Amino Acid Ester Prodrugs Research Articles

Anti-Inflammatory Immunomodulatory Activity of Valacyclovir on the in Vitro Activated Mammalian Macrophages.

Aciclovir, often known as acyclovir, is a nucleoside analog that exhibits antiviral activity in vitro against human herpesvirus 6 (HHV-6), cytomegalovirus (CMV), varicella-zoster virus (VZV), and herpes simplex virus (HSV). Valacyclovir is an amino acid ester prodrug of acyclovir. We examined valacyclovir, which is also an anti-viral agent, for its effects on inflammation. Mammalian Macrophages were activated by lipopolysaccharide (LPS) in the presence of a concentration range of Valacyclovir. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12p40 enzyme-linked immunosorbent assay (ELISA) was performed to measure the production levels of these pro-inflammatory cytokines. Our results suggest that Valacyclovir had anti-inflammatory activity on the LPS-activated mammalian macrophages. Valacyclovir has the potential to be utilized in the clinical setting as an anti-viral drug molecule with anti-inflammatory properties. Future studies are needed to further confirm its activities on different immune system cell types.

Inhibition of BPHL inhibits proliferation in lung carcinoma cell lines.

Lung cancer is one of the most common human malignant tumors and the leading cause of cancer death worldwide. Biphenyl hydrolase-like (BPHL) is a gene encoding the human BPHL enzyme, a serine hydrolase that catalyzes the hydrolytic activation of amino acid ester prodrugs of nucleoside analogs such as valacyclovir and valganciclovir. However, the role of BPHL in lung cancer is still unknown. In this study, we assessed the effect BPHL knockdown on the proliferation, apoptosis, colony formation, metastasis, and cell cycle of cancer cells. BPHL knockdown NCI-H1299 and A549 cells demonstrated decreased proliferation, as measured by Celigo cell counting. The MTT assay results were consistent with Celigo cell counting. Caspase 3/7 activity increased significantly in the NCI-H1299 and A549 cells after shBPHL knockdown. Decreased colony formation in the NCI-H1299 and A54 cells after shBPHL knockdown, as measured by crystal violet staining. Transmigration assay using a Transwell demonstrated that there were significantly fewer migrating cells in the lower chamber in the BPHL knockdown NCI-H1299 and A549 cells. Cell cycle analysis by Propidium Iodide (PI) staining and fluorescence activated cell sorter (FACS). We also explored the effect of BPHL knockdown on tumor growth in a mouse model of tumor implantation in nude mice. We found that the knockdown of BPHL gene expression by short hairpin RNA (shRNA) leads to a decrease in proliferation, colony formation, and metastasis and an increase in apoptosis in two lung adenocarcinoma (LUAD) cell lines in vitro. BPHL knockdown induces decreased tumor growth, colony formation, and metastasis; increased apoptosis; and altered cell cycle destruction. BPHL knockdown results in decreased tumor growth in vivo. Moreover, BPHL knockdown A549 cells demonstrated slower growth compared to control cells upon implantation in nude mice, confirming the in vitro findings. In this study, the data indicate that BPHL potentially promotes proliferation, inhibits apoptosis, and increases colony formation and metastasis in lung cancer. Overall, our study suggests that BPHL may be a gene that promotes tumor growth in lung cancer.

Cationic Ester Prodrugs of Curcumin with N,N-dimethyl Amino Acid Promoieties Improved Poor Water Solubility and Intestinal Absorption

Although curcumin (Cur) has powerful pharmacological effects, its use in medicine has not been established yet. The oral bioavailability (BA) of Cur is limited because of its poor water solubility. The purpose of this study was to confirm whether cationic N,N-dimethyl amino acid esters of Cur could act as prodrugs and improve its water solubility and oral bioavailability. Two N,N-dimethyl amino acid esters of Cur were synthesized. The hydrolysis profile of the esters was evaluated using rat and human microsomes. A pharmacokinetic study after oral administration of the Cur ester derivatives was performed in rats and compared to the administration of suspended or dissolved Cur formulation. The anti-inflammatory effects of the Cur derivatives were evaluated using macrophage RAW 264.7 stimulated with lipopolysaccharide. Cur ester derivatives showed > 200mM water solubility. The derivatives were reconverted to the parent compound (Cur) after cleavage of the ester bonds by microsomal esterase, indicating that the compounds could act as Cur prodrugs. The Cur prodrugs enhanced the absolute oral bioavailability of Cur by a 9- and threefold increase of suspended and dissolved Cur administration, respectively, thereby improving intestinal absorption. Cur prodrugs strongly attenuated COX2, iNOS, and ERK phosphorylation. The cationic N,N-dimethyl amino acid ester prodrugs of Cur improved the water solubility of Cur and enhanced oral bioavailability in rats. These Cur prodrugs may be good candidates for developing medicinal options previously unavailable due to the poor water solubility and oral BA of Cur.

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity.

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides.

Improving Drug Delivery While Tailoring Prodrug Activation to Modulate Cmax and Cmin by Optimization of (Carbonyl)oxyalkyl Linker-Based Prodrugs of Atazanavir.

Structure-property relationships associated with a series of (carbonyl)oxyalkyl amino acid ester prodrugs of the marketed HIV-1 protease inhibitor atazanavir (1), designed to enhance the systemic drug delivery, were examined. Compared to previously reported prodrugs, optimized candidates delivered significantly enhanced plasma exposure and trough concentration (Cmin at 24 h) of 1 in rats while revealing differentiated PK paradigms based on the kinetics of prodrug activation and drug release. Prodrugs incorporating primary amine-containing amino acid promoieties offered the benefit of rapid bioactivation that translated into low circulating levels of the prodrug while delivering a high Cmax value of 1. Interestingly, the kinetic profile of prodrug cleavage could be tailored for slower activation by structural modification of the amino terminus to either a tertiary amine or a dipeptide motif, which conferred a circulating depot of the prodrug that orchestrated a sustained release of 1 along with substantially reduced Cmax and a further enhanced Cmin.

Pharmacokinetics of gemcitabine and its amino acid ester prodrug following intravenous and oral administrations in mice

Gemcitabine is an intravenously administered anti-cancer nucleoside analogue. Systemic exposure following oral administration of gemcitabine is limited by extensive first-pass metabolism via cytidine deaminase (CDA) and potentially by saturation of nucleoside transporter-mediated intestinal uptake. An amino acid ester prodrug of gemcitabine, 5′-l-valyl-gemcitabine (V-Gem), was previously shown to be a substrate of the intestinally expressed peptide transporter 1 (PEPT1) and stable against CDA-mediated metabolism. However, preliminary studies did not evaluate the in vivo oral performance of V-Gem as compared to parent drug. In the present study, we evaluated the pharmacokinetics and in vivo oral absorption of gemcitabine and V-Gem following intravenous and oral administrations in mice. These studies revealed that V-Gem undergoes rapid systemic elimination (half-life < 1 min) and has a low oral bioavailability (<1%). Most importantly, the systemic exposure of gemcitabine was not different following oral administration of equimolar doses of gemcitabine (gemcitabine bioavailability of 18.3%) and V-Gem (gemcitabine bioavailability of 16.7%). Single-pass intestinal perfusions with portal blood sampling in mice revealed that V-Gem undergoes extensive activation in intestinal epithelial cells and that gemcitabine undergoes first-pass metabolism in intestinal epithelial cells. Thus, formulation of gemcitabine as the prodrug V-Gem does not increase systemic gemcitabine exposure following oral dosing, due, in part, to the instability of V-Gem in intestinal epithelial cells.

Bioactivatable Pseudotripeptidization of Cyclic Dipeptides To Increase the Affinity toward Oligopeptide Transporter 1 for Enhanced Oral Absorption: An Application to Cyclo(l-Hyp-l-Ser) (JBP485).

The cyclic dipeptides generally present lower affinity toward intestinal oligopeptide transporter 1 (PEPT1) than the linear dipeptides. JBP485 (cyclo(l-Hyp-l-Ser)) is a low-affinity substrate of PEPT1 with poor oral bioavailability. However, JBP923 (l-Hyp-l-Ser) is a high-affinity substrate of PEPT1 with high oral absorption. We hypothesize that the bioactivatable pseudo-tripeptidization prodrug strategy is promising to increase the affinity of cyclic dipeptides toward PEPT1. To test our hypothesis, we design five amino acid ester prodrugs of JBP485. Compared with JBP485, the optimal prodrug (JBP485-3-CH2-O-valine, J3V) demonstrates improved affinity of PEPT1, oral bioavailability in rats and beagle dogs. Moreover, J3V can dose-dependently protect against liver injury. Additionally, J3V is stable in the gastrointestinal tract, beneficial to the PEPT1-mediated membrane transport, and is bioactivated in the enterocytes and hepatic cells, essential to elicit its bioactivity. In summary, the bioactivatable pseudo-tripeptidization strategy shows potential in increasing affinity of PEPT1 to enhance oral bioavailability of cyclic dipeptides.

Design, Synthesis, and Pharmacokinetic Evaluation of Phosphate and Amino Acid Ester Prodrugs for Improving the Oral Bioavailability of the HIV-1 Protease Inhibitor Atazanavir.

Phosphate and amino acid prodrugs of the HIV-1 protease inhibitor (PI) atazanavir (1) were prepared and evaluated to address solubility and absorption limitations. While the phosphate prodrug failed to release 1 in rats, the introduction of a methylene spacer facilitated prodrug activation, but parent exposure was lower than that following direct administration of 1. Val amino acid and Val-Val dipeptides imparted low plasma exposure of the parent, although the exposure of the prodrugs was high, reflecting good absorption. Screening of additional amino acids resulted in the identification of an l-Phe ester that offered an improved exposure of 1 and reduced levels of the circulating prodrug. Further molecular editing focusing on the linker design culminated in the discovery of the self-immolative l-Phe-Sar dipeptide derivative 74 that gave four-fold improved AUC and eight-fold higher Ctrough values of 1 compared with oral administration of the drug itself, demonstrating a successful prodrug approach to the oral delivery of 1.

PEPT1-mediated prodrug strategy for oral delivery of peramivir

Peramivir was a novel and highly potent neuraminidase (NA) inhibitor for the treatment of influenza A and B. However, it exhibited a very low oral bioavailability (only 3%) due to the high polarity (log P of −1.4) and the low membrane permeability across the intestine. To utilize the PEPT1-mediated prodrug strategy to improve the oral absorption and develop the oral alternative, seven amino acid ester prodrugs and seven amino acid amide prodrugs have been synthesized. The permeability of these prodrugs across Caco-2 cells were screened. Peramivr-(CH2)2-l-Val and Peramivir-l-Ile were of the highest permeability in ester prodrugs and amide prodrugs, respectively, and then they were selected for further studies. Glycylsarcosine (gly-sar) uptake by Caco-2 could be inbihited by Peramivir-(CH2)2-l-Val and Peramivir-l-Ile in a concentration-dependent manner, and the IC50 was 1.34 ± 0.31 mM and 1.78 ± 0.48 mM, respectively. The direct uptake of Peramivir-(CH2)2-l-Val and Peramivir-l-Ile in MDCK-PEPT1 cells were significantly higher than in MDCK mock cells, and could be markedly inhibited by gly-sar. The uptake of Peramivir-(CH2)2-l-Val and Peramivir-l-Ile (0.01 to 50 mM) in MDCK-hPEPT1 cells conformed to Michaelis–Menten Equation. The oral bioavailability of peramivir was 65.3% and 37.3% after the oral administration of Peramivir-(CH2)2-l-Val and Peramivir-l-Ile to rats, respectively. The oral absorption and bioactivation of Peramivir-(CH2)2-l-Val was rapid and extensive, and no Peramivir-(CH2)2-l-Val was found in plasma. Because the amide bond was relatively stable, Peramivir-l-Ile could not be totally converted to the parent drug in vivo. Peramivir-(CH2)2-l-Val with good oral profiles and rapid bioactivation might be a promising prodrug for the further clinic development. The present study also corroborated the idea that the PEPT1-mediated prodrug approach has enormous promise for improving the oral absorption of poorly absorbed drug.

Controlled non-invasive iontophoretic delivery of triamcinolone acetonide amino acid ester prodrugs into the posterior segment of the eye

This study investigated short duration transscleral iontophoretic delivery of four triamcinolone acetonide (TA) amino acid ester prodrugs (TA-AA) (alanine, Ala; arginine, Arg; isoleucine, Ile and lysine, Lys) using whole porcine eyes globes in vitro. Post-iontophoretic biodistribution of TA was quantified by UHPLC-MS/MS in the different ocular compartments (cornea, aqueous humor, sclera, ciliary body, choroid and retinal pigmented epithelium (RPE), neural retina and vitreous humor). Transscleral iontophoresis (3 mA/cm2 for 10 min) increased total drug delivery of the TA-AA prodrugs by 14-30-fold as compared to passive diffusion. The TA-AA prodrugs had distinct biodistribution profiles – the penetration depth achieved was dependent on their physicochemical properties (e.g. lipophilicity for TA-Ile) and susceptibility to hydrolysis (e.g. TA-Arg). Intraocular drug distribution was also influenced by prodrug binding to melanin (TA-Lys). Interestingly, under conditions of equivalent charge (6 mA/cm2 for 5 min vs. 1.5 mA/cm2 for 20 min, i.e. 1.44 C respectively) the longer duration (20 min) at lower current density resulted in ∼6 times more TA delivery into the vitreous humor. Overall, the study provided further evidence of the potential of transscleral iontophoresis for the non-invasive treatment of posterior segment inflammatory diseases.

Identification of human, rat and mouse hydrolyzing enzymes bioconverting amino acid ester prodrug of ketoprofen

Alkyl ester prodrugs are well known to be bioconverted by carboxylesterases, particularly in rodents’ by first-pass metabolism in the systemic circulation and liver. However, the bioconversion of structurally more complex esters with polar functional groups is less well understood, especially in humans. Therefore, it is not clear if ester prodrugs can be utilized for targeted drug delivery. In the present study a brain-targeted ester prodrug (1) of ketoprofen, utilizing the l-type amino acid transporter 1 (LAT1) was prepared and the enzymes involved in its metabolism in human plasma and liver S9 subcellular fraction as well as rat brain S9 fraction were identified. Furthermore, species differences among mouse, rat and human plasma and liver S9 fraction were compared. The results showed that bioconversion of the ester prodrug was much faster in mouse plasma compared to human, while it’s half-life in rat plasma was closer to the one of human. Moreover, both rodent species showed more efficient bioconversion in the liver S9 fractions compared to human and relatively efficient bioconversion in the brain S9 fractions. More specifically, butyrylcholinesterase (BChE) and paraoxygenase 1 (PON1) were the main hydrolyzing enzymes of the prodrug 1 in human plasma, while carboxylesterases 1 and 2 (CES1 and CES2) as well as PONs were the main bioconverting enzymes in human liver S9 fractions. In rat brain S9 fraction, acetylcholinesterase (AChE) was hydrolyzing the prodrug 1, although also other unidentified metal-and pH-dependent enzyme(s) were recognized to be participating to the total bioconversion of the compound 1 in the brain.

Intestinal absorption and activation of decitabine amino acid ester prodrugs mediated by peptide transporter PEPT1 and enterocyte enzymes

Decitabine (DAC), a potent DNA methyltransferase (DNMT) inhibitor, has a limited oral bioavailability. Its 5′-amino acid ester prodrugs could improve its oral delivery but the specific absorption mechanism is not yet fully understood. The aim of this present study was to investigate the in vivo absorption and activation mechanism of these prodrugs using in situ intestinal perfusion and pharmacokinetics studies in rats. Although PEPT1 transporter is pH dependent, there appeared to be no proton cotransport in the perfusion experiment with a preferable transport at pH 7.4 rather than pH 6.5. This suggested that the transport was mostly dependent on the dissociated state of the prodrugs and the proton gradient might play only a limited role. In pH 7.4 HEPES buffer, an increase in Peff was observed for L-val-DAC, D-val-DAC, L-phe-DAC and L-trp-DAC (2.89-fold, 1.2-fold, 2.73-fold, and 1.90-fold, respectively), compared with the parent drug. When co-perfusing the prodrug with Glysar, a known substrate of PEPT1, the permeabilities of the prodrugs were significantly inhibited compared with the control. To further investigate the absorption of the prodrugs, L-val-DAC was selected and found to be concentration-dependent and saturable, suggesting a carrier-mediated process (intrinsic Km: 7.80 ± 2.61 mM) along with passive transport. Determination of drug in intestinal homogenate after perfusion further confirmed that the metabolic activation mainly involved an intestinal first-pass effect. In a pharmacokinetic evaluation, the oral bioavailability of L-val-DAC, L-phe-DAC and L-trp-DAC were nearly 1.74-fold, 1.69-fold and 1.49-fold greater than that of DAC. The differences in membrane permeability and oral bioavailability might be due to the different stability in the intestinal lumen and the distinct PEPT1 affinity which is mainly caused by the stereochemistry, hydrophobicity and steric hindrance of the side chains. In summary, the detailed investigation of the absorption mechanism by in vivo intestinal perfusion and pharmacokinetic studies showed that the prodrugs of DAC exhibited excellent permeability and oral bioavailability, which might be attributed to a hybrid (partly PEPT1-mediated and partly passive) transport mode and a rapid activation process in enterocytes.

Short-duration ocular iontophoresis of ionizable aciclovir prodrugs: A new approach to treat herpes simplex infections in the anterior and posterior segments of the eye

The objective was to investigate (trans)corneal and transscleral iontophoresis of biolabile amino acid ester prodrugs of aciclovir (ACV-X, X = Arg, Gly and Trp) as a means to increase ocular bioavailability of ACV. Prodrugs displayed tissue-dependent susceptibility to hydrolysis. Iontophoresis of ACV-Arg, ACV-Gly and ACV-Trp (5 mM, 0.5 mA/cm2) for 5 min followed by 55 min passive diffusion resulted in appreciable corneal deposition (21.5 ± 5.1, 14.1 ± 2.0 and 5.3 ± 0.6 nmol/cm2, respectively) and transcorneal permeation (13.9 ± 1.6, 10.9 ± 1.8 and 5.7 ± 0.5 nmol/cm2, respectively) of ACV species. In contrast, passive delivery of ACV across porcine cornea after 1 h was < LOQ (i.e. <0.125 nmol/cm2). Transscleral permeation of ACV-Arg, ACV-Gly and ACV-Trp (9 mM, 1.25 mA/cm2) after iontophoresis for 5 min was 20.4 ± 3.8, 12.3 ± 0.3 and 8.4 ± 0.4 nmol/cm2, respectively – far superior to passive delivery which was again < LOQ. Using intact porcine eye globes, 5 min transscleral iontophoresis of ACV-Gly at 3.75 mA/cm2 resulted in considerable delivery of ACV species to the choroid/retina and vitreous humour (5.7 ± 2.3 and 11.7 ± 3.7 nmol/cm2, respectively). Furthermore, the average concentration of ACV species in the whole eyeball (4.5 ± 1.6 nmol/cm3) was significantly higher than the IC50 of ACV against HSV-1 (<0.22 nmol/cm3), demonstrating the potential application for the treatment of ocular HSV infections.

Amino Acid Ester Prodrugs of Nucleoside and Nucleotide Antivirals.

The review covers basic principles of the prodrug strategy applied to antiviral nucleoside drugs or drug candidates. Specific role of amino acids as promoieties is explained with respect to transport mechanisms, pharmacokinetics and a low toxicity of compounds. Synthetic approaches to the most important representatives (compounds under clinical investigations or available on the market) are described, including valacyclovir, valganciclovir, valomaciclovir stearate, valcyclopropavir, valtorcitabine, valopicitabine and several attempts to amino acid modifications of antiretroviral nucleosides. A special attention is paid to acyclic nucleoside phosphonates, where the phosphonic acid residue is esterified with a side-chain hydroxyl group of appropriate amino acid (serine, tyrosine) which can be used as single amino acid or as a part of dipeptides further modified on the terminal carboxyl function. The most advantageous pharmacokinetic profile and the best oral bioavailability were found in tyrosinebased prodrugs. Studies were performed successfully on 1-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl]cytosine (cidofovir), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and some (R)-2- (phosphonomethoxy)propyl and 2-(phosphonomethoxy)ethyl derivatives including adefovir.

Targeted intracorneal delivery—Biodistribution of triamcinolone acetonide following topical iontophoresis of cationic amino acid ester prodrugs

The aim was to investigate intracorneal iontophoresis of biolabile triamcinolone acetonide (TA) amino acid ester prodrugs (TA-AA). Arginine and lysine esters of TA (TA-Arg and TA-Lys, respectively) were synthesized and characterized; quantification was performed by HPLC-UV and UHPLC–MS/MS. The aqueous solubility of the prodrugs (at pH 5.5) was ∼1000-fold greater than TA. Anodal iontophoresis (10min at 3mA/cm2) of TA-AA was investigated using isolated porcine cornea. Although no statistically significant difference was observed in total intracorneal delivery of TA (468.25±59.70 and 540.85±79.16nmolTA/cm2, for TA-Arg and TA-Lys, respectively), the different susceptibilities of the prodrugs to hydrolysis influenced intracorneal biodistribution. Quantification of TA in twenty-five 40μm thick corneal lamellae revealed significantly deeper penetration of TA following TA-Lys iontophoresis. Its superior resistance to hydrolysis enabled sustained electromigration into the deeper cornea suggesting judicious prodrug selection might enable targeted regioselective drug delivery. The intracorneal biodistribution following anodal iontophoresis of TA-Arg (2.3mM; 10min, 3mA/cm2) was visualized by full field optical coherence tomography providing qualitative confirmation of the extensive intracorneal penetration of TA. Short duration iontophoresis of TA-AA prodrugs may improve deep corneal bioavailability and efficacy in vivo, constituting a “single-shot” treatment option for corneal allograft rejection.

Topical iontophoretic delivery of ionizable, biolabile aciclovir prodrugs: A rational approach to improve cutaneous bioavailability

The objective was to investigate the topical iontophoretic delivery of a series of amino acid ester prodrugs of aciclovir (ACV-X, where ACV=aciclovir and X=Arg, Gly, Ile, Phe, Trp and Val) as a means to enhance cutaneous delivery of ACV. The newly synthesized prodrugs were characterized by 1H NMR and high resolution mass spectrometry. Analytical methods using HPLC–UV were developed for their quantification and each method was validated. Investigation of solution stability as a function of pH showed that all ACV-X prodrugs were relatively stable in acid conditions at pH 2.0 and pH 5.5 for up to 8h but susceptible to extensive hydrolysis at pH 7.4 and under alkaline conditions (pH 10). No ACV-X hydrolysis was observed after contact for 2h with the external surface of porcine stratum corneum. However, there was significant hydrolysis following contact with the dermal surface of dermatomed porcine skin, in particular, for ACV-Arg. Passive transport of ACV and ACV-X prodrugs from aqueous solution after 2h was below the limit of detection. Iontophoresis of ACV at 0.5mA/cm2 for 2h led to modest ACV skin deposition (QDEP,ACV) of 4.6±0.3nmol/cm2. In contrast, iontophoresis of ACV-X prodrugs under the same conditions produced order of magnitude increases in cutaneous deposition of ACV species, that is, QDEP,TOTAL=QDEP,ACV+QDEP,ACV-X. QDEP,TOTAL for ACV-Gly, ACV-Val, ACV-Ile, ACV-Phe, ACV-Trp and ACV-Arg was 412.8±44.0, 358.8±66.8, 434.1±68.2, 249.8±81.4, 156.1±76.3, 785.9±78.1nmol/cm2, respectively. The extent of bioconversion of ACV-X to ACV in the skin was high and the proportion of ACV present ranged from 81% to 100%. The skin retention ratio, a measure of the selectivity of ACV species for deposition over permeation after iontophoretic delivery of ACV-X prodrugs, was dependent on both the rate of transport and the susceptibility to hydrolysis of the prodrugs. Skin deposition of ACV and its six prodrugs were investigated further as a function of current density (0.125, 0.25 and 0.5mA/cm2); the effect of duration of current application (5, 10, 30, 60 and 120min) was evaluated using ACV-Arg and ACV-Ile. Iontophoresis of ACV-Arg and ACV-Ile at 0.25mA/cm2 for only 5min resulted in the deposition of appreciable amounts of ACV (36.4±5.7nmol/cm2 and 40.3±6.1nmol/cm2, respectively), corresponding to supra-therapeutic average concentrations in skin against HSV-1 or HSV-2. The results demonstrated that cutaneous bioavailability of ACV could be significantly improved after short-duration iontophoresis of ionizable, biolabile ACV-X prodrugs.

Cutaneous biodistribution of ionizable, biolabile aciclovir prodrugs after short duration topical iontophoresis: Targeted intraepidermal drug delivery

The objective was to determine the cutaneous biodistribution of aciclovir (ACV), that is, the amount of drug as a function of depth within the skin following topical iontophoretic administration of amino acid ester prodrugs of ACV (ACV-X, where X=Arg, Ile or Val). The results were compared to those obtained with marketed formulations of aciclovir and penciclovir (PCV), and following topical iontophoresis of ACV. Quantification of molecules as a function of position in the skin was achieved by snap-freezing and cryotoming skin samples to obtain coarse or fine lamellae with a thickness of either 100μm or 20μm. The molecules – ACV, ACV-X or PCV – were extracted and quantified by validated UHPLC-MS/MS analytical methods. Passive delivery of ACV or PCV from marketed cream and ointment formulations after application for 60min resulted in modest cutaneous deposition (QDEP,ACV and QDEP,PCV<2nmol/cm2). Moreover, ACV and PCV were found mainly in the stratum corneum or superficial viable epidermis. The levels in the deeper skin layers (100–200μm), corresponding to the basal epidermis and adjacent area, where the virus would be found, were negligible. In contrast, iontophoresis of ACV-Ile or ACV-Arg for only 10min at 0.25mA/cm2 resulted in much greater deposition of ACV species at the same skin depths (100–200μm): for ACV-Ile – QDEP,ACV and QDEP,ACV-Ile were 17.2±6.9nmol/cm2 and 8.2±1.3nmol/cm2, respectively; in the case of ACV-Arg there was complete bioconversion and only ACV was recovered (QDEP,ACV was 41.2±9.2nmol/cm2). In the higher resolution biodistribution studies, with 20μm lamellae, short duration iontophoresis at 0.25mA/cm2 for only 5min of ACV-Arg or ACV-Ile still enabled considerable amounts of ACV species to be delivered to the basal epidermis and neighboring layers (QDEP,ACV was 4.0±1.6nmol/cm2 and 6.1±1.7nmol/cm2, respectively). Iontophoresis of ACV under the same conditions resulted in negligible ACV deposition in these layers. Subsequent studies into the iontophoresis of ACV-Ile using a hydrogel revealed no statistically significant differences between QDEP,ACV in full-thickness porcine and human skins (13.4±1.5nmol/cm2 and 12.4±1.4nmol/cm2, respectively), validating the porcine model. In order to improve ACV-Ile stability, dry fast dissolving thin film formulations were also prepared. These were placed in contact with conducting hydrogels in situ immediately prior to iontophoresis. In addition to the improved stability, iontophoretic delivery of ACV-Ile from the thin film was also superior to that from hydrogel alone. Following iontophoresis of ACV-Ile using fast dissolving films, QDEP,ACV was 29.1±1.0nmol/cm2, and the amount in the basal epidermis area was 2.7±0.1nmol/cm2. Short duration iontophoresis for 5min of ACV-Ile from aqueous solution, hydrogel or fast dissolving film resulted in supra-therapeutic concentrations of ACV in the basal epidermis and its adjacent area.

Amino acid ester prodrugs conjugated to the α-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1)

L-type amino acid transporter (LAT1) is an intriguing target for carrier-mediated transport of drugs as it is highly expressed in the blood–brain barrier and also in various types of cancer. Several studies have proposed that in order for compounds to act as LAT1 substrates they should possess both negatively charged α-carboxyl and positively charged α-amino groups. However, in some reports, such as in two recent publications describing an isoleucine–quinidine ester prodrug (1), compounds having no free α-carboxyl group have been reported to exhibit high affinity for LAT1 in vitro. In the present study, 1 was synthesized and its affinity for LAT1 was evaluated both with an in situ rat brain perfusion technique and in the human breast cancer cell line MCF-7 in vitro. 1 showed no affinity for LAT1 in either model nor did it show any affinity for LAT2 in an in vitro study. Our results confirm the earlier reported requirements for LAT1 substrates. Thus drugs or prodrugs with substituted α-carboxyl group cannot bind to LAT with high affinity.

Synthesis and in vitro stability of amino acid prodrugs of 6-β-naltrexol for microneedle-enhanced transdermal delivery

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a–f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2=2.2±0.1h).

Synthesis and characterization of valyloxy methoxy luciferin for the detection of valacyclovirase and peptide transporter

An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner.