

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19

ACCEPTED MANUSCRIPT 1

to use carbohydrate receptors on the macrophage surface [151, 152] in therapy of diseases

2

connected with the macrophage function, lysosomal storage diseases [153] or HIV infections

3

[154].

4 5

2.3.4. Strategies based on the specific tumor microenvironment

RI PT

6 Strategies to improve or complement the anticancer drugs distribution within tumors hold

8

promise to increasie antitumor effects without corresponding increases in normal tissue

9

toxicity. A conjugate of vitamin E and paclitaxel (PTX-S-S-VE, Fig. 15) was synthesized to

10

increase the hydrophobic properties of the paclitaxel particle. This provides the ability to

11

deliver the active substance in the form of nanocarriers (nanoemulsions), which can circulate

12

long in the blood. In addition, increased level of glutathione in the tumor environment is

13

conducive to cleave the disulfide bond and release of the active anti-cancer drug. Further, this

14

conjugate has a greater antitumor activity against KB-3-1 cell line xenograft tumor than the

15

parent compound. Therefore, the described modification has favorable influence the

16

cytotoxicity [155].

M AN U

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7

Another form of the development of effective anticancer drugs is a synthesis of hypoxia-

18

activated prodrugs. These prodrugs are activated in the absence of oxygen [156]. Given a

19

central role in tumour progression and resistance to therapy, tumour hypoxia might well be

20

considered the best validated target that has not been yet exploited in anticancer therapy. The

21

compound

22

bromoethyl)phosphorodiamidic acid (1-methyl-2-nitro-1H-imidazol-5-yl)methyl ester, Fig.

23

16a) [157-159]. The drugs such as docetaxel and doxorubicin had minimal activity in hypoxic

24

regions, but the combination of TH-302 and chemotherapy resulted in increased expression of

25

γH2AX and cleaved caspases in regions both proximal and distal to blood vessels [156]. The

26

mechanism of prodrugs activation it consists of the enzymatic reduction by one- or two-

27

electron reductases (Fig. 16b). Reductases that catalyse concerted two-electron reductions fall

28

into two broad groups. The first are haemoproteins such as cytochrome P450s (CYPs),

29

especially CYP3A4, and CYP2S1. The best studied enzyme of a second group of two-electron

30

reductases is NAD(P)H dehydrogenase 1 (NQO1), which catalyses the facile two-electron

31

reduction of quinones including e.g. apaziquone and the aziridinylbenzoquinone to their

32

hydroquinones and the dinitrobenzamide to its active 4-hydroxylamine metabolite. The one-

33

electron reductase inducible nitric oxide synthase (iNOS) is also upregulated under hypoxia,

34

and can similarly catalyse the two-electron reduction [157]. Also the combination of

most

advanced

in

clinical

testing

is

TH-302

(N,N’-bis(2-

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17

20

ACCEPTED MANUSCRIPT gemcitabine and TH-302 has given encouraging results for therapy of human pancreatic

2

cancer [156]. Because gemcitabine is a nucleoside analogue commonly used in cancer therapy

3

the addition of a phosphoramidate motif to the gemcitabine can protect it against many of the

4

key cancer resistance mechanisms. Among the synthesized compounds, Acelarin (NUC-1031,

5

Fig. 17) was shown to be potent in vitro. It is an example of ProTides technology and

6

generates promising new anticancer agents in clinical development [160].

RI PT

1

The efficacy of drugs that alkylate the O6-position of guanine is inhibited by O6-

8

alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor

9

DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors,

10

while sparing normal tissues where this protein serves a protective function. To impart tumor

11

selectivity

12

purin-2-yl)carbamate as the hypoxia targeted prodrug of 6((3-((dimethylamino)methyl)-

13

benzyl)oxy)-9H-purin-2-amine (Fig. 18) was synthesized. This prodrug (derivative of O6-

14

benzylguanine) enhances the cytotoxic effect of laromustine under hypoxic and normoxic

15

conditions. Hence the conclusion that the combination of the other factors that rely on

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alkylation of the O6-position of guanine residues in DNA for their activity, may be useful in a

17

clinical setting [161].

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2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-

The high level of reactive oxygen species (ROS) in cancer cells has been exploited for

19

developing novel therapeutic strategies to preferentially kill cancer cells. Cohen’s group

20

reported the first H2O2-activated matrix metalloproteinase inhibitor (MMPi) by protecting the

21

hydroxyl group of the zinc-binding group with a boronic ester. For developing ROS-activated

22

anticancer prodrugs the boronic acids/esters can be used. These ROS-activated prodrugs

23

demonstrated selective cytotoxicity towards cancer cells [162].

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Interesting approach is that the tumor-localised bacteria may be conceived as a tumor-

25

specific enzymatic reservoir capable of local activation of prodrugs or the native expression of

26

such therapeutic genes by bacterium that may be sufficient to mediate prodrug activation. So

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it may be possible to use multiple prodrugs (e.g. fludarabine phosphate) in conjunction with

28

bacteria (e.g. Escherichia coli) to treat cancer [163].

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3. Conclusion

31 32

Although many years have passed since the introduction of the notion of prodrug, this

33

term is becoming more and more important and it does not refer only to the active substance

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any more but also to the form of therapy, the dosage form or the method of delivering the 21

ACCEPTED MANUSCRIPT active substance to the therapeutic target. An increase in searching for prodrugs also results

2

from the necessity to improve properties of substances that are already known and broadly

3

used in medicine. The introduction of new active substances is costly and it requires many

4

years of research and higher awareness of possible adverse effects observed in distant chronic

5

therapy makes it necessary to exercise a high degree of caution. The presented concepts of

6

looking for prodrugs are mostly based on two/three strategies: synthesis of prodrugs,

7

development of system, their effective administration using e.g. carriers or the use of

8

knowledge about the route of their activation. Prodrugs are thought to give the possibility

9

developing selective drugs towards the therapeutic target, e.g. a cancer cell or a micro-

10

organism. Such an approach is made possible by knowledge on pathogenesis of diseases at the

11

molecular level, metabolism of healthy and pathogenically changed cells and micro-organism

12

metabolism (bacteria, viruses, fungi, protozoa etc.). The development of biochemical, genetic

13

and microbiological research gives the direction to pharmacy. A lot of drugs which have been

14

used for years are still tested in terms of their metabolism and molecular mechanism of action,

15

providing new knowledge on the active substance. A range of them turn out to meet the

16

criteria for a prodrug. Prodrugs are being looked form in practically all groups of

17

pharmacological drugs. For the purposes of this article, the development of the concept of

18

searching from prodrugs as regards selected antiviral drugs proving that chemical

19

modifications of known structures are a very numerous group together with synthesis of new

20

structures sensitive to appropriate enzymes. It seems that this form of searching brings the

21

most benefits in the form of implementations as it is based on the knowledge of

22

pharmacological properties (including toxicological ones) of substances which have been

23

used for years. Apart from this, it applies to a broad range of applications and it is of

24

considerable importance in designing drugs and producing them on a scale which is

25

appropriate for the industry. New strategies (e.g. ADEPT, GDEPT, LEAPT), which are based

26

to a greater extent on individualization of therapy are not introduced so quickly as the

27

possibilities of assessing both effectiveness and safety of their use are limited. Hence, the

28

number of publications is not as numerous as in the case of synthesis of new structures or

29

modifications of the known structures and they are still a novelty. Apart from this, application

30

of enzymes, antibodies or genes (proteins, DNA) involves numerous analytical limitations

31

which influence the quality of the medication, its homogeneity, stability, purity and

32

repeatability of the composition. The process of validating the production and the assessment

33

of the quality of the finished product is not only expensive but it also requires the application

34

of very specific and specialized analytical methods. However, it is worth showing routes for

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searching new structures and their synthesis as well as new therapeutic strategies with the

2

application of prodrugs, waiting for success and extension of knowledge on the fate of the

3

drug in the system and molecular explanation of activity of xenobiotics.

4 Conflict of interest

6

The authors confirm that this article content has no conflict of interest.

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References

9 [1]

K.M. Huttunen, H. Raunio, J. Raunio, Prodrugs-from serendipity to rational design, Pharmacol. Rev. 63 (2011) 750–771.

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

[2]

J. Rautio, H. Kumpulainen, T. Heimach, R. Oliyai, T. Järvinen, J. Savolainen, Prodrugs: design and clinical applications, Nature Rev. Drug. Discov. 7 (2008) 255– 270. P. Ettmayer, G.L. Amidon, B. Clement, B. Testa, Lessons learned from marketed and investigational prodrugs, J. Med. Chem. 47 (2004) 2393–2404. D.H. Jornada, G.F dos Santos Fernandez, D.E. Chiba, T.R. Ferreira de Melo, J.L. dos Santos, M.C. Chung, The prodrug approach: a successful tool for improving drug solubility, Molecules 21 (2016) doi:10.3390/molecules21010042. A. Albert, Chemical aspects of selective toxicity, Nature, 182 (1958) 421–423. B. Testa, Prodrug research: futile or fertile?, Biochem. Pharmacol. 68 (2004) 2097– 2106. V.J. Stella, R.T. Borchardt, M.J. Hageman, R. Oliyai, H. Maag, J. Tilley, Prodrugs: Challenges and Rewards, Part 1 & 2, Springer Science + Business Media, New York, 2007. J.B. Zawilska, J. Wojcieszak, A.B. Olejniczak, Prodrugs: A challenge for the drug development, Pharmacol. Rep. 65 (2013) 1–14. V.J. Stella, Prodrugs: Some thoughts and current issues, J. Pharm. Sci. 99 (2010) 4755–4765. B. Testa, Prodrugs: Bridging pharmacodynamic/pharmacokinetic gaps, Curr. Opin. Chem. Biol. 13 (2009) 338–344. R. Karaman, Prodrugs design based on inter- and intramolecular chemical processes, Chem. Biol. Drug Des. 82 (2013) 643–668. N.R. Srinivas, The rationality for using prodrug approach in drug discovery programs for new xenobiotics: opportunities and challenges, Eur. J. Drug Metab. Pharmacokinet. 36 (2011) 49–59. R. Silverman, The organic chemistry of drug design and drug action, Ed. II, Elsevier Academic Press, Burlington, 2004. N. Das, M. Dhanawat, B. Dash, R.C. Nagarwal, S.K. Shrivastava, Codrug: An efficient approach for drug optimization, Eur. J. Pharm. Sci. 41 (2010) 41, 571–588. A. Stańczak, A. Ferra, Prodrugs and soft drugs, Pharmacol. Rep. 58 (2006) 599-613.

[7]

[8] [9] [10] [11] [12]

[13] [14] [15]

M AN U

TE D

[5] [6]

EP

[4]

AC C

[3]

SC

10 11

23

ACCEPTED MANUSCRIPT

[21]

[22] [23]

[24]

[25]

[26]

[27] [28]

[29] [30]

[31] [32]

RI PT

[20]

SC

[19]

M AN U

[18]

TE D

[17]

G.R. Kokil, P.V. Rewatkar, Bioprecursor prodrugs: molecular modification of the active principle, Mini Rev. Med. Chem. 10 (2010) 1316–1330. C.G. Wermuth, Designing prodrugs and bioprecursors, in: G. Jolles, K.R. Woolridge, Drug Design: Fact or Fantasy, Academic Press, London, 1984; pp. 47–72. B.M. Liederer, R.T. Borchardt, Enzymes involved in the bioconversion of ester-based prodrugs, J. Pharm. Sci. 95 (2006) 1177–1195. S.D. Clas, R.I. Sanchez, R. Nofsinger, Chemistry-enabled drug delivery (prodrugs): recent progress and challenges, Drug Discov. Today 19 (2014) 79–87. C.E. Wheelock, T.F. Severson, B.D. Hammock, Synthesis of new carboxylesterase inhibitors and evaluation of potency and water solubility, Chem. Res. Toxicol. 14 (2001) 1563–1572. S. Benchairt, C.L. Morton, J.L. Hyatt, P. Kuhn, M.K. Danks, M.K. Potter, M.R. Redinbo, Crystal structure of human carboxylesterase 1 complexed with the Alzheimer’s drug tacrine: From binding promiscuity to selective inhibition, Chem. Biol. 10 (2003) 341–349. T. Satoh, M. Hosokawa, Structure, function and regulation of carboxylesterases, Chem. Biol. Interact. 162 (2006) 195–211. T. Satoh, P. Taylor, W.F. Bosron, S.P. Sanghani, M. Hosokawa, B.N. La Du, Current progress on esterases: from molecular structure to function, Drug Metab. Dispos. 30 (2002) 488–493. M. Miwa, M. Ura, M. Nishida, N. Sawada, T. Ishikawa, K. Mori, N. Shimma, I. Umeda, H. Ishitsuka, Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue, Eur. J. Cancer. 34 (1998) 1274–1281. P.D. Senter, H. Marquardt, B.A. Thomas, B.D. Hammock, I.S. Frank, H.P. Svensson, The role of rat serum carboxylesterase in the activation of paclitaxel and camptothecin prodrugs, Cancer Res. 7 (1996) 1471–1474. Y. Yoshigae, T. Imai, M. Taketani, M. Otagiri, Characterization of esterases involved in the stereoselective hydrolysis of ester-type prodrugs of propranolol in rat liver and plasma, Chirality 11 (1999) 10–13. D.M. Quinn, Acetylcholinesterse: Enzyme structure, reaction dynamics, and virtual transition states, Chem. Rev. 87 (1987) 955–979. R.V. Tak, D. Pal, H. Gao, S. Dey, A.K. Mitra, Transport of acyclovir ester prodrugs through rabbit cornea and SIRC-rabbit corneal epithelial cell line, J. Pharm. Sci. 90 (2001) 1505–1515. M. Nakamura, E. Shirasawa, M. Hikida, Characterization of esterases involved in the hydrolysis of dipivefrin hydrochloride, Ophtalmic. Res. 25 (1993) 46–51. E.H. Sklan, A. Lowenthal, M. Korner, Y. Ritoy, D.M. Landers, T. Rankinen, C. Bouchard, A.S. Leon, T. Rice, D.C. Rao, J.H. Wilmore, J.S. Skinner, H. Soreg, Acetylcholinesterase/paraoxonase genotype and expression predict anxiety scores in health, risk factors, exercise training, and genetics study, Proc. Natl. Acad. Sci. USA 101 (2004) 5512–5517. B. Fuhrman, A. Partoush, M. Aviram, Acetylcholine esterase protects LDL against oxidation, Biochem. Biophys. Res. Commun. 322 (2004) 974–978. H. Sun, Y.P. Pang, O. Lockridge, S. Brimijoin, Re-engineering butyrylcholinesterase as a cocaine hydrolase, Mol. Pharmacol. 62 (2002) 220–224.

EP

[16]

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

24

ACCEPTED MANUSCRIPT

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

[48] [49]

RI PT

[37]

SC

[36]

M AN U

[35]

TE D

[34]

O. Lockridge, Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine, Pharmacol. Ther. 47 (1990) 35–60. E. Masson, M.T. Froment, P.L. Fortier, J.E. Visicchio, C.F. Bartels, O. Lockridge, Butyrylcholineste-rase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin related neutral esters, Biochim. Biophys. Acta 1387 (1998) 41–52. O. Lockridge, N. Mottershow-Jackson, H.W. Eckerson, B.N. La Du, Hydrolysis of diacetylmorphine (heroin) by human serum cholinesterase, J. Pharmac. Exp. Ther. 215 (1980) 1–8. A. Tunek, E. Levin, L.A. Svensson, Hydrolysis of 3H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro, Biochem. Pharmacol. 37 (1988) 3867–3876. C. Meyers, O. Lockridge, B.N. La Du, Hydrolysis of methylprednisolone acetate by human serum cholinesterase, Drug Metab. Dispos. 10 (1982) 279–280. J.F. Gilmer, L.M. Moriarty, M.N. Lally, J.M. Clancy, Isosorbide-based aspirin prodrugs: II. Hydrolysis kinetics of isosorbide diaspirinate, Eur. J. Pharm. Sci. 16 (2002) 297–304. P. Gajewski, M. Tomaniak, K.J. Filipiak, Paraoksonaza 1 - co o niej obecnie wiadomo? Paraoxonase 1 - what do we know today?, Folia Cardiologica 10 (2015) 183–189 (in polish). D. Litvinov, H. Mahini, M. Garelnabi, Antioxidant and anti-inflammatory role of paraoxonase 1: implication in arteriosclerosis diseases, N. Am. J. Med. Sci. 4 (2012) 523–532. P.M. Dansette, J. Rosi, G. Bertho, D. Mansuy, Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer, Chem. Res. Toxicol. 25 (2012) 348–356. D.I. Draganov, B.N. La Du, Pharmacogenetics of paraoxonases: A brief review, Naunyn-Schmiedeberg's Arch. Pharmacol. 369 (2004) 78–88. K. Tougou, A. Nakamura, S. Watanabe, Y. Okuyama, A. Morino, Paraoxonase has a major role in the hydrolysis of prulifloxacin (NM441), a prodrug of a new antibacterial agent, Drug Metab. Dispos. 26 (1998) 355–359. A. Salvi, P.A. Carrupt, J.M. Mayer, B. Testa, Esterase-like activity of human serum albumin toward prodrug esters of nicotinic acid, Drug Metab. Dispos. 25 (1997) 395– 398. U. Kragh-Hansen, Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin-ligand complexes, Biochim. Biophys. Acta 1830 (2013) 5535–5544. K.W. Nti-Addae, J.S. Laurence, A.L. Skinner, V.J. Stella, Reversion of sulfenamide prodrugs in the presence of free thiol containing proteins, J. Pharm. Sci. 100 (2011) 3023–3027. R.M. Laethem, T.A. Blumenkopf, M. Cory, L. Elwell, C.P. Moxham, P.H. Ray, L.M. Walton, G.K. Smith, Expression and characterization of human pancreatic preprocarboxypeptidase A1 and preprocarboxypeptidase A2, Arch. Biochem. Biophys. 332 (1996) 8–18. R.S. Sidhu, A.H. Blair, Human liver aldehyde dehydrogenase. Esterase activity, J. Biol. Chem. 250 (1975) 7894–7898. J.A. Verpoorte, S. Mehta, J.T. Edsall, Esterase activities of human carbonic anhydrases B and C, J. Biol. Chem. 242 (1967) 4221–4229.

EP

[33]

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

25

ACCEPTED MANUSCRIPT

[55] [56]

[57] [58] [59]

[60]

[61] [62] [63] [64] [65] [66]

[67] [68]

RI PT

[54]

SC

[53]

M AN U

[52]

TE D

[51]

H. Tsunematsu, S. Yoshida, K. Horie, M. Yamamoto, Synthesis and the stereoselective enzymatic hydrolysis of flurbiprofen-basic amino acid ethyl esters, J. Drug Target. 2 (1995) 517–525. T. Tsujita, K. Shirai, Y. Saito, H. Okuda, Relationship between lipase and esterase, Prog. Clin. Biol. Res. 344 (1990) 915–933. F.J. Gonzalez, R.H. Tukey, Drug metabolism, in: Goodman and Gilman's The Pharmacological Basis of Therapeutics, The McGraw-Hill Companies Inc., New York, 2006, pp. 71–91. B.; Testa, S.D. Kramer, The biochemistry of drug metabolism-an introduction: Part 2. Redox reactions and their enzymes, Chem. Biodivers. 4 (2007) 257–405. S.D. Kramer, B. Testa, The biochemistry of drug metabolism–an introduction: Part 6. Inter-individual factors affecting drug metabolism, Chem. Biodivers. 5 (2008) 2465– 2478. D.A. Sica, T.W. Gehr, S. Ghosh, Clinical pharmacokinetics of losartan, Clin. Pharmacokinet. 44 (2005) 797–814. P.J. Neuvonen, Drug interactions with HMG-CoA reductase inhibitors (statins): the importance of CYP enzymes, transporters and pharmacogenetics, Curr. Opin. Investing. Drugs 11 (2010) 323–332. L. Wallentin, P2Y(12) inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use, Eur. Heart J. 30 (2009) 1964–1977. H. Caplain, F. Donat, C. Gaud, J. Necciari, Pharmacokinetics of clopidogrel, Semin. Thromb. Hemos. 25 (1999) 25–28. M. Tang, M. Mukundan, J. Yang, N. Charpentier, E.L. LeCluyse, C. Black, D. Yang, D. Shi, B. Yan, Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carboxylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol, J. Pharmacol. Exp. Ther. 319 (2006) 1467–1476. M. Kazui, Y. Nishiya, T. Ishizuka, K. Hagihara, N.A. Farid, O. Okazaki, T. Ikeda, A. Kurihara, Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab. Dispos. 38 (2010) 92–99. H.S. Smith, Opioid metabolism, Mayo Clin. Proc. 84 (2009) 613–624.

EP

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

[50]

K.M. Huttunen, N. Mähönen, H. Raunio, J. Rautio, Cytochrome P450 activated prodrugs: targeted drug delivery., Curr. Med. Chem. 15 (2008) 2346–2365. V. Jordan, New insights into the metabolism of tamoxifen and its role in the treatment and prevention of breast cancer, Steroids 72 (2007) 829–842. T. Imai, Human carboxylesterase isozymes: catalytic properties and rational drug design, Drug Metab. Pharmacokinet. 21 (2006) 173–185. T. Satoh, M. Hosokawa The mammalian carboxylesterases: From molecules to functions, Annu. Rev. Pharmacol. Toxicol. 38 (1998) 257–288. B. Bukowska, D. Pieniążek, K. Hutniki, W. Duda, Acetylo- i butyrylocholinoesteraza - budowa, funkcje i ich inhibitory. Acetyl- and butyrylcholinesterase - structure, functions and their inhibitors, Curr. Top. Biophys. 30 (2007) 11–23 (in polish). V.N. Talesa, Acetylcholinesterase in Alzheimer’s disease, Mech. Ageing Dev. 122 (2001) 1961–1969. K. Biggadike, R.M. Angell, C.M. Burgess, R.M. Farrell, A.P. Hancock, A.J. Harker, W.R. Irving, C. Ioannou, P.A. Procopiou, R.E. Shaw, Y.E. Solanke, O.M. Singh, M.A.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

26

ACCEPTED MANUSCRIPT 1 2 3 4 5 6

[69]

Snowden, R.J. Stubbs, S. Walton, H.E. Weston, Selective plasma hydrolysis of glucocorticoid gamma-lactones and cyclic carbonates by the enzyme paraoxonase: An ideal plasma inactivation mechanism, J. Med. Chem. 43 (2000) 19–21. T. Imai, M. Imoto, H. Sakamoto, M. Hashimoto, Identification of esterases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption, Drug Metab. Dispos. 33 (2005) 1185–1190.

[70]

28 29 30 31

[78]

M. Krečmerová, R. Pohl, M. Masojídková, J. Balzarini, R. Snoeck, G. Andrei, N4Acyl derivatives as lipophilic prodrugs of cidofovir and its 5-azacytosine analogue, (S)-HMPM-5-azaC: Chemistry and antiviral activity, Bioorg. Med. Chem. 22 (2014) 2896–2906.

32 33 34

[79]

C. Meier, U. Görbig, C. Müller, J. Balzarini, cyclo-Sal-PMEA and cycloAmb-PMEA: potentially new phosphonate prodrugs based on the cyclo-Sal-Pronucleotide approach, J. Med. Chem. 48 (2005) 8079–8086.

35 36 37 38 39 40 41 42 43 44 45 46 47

[80]

[75]

[76]

[77]

[81]

[82]

SC

M AN U

[74]

TE D

[73]

EP

[72]

AC C

[71]

R. Böttger, D. Knappe, R. Hoffmann, Readily adaptable release kinetics of prodrugs using protease-dependent reversible PEGylation, J. Control. Rel. 230 (2016) 88–94. K. Beaumont, R. Webster, I. Gardner, K. Dack, Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: challenges to the discovery scientist, Curr. Drug Metab. 4 (2003) 461–485. B. Butora, N. Qi, W. Fu, T. Nguyen, H.C. Huang, I.W. Davies, Cyclic-disulfide-based prodrugs for cytosol-specific drug delivery, Angew. Chem. Int. Ed. 53 (2014) 14046– 14050. F. Pertusati, M. Serpi, C. McGuigan, Medicinal chemistry of nucleoside phosphonate prodrugs for antiviral therapy, Antiviral. Chem. Chemother. 22 (2012) 181–203. S.J. Hurwitz, R.F. Schinazi, Prodrug strategies for improved efficacy of nucleoside antiviral inhibitors, Curr. Opin., 8 (2013) 556–564. L. Lou, Advances in nucleotide antiviral development from scientific discovery to clinical applications: Tenofovir disoproxyl fumarate for Hepatitis B, J. Clin. Transl. Hepatol. 1 (2013) 33–38. A. Antela, C. Aquiar, J. Comston, B.M. Hendry, M. Boffito, P. Mallon, V. PourcherMartinez, G. Di Perri, The role of tenofovir alafenamide in future HIV management. HIV Medicine 17 (2016) 4–16. A.S. Ray, M.W. Fordyce, M.J.M. Hitchcok, Tenofovir alafenamide: A novel prodrug of tenofovir for the treatment of Human Immunodeficiency Virus, Antiviral Res. 125 (2016) 63–70.

RI PT

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

S. Benzaria, H. Pélicano, R. Johnson, G. Maury, J.L. Imbach, A.M. Aubertin, G. Obert, G. Gosselin, Synthesis, in vitro antiviral evaluation, and stability studies of bis(S-aryl-2-thioethyl) ester derivatives of 9[2-(phosphonomethoxy)ethyl]adenine (PMEA) as potential PMEA prodrugs with improved oral bioavailability, J. Med. Chem. 39 (1996) 4958–4965. C. Ballatore, C. McGuigan, E. De Clerq, J. Balzarini, Synthesis and evaluation of novel amidate prodrugs of PMEA and PMPA. Bioorg. Med. Chem. Lett. 11 (2001) 1053–1056. M.D. Erion, K.R. Reddy, S.H. Boyer, M.C. Matelich, J. Gomez-Galeno, R.H Lemus, B.G. Ugarkar, T.J. Colby, J. Schanzer, P.D. Van Poelie, Design, synthesis, and characterization of a series of cytochrome P450 3A-activated prodrugs (HepDirect prodrugs) useful for targeting phosphor(on)ate-based drugs to the liver, J. Am. Chem. Soc. 126 (2004) 5154–5163. 27

ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

[83]

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

[87]

[84]

[90]

[91]

[92]

[93]

[94]

M AN U

TE D

[89]

EP

[88]

D. Dibliková, M. Kopečná, B. Školová, M. Krečmerová, J. Roh, A. Hrabálek, K. Vávrová, Transdermal delivery and cutaneous targeting of antivirals using a penetration enhancer and lysolipid prodrugs, Pharm. Res. 31 (2014) 1071–1081. T. Gollnest, T.D. de Oliveira, D. Schols, J. Balzarini, C. Meier, Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals, Nat. Commun. 6 (2015) 8716. Doi: 10.1038/ncomms8716. C. Li, D.C. Quenelle, M.N. Prichard, J.C. Drach, J. Zemlicka, Synthesis and antiviral activity of 6-deoxycyclopropavir, a new prodrug of cyclopropavir, Bioorg. Med. Chem. 20 (2012) 2669–2674. A. Diez-Torrubia, S. Cabrera, S. de Castro, C. Garcia-Aparicio, D. Mulder, I. De Meester, M.J. Camarasa, J. Balzarini, S. Velázquez Novel water-soluble prodrugs of acyclovir cleavable by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme, Eur. J. Med. Chem. 70 (2013) 456–468. A.D. Vadlapudi, R.K. Vadlapatla, D. Kwatra, R. Earla, S.K. Samanta, D. Pal, A.K. Mitra, Targeted lipid based drug conjugates: A novel strategy for drug delivery, Int. J. Pharm. 434 (2012) 315–324. A.D. Vadlapudi, K. Cholkar, R.K. Vadlapatla, A.K. Mitra, Aqueous nanomicellar formulation for topical delivery of biotinylated lipid prodrug of acyclovir: Formulation development and ocular biocompatibility, J. Ocul. Pharmacol. Ther. 30 (2014) 49–58. A.D. Vadlapudi, R.K. Vadlapatla, R. Earla, S. Sirimulla, J.B. Bailey, D. Pal, A.K. Mitra, Novel biotinylated lipid prodrugs of acyclovir for the treatment of Herpetic Keratitis (HK): Transporter recognition, tissue stability and antiviral activity, Pharm. Res. 30 (2013) 2063–2076. K.G. Janoria, S.H. Boddu, Z. Wang, D.K. Paturi, S. Samanta, D. Pal, A.K. Mitra, Vitreal pharmacokinetics of biotinylated ganciclovir: Role of sodium-dependent

AC C

[86]

SC

RI PT

[85]

M.J. Sofia, Nucleotide prodrugs for HCV therapy, Antivir. Chem. Chemother. 22 (2011) 23–49. L. Bondada, M. Detorio, L. Bassit, S. Tao, C.M. Montero, T.M. Singletary, H. Zhang, L. Zhou, J.H. Cho, S.J. Coats, R.F. Schinazi, Adenosine dioxolane nucleoside phosphoramidates as antiviral agents for human immunodeficiency and Hepatitis B viruses, ACS Med. Chem. Lett. 4 (2013) 747–751. T.K. Warren, R. Jordan, M.K. Lo, A.S. Ray, R.L. Mackman, V. Soloveva, D. Siegel, M. Perron, R. Bannister, H.C. Hui, N. Larson, R. Strickley, J. Wells, K.S. Stuthman, S.A. Van Tongeren, N.L. Garza, G. Donnelly, A.C. Shurtleff, C.J. Retterer, D. Gharaibeh, R. Zamani, T. Kenny, B.P. Eaton, E. Grimes, L.S. Welch, L. Gomba, C.L. Wilhelmsen, D.K. Nichols, J.E. Nuss, E.R. Nagle, J.R. Kugelman, G. Pakacios, E. Doerffler, S. Neville, E. Carra, M.O. Clarke, L. Zhang, W. Lew, B. Ross, Q. Wang, K. Chun, L. Wolfe, D. Babusis, Y. Park, K.M. Stray, I. Trancheva, J.Y. Feng, O. Barauskas, Y. Xu, P. Wong, M.R. Braun, M. Flint, L.K. McMullan, S.S. Chen, R. Fearns, S. Swaminathan, D.L. Mayers, C.F. Spiropoulou, W.A. Lee, S.T. Nichol, T. Cihlar, S. Bavari, Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys, Nature 531 (2016) 381–385. C. McGuigan, C. Bourdin, M. Derudas, N. Hamon, K. Hinsinger, S. Kandil, K. Madela, S. Meneghesso, F. Pertusati, M. Serpi, M. Slusarczyk, S. Chamberlain, A. Kolykhalov, J. Vernacio, C. Vanpouille, A. Introini, L. Margolis, J. Balzarini, Design, synthesis and biological evaluation of phosphoradiamidate prodrugs of antiviral and anticancer nucleosides, Eur. J. Med. Chem. 70 (2013) 326–340.

28

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35 36 37 38 39 40 41 42 43 44 45 46

[99]

[100]

[101] [102]

EP

[103]

RI PT

[98]

SC

[97]

M AN U

[96]

TE D

[95]

multivitamin transporter expressed on retina, J. Ocul. Pharmacol. Ther. 25 (2009) 39– 49. M.R. Gokulgandhi, M. Barot, M. Bagui, D. Pal, A.K. Mitra, Transporter-targeted lipid prodrugs of cyclic cidofovir: A potential approach for the treatment of cytomegalovirus retinitis, J. Phar. Sci. 101 (2012) 3249–3263. H. Sabit, A. Dahan, J. Sun, C.J. Provoda, K.D. Lee, J.H. Hilfinger, G.L. Amidon, Cytomegalovirus protease targeted prodrug development, Mol. Pharmaceutics 10 (2013) 1417–1424. H. Guo, S. Sun, Z. Yang, Y. Tang, Y. Wang, Strategies for ribavirin prodrugs and delivery systems for reducing the side-effects hemolysis and enhancing their therapeutic effect, J. Control. Release. 209 (2015) 27–36. S.D. Dong, C.C. Lin, M. Schroeder, Synthesis and evaluation of new phosphorylated ribavirin prodrug, Antiviral Res. 99 (2013) 18–26. S.J. Coats, E.C. Garnier-Amblard, F. Amblard, M. Ehteshami, S. Amiralaei, H. Zhang, L. Zhou, S.R. Boucle, X. Lu, L. Bondada, J.R. Shelton, H. Li, P. Liu, C. Li, J.H. Cho, S.N. Chavre, S. Zhou, J. Mathew, R.F. Schinazi, Chutes and ladders in hepatitis C nucleoside drug development, Antiviral Res., 102 (2014) 119–147. Y.L. Chen, N.A. Ghafar, R. Karuna, Y. Fu, S.P. Lim, W. Schul, F. Gu, M. Herve, F. Yokohama, G. Wang, D. Cerny, K. Fink, F. Blasco, P.Y. Shi, Activation of peripheral blood mononuclear cells by Dengue Virus infection depontentiates balapiravir, J. Virol. 88 (2014) 1740–1747. Y.L. Chen, F. Yokokawa, P.Y. Shi, The search for nucleoside/nucleotide analog inhibitors of dengue virus, Antiviral Res. 122 (2015) 12–19. P.L. Beaulieu, J. De Marte, M. Garneau, L. Luo, T. Stammers, C. Telang, D. Wernic, G. Kukoli, J. Duan, A prodrug strategy for oral delivery of poorly soluble HCV NS5B thumb pocket 1 polymerase inhibitor using self-emulsifying drug delivery systems (SEDDS), Bioorg. Med. Chem. Lett. 25 (2015) 210–215. S. Venkatraman, F. Velazquez, S. Gavalas, W. Wu, K.X. Chen, A.G. Nair, F. Bennelt, Y. Huang, P. Pinto, Y. Jiang, O. Selyutin, B. Vibulbhan, Q. Zeng, C. Lesburg, J. Duca, L. Heimark, H.C. Huang, S. Agrawal, C.K. Jiang, E. Ferrari, C. Li, J. Kozlowski, S. Rosenblum, N.Y. Shih, F.G. Njoroge, Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics, Bioorg. Med. Chem. 22 (2014) 447– 458.

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

[104] O. Sari, V. Roy, M. Métifiot, C. Marchand, Y. Pommier, S. Bourg, P. Bonnet, R.F. Schinazi, L.A. Agrofoglio, Synthesis of dihydropyrimidine α,γ-diketobutanoic acid derivatives targeting HIV integrase, Eur. J. Med. Chem. 104 (2015) 127–138. [105] V. Nair, M. Okello, Integrase inhibitor prodrugs: Approaches to enhancing the antiHIV of β-diketo acids, Molecules 20 (2015) 12623–12651. [106] G. Xu, H.L. McLeod, Strategies for enzyme/prodrug cancer therapy, Clin. Cancer Res. 7 (2001) 3314–3324. [107] A. Stańczak, M. Szumilak, Proleki w terapii nowotworów. Część II. Strategie ADEPT i V/GDEPT. Prodrugs in cancer therapy. Part II. ADEPT and V/GDEPT strategies, Farm. Przegl. Nauk. 5 (2010) 11–16 (in polish). [108] K.D. Bagshawe, S.K. Sharma, R.H. Begent, Antibody-directed enzyme prodrug therapy (ADEPT) for cancer, Expert Opin. Biol. Ther. 4 (2004) 1777–1789.

29

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

[109] K.D. Bagshawe, Antibody-directed enzyme prodrug therapy (ADEPT) for cancer, Expert Rev. Anticancer Ther. 6 (2006) 1421–1431. [110] F. Kratz, I.A. Müller, C. Ryppa, A. Warnecke, Prodrug strategies in anticancer chemotherapy, Chem. Med. Chem. 3 (2008) 20–53. [111] K. Hida, J. Hanes, M. Ostermeier Directed evolution for drug and nucleic acid delivery, Adv. Drug Deliv. Rev. 59 (2007) 1562–1578. [112] I. Niculescu-Duvaz, C.J. Springer, Antibody-directed enzyme prodrug therapy (ADEPT): a review, Adv. Drug Deliv. Rev. 26 (1997) 151–172. [113] P.D. Senter, J.C. Springer, Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates, Adv. Drug Deliv. Rev. 53 (2001) 247–264. [114] P.D. Senter, Activation of prodrugs by antibody-enzyme conjugates: a new approach to cancer therapy, FASEB J. 4 (1990) 188–193. [115] V.M. Vrudhula, P.D. Senter, K.J. Fischer, P.M. Wallace, Prodrugs of doxorubicin and melphalan and their activation by a monoclonal antibody-penicillin-G amidase conjugate, J. Med. Chem. 36 (1993) 919–923. [116] K. Bosslet, J. Czech, D. Hoffmann, Tumor-selective prodrug activation by fusion protein-mediated catalysis, Cancer Res. 54 (1994) 2151–2159. [117] V.M. Vrudhala, H.P. Svensson, P.D. Senter, Cephalosporin derivatives of doxorubicin and melphalan and their activation by monoclonal antibody-lactamase conjugates, J. Med. Chem.38 (1995) 1380–1385. [118] R.P. Alexander, N.R. Beeley, M. O'Driscoll, F.P. O’Neill, T.A. Millican, A.J. Pratt, F.W. Willenbrock, Cephalosporin nitrogen mustard carbamate prodrugs for „ADEPT”, Tetrahedron Lett. 32 (1991) 3269–3272. [119] G.R. Melton, R.F. Sherwood, Antibody-enzyme conjugates for cancer therapy, J. Natl. Cancer Inst. 88 (1996) 153–156. [120] P.M. Wallace, J.F. Macmaster, V.F. Smith, W.L. Cosand, Intratumoral generation of 5-fluorouracil mediated by an antibody-cytosine deaminase conjugate in combination with 5-fluorocytosine, Cancer Res. 54 (1994) 2719–2723. [121] P.D. Senter, M.G. Saulnier, G.J. Schreiber, D.L. Hirschberg, D.L. Brown, I. Hellström, K.E. Hellström, Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate, Proc. Natl. Acad. Sci. USA 85 (1988) 4842–4846. [122] K.N. Syrigos, G. Rowlinson-Busza, A.A. Epenetos, In vitro cytotoxity following specific activation of amygdalin by β-glucosidase conjugated to a bladder cancerassociated monoclonal antibody, Int. J. Cancer 78 (1998) 712–719. [123] O. Greco, G.U. Dachs, Gene directed enzyme/prodrug therapy of cancer: Historical appraisal and future prospectives, J. Cell. Physiol. 187 (2001) 22–36. [124] J. Sirhan, R. Karaman, Gene directed enzyme prodrug therapy (GDEPT), in: Karaman, R. Prodrugs design – a new era, Nova Publisher, USA, 2014, pp. 210–232. [125] H. Wang, X.L. Zhou, W. Long, J.J. Liu, F.Y. Fan, A fusion protein of RGD4C and βlactamase has a favorable targeting effect in its use in antibody directed enzyme prodrug therapy, Int. J. Mol. Sci. 16 (2015) 9625–9634. [126] P. Wentworth, A. Datta, D. Blakley, T. Boyle, L.J. Partridge, G.M. Blackbum, Toward antibody-directed “abzyme” prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and it in vitro application to human tumor cell killing, Proc. Natl. Acad. Sci. USA 93 (1996) 799–803.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

30

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

[127] A. Mayer, R.J. Francis, S.K. Sharma, B. Tolner, C.J. Springer, J. Martin, G.M. Boxer, J. Bell, A.J. Green, J.A. Hartley, C. Cruickshank, J. Wren, K.A. Chester, R.H.J. Begent, A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody-enzyme fusion protein MFECP1 and a bis-iodo phenol mustard prodrug, Clin. Cancer Res. 12 (2006) 6509–6516. [128] A. Mayer, R. Francis, S.K. Sharma, G. Sully, S. Parker, B. Tolner, N. Griffin, N. Germain, P. Beckett, G. Boxer, A. Green, A. Chester, R.H. Begent, A phase I/II trial of antibody directed enzyme prodrug therapy (ADEPT) with MFECP1 and ZD2767P, Br. J. Cancer 91 (2004) 8–23. [129] H. Aloysius, L. Hu, Targeted prodrug approaches for hormone refractory prostate cancer, Med. Res. Rev. 35 (2015) 554–585. [130] D.C. Osipovitch, A.S. Parker, C.D. Makokha, J. Desrosier, W.C. Kett, L. Moise, C. Bailey-Kellogg2, K.E. Griswold, Design and analysis of immune-evading enzymes for ADEPT therapy, Protein. Eng. Des. Sel. 25 (2012) 613–623. [131] C.J. Springer, I. Niculescu-Duvaz Gene-directed enzyme prodrug therapy (GDEPT): choice of prodrugs, Adv. Drug Deliv. Rev. 22 (1996) 351–364. [132] D.J. Kerr, L.S. Young, P.F. Searle, A. McNeish, Gene directed enzyme prodrug therapy for cancer, Adv. Drug Deliv. Rev. 26 (1997) 173–184. [133] M. Rooseboom, J.N. Commandeur, N.P.E. Vermeulen, Enzyme-catalyzed activation of anticancer prodrugs, Pharmacol. Rev. 56 (2004) 53–102. [134] J. Zhang, V. Kale, M. Chen, Gene-directed enzyme prodrug therapy, AAPS J. 17 (2015) 102–110. [135] I.V. Alekseenko, E.V. Snezhkov, I.P. Chernov, V.V. Pleshkan, V.K. Potapov, A.V. Sass, G.S. Monastyrskaya, E.P. Kopantzev, T.V. Vinogradova, Y.V. Khramtsov, A.V. Ulasov, A.A. Rosenkranz, A.S. Sobolev, O.A. Bezborodova, A.D. Plyutinskaya, E.R .Nemtsova, R.I. Yakubovskaya, E.D. Sverdlov, Therapeutic properties of a vector carrying the HSV thymidine kinase and GM-CSF genes and delivered as a complex with a cationic copolymer, J. Trans. Med. 4 (2015) 13:78 doi: 10.1186/s12967-0150433-0. [136] F. Husseini, J.P. Delord, C. Fournel-Federico, J. Guitton, P. Erbs, M. Homerin, C. Halluard, C. Jemming, C. Orange, J.M. Limacher, J.E. Kurtz, Vectorized gene therapy of liver tumors: oroof of concept of TG4023 (MVA-FCU1) in combination with flucytosine, Annal. Oncol. (2016) Doi: 10.1093/annonc/mdw440. [137] A.D. William, Suicide gene therapy, J. Biomed. Biotechnol. 1 (2003) 48–70. [138] C. Avendano, C.J. Menendez, Medicinal chemistry of anticancer drugs, Elsevier B.V., Oxford, 2008. [139] S.H. Chen, H.D. Shine, J.C. Goodman, R.G. Grossman, S.L. Woo, Gene therapy for brain tumors: Regression of experimental gliomas by adenovirus-mediated gene transfer in vivo, Proc. Natl. Acad. Sci. USA 91 (1994) 3054–3057. [140] C.A. Mullen, M. Kilstrup, R.M. Blaese, Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system, Proc. Natl. Acad. Sci. USA 89 (1992) 33–37. [141] X. Guo, T.R. Evans, S. Somanath, A.L. Armesilla, J.L. Darling, A. Schatzlein, J. Cassidy, W. Wang, In vitro evaluation of cancer-specific NF-κB-CEA enhancer– promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines, Br. J. Cancer 97 (2007) 745–754.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

31

ACCEPTED MANUSCRIPT [142] C.R. Miller, C.R. Williams, D.J. Buchsbaum, G.Y. Gillespie, Intratumoral 5fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas, Cancer Res. 62 (2002) 773–780.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

[143] J. Hlavaty, H. Petznek, H. Holzmüller, A. Url, G. Jand, A. Berger, B. Salmons, W.H. Günzburg, M. Renner, Evaluation of a gene-directed enzyme-prodrug therapy (GDEPT) in human pancreatic tumor cells and their use in vivo models for pancreatic cancer, PLoS ONE 7 (2012) e40611. Doi:10.1371/journal.pone.0040611. [144] A.V. Patterson, M.P. Saunders, O. Greco, Prodrugs in genetic chemoradiotherapy, Curr. Pharm. Des. 9 (2003) 2131–2154. [145] L. Chen, D.J. Waxman, Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer, Curr. Pharm. Des. 8 (2002) 1405–1416. [146] G. Bonifert, L. Folkes, C. Gmeiner, G. Dachs, O. Spadiut, Recombinant horseradish peroxidase variants for targeted cancer treatment, Cancer Mde. 5 (2016) 1194–1203. [147] M. Matuskova, Z. Kozovska, L. Toro, E. Durinikova, S. Tyciakova, Z. Cierna, R. Bohovic, L. Kucerova, Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases, J. Exp. Clin. Cancer Res. 9 (2015) 34:33 Doi: 10.1186/s13046-0150149-2. [148] I. Amara, W. Touati, P. Beaune, I. de Waziers, Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors, Biochimie 105 (2014) 4–11. [149] P. Garnier, X.T. Wang, M.A. Robinson, S. van Kasteren, A.C. Perkins, M. Frier, A.J. Faibanks, B.G. Davis, Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs, J. Drug Target. 18 (2010) 794–802. [150] M.A. Robinson, S.T. Charlton, P. Garnier, X.T. Wang, S.S. Davis, A.C. Perkins, M. Frier, R. Duncan, T.J. Savage, D.A. Wyatt, S.A. Watson, B.G. Davis, LEAPT: Lectindirected enzyme-activated prodrug therapy, Proc. Natl. Acad. Sci. USA 101 (2004) 14527–14532. [151] G.D. Brown, S. Gordon, Immune recognition: A new receptor for β-glucans, Nature 413 (2001) 36–37. [152] S.J. Lee, S. Evers, D. Roeder, A.F. Parlow, L. Risteli, Y.C. Lee, T. Feizi, H. Langen, M.C. Nussenzweig, Mannose receptor-mediated regulation of serum glycoprotein homeostasis, Science 295 (2002) 1898–1901. [153] E. Beutler, Gaucher disease, Blood Rev. 2 (1988) 59–70. [154] S. Aquaro, P. Bagnarelli, T. Guenci, A. De Luca, M. Clementi, E. Balestra, R. Caliò, C.F. Perno, Long‐term survival and virus production in human primary macrophages infected by human immunodeficiency virus, J. Med. Virol. 68 (2002) 479–488. [155] Q. Fu, Y. Wang, Y. Ma, D. Zhang, J.K. Fallon, X. Yang, D. Liu, Z. He, F. Liu, Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly, Sci. Rep. 5 (2015) 12023; doi: 10.1038/srep12023. [156] Q. Tan, J.K. Saggar, M. Yu, M. Wang, I.F. Tannock, Mechanisms of drugs resistance related to the microenvironment od solid tumors and possible strategies to inhibit them, Cancer J. 21 (2015) 254–262. [157] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy, Nature Rev. 11 (2011) 393–410.

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[158] J.D. Sun, Q. Liu, J. Wang, D. Ahluwalia, D. Ferraro, Y. Wang, J.X. Duan, W.S. Ammons, J.G. Curd, M.D. Matteuci, C.P. Hart, Selective tumor hypoxia targeting by huypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer, Clin. Cancer Res. 18 (2012), 758–770. [159] J.K. Saggar, I.F. Tannock, Activity of the hypoxia-activated pro-drug TH-302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of vhemotherapy, Int. J. Cancer 134 (2014) 2726–2734. [160] M. Slusarczyk, M.H. Lopez, J. Balzarini, M. Mason, W.G. Jiang, S. Blagden, E. Thompson, E. Ghazaly, C. McGuigan, Application of ProTide Technology to gemcitabine: a successful approach to overcome the key cancer resistance mechanisms leads to a new agent (NUC-1031) in clinical development, J. Med. Chem. 57 (2014) 1531–1542. [161] R. Zhu, H.A. Seow, R.P. Baumann, K. Ishiguro, P.G. Penketh, K. Shyam, A.C. Sartorelli, Design of a hypoxia-activated prodrug inhibitor of O6-alkylguanine-DNA alkyltransferase, Bioorg. Med. Chem. Lett. 22 (2012) 6242–6247. [162] X. Peng, V. Gandhi, ROS-activated anticancer prodrugs: a new strategy for tumorspecific damage, Ther. Deliv. 3 (2012) 823–833. [163] P. Lehouritis, M. Stanton, F.O. McCarthy, M. Jeavons, M. Tangney, Activation of multiple chemotherapeutic prodrugs by the natural enzymolome of tumor-localised probiotic bacteria, J. Control. Rel. 222 (2016) 9–17.

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ACCEPTED MANUSCRIPT Figures Fig. 1. Simplified diagram of the activation of prodrugs. Fig. 2. Metabolism of clopidogrel. Fig. 3. Prodrugs selected from the phosphate group. Fig. 4. Metabolism of adefovir dipivoxil.

Fig. 6. Metabolism of GS-5734 - compound active for Ebola virus.

RI PT

Fig. 5. General scheme of the design prodrugs of phosphate moiety.

Fig. 7. Chemical structure of triphosphate - analogue of 2’,3’-didehydro-3-deoxythymidine.

Fig. 9. Examples of amino acid analogs of acyclovir.

SC

Fig. 8. Metabolism of 6-deoxycyclopropavir.

Fig. 10. Examples of chemical combination with the active compounds biotin antiviral (acyclovir ACV, ganciclovir - GSV, cidofovir).

M AN U

Fig. 11. Modifications to the structure of ribavirin.

Fig. 12. Prodrugs selected from the group of inhibitors of HCV polymerase. Fig. 13. Simplified diagram of ADEPT therapy. Fig. 14. Simplified diagram of GDEPT therapy.

Fig. 15. Chemical structure the conjugate of vitamin E and paclitaxel.

TE D

Fig. 16. Mechanism of bioreductive prodrugs activation (e.g. TH-302).

Fig. 17. Chemical structure of gemcitabine ProTides: Acelarin (NUC-1031).

AC C

EP

Fig. 18. Chemical structure of O6-benzylguanine derivatives.

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Fig. 1

ACCEPTED MANUSCRIPT

O

O

CYP1A2 CYP2B6 CYP2C19

O

N

O

Cl

S

Clopidogrel

N HOOC HO-S

Cl

Cl

2-Oxo-clopidogrel GSH

PON-1 or -3

85% O

O

OH

O

O

N HOOC HS

Cl

SR26334

N

HOOC HS

Cl

Cl

R-130964 active

M AN U

"Endo"-isomer active

EP

TE D

Fig. 2

AC C

O

SC

N

O

RI PT

S

S

O

N O

CES-1

CYP2B6 CYP2C9 CYP2C19 CYP3A4

ACCEPTED MANUSCRIPT NH2 N

HOOC

N

N

NH2 N

N

O

O

N

O O

O

O O

O

O O

O

O

O

Adefovir dipivoxil

O

TDF, tenofovir disoproxil fumarate

NH2

N N

SC

NH2 N N

N

N

N

N

O

O

M AN U

O

O O

P

O

O

H3C(H2C)15O

N

O

RI PT

O

P

N

COOH

O

P

OH

N H

O

Hexadecyloxy-tenofovir

P

O

TAF, isopropylaninyl monoamidate-tenofovir

NH2

O NH

TE D

N

N

O O

O

O

O

O O

P

OH

O

HO

Hexadecyloxy-cidofovir

AC C

N

(CH2)20CH3 O O O

O

O

P

OH

O

P O

N

N N

O O R1: H or CH2CH2COOCH(CH2)2

OH

N4-Docosanoyl(behenoyl)-cidofovir

N H

R1

O HO

F

NH

N

N

O

Sofosbuvir

O

HN

N

O

OH

EP

H3C(H2C)15O

O

P

N H

Phosphoramidate of dioxolane adenosine nucluoside

Fig. 3

ACCEPTED MANUSCRIPT NH2 N N

N

N

N

O

O

O

HO

- (CH3)3CCOOH

O

O

Spontaneous P

O

HO

- HCHO

Adefovir dipivoxil

Esterase

- (CH3)3CCOOH

Spontaneous

N

M AN U

N

SC

- HCHO

NH2

N

O

O HO

P

Adefovir

OH

AC C

EP

TE D

Fig. 4

O

O

O

N

P

O

O O

O

RI PT

Esterase

P O

N

N

O

O O

N

N

N

O

O

NH2

NH2

N N

ACCEPTED MANUSCRIPT X

X

R1

NH

O

O

Nucleobase

-O-sugar or -CH2-O-CH2-CH2-

P O

-CH2-O-CH2-CH2-

X: O-(CH2)2-S-CO-R2 R1

X: OAr , phosphonamidate X: NH-CH(R3)-COOR4, phosphonodiamidate

O

or O-sugar (cyclic)

SATE

ProTides

O P

O

or -CH2-O-CH2-CH2-

Nucleobase

O

O P

O

M AN U

HepDirect

TE D

Fig. 5

EP

Ar

-O-sugar -

-O-sugar or

SC

O

Nucleobase

S

AC C

R2

P

O

RI PT

O

-O-sugar or

-CH2-O-CH2-CH2-

cycloSal

Nucleobase

ACCEPTED MANUSCRIPT

NH2

NH2

N

N

O N H

O

O

-

N

O

P O

O

CN

O

O-

O

OH

HO

NH2

O O

-

OH

Nucleoside monophosphorate

TE D EP

O

P

O

O-

P

O

-

N

O

O

HO

CN OH

Nucleoside triphosphorate

Fig. 6

AC C

p O-

CN HO

O

O

O

M AN U

O-

N

O

SC

N

N O P

OH

Alanine metabolite NH2

O

CN HO

GS-5734

-

N

O

P

N H

RI PT

O

O

ACCEPTED MANUSCRIPT O O

R

O

P

O O

P OH

NH

P

O

OH

O

R

N

O

SC

O

O O

RI PT

O O

O

M AN U

Triphosphate analogue of 2'3'-didehydro-3-deoxythymidine (Stavudine, d4T)

AC C

EP

TE D

Fig. 7

ACCEPTED MANUSCRIPT O N

N

N

Xanthine oxidase

HO

NH

HO

N

N

N

NH2

OH

N

NH 2

OH

AC C

EP

TE D

M AN U

SC

Fig. 8

RI PT

Cyclopropavir

6-Deoxycyclopropavir

ACCEPTED MANUSCRIPT

O N

N

O

NH

O N

N H

(Pro-Val)2-H

H-Val-Pro-Val

O

O

Tripeptide ester

M AN U

SC

Tetrapeptide amide

TE D

Fig 9

EP

N

N

RI PT

O

NH

AC C

HO

N

O

NH2

ACCEPTED MANUSCRIPT O N

NH

O H HN

N

NH2

S H

N H

O

N

O

O

Biotin-ACV

RI PT

O

N

O

O

S N H

O

NH2

Biotin-12-Hydroxystearic acid-ACV

H

M AN U

HN

N

SC

O

H

N

O

O

NH

O N

NH

O

H

O

HN N H

N

NH2

HO

H

Biotin-GCV

TE D

O

S

N

O

NH2 N

O

HN

N

NH

O

H H N

AC C

EP

H

S

O O n

O

Biotin-C2-Cidofovir Biotin-C6-Cidofovir Biotin-C12-Cidofovir

Fig 10

P

O

O

ACCEPTED MANUSCRIPT

HN

NH2

N O

N

N

HO

O

NH2

N

N

O

N

HO

O R

O

O

NH2

N

N

O

P

N

O

OH

Levovirin

Hemoglobin

O N H

O

P

O

O

NH2

N

N

Aloxyalkylphosphonate ester

HO

O

N

Polymer

OH

OH

SC

Taribavirin

Lys

HO

OH

HO

M AN U

HO

RI PT

OH

OH

TE D AC C

EP

Fig. 11

N

N

OH

n

Hb-RBV

NH2

N

O HO

O

Polymer-RBV

ACCEPTED MANUSCRIPT

NH

NH2 O

O O

N O O

O

O

O

N

F

N

N3 O

O

O

RI PT

O

N

Cl

HN N

Balapiravir

F

O

O

OH O

N

N N

O

O O

M AN U

O

SC

Ester product of tricyclic indole derived inhibitor of HCV

F

O O

OH

TE D

Dihydropyrimidine α,γ-diketobutanoic acid derivative

AC C

EP

Fig. 12

O

F

Pyridinone α,γ-diketobutanoic acid derivative

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 13

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig 14

ACCEPTED MANUSCRIPT

O O

O O O

OH O

O O

O

O

O

S S O

O O

M AN U

PTX-S-S-VE

RI PT

NH

OH

SC

O

O

AC C

EP

TE D

Fig. 15

ACCEPTED MANUSCRIPT a) N

N

N

H N

O P

CH3

Br

e

-

O2N N

Br

O

.O2

N O H

O2

TH-302

b)

[Prodrug] .O2

.-

M AN U

Prodrug

X

.O2

O2

EP

Y

O2

TE D

R. + Drug

AC C

-O

H N P

N O H

SC

Two-electrone reductase One-electrone reductase

Br

P

CH3

N O H

Br

H N

RI PT

O2N

Fig. 16.

Potential active species

Z

Br Br

RI PT

ACCEPTED MANUSCRIPT

NH2 O P

N O N

NH F

BnO CH3 HO

F

M AN U

O

O

SC

O

TE D

Acelarin (NUC-1031)

AC C

EP

Fig. 17.

H3C

N

O

CH3 H3C

H3C N

N

CH3

CH3 O

N O H

N

N

N H

RI PT

ACCEPTED MANUSCRIPT

SC M AN U

Prodrug

EP

TE D

Fig. 18.

N

N

H2N

O2N

AC C

O

N

N H

ACCEPTED MANUSCRIPT Highlights Prodrugs are a wide group of substances of low or no pharmacological activity.

•

Prodrugs can be activated chemically or enzymatically.

•

More and more of all marketed medicines can be classified as prodrugs.

•

The biochemistry, genetics and microbiology gives the direction to pharmacy.

AC C

EP

TE D

M AN U

SC

RI PT

•