Synthesis and in vitro anti-HIV activities of didanosine prodrugs

A series of prodrugs of didanosine were synthesized in an effort to enhance the anti-HIV activity. The 5'-OH function of didanosine was esterified with different aryl piperazine acetic acid derivatives and evaluated for anti-HIV-1 activity in MT-4 cell line using the MTT assay method. Among the synthesized compounds, (tetrahydro-5-(1,6-dihydro-6-oxopurin-9-yl)furan-2-yl)methyl 2-(4-(4-chlorophenyl)piperazin-1-yl)acetate (4b) was found to be the most potent compound with EC50 of 0.64 microM and was not toxic to the MT-4 cells up to 1000 microM with a selectivity index of > 1562. Compound 4b was found to be seven times more potent than the parent drug didanosine (EC50 of 4.8 microM) in vitro. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t1/2 ranging from 20-60 min.     

Simple mono-derivatisation of the aryl moiety of D4A and DDA-based phosphoramidate prodrugs significantly enhances their anti-HIV potency in cell culture.

Simple mono-derivatisation of the aryl moiety of some phosphoramidate pronucleotide derivatives of d4A and ddA served to increase the lipophilicity of these membrane-soluble prodrugs. A concomitant and significant enhancement of potency against HIV-1 and HIV-2 in vitro was observed for the ddA- and d4A-based prodrugs compared to the original underivatised prodrugs.     

Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the V_max value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker.

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.     

Synthesis, biological evaluation and kinetic studies of glyceride prodrugs of diclofenac

The prodrugs (glyceride derivatives) 3a and 3b of diclofenac were prepared by reacting 1, 2, 3-trihydroxy propane-1,3-dipalmitate/stearate with the acid chloride of diclofenac as potential prodrugs to reduce the gastrointestinal toxicity associated with them. These prodrugs were evaluated for their ulcerogenicity, anti-inflammatory and analgesic activity. It was found that the prodrugs were significantly less irritating to the gastric mucosa as indicated by severity index of 0.86, 0.78 compared to 1.6 of diclofenac. The prodrugs 3a and 3b showed better anti-inflammatory and analgesic activity than the parent drugs. The hydrolysis of prodrugs 3a and 3b were studied at pH 3, 4, 5 and 7.4. The HPLC analysis showed that the prodrugs were resistant to hydrolysis at pH 3, 4 and 5 indicating that they did not hydrolyze in acidic environment, whereas at pH 7.4 the prodrugs readily released the parent drug in significant quantities. The plasma levels of diclofenac were also analyzed by HPLC in rats after single oral dose of the prodrugs. The results indicated that the parent drugs were readily released. The concentration of diclofenac during the study was found higher in animals treated with prodrugs 3a and 3b compared with animals treated with diclofenac. The concentration of diclofenac was found to be 38.59, 33.6 and 30.36 microg/ml in animals treated with prodrugs 3a, 3b and diclofenac respectively. In conclusion, all these studies indicated that the glyceride prodrugs of diclofenac might be considered as potential biolabile prodrugs of diclofenac.     

Syntheses of cyclic prodrugs of RGD peptidomimetics with various macrocyclic ring sizes: evaluation of physicochemical, transport and antithrombic properties.

The objective of this work was to synthesize cyclic prodrugs 1a-d of RGD peptidomimetics 2a-d with various ring sizes (n[CH2] = 1, 3, 5 and 7) and to evaluate the effect of ring size on their transport, physicochemical, enzymatic stability, and antithrombic properties. The syntheses of cyclic prodrugs 1a-d were achieved by converging two key intermediates, Boc-Phe-O-CH2-OCO-OpNP (5) and H2N-(CH2)n-CO-Asp(OBzl)-OTce (8a-d), to give linear precursors Boc-Phe-O-CH2-OCO-HN-(CH2)n-CO-Asp(OBzl)-OTce (9a-d). The N- and C-terminus protecting groups were removed from 9a-d to give 10a-d. Linear precursors 10a-d were cyclized, and the remaining Bzl-protecting group was removed to produce cyclic prodrugs 1a-d in around 20% overall yield. The linear RGD peptidomimetics (2a-d) were synthesized using standard Boc-amino acid chemistry by solution-phase method. Increasing the ring size by adding methylene groups also increases the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics. The transport properties of cyclic prodrugs 1c and 1d were 2.6- and 4.4-fold better than those of parent compounds 2c and 2d, respectively. These results suggest that increasing the hydrophobicity of the cyclic prodrugs and parent RGD peptidomimetics enhanced their transport properties. The hydrodynamic radii of the cyclic prodrugs were also smaller than those of their respective parent compounds, suggesting that the change in size may contribute to their transport properties. The chemical stability of the cyclic prodrugs was affected by the ring size, and the cyclic prodrug with the larger ring size (i.e. 1d) was more stable than the smaller one (i.e. 1a). All the cyclic prodrugs were more stable at pH 4 than at pH 7 and 10. Prodrug-to-drug conversion could be induced by isolated esterase as well as esterase found in human plasma. An increase in the length of methylene group (n[CH2] = 1, 3, 5, 7) enhanced the antithrombic activity of the prodrugs and the parent compounds. In summary, the ring size of cyclic prodrugs affected their transport, physicochemical, and antithrombic properties.     

Synthesis and assessment of first-generation polyamidoamine dendrimer prodrugs to enhance the cellular permeability of P-gp substrates

The aim of this study is to evaluate the potential use of first-generation (G1) polyamidoamine (PAMAM) dendrimers as drug carriers to enhance the permeability, hence oral absorption, of drugs that are substrates for P-glycoprotein (P-gp) efflux transporters. G1 PAMAM dendrimer-based prodrugs of the water-insoluble P-gp substrate terfenadine (Ter) were synthesized using succinic acid (suc) or succinyl-diethylene glycol (suc-deg) as a linker/spacer (to yield G1-suc-Ter and G1-suc-deg-Ter, respectively). In addition, the permeability of G1-suc-deg-Ter was enhanced by attaching two lauroyl chains (L) to the dendrimer surface (L2-G1-suc-deg-Ter). All of the G1 dendrimer-terfenadine prodrugs were more hydrophilic than the parent drug, as evaluated by drug partitioning between 1-octanol and phosphate buffer at pH 7.4 (log K(app)). The influence of the dendrimer prodrugs on the integrity and viability of human Caucasian colon adenocarcinoma cells (Caco-2) was determined by measuring the transepithelial electrical resistance (TEER) and leakage of lactate dehydrogenase (LDH) enzyme, respectively. The LDH assay indicated that the dendrimer prodrugs had no impact on the viability of Caco-2 cells up to a concentration of 1 mM. However, the IC(50) of the prodrugs was lower than that of G1 PAMAM dendrimer because of the high toxicity of terfenadine. Measurements of the transport of dendrimer prodrugs across monolayers of Caco-2 cells showed an increase of the apparent permeability coefficient (P(app)) of terfenadine in both apical-to-basolateral (A --> B) and basolateral-to-apical (B --> A) directions after its conjugation to G1 PAMAM dendrimer. The A --> B P(app) of the dendrimer prodrugs was significantly greater than B --> A P(app). The surface-modified dendrimer prodrug L2-G1-suc-deg-Ter showed the highest A --> B permeability among the conjugates.     

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 of phosphonate 3-phthalidyl esters as prodrugs for potential intracellular delivery of phosphonates.

A new prodrug approach for intracellular delivery of phosphonates was developed via the synthesis of 3-phthalidyl esters of 1-naphthalenemethylphosphonate. This approach is advantageous over the traditional acyloxymethyl phosphonate prodrugs, because these prodrugs do not generate formaldehyde and have improved plasma half-lives.     

Water soluble prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one

Fourteen prodrugs of the antitumor agent 3-[(3-amino-4-methoxy)phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one (1) were prepared to improve its water solubility and potency. These prodrugs include alpha-amino acid (1a-1h), aliphatic amino acid (1i-1l), phosphoramidate (1m), and phosphate (1n) derivatives. All of the prodrugs showed improved water solubility. A number of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) exhibited more potent antitumor activity compared to the parent compound (1). The phosphate prodrug 1n also offered a potent antitumor activity, but the phosphoramidate 1m did not show any antitumor activity in vivo. None of the prodrugs exhibited significant toxicities in mice. These results indicate that the design and preparation of the amino acid prodrugs (1a, 1b, 1d-1f, 1h, 1j, and 1k) and phosphate prodrug (1n) are beneficial for enhancing the antitumor activity of 1. The similar approaches may be used to improve water solubility and bioactivity of other poorly soluble aromatic amines.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Effect of the acyl groups on O-->N acyl migration in the water-soluble prodrugs of HIV-1 protease inhibitor

To improve the low water-solubility of HIV-1 protease inhibitors KNI-272, -279 and -727, we previously reported the water-soluble prodrugs of these inhibitors based on O-->N intramolecular acyl migration reaction. These prodrugs were rapidly converted to the corresponding parent drugs under physiological conditions. To understand the steric and electrostatic effects of O-acyl moiety on the migration rate, we examined several types of prodrug. A remarkably slow migration was observed in the benzoyl-type prodrugs, and Hammett plot of migration rate constants of p-substituted benzoyl-type prodrugs gave a linear free energy relationship.     

Chlorzoxazone esters of some non-steroidal anti-inflammatory (NSAI) carboxylic acids as mutual prodrugs: Design,synthesis, pharmacological investigations and docking studies

The discovery of the inducible isoform of cyclooxygenase enzyme (COX-2) spurred the search for anti-inflammatory agents devoid of the undesirable effects associated with classical NSAIDs. New chlorzoxazone ester prodrugs (6–8) of some acidic NSAIDs (1–3) were designed, synthesized and evaluated as mutual prodrugs with the aim of improving the therapeutic potency and retard the adverse effects of gastrointestinal origin. The structure of the synthesized mutual ester prodrugs (6–8) were confirmed by IR, ¹H NMR, mass spectroscopy (MS) and their purity was ascertained by TLC and elemental analyses. In vitro chemical stability revealed that the synthesized ester prodrugs (6–8) are chemically stable in hydrochloric acid buffer pH 1.2 as a non-enzymatic simulated gastric fluid (SGF) and in phosphate buffer pH 7.4 as non-enzymatic simulated intestinal fluid (SIF). In 80% human plasma, the mutual prodrugs were found to be susceptible to enzymatic hydrolysis at relatively faster rate (t_1/2 ≈ 37 and 34 min for prodrugs 6 and 7, respectively). Mutual ester prodrugs (6–8) were evaluated for their anti-inflammatory and muscle relaxation activities. Scanning electromicrographs of the stomach showed that the ester prodrugs induced very little irritancy in the gastric mucosa of rats after oral administration for 4 days. In addition, docking of the mutual ester prodrugs (6–8) into COX-2 active site was conducted in order to predict the affinity and orientation of these prodrugs at the enzyme active site.     

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.     

Modulating paclitaxel bioavailability for targeting prostate cancer

Four novel water-soluble peptide-paclitaxel conjugates were designed and synthesized as prostate-specific antigen (PSA)-activated prodrugs for prostate cancer therapy. These prodrugs were composed of a peptide, HSSKLQ or SSKYQ, each of which is selectively cleavable by PSA; a self-immolative linker, either para-aminobenzyl alcohol (PABS) or ethylene diamine (EDA); and the parent drug, paclitaxel. Introduction of a PABA or EDA linker between the peptide and paclitaxel in prodrugs 2-5 resulted in products with an increased rate of hydrolysis by PSA. The stability of prodrugs 2 and 3, with the PABA linker, was poor in the serum-containing medium because of the weak carbonate bond between the PABA and paclitaxel; however, this disadvantage was overcome by introducing a carbamate bond using an EDA linker in prodrugs 4 and 5. Thus, the incorporation of an EDA linker increased both the stability and PSA-mediated activation of these prodrugs. The cytotoxicity of each prodrug, as compared to paclitaxel, was determined against a variety of cell lines, including the PSA-secreting CWR22Rv1 prostate cancer cell line. The EDA-derived prodrug of paclitaxel 5 was stable and capable of being efficiently converted to an active drug that killed cells specifically in the presence of PSA, suggesting that this prodrug and similarly designed PSA-cleavable prodrugs may have potential as prostate cancer-specific therapeutic agents.     

Synthesis, hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.     

Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. Didanosine was analyzed using a 150 mm × 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20°C, 4°C and −20°C.     

Synthesis,hydrolysis studies and phamacodynamic profiles of amide prodrugs of dexibuprofen with amino acids

The present investigation deals with the synthesis of novel prodrugs of dexibuprofen with amino acids with an aim to achieve potent anti-inflammatory activity and less gastrointestinal toxicity. Structures of synthesized compounds were confirmed by spectral and elemental analyses. In vitro hydrolytic studies in simulated intestinal fluid, 80% plasma and rat faecal matter showed satisfactory release of dexibuprofen due to enzymatic cleavage. The synthesized prodrugs were evaluated for anti-inflammatory activity, analgesia, ulcerogenicity and histopathology. The anti-inflammatory activity of dexibuprofen was 43.3% whereas an improved value of 73.4, 77.3, 72.8 and 64.5% was observed for the synthesized prodrugs. The percentage analgesia of the prodrugs increased, whereas a decrease in the mean ulcer index values than dexibuprofen was observed. The histopathological studies revealed less ulceration in the gastric region when treated with prodrugs. Thus, the prodrugs were proved to be better in action as compared with the parent drug.     

Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity.

The device of new hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi- and trivalent cluster glycosides (K(GN)(2) and K(2)(GN)(3), respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10-fold higher than that of the parent drug (52+/-6% and 62+/-3% vs. 4.8+/-0.7% of the injected dose for PMEA) and could be attributed for 90% to parenchymal cells. Accumulation of the PMEA prodrugs in extrahepatic tissue (e.g., kidney, skin) was substantially reduced. The ratio of parenchymal liver cell-to-kidney uptake-a measure of the prodrugs therapeutic window-was increased from 0.058 +/- 0.01 for PMEA to 1.86 +/- 0.57 for K(GN)(2)-PMEA and even 2.69 +/- 0.24 for K(2)(GN)(3)-PMEA. Apparently both glycosides have a similar capacity to redirect (antiviral) drugs to the liver. After cellular uptake, both PMEA prodrugs were converted into the parent drug, PMEA, during acidification of the lysosomal milieu (t(1/2) approximately 100 min), and the released PMEA was rapidly translocated into the cytosol. The antiviral activity of the prodrugs in vitro was dramatically enhanced as compared to the parent drug (5- and 52-fold for K(GN)(2)-PMEA and K(2)(GN)(3)-PMEA, respectively). Given the 15-fold enhanced liver uptake of the prodrugs, we anticipate that the potency in vivo will be similarly increased. We conclude that PMEA prodrugs have been developed with greatly improved pharmacokinetics and therapeutic activity against viral infections that implicate the liver parenchyma (e.g., HBV). In addition, the significance of the above prodrug concept also extends to drugs that intervene in other liver disorders such as cholestasis and dyslipidemia.