Amino acids as promoieties in prodrug design and development

Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

ADR-12406; No of Pages 16

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Advanced Drug Delivery Reviews

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Amino acids as promoieties in prodrug design and development☆

Balvinder S. Vig a,⁎, Kristiina M. Huttunen b, Krista Laine b, Jarkko Rautio b

a Bristol-Myers Squibb Company, USAb University of Eastern Finland, Finland

☆ This review is part of the Advanced Drug Delivery Re⁎ Corresponding author at: Bristol-Myers Squibb Com

E-mail address: [email protected] (B.S. Vig).

0169-409X/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.addr.2012.10.001

Please cite this article as: B.S. Vig, et al., Amidx.doi.org/10.1016/j.addr.2012.10.001

a b s t r a c t
a r t i c l e i n f o
Article history:Received 20 June 2012Accepted 16 October 2012Available online xxxx

Keywords:SolubilityPermeabilityHydrolysisStabilityDrug deliveryTargetingTransportersFormulationChallenges

Prodrugs are biologically inactive agents that upon biotransformation in vivo result in active drug molecules.Since prodrugs might alter the tissue distribution, efficacy and the toxicity of the parent drug, prodrug designshould be considered at the early stages of preclinical development. In this regard, natural and syntheticamino acids offer wide structural diversity and physicochemical properties. This review covers the use ofamino acid prodrugs to improve poor solubility, poor permeability, sustained release, intravenous delivery,drug targeting, andmetabolic stability of the parent drug. In addition, practical considerations and challenges as-sociated with the development of amino acid prodrugs are also covered.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Amino acids as promoieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Structural diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Amino acid prodrug linkage chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Solubility of amino acids and their prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Chemical stability of amino acid prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.5. Enzymatic stability of amino acid prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Amino acid prodrugs in drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. Prodrugs for improved oral drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1.1. Overcoming poor aqueous solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1.2. Utilization of intestinal transporters for oral drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.2. Sustained release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Prodrugs for intravenous delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.4. Drug targeting by amino acid prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.5. Enhanced metabolic stability by amino acid prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Practical considerations and challenges associated with the development of amino acid prodrugs for oral delivery . . . . . . . . . . . . . . . 04.1. Discovery and screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.3. Analytical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.4. Formulation/stability challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.5. Safety and toxicology assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

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2 B.S. Vig et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

1. Introduction

Prodrugs are bioreversible derivatives of pharmacologically activeagents that must undergo an enzymatic and/or chemical transforma-tion in vivo to release the active parent drug, which can then elicit itsdesired pharmacological effect (Fig. 1). In most cases, prodrugs aresimple chemical derivatives that are one or two chemical or enzymat-ic steps away from the active parent drug. Prodrugs have offered aversatile approach to enhance the clinical usefulness of many phar-macological agents in the past by improving their physicochemical,biopharmaceutical or pharmacokinetic properties [1,2], and as manyas 20% of all small molecule drugs approved during the period2000–2008 can be classified as prodrugs [3,4]. Moreover, dozens ofprodrugs are currently undergoing clinical trials. Despite these highnumbers, pharmaceutical scientists have only begun to understandthe full potential of prodrug approach in modern drug discoveryand development, and many novel prodrug innovations are awaitingtheir discovery.

Increasingly, prodrug approach is becoming an integral part ofdrug delivery and discovery stratagem. Since prodrugs might alterthe tissue distribution, efficacy and the toxicity of the parent drug,prodrug design should be considered at the early stages of preclinicaldevelopment. The choice of the promoiety is largely dependent onthe objective of the prodrug approach e.g., improving solubility andpermeability as well as functional groups on parent drug that areamenable for prodrug derivatization. Literature is laden with exam-ples of prodrugs and promoieties with most common being alkyland phosphate prodrugs.

Amino acid prodrugs have a proven track of improving oral deliveryof the drugs that have poor solubility and permeability. Introducing anamino acid, either a natural or its derivative, to a parent drug usually in-creases the water solubility by several orders of magnitude through anionized carboxylate anion or ammonium cation. Moreover, varioustransporters needed for absorption of amino acids and oligopeptidesare expressed in the brush-border membranes of intestinal epithelialcells and are found to play a significant role in the absorption of severalamino acid prodrugs. For example, the enhanced oral bioavailability ofclinically used valacyclovir and valganciclovir, amino acid esterprodrugs of acyclovir and ganciclovir, respectively, has been attributedto their enhanced intestinal transport via H+-coupled peptide trans-porter 1 (PEPT1) [5,6]. More recently, amino acid prodrugs have provid-ed controlled drug release in the form of lisdexamfetamine dimesylatethat is a L-lysine amino acid amide prodrug of D-amphetamine [7].

Fig. 1. Amino Acid (AA) prodrugs

Please cite this article as: B.S. Vig, et al., Amino acids as promoieties in prdx.doi.org/10.1016/j.addr.2012.10.001

Amino acid prodrugs have proven commercial and regulatory track,which is beneficial in bringing such drugs to the patients. In addition,a number of preclinical examples of using amino acids as promoietiesfor other drug delivery purposes such as parenteral and targeted drugdelivery have been described in the literature.

Despite the success of various amino acid prodrugs, surprisingly noother amino acid prodrug reviews except a recent editorial [8] havebeen published to date. Therefore, we hope that this timely reviewusing both marketed and investigational amino acid prodrugs as exam-ples will be of interest for scientists working in the area of drug discov-ery and development as well as those working in clinic.

2. Amino acids as promoieties

A wide variety of promoieties have been used to overcome liabil-ities associated with drugs [2]. The selection of promoiety dependson the purpose of the prodrug, type of functional groups availableon the parent drug, chemical and enzymatic conversion mechanismsof prodrug to parent drug, safety of the promoiety, and ease ofmanufacturing. In this regard, amino acids as promoieties offer sever-al advantages (Table 1).

2.1. Structural diversity

In general terms, amino acids are molecules containing an aminegroup, a carboxylic acid group and a side-chain that varies between dif-ferent amino acids (Fig. 2). The 20 natural amino acids are commonlyfound in proteins and are also referred as alpha amino acids. In addition,selenocysteine and pyrrolysine are often referred as the natural aminoacids. Besides the 22 natural amino acids, a variety of other amino acids(e.g., β-alanine, α-aminobutyric acid, γ-aminobutyric acid, ornithine)are also found in minor amounts in proteins and in non-protein com-pounds. The α-amino acids differ in the nature of the side-chain(R group) attached to their α-carbon, which can vary in size, rangingfrom just one hydrogen atom in glycine to a large heterocyclic group intryptophan. α-Amino acids are typically divided into three categories,based on the properties of their side-chain (Fig. 2); the first containingα-amino acidswith relatively nonpolar R groups (glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, tryptophan, andmethionine),the second containingα-amino acids with uncharged but polar R groups(serine, threonine, cysteine, tyrosine, asparagine, glutamine), and thethird containing α-amino acids with charged R groups (aspartic acid,glutamic acid, lysine, arginine, histidine).

concept in oral drug delivery.

odrug design and development , Adv. Drug Deliv. Rev. (2012), http://



Table 1Advantages of using amino acids as promoieties.

Advantage Rationale

Large structural diversity ▪ 20 common natural amino acids▪ A number of other naturally occurring amino acids▪ Extensive array of synthetic amino acids

Wide-range of functional groups for attachment to parent drug ▪ α-Amine and α-carboxylic group▪ Side chain functional groups e.g., amine, carboxylic acid, alcohol, and thiol

Well established prodrug chemistry ▪ Commonly used linkage chemistry between amino acid and parent including ester,amide, carbonate, carbamate

Commercial availability ▪ Large number of amino acid suppliers availableFewer safety concerns ▪ Amino acids are building blocks for proteins and are generally regarded as safe.Substrates for various intestinal influx transporters ▪ Can potentially target carrier-mediated transporters for improved delivery across

cell membranesAvailability of commercially successful amino acid prodrugs ▪ Many amino acid prodrugs have been commercially developed and helping patients.

▪ Commercial and regulatory precedence in developing amino acid prodrugsUsed to improve pharmaceutical properties of marketed drugsor difficult compounds

▪ Improved pharmaceutical properties of already marketed compounds e.g., valacyclovir,valganciclovir and new chemical entities e.g., brivanib alaninate, LY354740

▪ Proven to improve solubility, permeability, sustained release, targeting transporter,overcoming resistance, etc.

3B.S. Vig et al. / Advanced Drug Delivery Reviews xxx (2012) xxx–xxx

All α-amino acids, except glycine, have a chiral α-carbon and canexist in two optical isomers, L- and D-form. The L-form of amino acidsoccurs naturally and prodrugs utilizing these amino acids are generallyactivated by naturally occurring enzymes. The L- and D-amino acidprodrugs tend to have very similar physicochemical properties but thelatter is generally more stable to hydrolysis by naturally occurring en-zymes [9]. This property of amino acid is often utilized by medicinalchemists to develop stable amino acid prodrugs. In addition to thenatural amino acids and their D-forms, extensive arrays of syntheticamino acids and di-/tri-peptides are commercially available for medic-inal chemists as promoieties [10]. Amino acids as promoieties perhapsoffer the most structural diversity and expanse of physicochemicalproperties.

2.2. Amino acid prodrug linkage chemistry

Most amino acid prodrugs are either esters or amides, in whichα-amine or α-carboxylic group of amino acid is attached to parent

Fig. 2. Natural amino acid


functional groups (hydroxyl, amine, and carboxyl). α-Amine orα-carboxylic group of amino acid can also be linked to parent drug viacarbonate and carbamate links. The use of amino acid side chains,which offer wealth of functional groups (e.g., amine, carboxylic acid, al-cohol, thiol), as prodrug handles presents tremendous opportunities inprodrug design. The prodrug linkage structures for the most commonfunctionalities are illustrated in Fig. 3. While in most cases amino acidsare directly conjugated to the parent drugs, bifunctional linkers havebeen used to further increase the structural diversity and expand thetypes of parent drugs that could be linked to amino acids [11,12].

2.3. Solubility of amino acids and their prodrugs

Introducing an amino acid, either a natural or its derivative, to aparent drug usually increases the water solubility by several orders ofmagnitude through an ionized carboxylate anion or ammonium cation.Consequently, there are several amino acid ester prodrugs that are in-vestigated as water soluble derivatives for oral administration [13–16].

s structural diversity.




Fig. 3. Typical links between amino acid promoieties and parent drugs.


Amino acid esters [17,18], amino acid amides [19–22] or other aminoacid examples (amidoximes [23], ureas [24]) have been investigatedfor parenteral use, albeit to a lesser extent.

All natural α-amino acids except proline have a primary α-aminegroup. In proline, the side-chain links to theα-amino group and, there-fore, it is the only amino acid containing a secondary amine at this posi-tion. The pKa of theα-amino group is typically in the range of 8.8 to 10.6(Table 2). Theα-amino acids contain an acidicα-carboxyl groupwith apKa typically between 1.7 and 2.6 (Table 2). The dissociation constant ofboth theα-amino andα-carboxyl groups are affected by each other andthe side chain. The α-amino group in its charged state is strongly elec-tron withdrawing and makes the α-carboxyl group more acidic, there-by lowering its pKa as compared with typical carboxylic acid pKa ofapproximately 4.5.

At physiological pH, the predominant form of α-amino acids con-tains both a negative carboxylate and a positive ammonium group.This molecular state is known as a zwitterion, and has minimum sol-ubility at the isoelectric point (mid-point between 2 pKas, Fig. 4).

Table 2Properties of naturally occurring amino acids.

Amino acid Code Mol. wt Hydropathy Charge

Polar and chargedArginine R 174.20 Hydrophilic +Histidine H 155.16 Moderate +Lysine K 146.19 Hydrophilic +Aspartate D 133.10 Hydrophilic -Glutamate E 147.13 Hydrophilic -

Polar and not chargedSerine S 105.09 Hydrophilic NThreonine T 119.12 Hydrophilic NAsparagine N 132.12 Hydrophilic NGlutamine Q 146.15 Hydrophilic NCysteine C 121.16 Moderate N

Non polarGlycine G 75.07 Hydrophobic NAlanine A 89.09 Hydrophobic NProline P 115.13 Hydrophobic NValine V 117.15 Hydrophobic NIsoleucine I 131.17 Hydrophobic NLeucine L 131.17 Hydrophobic NMethionine M 149.21 Moderate NPhenylalanine F 165.19 Hydrophobic NTryptophan W 204.22 Hydrophobic NTyrosine Y 181.19 Hydrophobic N

pKa values from Introduction to Organic and Biochemistry, 4th ed. William H. Brown, Brooks


However, in the most α-amino acid prodrugs either the carboxylicacid or amine group is linked to a parent drug abolishing zwitterionicfunction. Exceptions are α-amino acids that have an additionalfunctional group in the side chain amenable to prodrug formation(e.g., –SH in cysteine, –OH in tyrosine, –NH2 in lysine, –COOH in asparticand glutamic acid) and, thus, they can maintain a double charge.

Water solubility ofα-amino acids themselves is largely a function ofthe polar or nonpolar nature of the side chain (Table 2). An increase inhydrocarbon content of the side chain (R group) from glycine (R=H)to valine R=(CH(CH3)2) and leucine R=(CH2CH(CH3)2) decreaseswater solubility. Consequently, amino acids with shorter hydrocarbonside chain resulted in higher aqueous solubility as compared to aminoacids with longer hydrocarbon side chains in a series of α-amino acidamide prodrugs of dapsone [21]. Moreover, α-amino acids with un-charged but polar R groups are generally more soluble than the corre-sponding α-amino acids having nonpolar R groups. This solubilitydata is in line with the observation from several studies where phenyl-alanine, as a promoiety, resulted in the lowest soluble prodrug in a

pKa, NH2 pKa, COOH pKa, R Calculated solubility molality

9.04 2.01 12.48 1.1259.10 1.77 6.10 0.2818.95 2.18 10.53 1.6879.82 2.10 3.86 0.0389.47 2.10 4.07 0.060

9.15 2.21 3.4809.10 2.09 0.8228.80 2.02 0.1909.13 2.17 0.291

10.25 1.86 8.00 0.211

9.78 2.35 3.3709.87 2.35 1.867

10.60 2.00 11.2989.72 2.29 0.5019.76 2.32 0.2429.74 2.33 0.1679.20 2.28 0.3759.24 2.58 0.1709.39 2.38 0.0659.11 2.20 10.07 0.003

/Cole Publishing Company, 1987, p. 356. Calculated solubilities from [25].




Fig. 4. Theoretical pH–solubility profile of an amino acid prodrug with free α-aminogroup (7.13 pKa) and an amino acid prodrug with a free α-carboxylic group (3.56 pKa),both with an intrinsic solubility if 0.1 mg/ml.

Fig. 6. pH–Stability profile of DMMTX at 37 °C. Data from reference [29].


series of amino acid prodrugs [21,26]. Among the α-amino acids withionizable R groups, lysine renders relatively high water solubility overthe wide pH range due to the ionized ε-amine regardless of prodrugtype (e.g., ester or amide) [21,27].

Introduction of amino acid generally introduces a charge to themolecule; consequently, the molecule exhibits a pH-dependent solu-bility. The net ionization and thus the pH–solubility profile of themolecule will depend on the number of ionic groups present andtheir pKa. Esterification of the α-carboxylic group of an amino acidby a non-ionic parent drug typically reduces the pKa of the α-aminefrom approximately 9.5 to 7. Whereas amidation of the α-aminogroup of an amino acid by a non-ionic parent drug typically increasesthe pKa of the α-carboxyl from approximately 2.2 to 3.5. Theoretical(using Henderson–Hasselbalch equation) pH–solubility profile of anamino acid prodrug with free α-amino group (pKa 7.13) and anamino acid prodrug with a free α-carboxylic group (pKa 3.56), bothwith an intrinsic solubility of 0.1 mg/ml, is shown in Fig. 4. Sinceionization and water solubility of amino acid prodrugs are highly-

Fig. 5. General scheme for chemical hydrolysis


dependent on the gastric pH, it is important to characterize pH–solubility profile of an amino acid prodrug, and its impact of the bio-availability as the molecule passes through various portions of theGI-tract.

2.4. Chemical stability of amino acid prodrugs

Amino acids prodrugs are designed such that they have reasonablygood chemical stability and are converted (or activated) to parentdrug by enzymes. The high chemical stability reduces the conversionof the prodrug to parent during the shelf life and normal handling ofthe prodrug, while activation by enzyme allows for rapid and quanti-tative conversion to the parent drug. Amino acid ester and amideprodrugs are hydrolyzed by the same acid or base catalyzedmechanism(Fig. 5) and by a number of endogenous hydrolases [28]. However, as ageneral rule, amide bond is more stable than the ester bond to chemicaland enzymatic hydrolysis. A “U” shape pH–stability profile that is typi-cal of most ester and amide amino acid prodrugs is shown in Fig. 6 [29].Generally, ester and amide prodrugs are least stable at the high pH andvery low pH and show pH of maximum stability in-between.

Indeed, some amino acid ester prodrugs display low chemical sta-bility in aqueous solutions as represented by esters of metronidazole

of amino acid ester and amide prodrugs.





[17,30], acyclovir [31] and acetaminophen [32]. The reason for lowstability is due partly to the neighboring amino group which can par-ticipate in intramolecular catalysis, and partly to the strong electron-withdrawing effects of the protonated amino group that activatesthe ester linkage [30,33]. This facilitating effect of the amino groupfor ester bond hydrolysis can be successfully diminished by extendingthe amino group from the ester bond linkage [34,35]. For example,aminomethylbenzoate esters of metronidazole [35], hydrocortisone,prednisolone andmethylprednisolone [36] showed high chemical sta-bility and yet retained a rapid rate of enzymatic ester hydrolysis. Alsousing the aspartic acid as the promoiety a more chemically stableprodrug was obtained among the floxuridine amino acid esters. Theimproved chemical stability of these prodrugs was potentially due tothe negatively charged side chain that may hinder hydroxide ion ca-talysis of the ester bond [37].

The amide bond is less polarized than the ester bond due to thelower electronegativity of the nitrogen compared to the correspond-ing oxygen, thus, rendering the amide bond more resistant than theester bond to chemical hydrolysis. This is illustrated by amino acidamides of dapsone, which irrespective of the amino acid promoiety(glycine, alanine, leucine, phenylalanine, lysine) had the projectedshelf-lives (t90%) greater than 2 years at pH 4 [21].

The amino acid promoiety influences the chemical stability ofprodrugs in a profound manner. In general, short chain aliphatic aminoacids (glycine, alanine) and the secondary amino acid proline result inester and amide prodrugs that undergo faster chemical hydrolysis inaqueous solutions than prodrugs with branched aliphatic amino acids(valine, isoleucine) or aromatic amino acids (phenylalanine). Possiblecontributing factors to the differences are both small variations in thepKa values of α-amino group and steric hindrance at the reactive site.For example, the presence of a highly ionized α-amine group in proline(pKa 10.60) in the acyl portion of the ester prodrugs is capable of in-creasing the chemical hydrolysis rate of ester bondsmaking prolyl estersthe least chemically stable among series of amino acid esters [37–39].The short half-life may be desirable, as in the case of chewable prolineester prodrug of acetaminophen that has very short half life (about4 min) at pH 7.4, and that is expected to quickly convert into acetamin-ophen after swallowing [32]. The influence of the steric factors on chem-ical hydrolysis is aptly illustrated by the amino acid amides of dapsone.The rates of hydrolysis of prodrugs decreased in the order glycine>alanine>phenylalanine>lysine>leucine which was better explainedby steric factors at the amide bond than differences in the pKa values[21]. Steric factors may also explain that incorporating the L-valineamino acid possessing a branched side chain has led to the developmentof twomarketed and several clinically tested L-valine prodrugs which allshow reasonable good chemical stability over the physiological pHrange. One of these prodrugs, valacyclovir showed maximal stability atpH under 4 and hydrolyzed only 2% after a period of 24 h [40]. Finally,the stereochemistry of amino acids appeared to have a slight [39] or noeffect [38,41] on the chemical stability of amino acid prodrugs. Althoughthere are some general rules regarding the chemical stability of aminoacid prodrugs, it is important to characterize pH–stability profile ofamino acid prodrugs and determine pH of maximum stability, which isvery important in guiding formulation choices and predicting stabilitychallenges.

2.5. Enzymatic stability of amino acid prodrugs

The enzymatic hydrolysis of amino acid esters and amides by var-ious hydrolases (e.g., esterases and/or peptidases) is far more effec-tive than chemical hydrolysis. The half-lives of prodrugs are usuallyseveral orders of magnitude shorter in blood or tissue homogenatesthan in aqueous solutions [28]. Often the activating enzymes areunidentified. However, it has been suggested that the biphenylhydrolase-like protein, human valacyclovirase (VACVase), is respon-sible at least partly for hydrolysis of the prodrugs valacyclovir and


valganciclovir, and might be involved in the activation of other aminoacid prodrugs as well [42–44]. As the specificity of this enzyme residesmainly in the amino acid acyl promoiety and to a less extent in thealcohol moiety of a parent drug and that theα-amino group in the sub-strate is important for activity, the enzyme can be better defined asan α-amino acid ester prodrug-activating enzyme [44,45]. VACVaseprefers small, hydrophobic (valine, proline), and aromatic side chains(phenylalanine) over the charged amino acids (lysine, aspartic acid).It has shown clear stereoselectivity for L-valine over D-valine prodrugsirrespective of the parent drug while exhibiting comparable hydrolyticactivity toward D-phenylalanine and L-phenylalanine esters as indicatedin a study of various nucleoside analogue prodrugs [43].

The amino acid amides are very poor substrates of VACVase due toboth low affinity and the much stable amide bond. Therefore, amideprodrugs require another hydrolytic activation mechanism. As anexample, various amino acid amide prodrugs of dapsone were all sub-strates of leucine aminopeptidase, with decreasing specificity constants(kcat/Km) following the order leucine≈alanine>phenylalanine>lysine≈glycine [21]. In addition, only L-phenylalanine was a substratein the presence of chymotrypsin while only L-lysine was a substrate inthe presence of trypsin. Also these amino acid amide prodrugs showedstereoselectivity, as L-amino acid derivatives were converted to dap-sone more rapidly than the corresponding D-amino acid derivatives.α-Amino acid prodrugs have also been studied for targeting specific en-zymes overexpressed in cancer tissues. The L-prolylamide prodrug ofthe antitumor drug melphalan was shown to be a good substrate ofprolidase, an enzyme overexpressed in melanoma. For comparisonD-propylamide was a poor substrate of prolidase [20].

3. Amino acid prodrugs in drug delivery

Prodrug approach is rather a common way to increase the dissolu-tion rate and solubility of poorly soluble (solubilityb10 μM) drugs thathave limited oral absorption or unsatisfactory solubility for parenteraldelivery [46,47]. As discussed above, amino acids are excellentpromoieties to increase the aqueous solubility of the parent drug. To in-crease thedissolution rate further amino acid prodrugs canbe convertedto their salt forms. In the following sections, we will present few exam-ples of commercial as well as investigational amino acid prodrugs thathave been designed to improve 1) oral bioavailability, 2) sustaineddrug delivery, 3) intravenous drug delivery, 4) to target drugs to theirsite of action and 5) improve enzymatic stability.

3.1. Prodrugs for improved oral drug delivery

3.1.1. Overcoming poor aqueous solubilityBrivanib alaninate (BMS-582664) is an investigational amino acid

ester prodrug of brivanib (BMS-540215), a selective dual inhibitor ofvascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblastgrowth factor receptor 1 (FGFR-1), currently under developmentfor treatment of hepatocellular carcinoma and colorectal cancer [48].Brivanib has very low aqueous solubility (b1 μg/ml at pH 6.5), whichis believed to contribute to its solubility/dissolution rate-limited oralbioavailability (22–88% in mice, rats, dogs, and monkeys), particularlyat high doses. The L-alanyl ester prodrug of brivanib has very highaqueous solubility (73 mg/ml at pH 5.8), which resulted in improvedoral bioavailability of brivanib (52–97% in several animal models)[14,49]. In addition, administration of the prodrug resulted in dose-proportional increase in exposure of brivanib in animals. Brivanibalaninate is rapidly and completely converted to brivanib by variousesterases most probably during the absorption (Fig. 7) and it thereforeoffers an excellent way to deliver brivanib orally [50].

CAM-4562 is a dimethyl glycine ester of CAM-4451, a selectivenonpeptide NK1 neurokinin receptor antagonist that is used as anantidepressant, antiemetic, and anxiolytic agent [13]. CAM-4451 hasvery low aqueous solubility (b2 μg/ml) and consequently poor oral




Fig. 7. Bioconversion of brivanib alaninate to brivanib by esterases.


bioavailability [13,15]. While amino acid leucine has a modest in-crease in solubility (0.1 mg/ml) of CAM-4451, the dimethyl glycineprodrug, CAM-4562 has 1500-fold greater solubility (3 mg/ml) andnearly 3-times better oral bioavailability (39%) than that of its parentdrug [13]. Moreover, the dimethyl glycine prodrug showed more se-lective hydrolysis to the parent drug by aminopeptidases at thebrush border membrane of the gastrointestinal tract (Fig. 8), whereasthe leucine ester was more susceptible for hydrolysis before reachingthe brush border membrane, which may cause potential precipitationproblems before absorption.

CEP-7055 is a dimethyl glycine ester and CEP-5214, a pan inhibitorof VEGFR tyrosine kinases with antitumor activity [51]. CEP-5214 hasvery low aqueous solubility (b10 μg/ml), which is believed to be re-sponsible for its poor oral bioavailability [13,15]. The HCl salt of the di-methyl glycine ester prodrug, CEP-7055, has yielded even 4000-foldgreater solubility (40 mg/ml), which has resulted in improved bioavail-ability ranging from 15 to 20%. Like CAM-4451, CEP-7055 is hydrolyzedto the active parent compound during the absorption in the enterocytesby aminopeptidases (Fig. 8). CEP-7055 has advanced to clinical trials asa chemotherapeutic agent for glioblastoma and colon cancer in combi-nation therapy [15,51,52].

QC12 is an amino acid prodrug of quercetin (3′,4′,3,5,7-tetra-hydroxyflavone), where one of the phenol groups of the parent drugis attached to amino acid glycine via a carbamate bond [16,53]. Querce-tin is a naturally occurring flavonoid with many biological activities,including inhibition of tyrosine kinases [54]. However, it is very waterinsoluble, which has required dissolution of this anti-tumor agent indimethylsulphoxide (DMSO) in clinical trials. QC12 has multifold

Fig. 8. Bioconversion of amino acid ester prodrugs, CAM-4562 and CEP-7055 to th


increased aqueous solubility compared to quercetin. It is bioactivatedby cellular hydrolyzing enzymesmost probably before or during the ab-sorption (Fig. 9). Recently, quercetin has been proposed to act as aprodrug itself, since it is rapidly transformed into an active metabolites,3,4-dihydroxyphenylacetic acid (DOPAC) and m-hydroxyphenylaceticacid (m-HPAA) by intestinal microflora [55].

3.1.2. Utilization of intestinal transporters for oral drug deliveryAfter oral administration, most of the compounds are absorbed pas-

sively either by transcellular (neutral small molecules) or paracellular(charged small compounds) route across the mucosal enterocyticmembrane [56,57]. Since amino acids are charged at physiologicalfluids, they have low membrane permeability and are therefore spe-cifically transported across the lipid bilayers by energy-dependentamino acid and peptide transporters. Amino acid prodrugs, which aredesigned to be absorbed by these amino acid and peptide transportershave to mimic structural features of natural substrates of these trans-porters [58,59]. The intestinal mucosa expresses several amino acidand peptide transporters, of which the most abundant transporters arethe intestinal proton-coupled peptide transporter 1 (PepT1, SLC15A1)and Na+-dependent neutral amino acid transporter (ATB0,+, SLC6A14).Both of these transporters are relatively highly expressed on the apicalsurface of the enterocytes and have relatively high capacity, whichensures rapid uptake of their substrates. Since they both recognize awide range of substrates, they are ideal transporters to carry aminoacid and peptide prodrugs across the mucosal membrane [58,60].

L-Valyl ester prodrugs are probably the first commercial examplesof amino acid prodrugs that utilize intestinal transporters to increase

eir parent drugs CAM-4451 and CEP-5214, respectively, by aminopeptidases.




Fig. 9. Bioconversion of a carbamate prodrug of quercetin, QC12 by cellular hydrolyzing enzymes and the proposed biotransformation to the active metabolites, DOPAC andm-HPAAby intestinal microflora.


permeability and consequently poor oral bioavailability of their par-ent drugs. Valacyclovir (Valtrex®) was the pioneer of L-valyl esterprodrugs, which achieved 3–5-times higher oral bioavailability(>60%) [61] than that of its parent drug, acyclovir (10–20%) [62,63].Valacyclovir is absorbed by the PepT1 [5] and ATB0,+ transporters[64], and is then rapidly hydrolyzed to acyclovir predominantly bybiphenyl hydrolase-like protein (valacyclovirase) [42]. Acyclovir,like all nucleosides, is actually a pre-prodrug or double prodrug, sinceafter the hydrolysis of the valine promoiety, acyclovir is phosphorylatedto the corresponding monophosphate selectively by virus-specific thy-midine kinase and subsequently converted to di- and triphosphatesby cellular kinases (Fig. 10) [65,66]. Finally acyclovir triphosphate actsas an antiherpetic agent by inhibiting viral DNA replication.

Soon after the discovery of valacyclovir, valganciclovir (Valcyte®)was designed. Valganciclovir is also absorbed by the PepT1 [6] andATB0,+ transporters [67] afterwhich it is bioactivated by valacyclovirase(Fig. 10) [42]. The oral bioavailability of ganciclovir is approximately60%, which is almost 10-times higher than that from oral ganciclovir(6–8%) [68,69]. The success of valacyclovir and valganciclovir hasproved that L-valine prodrug approach is a very efficient option to im-prove the oral bioavailability by utilizing amino acid transporters.Therefore, there are several other L-valyl ester prodrugs, including nu-cleoside analogues levovirin valinate, valopicitabine, and valtorcitabine,currently undergoing clinical evaluation [70].

There are also fewexamples of amino acid amideprodrugs, designedto be absorbed by intestinal transporters. One of these is midodrine(ProAmatine®, Gutron®), a glycine prodrug of desglymidodrine(DMAE), in which the glycine promoiety is attached to an aminegroup of DMAE with an amide bond (Fig. 11). The oral bioavailabilityof midodrine is 93%, which is 2-times higher than the correspondingvalue for DMAE. This prodrug is absorbed mainly via the PepT1 trans-porter [71] and bioconverted by yet unknown peptidases in the liverand systemic circulation [72]. The released DMAE acts then peripherallyas a selectiveα1-receptor agonist. Midodrine has been used successful-ly in the treatment of neurogenic orthostatic hypotension, and more

Fig. 10. Bioconversion of valacyclovir and valganciclovir to their parent drugs by valacycloviby cellular kinases.


recently in the treatment of dialysis hypotension and it hasminimal car-diac and CNS effects.

LY544344 (or talaglumetad) is another example of amino acidamide prodrugs, where the alanyl promoiety is attached to the aminegroup of the parent drug, LY354740 (or eglumetad). LY354740, wasan investigational drug, which was expected to be useful in the treat-ment of many psychiatric disorders, including psychosis, anxiety, anddrug withdrawal [73]. However, due to its low membrane perme-ability LY354740 had limited oral bioavailability (ca. 10% in animaland humans studies). The alanyl prodrug, LY544344, showed high affin-ity for the PepT1 transporter and improved oral bioavailability (ca. 85%)in various animal models [74,75]. Furthermore, LY544344 had an idealbioactivation profile, since it was extensively (more than 97%) andrapidly hydrolyzed during the absorption (Fig. 12). In clinical trials,LY544344 increased systemic LY354740 exposure by 13-fold comparedto equivalent LY354740 doses. However, the clinical development ofLY544344 was recently discontinued.

Gabapentin and baclofen are structural analogs of GABA (gammaamino butyric acid) used for the treatment of various neurological dis-orders. Both gabapentin and baclofen have structural features ofamino acids. They are both absorbed in the upper small intestine by alow-capacity solute transporter localized in the upper small intestine,possibly an L-type amino acid transporter. The XP13512 and XP19986are novel prodrugs of gabapentin and baclofen, respectively, desiredto overcome pharmacokinetic limitations of the parent drugs (Fig. 13)[76,77]. Transport of these prodrugs is mediated by monocarboxylatetransporter type 1 (MCT-1) and sodium-dependentmultivitamin trans-porter (SMVT) [76,77]. These prodrugs further exemplify the utility ofamino acid prodrugs targeted to transporters other then oligopeptidetransporters expressed in the gastrointestinal tract.

3.2. Sustained release

One of the most studied fields in drug development is long-actingpharmacological agents. In sustained release the drug is released over

rase and to their corresponding triphosphates firstly by thymidine kinase and secondly




Fig. 11. Bioconversion of an amide prodrug midodrine by peptidases.

OH

ONH

O

OO

O

Cl

OH

OH2N

Cl

R-baclofenXP19986

OH

ONH

O

OO

OOH

OH2N

GabapentinXP13512

Fig. 13. Bioconversion of an XP13512 and XP19986 by cellular hydrolyzing enzymes.


a period of time in a controlled manner to maximize efficacy and/orminimize side effects. Unlike formulation-based sustained release for-mulations such as liposomes, microspheres, hydrogels, the sustainedduration of action achieved by prodrugs is the result of controlled bio-transformation reaction. Depending upon the prodrug approach, thebioactivation of sustained release prodrugs can occur during the oralabsorption in enterocytes, in the systemic circulation, or in targetcells. Due to the consistent rate of hydrolysis and delivery, these pro-drugs are expected to be less vulnerable to absorption-related changeswhen compared with formulation-based delivery systems. Amino acidamides offer a great opportunity to design sustained release pro-drugs, since amide bonds are usually more resistant towards chemical/enzymatic hydrolysis than the corresponding ester bonds. SimilarlyD-amino acid ester prodrugs that are generally more resistant to enzy-matic hydrolysis than the corresponding L-amino acid ester prodrugscould be used for sustained delivery.

Lisdexamfetamine dimesylate (LDX; Vyvanse®) is a L-lysine aminoacid amide prodrug of D-amphetamine, which is used as a psychostimu-lant for attention deficit/hyperactivity disorder (ADHD) in childrenaged 6–12 as well as in adults [78]. LDX is absorbed primarily as an in-tact prodrugmost-likely by PepT1 and/or amino acid transporters and itreleases the active parent drug, amphetamine, mainly in red blood cells(Fig. 14) [7]. Extended release and consequent long duration of actionallows once-daily dosing of LDX. The major advantage of LDX is thatbeing a sustained-release prodrug, it has less abuse potential thanother amphetamines if inhaled or injected [79].

3.3. Prodrugs for intravenous delivery

Injectable drugs gain rapid and complete entry into systemic circula-tion. However, compared to oral formulations, they have unique re-quirements such as stability, sterility and lack of particles that maycause lung emboli or other venous blockages. Formolecules that are for-mulated as injectables, sufficient aqueous solubility is required. Formolecules that do not have sufficient aqueous solubility, often organicsolvents, solubilizers, and surfactants are used to improve drug solubil-ity in the formulation.When formulation approaches have been proveninsufficient, harmless, or even toxic, prodrug strategies have been usedto improve aqueous solubility of the drug therebymaking them amena-ble for parenteral delivery. In this regard, amino acid prodrugs have

Fig. 12. Bioconversion of an amide prodrug of LY354740 by cellular hydrolyzingenzymes.


been utilized to improve aqueous solubility of the parenterally adminis-tered drugs.

Bundgaard et al. [17] synthesized several amino acid esterprodrugs of poorly soluble drug, metronidazole, for parenteral use.Metronidazole, an antimicrobial drug, is effective against anaerobicbacteria and certain parasites, and is generally administered via oralroute. However, its intravenous administration is often needed toachieve a rapid onset of action. The hydrochloride salt of metronidazoleN,N-dimethylglycinate (Fig. 15a) was highly soluble (>0.5 g/ml) andproved to be the most promising candidate for parenteral delivery.This amino acid prodrug released metronidazole rapidly and quantita-tively in 80 % human plasma and in the whole human blood withhalf-lives (t1∕2) of 12 min and 9.5 min, respectively. Additionally, arapid conversion of prodrug into parent drug was observed after its in-travenous administration in beagle dogs [30]. Unfortunately, the highinstability of metronidazole N,N-dimethylglycinate limited its formula-tion as a ready-to-use solution, even at refrigerated temperatures. Anattempt to increase the chemical stability of amino acid ester prodrugsof metronidazole was made by incorporating a phenyl group betweenthe ester moiety and the electron-withdrawing amino group [33,35].The aminomethylbenzoate esters of metronidazole (Fig. 15b and c)had high solubility (>0.1 g/ml) and chemical stability in aqueoussolution, whereas they readily released metronidazole in 80% humanplasma andwhole blood with half-lives of 0.4 min and 0.6 min, respec-tively. In fact, the calculations based on their stability studies at elevatedtemperatures showed that the shelf-lives (t90%) of 12 to 14 years can beachieved for aminomethylbenzoate esters at +25 °C. These estimatedshelf-lives can, however, be limited by precipitation of formedmetroni-dazole during storage.

Rapamycin an immunosuppressant and chemotherapeutical drugused to prevent rejection after organ transplantation, has lowaqueous solubility and poor oral bioavailability. Rapamycin-28-N,N-dimethylglycinate, an amino acid ester prodrug, resulted in goodaqueous solubility >50 mg/ml, which allowed for parenteral delivery[80] of the molecule (Fig. 16). However, this prodrug had inadequatechemical stability to be formulated as ready-to-use formulation. Itspreclinical pharmacokinetic evaluation in mice at the intravenousdoses of 10–100 mg/kg revealed that the detectable plasma con-centrations of rapamycin persisted for 5–12 h after administration,serving as an effective slow-release delivery system for the parentdrug [81].

One example of amino acid amide prodrug for intravenous deliveryis an investigational mono-substituted L-lysine amide of bacteriostaticand antileprosy agent, dapsone (diamino-diphenyl sulfone) [82]. Dap-sone itself has low aqueous solubility, approximately 0.16 mg/ml [21].Converting dapsone into an amino acid HCl salt increases solubility toover 65 mg/ml, even at pH 7.4 [21]. The L-lysinyl amide of dapsoneshows good predicted self-life (>2 years at pH 4) and is rapidly and




Fig. 14. Sustained release of amphetamine from its amide prodrug, lisdexamfetamine in red blood cells.


quantitatively hydrolyzed in systematic circulation after intravenousadministration. Therefore it serves as a good prodrug example forother poorly soluble amines (Fig. 17).

Carbamazepine is an anticonvulsant drug that has relatively pooraqueous solubility (0.12 mg/ml). A lack of the commercially availableintravenous formulation has led to the preparation of N-glycyl prodrugof carbamazepine (Fig. 18), where an amino acid glycine has beenlinked to the urea nitrogen of parent drug forming peptide-likeacyl-urea functionality [24]. The intrinsic aqueous solubility of neutralN-glycyl carbamazepine was observed to be 4.4 mg/ml and it waspredicted to exceed 50 mg/ml at pH 4.0. The prodrug demonstratedrapid and complete conversion to parent carbamazepine (t1∕2 about1 min) after intravenous administration in rats [83], but due to itschemical instability (t90%=5.9 days at pH 4.0, +25 °C) the parenteralformulation of prodrug was limited only to the freeze-dried productfor reconstitution prior the use.

Another carbamazepine prodrug, N-cysteamine carbamazepine,has also been developed for intravenous use [84]. The aqueous solu-bility of N-cystamine derivative exceeded 100 mg/ml at pH 2.6, andits predicted shelf-life (t90%) was 320 days at pH 4.0 (+25 °C). Afterintravenous dose of N-cysteamide prodrug in rats, parent carbamaze-pine was immediately seen in plasma, whereas no intact prodrug wasobserved, suggesting that the conversion of prodrug to parent drugoccurred rapidly and most probably via reaction with glutathione[84] (Fig. 19).

3.4. Drug targeting by amino acid prodrugs

Ever since Paul Ehrlich coined the term “magic bullet”, the searchfor compounds that selectively target diseased cells without affectingnormal cells has been both a major opportunity and challenge [85]. Inprodrug design, drug targeting can, in general, be achieved either byselectively transporting a prodrug to the site of drug action or by

a)

b)

c)

HCI

HCI

HCI

Fig. 15. Chemical structures of metronidazole prodrugs for intravenous delivery.


site-selective activation of a prodrug. Site-directed drug deliveryafter systemic administration is a very challenging task, due to vari-ous complex and unpredictable barriers in the body. Nevertheless,amino acid prodrugs have demonstrated some success in the areasof ocular, cancer and brain drug targeting.

Amino acid prodrug strategies utilizing endogenous nutrienttransporters that are over expressed in certain organs have becomean attractive approach for improved and targeted drug delivery. Asdescribed earlier, oral absorption of several compounds has been im-proved by targeting them to PepT1 expressed in gastrointestinal tract.Similarly, PepT1 is implicated in improved ocular bioavailability ofvarious dipeptide prodrugs of antiviral drugs such as acyclovir [86]and ganciclovir [87,88]. For example, when the ocular absorption pro-files of acyclovir prodrugs were studied in rabbits, the area under thecurves for L-serine and L-valine ester prodrugs of acyclovir was ap-proximately two-fold higher compared to parent acyclovir.

Higher expression of PepT1 in cancer cells over normal cells wasalso sufficient to allow amino acid ester prodrugs of the antineoplas-tic drug, floxuridine, to be selectively accumulated in cancer cells [37].On the other hand, approximately 5-fold higher accumulation of va-line ester prodrug of SN-38 in human breast cancer cell line, MCF7cells, compared to SN-38 was not attributed due to uptake by PepT1[89]. The improved intracellular uptake was attributed to variousamino acid transporters such as ATB0,+, ATA1, ATA2, and ASCT2. Inaddition, the relationship of the valine prodrug of SN-38 with variousefflux transporters was diverse but not significant. Whereas the rec-ognition of the prodrug by P-gp and MRP1 was appreciably de-creased, the involvement of MRP2 and BCRP was slightly increased,ruling each out.

Selective expression of large neutral amino acid transporter 1(LAT1) at the blood–brain barrier has been utilized to target severalamino acid prodrugs of ketoprofen [90,91] and valproic acid [92] tothe central nervous system (CNS). The L-tyrosine and L-lysine prodrugsof ketoprofen showed concentration-dependent, yet saturable, brainuptake in in situ rat brain perfusion experiments. The uptake was in-hibited by 2-aminobicyclo(2, 2, 1)heptane-2-carboxylic acid (BCH), awell-known LAT1 inhibitor, suggesting that the brain uptake of prodrugswas mediated by LAT1 (Fig. 20). Furthermore, after bolus injection of

Fig. 16. Chemical structures of rapamycin and its 28-N,N-diglycinyl ester prodrug.




Fig. 17. Bioconversion of an amino acid amide prodrug of dapsone.

Fig. 18. Conversion of N-glycyl carbamazepine to carbamazepine in vivo.

Fig. 19. Suggested conversion mechanism of N-cysteamine carbamazepine to parent drug by nucleophilic attack with glutathione.


lysine prodrug in rats, both prodrug andketoprofenwere detected in therat whole brain tissue. In in vivo microdialysis in rat striatum studies,lysine prodrug was able to deliver ketoprofen to its site of action, i.e.,into the intracellular compartments of brain cells

Enzymes that are unique in a tissue, or present at a higher concen-tration compared to other tissues, can be exploited for site-selectiveprodrug conversion and hence targeting. For example, some tumortissues have been shown to evoke increased prolidase activity com-pared to normal tissues, which could be used to target prodrugsdesigned to be activated by this enzyme. Proline prodrugs of bothmelphalan [93] and methotrexate [94] (Fig. 21) were designed as

Fig. 20. Chemical structures and activation of LAT1-target


substrates for a specific cytosolic imidodipeptidase, prolidase, toovercome their resistance by a number of cancer cells. Both prodrugsshowed increased cytotoxicity for breast cancer cell line MDA-MB-231 compared with parent drugs suggesting that the proline prodrugapproach may overcome the resistance associated with parent drugsfor at least this specific cell line. The increased cytotoxicity was attrib-uted to two mechanisms; first, proline prodrugs were more effective-ly transported into the MDA-MB-231 cells likely by transported-mediated mechanisms, and secondly, higher prolidase activity inMDA-MB-231 cells than in normal cells may contribute to the effectiverelease of the parent drugs at the site of drug action. Most recently,

ed ketoprofen prodrugs for improved brain delivery.




Fig. 21. Hydrolysis of proline prodrug of methotrexate.


Tsume et al., showed feasibility of cathepsin D to activate amino acid/dipeptide monoester prodrugs of floxuridine [95].

3.5. Enhanced metabolic stability by amino acid prodrugs

Amino acid prodrug strategy has been successfully used to enhancemetabolic stability of nucleoside drugs. 2-Bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) is a potent and selectiveinhibitor of human cytomegalovirus (HCMV), but it lacks clinical utilitydue to rapid in vivo metabolism. The N-glycosidic bond of BCDRB iscleaved by two enzyme 8-oxoguanine DNA glycosylase (OGG1) andN-methylpurine DNA glycosylase (MPG), rendering the molecule inac-tive (Fig. 22) [96]. Amino acid ester prodrugs of BDCRB were synthe-sized to improve its metabolic stability and enhance in vitro potencyand systemic exposure [97]. To this end, L-Asp-BDCRB showed potentand selective antiviral activity in addition to favorable stability as com-pared to BDCRB (Fig. 22). L-Asp-BDCRB, was rapidly absorbed after oraladministration to mice, and exhibited 5-fold greater half-life thanBDCRB [97].

Gemcitabine, a nucleoside anticancer agent, is a polar drug withlow membrane permeability and is administered intravenously. Fur-ther, cytidine functionality undergoes significant metabolism by cyti-dine deaminase, which cleaves the glycosidic bond rendering themolecule inactive (Fig. 23). A number of amino acid ester prodrugsof gemcitabine were synthesized to improve its oral permeability via

Fig. 22. BDCRB N-glycosidic bond cleavage by 8-oxoguanine DNA glycosylase (OGG1)and N-methylpurine DNA glycosylase (MPG) and bioevasion by enhanced stabilityvia conversion to L-Asp-BDCRB prodrug.


oligopeptide transporters and enhancemetabolic stability towards cy-tidine deaminase [41]. 5’-L-Isoleucyl-gemcitabine prodrug showedenhanced transport in cells expressing hPEPT1. This prodrug, unlikeVal-gemcitabine, showed slower bioconversion in Caco-2 cell homog-enates and in human plasma, and exhibited remarkable resistance tocytidine deaminase deactivation (Fig. 23). Together these propertieswould allow a longer systemic circulation half-life and facilitatetargeting of cells overexpressing the hPEPT1 transporter [41].

4. Practical considerations and challenges associated with thedevelopment of amino acid prodrugs for oral delivery

Development of amino acid prodrugs, like any other prodrugs, canpose challenges that should be considered during the discovery stageand throughout the development. These challenges are associatedwith synthesis, in vitro and in vivo screening, analytical and bioanalyticalmethods, formulation, metabolism, pharmacokinetic assessment, safetyand toxicology assessment, and regulatory considerations.

4.1. Discovery and screening

The prodrug development is an iterative process that involvessynthesis of prodrugs followed by in vitro and in vivo evaluationuntil a target prodrug profile (physicochemical, pharmacokinetic,and/or pharmacodynamic) is achieved. Discovery and developmentscientists employ a variety of tools to assess physicochemical andbiopharmaceutics performance of prodrugs (Table 3). It is importantthat in vitro and in vivo screens used to evaluate intended purpose

Fig. 23. Gemcitabine N-glycosidic bond cleavage by cytidine deaminase and improvedpermeability and stability via conversion to L-Ile-gemcitabine prodrug.




Table 3In vitro and in vivo tools to assess biopharmaceutical performance of prodrugs.

Assessment Screening tools

Solubility ▪ High-throughput solubility screens▪ pH-dependent solubility

Permeability ▪ PAMPA▪ Caco-2 (bidirectional)▪ Animal tissues▪ Cell lines expressing transporters of interest

Chemical stability ▪ Solution stability (e.g., pH-dependent,oxidative, photo stability)

▪ Solid state stabilityEnzymatic stability ▪ Cell homogenates

▪ Tissue homogenates▪ Pure enzymes

In vivo screening ▪ PK screen in animals▪ Portal vein studies


(e.g., improving solubility, permeability, stability) of prodrugs haverelevance to humans, to avoid surprises in the clinical studies. Charac-terization of oral prodrugs that are designed to convert to parent drugprior to reaching systemic circulation requires systematic evaluationof both prodrug (solubility, permeability, chemical stability, biocon-version, pharmacokinetics, etc.) and parent drug (protein binding,serum stability, metabolism, pharmacokinetics, etc.).

Solubility determination, although routine [98], can be ratherchallenging for prodrugs. This is due to instability of prodrug whendetermining equilibrium solubility. A significant conversion (>10%) ofprodrug (high solubility) to parent drug (low solubility) can complicatesolubility results. In such cases, apparent solubility (solubility at a timewhere there is no significant degradation of prodrug)may be used. Sim-ilarly, permeability determination [99] can be rather challenging forprodrugs. Caco-2 cells that are most commonly used to determinedrug permeability, express a number of esterases and metabolic en-zymes that can convert prodrug to parent drug either on the apicalside or in the cytosol. Therefore it may be necessary to evaluate perme-ability of both prodrug and parent drug [14]. At the same time Caco-2permeability model can be used to understand rate and extent ofprodrug bioconversion. For prodrugs that are designed to target a spe-cific transporter, in vitro cell culture models expressing transporter/sof interest should be used. Importance of such in vitro screens hasbeen demonstrated for the evaluation of amino acid prodrugs targetinghuman intestinal transporters such as hPEPT1 [5,6] and LAT1 [92].

One of the major challenges with prodrugs is difficulty inpredicting their rates of bioconversion in vivo [100]. The chemical sta-bility and hydrolysis mechanism of the prodrugs can be assessed andunderstood using well-established chemical kinetics principles. How-ever, predicting rates of bioconversion in vivo using in vitro tools andanimal screens is rather difficult. The large diversity of hydrolases andinterspecies differences makes extrapolation to humans difficult.Therefore it is important to select an animal species that most closelyrepresents humans. Further, within humans, prodrugs could exhibitinter individual variability in the rate of bioconversion due to factorssuch as age, gender, physiological conditions, and disease state.

Amino acids introduce a cation (typical pKa of α-amino group inamino acid prodrugs is 6.8–7.9) or anion (typical pKa of α-carboxylgroup in amino acid prodrugs is 3.5–4.3) to the parent drug, whichcould lead to pH-dependent solubility (Fig. 4) and consequently ab-sorption, and potential drug–drug interactions with gastric pH modi-fiers. This may be less of an issue if the intrinsic solubility of theprodrug is high, such that the entire dose in soluble under gastroin-testinal physiological conditions (BCS class I or III).

4.2. Synthesis

Amino acid prodrugs are expected to pose synthetic challengessimilar to other prodrugs. Amino acid prodrug linkage chemistry is


well-established, therefore many of the synthetic challenges will de-pend on the number and type of protecting groups required tomask the active groups on the amino acid and parent drug. Conver-sion of parent drug to amino acid prodrug will generally add at least2 synthetic steps. The additional synthetic steps and protectinggroups could increase the total number of impurities and degradationproduct that may require qualification in toxicology studies. Further,all amino acids except glycine, introduce a stereocenter to the prodrug.Depending on whether the parent drug has any stereocenters, thiscan result in 2n stereoisomers, where n is the number of sterocenters(red — brivanib). Together amino acid prodrugs could pose significantsynthetic, purification, and analytical challenges. Amino acid prodrugslike other prodrugs, increase the molecular weight of the parent,e.g., hydrochloride salt of valine ester will increase formula weight by135 Da. Therefore one needs to be mindful of the increase in dose dueto the “non-active” contribution of the prodrug relative to the bioavail-ability enhancement with prodrug.

4.3. Analytical

Amino acid prodrugs can pose significant analytical and bio-analytical challenges during quantitative analysis of prodrug, parentdrug, impurities, degradation byproducts, and metabolites in drugsubstance, formulations, and bioanalytical samples from matricessuch as blood, urine, and in vitro incubations [101]. The prodrug andparent molecule are expected to have very different physicochemicalproperties, and thus optimal liquid chromatography (LC) and massspectrometry (MS) conditions, can vary greatly among the analytes ofinterest. Analytical scientists have to balance between a single LC/MSmethod which may not be optimal and separate analytical methods,which could be time consuming. Since most amino acid prodrugs con-tain one ormore stereocenters, analysis of diastereomers could be chal-lenging and in many cases requires chiral method development [102].Further, it is important to evaluate stability of amino acid prodrugs invarious analytical matrices and necessary protocols should be devel-oped to ensure that prodrug does not degrade in the samples and dur-ing the analysis. This is important to accurately determine the amountof prodrug and parent drug as itmay impact analytical results and phar-macokinetic calculations.

4.4. Formulation/stability challenges

The stability challenges posed by amino acid prodrugs are no differ-ent than those posed by other prodrugs and are largely dependent onthe chemical stability of the linkage between the parent drug and thepromoiety. Prodrugs need to have a balance between good chemicalstability so that an appropriate shelf life could be assigned to the drugsubstance and the drug product, but at the same time they need to rap-idly and quantitatively convert to the parent drug in vivo. Sinceprodrugs are designed to have a chemically or enzymatically labile link-er,most prodrugs solutions are chemically not stable to be developed asready-to-use solution formulations for parenteral or oral (e.g., pediatricformulations) administration. Therefore most parenteral prodrugs areformulated as lyophilized products and oral prodrugs are formulatedas solid dosage forms or powder for reconstitution. In-terms of oralsolid dosage form (e.g., tablets and capsules), stability of prodrug couldlimit processing choice.Wet granulation is usually avoided for hydrolyz-able drugs. However, a careful evaluation of the process is required. Forexample, tablets and capsules manufactured by wet granulation weremore stable for prodrug, DMP 754, than those manufactured by thedry granulation process [103]. The authors attributed this to amore uni-form distribution of the pHmodifier, disodium citrate, used to maintainthe microenvironment pH where the prodrug was most stable [103].

Amino acid ester prodrugs introduce amine functionality in themol-ecule. Primary and secondary amines can undergo Maillard reactionwith excipients such as lactose. The degradation product in a Maillard





reaction can undergoAmadori rearrangements to formawide variety ofproducts. The Maillard reaction is base-catalyzed and could be acceler-ated if alkaline lubricants are used. The amorphous content of lactose,equilibrium moisture content, microenvironmental pH and salt/freebase form of the drug can contribute to the extent of Maillard reaction.Therefore excipients such as lactose should be avoided or used cau-tiously in amino acid ester prodrug formulations. Amino acid estersthat are basic in nature and substantially ionized and soluble at thesame pH as insoluble excipient of opposite charge can interact withsuch excipients. This interaction could influence drug's dissolution invitro, but may not have impact on in vivo bioavailability. This was dem-onstrated for brivanib alaninate, which was found to interact withcrosscarmellose sodium (CCS), a commonly used disintegrant, resultingin incomplete dissolution at pH 4.5 [104]. This effect was not observedwith crosspovidone, a non-ionic disintegrant. Althoughbrivanib alaninateinteracted with CCS in vitro, this interaction did not impact drug's bio-availability in vivo [104].

Majority of the amino acid prodrugs are simple esters and amidesand as such do not generate reactive impurities such as formaldehydeupon degradation upon storage. Amino acid prodrugs containing acarbamate linkage can release formaldehyde upon hydrolysis, whichcould result in secondary degradation byproducts that may requirequalification in non-clinical toxicology studies. Further, formaldehydecan crosslink the capsules, which can adversely affect the dissolution ofcapsule formulation [105]. Amino acid prodrugs could bring additionalstability challenges e.g., racemization and/or intramolecular cyclization.

4.5. Safety and toxicology assessment

The other major challenge is toxic potential of prodrug andpromoiety as compared to the parent drug [106]. This is especially im-portant when prodrug and promoiety exhibit different toxicities ascompared to the parent. Inmany cases, a comparative toxicology of par-ent and active drug may be evaluated [106]. Amino acids are buildingblocks for proteins and are generally regarded as safe [107]. However,there may be specific concerns around certain natural amino acids e.g.,tyrosine, phenylalanine and synthetic amino acids, which should betaken into account, especially for compoundswith high dose and chron-ic usage. Similarly the safety of the non-natural amino acids, if used aspromoiety, must be considered [108].

5. Conclusion

In conclusion, amino acid prodrugs have a proven track of improv-ing oral delivery of the drugs that have poor solubility and permeabil-ity. More recently, amino acid prodrugs have been successfully used toachieve sustained release, intravenous delivery, and improve meta-bolic stability. Importantly, amino acid prodrugs have proven com-mercial and regulatory track, which is beneficial in bringing suchdrugs to the patients. Development of amino acid prodrugs is not with-out challenges. Pharmaceutical scientists must pay special attention touse discovery screens, safety, synthetic, analytical, and formulation is-sues which could pose significant challenges in the development ofamino acid prodrugs. We hope that this timely review stimulates thecontinual research in use of amino acid prodrugs to overcome deliverychallenges posed by complex molecules currently under development.

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Amino acids as promoieties in prodrug design and development