Amino Acid Ester Prodrugs Research Articles

Novel l-amino acid ester prodrugs of azacitidine: Design, enzymatic synthesis and the investigation of release behavior

Abstract A facile, enzymatic synthesis protocol of l -amino acid ester prodrugs of azacitidine was developed. Firstly, transesterification of azacitidine with Boc protected vinyl aminocarboxylates was performed under the catalysis of subtilisin in anhydrous pyridine at 50 °C. A series of Boc-protected amino acid-azacitidine conjugates were prepared in good regioselectivity and yield. Various factors which influence the catalytic efficiency were systematically investigated. And then, the obtained azacitidine derivatives were subjected to a deprotection process to give l -amino acid ester prodrugs of azacitidine. In vitro hydrolysis of prodrugs showed that the amino acid ester prodrugs had obvious sustained release characteristic. These characteristics will have potential value for clinic application.

Synthesis, chemical and enzymatic hydrolysis, and aqueous solubility of amino acid ester prodrugs of 3-carboranyl thymidine analogs for boron neutron capture therapy of brain tumors

Various water-soluble l-valine-, l-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogs (3-CTAs), designated N5 and N5-2OH, were synthesized for Boron Neutron Capture Therapy (BNCT) of brain tumors since the water solubilities of the parental compounds proved to be insufficient in preclinical studies. The amino acid ester prodrugs were prepared and stored as hydrochloride salts. The water solubilities of these amino acid ester prodrugs, evaluated in phosphate buffered saline (PBS) at pH 5, pH 6 and pH 7.4, improved 48–6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3′-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5′-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5′-valine > 5′-glutamate > 5′-glycine. The rate of the hydrolysis of the 5′-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5′-glutamate ester prodrug of N5 and the 5′-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies.

The novel adenosine A2A antagonist prodrug MSX-4 is effective in animal models related to motivational and motor functions

Adenosine A2A and dopamine D2 receptors interact to regulate diverse aspects of ventral and dorsal striatal functions related to motivational and motor processes, and it has been suggested that adenosine A2A antagonists could be useful for the treatment of depression, parkinsonism and other disorders. The present experiments were performed to characterize the effects of MSX-4, which is an amino acid ester prodrug of the potent and selective adenosine A2A receptor antagonist MSX-2, by assessing its ability to reverse pharmacologically induced motivational and motor impairments. In the first group of studies, MSX-4 reversed the effects of the D2 antagonist eticlopride on a concurrent lever pressing/chow feeding task that is used as a measure of effort-related choice behavior. MSX-4 was less potent after intraperitoneal administration than the comparison compound, MSX-3, though both were equally efficacious. With this task, MSX-4 was orally active in the same dose range as MSX-3. MSX-4 also reversed the locomotor suppression induced by eticlopride in the open field, but did not induce anxiogenic effects as measured by the relative amount of interior activity. Behaviorally active doses of MSX-4 also attenuated the increase in c-Fos and pDARPP-32(Thr34) expression in nucleus accumbens core that was induced by injections of eticlopride. In addition, MSX-4 suppressed the oral tremor induced by the anticholinesterase galantamine, which is consistent with an antiparkinsonian profile. These actions of MSX-4 indicate that this compound could have potential utility as a treatment for parkinsonism, as well as some of the motivational symptoms of depression and other disorders.

Design, synthesis and in vitro evaluation of mono (2, 2, 2-trifluoroethyl) esters, mono l-amino acid ester prodrugs of acyclic nucleoside phosphonates as anti-HBV agents

Abstract A series of novel mono (2, 2, 2-trifluoroethyl) esters, mono l -amino acid ester prodrugs of acyclic nucleoside phosphonates was synthesized and their in vitro anti-HBVactivity was evaluated in HepG 2 2.2.15 cells. Compound 1d exhibited more potent anti-HBV activity and lower cytotoxicity than those of adefovir dipivoxil and alamifovir (MCC-478) with EC 50 and CC 50 values of 0.01 μmol/L and >8000 μmol/L respectively.

Small Neutral Amino Acid Ester Prodrugs of Acyclovir Targeting Amino Acid Transporters on the Cornea: Possible Antiviral Agents Against Ocular HSV-1 Infections.

The aim of this study was to characterize the affinity and permeability patterns of the amino acid ester prodrugs of acyclovir (ACV), L-alanine-ACV (AACV), L-serine-ACV (SACV), L-serine-succinate-ACV (SSACV) and L-cysteine-ACV (CACV) on rabbit primary corneal epithelial cell culture (rPCEC) and on rabbit cornea. Amino acid prodrugs of acyclovir, AACV, SACV, SSACV and CACV were synthesized in our laboratory. Chemical hydrolysis in aqueous buffer, enzymatic hydrolysis in corneal homogenates and transport across freshly excised rabbit cornea of these prodrugs were studied. SSACV inhibited the uptake of [(3)H] L-alanine on rPCEC and across the intact rabbit cornea. Lineweaver-Burk plot transformation revealed competitive inhibition between L-alanine and SSACV. In corneal tissue homogenate, the half lives of SSACV, SACV and CACV (t1/2) were observed to be 3.5 ± 0.4, 9.2 ± 0.6 and 1.8 ± 0.1 hr respectively, whereas AACV was readily converted to the active parent drug acyclovir exhibiting complete degradation before 5 min. Interestingly translocation of SACV across cornea was inhibited in the presence of 5 mM arginine (~51%), a specific substrate for cationic transport system and in presence of BCH (~38%), a substrate specific for large neutral amino acid transport system (LAT) or cationic and neutral amino acid transport system (B(0,+)). SACV exhibited higher permeability across cornea along with excellent antiviral activity against herpes simplex virus (HSV-1) and varicella-zoster virus (VZV) in comparison to ACV. Recognition by multiple transporters, stability in corneal homogenate and changes in physico-chemical properties contributed to the increased permeability of SACV across cornea.

Human Carboxymethylenebutenolidase as a Bioactivating Hydrolase of Olmesartan Medoxomil in Liver and Intestine

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys132 of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

Small Neutral Amino Acid Ester Prodrugs of Acyclovir Targeting Amino Acid Transporters on the Cornea: Possible Antiviral Agents against Ocular HSV-1 Infections

The aim of this study was to characterize the affinity and permeability patterns of the amino acid ester prodrugs of acyclovir (ACV), L-alanine-ACV (AACV), L-serine-ACV (SACV), L-serine-succinate-ACV (SSACV) and L-cysteine-ACV (CACV) on rabbit primary corneal epithelial cell culture (rPCEC) and on rabbit cornea. Amino acid prodrugs of acyclovir, AACV, SACV, SSACV and CACV were synthesized in our laboratory. Chemical hydrolysis in aqueous buffer, enzymatic hydrolysis in corneal homogenates and transport across freshly excised rabbit cornea of these prodrugs were studied. SSACV inhibited the uptake of [3H] L-alanine on rPCEC and across the intact rabbit cornea. Lineweaver-Burk plot transformation revealed competitive inhibition between L-alanine and SSACV. In corneal tissue homogenate, the half lives of SSACV, SACV and CACV (t1/2) were observed to be 3.5 ± 0.4, 9.2 ± 0.6 and 1.8 ± 0.1 hr respectively, whereas AACV was readily converted to the active parent drug acyclovir exhibiting complete degradation before 5 min. Interestingly translocation of SACV across cornea was inhibited in the presence of 5 mM arginine (~51%), a specific substrate for cationic transport system and in presence of BCH (~38%), a substrate specific for large neutral amino acid transport system (LAT) or cationic and neutral amino acid transport system (B0,+). SACV exhibited higher permeability across cornea along with excellent antiviral activity against herpes simplex virus (HSV-1) and varicella-zoster virus (VZV) in comparison to ACV. Recognition by multiple transporters, stability in corneal homogenate and changes in physico-chemical properties contributed to the increased permeability of SACV across cornea.

Simultaneous determination of didanosine and its amino acid prodrug, valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2-20,000 ng/mL for didanosine and 4-300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC-MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague-Dawley rats.

Synthesis, Transport and Pharmacokinetics of 5′-Amino Acid Ester Prodrugs of 1-β-d-Arabinofuranosylcytosine

Cytarabine (1-beta-d-arabinofuranosylcytosine, ara-C, 1) suffers from low oral bioavailability due to low intestinal membrane permeability and poor metabolic stability, and intravenous infusion is usually adopted as the clinical standard dosing administration. To develop an oral alternative for 1 and utilize the intestinal oligopeptide transporter 1 (PepT1), a series of 5'-amino acid ester derivatives of 1 was synthesized to clarify which modification was the most suitable to increase the oral bioavailability of 1. Their apical-to-basolateral permeability across Caco-2 cells and the antiproliferative activity with HL-60 cells were screened. 5'-Valyl prodrug 2 demonstrated the highest permeability and was selected for further study. Glycylsarcosine (gly-sar, a typical substrate of PepT1) uptake by Caco-2 cells can be inhibited by 2 in a concentration-dependent manner, and IC(50) for 2 was 2.18 +/- 0.12 mM. The uptake of 2 was markedly increased in the long-term leptin-treated Caco-2 cells compared with the control Caco-2 cells, and was significantly inhibited by the excess of gly-sar, but not by l-valine. A dose-proportional pharmacokinetics was observed in rats when 5, 15, 30 mg/kg doses of 2 (calculated as 1) were orally administered. The oral absolute bioavailability of 1 was 60.0% and 21.8% after 2 and 1 were orally administered to rats 30 mg/kg, respectively. Following oral administration of 15 mg/kg, the absorption and bioactivation of 2 were extensive and rapid, over 98% of prodrug hydrolysis occurring before appearance in the portal vein. The in vivo dispositions of 1-beta-D-arabinofuranosyluracil (ara-U), a deaminated product of 1, were investigated. Oral administration of 2 resulted in an increased 1/ara-U ratio (2.76) in the blood, much higher than that (1.25) after 1 orally taken. Overall, these results demonstrated that the PepT1-mediated absorption of 2 and the increased metabolic stability resulted in a dramatic increase in the oral bioavailability of 1 in rats and further corroborated the thought that prodrug design strategy targeting intestinal PepT1 was an important and promising strategy to improve oral bioavailability of poorly absorbed drugs.

5′- O-Aliphatic and amino acid ester prodrugs of (−)-β- d-(2 R,4 R)-dioxolane-thymine (DOT): Synthesis, anti-HIV activity, cytotoxicity and stability studies

A series of (−)-β- d-(2 R,4 R)-dioxolane-thymine-5′- O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (−)-β- d-(2 R,4 R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1 LAI in human peripheral blood mononuclear (PBM) cells as well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was observed for the compound 2– 4 (5′- O-aliphatic acid esters) without increase in cytotoxicity in comparison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0 and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the half-lives from 12.3 to 48.0 min.

Enhanced Cancer Cell Growth Inhibition by Dipeptide Prodrugs of Floxuridine: Increased Transporter Affinity and Metabolic Stability

Dipeptide monoester prodrugs of floxuridine were synthesized, and their chemical stability in buffers, resistance to glycosidic bond metabolism, affinity for PEPT1, enzymatic activation and permeability in cancer cells were determined and compared to those of mono amino acid monoester floxuridine prodrugs. Prodrugs containing glycyl moieties were the least stable in pH 7.4 buffer (t1/2 < 100 min). The activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of dipeptide monoester prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono amino acid monoester prodrugs (t1/2 ∼ 40 to 100 min). All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine. In general, the 5′-O-dipeptide monoester floxuridine prodrugs exhibited higher affinity for PEPT1 than the corresponding 5′-O-mono amino acid ester prodrugs. The permeability of dipeptide monoester prodrugs across Caco-2 and Capan-2 monolayers was 2- to 4-fold higher than the corresponding mono amino acid ester prodrug. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic ductal cell lines indicated that the dipeptide monoester prodrugs were equally as potent as mono amino acid prodrugs. The transport and enzymatic profiles of 5′-l-phenylalanyl-l-tyrosyl-floxuridine, 5′-l-phenylalanyl-l-glycyl-floxuridine, and 5′-l-isoleucyl-l-glycyl-floxuridine suggest their potential for increased oral uptake, delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil, as well as enhanced uptake and cytotoxic activity in cancer cells, attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action.

Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: Interaction with the transporters on Caco-2 cells

In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, l-alanine-ACV (AACV), l-serine-ACV (SACV), l-isoleucine-ACV (IACV), γ-glutamate-ACV (EACV) and l-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants ( λ z). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV ( p < 0.05). C max(T) (maximum concentration) of SACV was observed to be 39 ± 22 μM in plasma which is 2 times better than VACV and 15 times better than ACV. C last(T) (concentration at the last time point) of SACV was observed to be 0.18 ± 0.06 μM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts ( C max) and eliminated at varying rates ( λ z) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Ocular pharmacokinetics of acyclovir amino acid ester prodrugs in the anterior chamber: Evaluation of their utility in treating ocular HSV infections

Purpose To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits. Methods Stability of l-alanine–ACV (AACV), l-serine–ACV (SACV), l-isoleucine–ACV (IACV), γ-glutamate–ACV (EACV) and l-valine–ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose-dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques. Results Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5 mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants ( λ z). SACV and VACV exhibited approximately two-fold increase in area under the curve (AUC) relative to ACV ( p < 0.05). C last (concentration at the last time point) of SACV was observed to be 8 ± 2.6 μM in aqueous humor which is two and three times higher than VACV and ACV, respectively. Conclusions Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates ( k a) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC and high concentration at last time point ( C last) seems to be a promising candidate for the treatment of ocular HSV infections.

Discovery of Brivanib Alaninate ((S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), A Novel Prodrug of Dual Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1 Kinase Inhibitor (BMS-540215)

A series of amino acid ester prodrugs of the dual VEGFR-2/FGFR-1 kinase inhibitor 1 (BMS-540215) was prepared in an effort to improve the aqueous solubility and oral bioavailability of the parent compound. These prodrugs were evaluated for their ability to liberate parent drug 1 in in vitro and in vivo systems. The l-alanine prodrug 8 (also known as brivanib alaninate/BMS-582664) was selected as a development candidate and is presently in phase II clinical trials.

Synthesis and properties of a new water-soluble prodrug of the adenosine A 2A receptor antagonist MSX-2.

The compound L-valine-3-{8-[(E)-2-[3-methoxyphenyl)ethenyl]-7-methyl-1-propargyl­xan­thine-3-yl}propyl ester hydrochloride (MSX-4) was synthesized as an amino acid ester prodrug of the adenosine A2A receptor antagonist MSX-2. It was found to be stable in artificial gastric acid, but readily cleaved by pig liver esterase.

Transport of levovirin prodrugs in the human intestinal Caco-2 cell line

The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor.

Interaction of Intestinal Nucleoside Transporter hCNT2 with Amino Acid Ester Prodrugs of Floxuridine and 2-Bromo-5,6-dichloro-1-.BETA.-D-ribofuranosylbenzimidazole

Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. We studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB). Na(+)-dependent uptakes of [(3)H]-inosine and [(3)H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine (60-70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10-30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80-90% for adenosine and 60-80% for inosine). The amino acid site of attachment (3'- and 5'-monoesters) and stereochemistry (L- and D-amino acid esters), did not significantly affect the uptake of [(3)H]-inosine and [(3)H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs.

Nucleoside Ester Prodrug Substrate Specificity of Liver Carboxylesterase

Carboxylesterases are among the best characterized prodrug-hydrolyzing enzymes involved in the activation of several therapeutic carbamate and ester prodrugs. The broad specificity of these enzymes makes them amenable for designing prodrugs. Porcine liver carboxylesterase 1 specificity for amino acid esters of three nucleoside analogs [floxuridine, gemcitabine, and 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl) benzimidazole] was evaluated to assess optimal structural preferences for prodrug design. The amino acid promoiety and the esterification site influenced carboxylesterase hydrolysis rates up to 1164-fold and the binding affinity up to 26-fold. Carboxylesterase (CES) 1 exhibited high-catalytic efficiency hydrolyzing prodrugs containing a phenylalanyl moiety but was over 100-fold less efficient with valyl or isoleucyl prodrugs, regardless of the nucleoside or esterification site. CES1 catalytic efficiency was 2-fold higher with 5' phenylalanyl monoesters than the corresponding 3' esters of floxuridine. This preference was reversed with phenylalanyl gemcitabine prodrugs, evident from a 2-fold preference for 3' monoesters over 5' esters. The newly characterized esterase valacyclovirase was several hundred-fold more efficient (up to 19,000-fold) than carboxylesterase in hydrolyzing amino acid esters but similar in apparent binding affinity. The specific activities of the two enzymes with several amino acid ester prodrugs clearly suggest that initial hydrolysis rates are relatively low for prodrugs with isoleucyl, aspartyl, and lysyl promoieties for both enzymes compared with those with phenylalanyl, valyl, prolyl, and leucyl progroups. The low relative hydrolysis rates of isoleucyl, aspartyl, and lysyl prodrugs may facilitate prolonged systemic disposition of the nucleoside analogs for improved therapeutic action.

Floxuridine Amino Acid Ester Prodrugs: Enhancing Caco-2 Permeability and Resistance to Glycosidic Bond Metabolism

The aim of this study was to synthesize amino acid ester prodrugs of 5-fluoro-2'-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism. Amino acid ester prodrugs were synthesized and examined for their hydrolytic stability in human plasma, in Caco-2 cell homogenates, and in the presence of thymidine phosphorylase. Glycyl-L: -sarcosine uptake inhibition and direct uptake studies with HeLa/PEPT1 cells [HeLa cells overexpressing oligopeptide transporter (PEPT1)] were conducted to determine PEPT1-mediated transport and compared with permeability of the prodrugs across Caco-2 monolayers. Isoleucyl prodrugs exhibited the highest chemical and enzymatic stability. The prodrugs enhanced the stability of the glycosidic bond of floxuridine. Thymidine phosphorylase rapidly cleaved floxuridine to 5-fluorouracil, whereas with the prodrugs no detectable glycosidic bond cleavage was observed. The 5'-L: -isoleucyl and 5'-L: -valyl monoester prodrugs exhibited 8- and 19-fold PEPT1-mediated uptake enhancement in HeLa/PEPT1 cells, respectively. Uptake enhancement in HeLa/PEPT1 cells correlated highly with Caco-2 permeability for all prodrugs tested. Caco-2 permeability of 5'-L: -isoleucyl and 5'-L: -valyl prodrugs was 8- to 11-fold greater compared with floxuridine. Amino acid ester prodrugs such as isoleucyl floxuridine that exhibit enhanced Caco-2 transport and slower rate of enzymatic activation to parent, and that are highly resistant to metabolism by thymidine phosphorylase may improve oral delivery and therapeutic index of floxuridine.