Ontogeny of Hepatic Transporters and Drug-Metabolizing Enzymes in Humans and in Nonclinical Species.

The liver represents a major eliminating and detoxifying organ, determining exposure to endogenous compounds, drugs, and other xenobiotics. Drug transporters (DTs) and drug-metabolizing enzymes (DMEs) are key determinants of disposition, efficacy, and toxicity of drugs. Changes in their mRNA and protein expression levels and associated functional activity between the perinatal period until adulthood impact drug disposition. However, high-resolution ontogeny profiles for hepatic DTs and DMEs in nonclinical species and humans are lacking. Meanwhile, increasing use of physiologically based pharmacokinetic (PBPK) models necessitates availability of underlying ontogeny profiles to reliably predict drug exposure in children. In addition, understanding of species similarities and differences in DT/DME ontogeny is crucial for selecting the most appropriate animal species when studying the impact of development on pharmacokinetics. Cross-species ontogeny mapping is also required for adequate translation of drug disposition data in developing nonclinical species to humans. This review presents a quantitative cross-species compilation of the ontogeny of DTs and DMEs relevant to hepatic drug disposition. A comprehensive literature search was conducted on PubMed Central: Tables and graphs (often after digitization) in original manuscripts were used to extract ontogeny data. Data from independent studies were standardized and normalized before being compiled in graphs and tables for further interpretation. New insights gained from these high-resolution ontogeny profiles will be indispensable to understand cross-species differences in maturation of hepatic DTs and DMEs. Integration of these ontogeny data into PBPK models will support improved predictions of pediatric hepatic drug disposition processes. SIGNIFICANCE STATEMENT: Hepatic drug transporters (DTs) and drug-metabolizing enzymes (DMEs) play pivotal roles in hepatic drug disposition. Developmental changes in expression levels and activities of these proteins drive age-dependent pharmacokinetics. This review compiles the currently available ontogeny profiles of DTs and DMEs expressed in livers of humans and nonclinical species, enabling robust interpretation of age-related changes in drug disposition and ultimately optimization of pediatric drug therapy.

Human tissue-engineered skeletal muscle: a novel 3D in vitro model for drug disposition and toxicity after intramuscular injection.

The development of laboratory-grown tissues, referred to as organoids, bio-artificial tissue or tissue-engineered constructs, is clearly expanding. We describe for the first time how engineered human muscles can be applied as a pre- or non-clinical model for intramuscular drug injection to further decrease and complement the use of in vivo animal studies. The human bio-artificial muscle (BAM) is formed in a seven day tissue engineering procedure during which human myoblasts fuse and differentiate to aligned myofibers in an extracellular matrix. The dimensions of the BAM constructs allow for injection and follow-up during several days after injection. A stereotactic setup allows controllable injection at multiple sites in the BAM. We injected several compounds; a dye, a hydrolysable compound, a reducible substrate and a wasp venom toxin. Afterwards, direct reflux, release and metabolism were assessed in the BAM constructs in comparison to 2D cell culture and isolated human muscle strips. Spectrophotometry and luminescence allowed to measure the release of the injected compounds and their metabolites over time. A release profile over 40 hours was observed in the BAM model in contrast to 2D cell culture, showing the capacity of the BAM model to function as a drug depot. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which increased with increasing toxic insult. Taken together, we show that the BAM is an injectable human 3D cell culture model that can be used to measure release and metabolism of injected compounds in vitro.

Fexofenadine, a Putative In Vivo P-glycoprotein Probe, Fails to Predict Clearance of the Substrate Tacrolimus in Renal Recipients.

Whether the combined use of probe drugs for CYP3A4 and P-glycoprotein can clarify the relative contribution of these proteins to pharmacokinetic variability of a dual substrate like tacrolimus has never been assessed. Seventy renal recipients underwent simultaneous 8-h pharmacokinetic profiles for tacrolimus, the CYP3A4 probe midazolam, and the putative P-glycoprotein probe fexofenadine. Patients were genotyped for polymorphisms in CYP3A5, CYP3A4, ABCB1, ABCC2 and SLCO2B1, -1B1, and 1B3. Carriers of the ABCB1 2677G>A polymorphism displayed lower fexofenadine Cmax (-66%; P = 0.012) and a trend toward higher clearance (+157%; P = 0.078). Predictors of tacrolimus clearance were CYP3A5 genotype, midazolam clearance, hematocrit, weight, and age (R2 = 0.61). Fexofenadine pharmacokinetic parameters were not predictive of tacrolimus clearance. In conclusion, fexofenadine pharmacokinetics varied considerably between renal recipients but most of this variability remained unexplained, with only minor effects of genetic polymorphisms. Fexofenadine cannot be used to assess in vivo CYP3A4-P-glycoprotein interplay in tacrolimus-treated renal recipients.

Determinants of the Magnitude of Interaction Between Tacrolimus and Voriconazole/Posaconazole in Solid Organ Recipients.

Administration of azole antifungals to tacrolimus-treated solid organ recipients results in a major drug-drug interaction characterized by increased exposure to tacrolimus. The magnitude of this interaction is highly variable but cannot currently be predicted. We performed a retrospective analysis of 126 solid organ recipients (95 lung, 31 kidney) co-treated with tacrolimus and voriconazole (n = 100) or posaconazole (n = 26). Predictors of the change in tacrolimus dose-corrected trough concentrations (C/D) between baseline and tacrolimus-azole co-therapy were assessed using linear mixed modeling. Patients were genotyped for relevant polymorphisms in CYP3A4, CYP3A5, MDR1, CYP2C19, POR, and UGT1A4. Tacrolimus C/D increased by a factor 5.0 ± 2.7 (range 1.0-20.2) for voriconazole and 4.4 ± 2.6 (range 0.9-18.0) for posaconazole, suggesting that a 66% dose reduction is insufficient for the majority of patients. Change in C/D was blunted in CYP3A5 expressors (estimated effect: -43%, p = 0.017) and affected by hematocrit (+8% per %, p = 0.004), baseline C/D (-14% per 100% increase, p < 0.001), and age (+1%, p = 0.008). However, the final model explained only 22% of interindividual variability in C/D change. In conclusion, CYP3A5 genotype and several clinical variables were identified as modulators of the tacrolimus-azole interaction, but these did not permit accurate predictions in individual patients.

Effect of ABCB1 diplotype on tacrolimus disposition in renal recipients depends on CYP3A5 and CYP3A4 genotype.

The relevance of most genetic polymorphisms beyond CYP3A5*1 on tacrolimus disposition remains unclear. We constructed a predictive mixed model for tacrolimus dose-corrected trough concentration (C0/dose) at months 3, 12 and 24 after transplantation in a retrospective cohort of 766 predominantly Causasian adult renal recipients (n=2042 trough concentrations). All patients were genotyped for 32 single-nucleotide polymorphisms with a proven or possible relevance to tacrolimus disposition based on the previous studies. Of these, ABCB1, ABCC2, OATP1B1, COMT, FMO, PPARA and APOA5 were analyzed as (functional) diplotype groups. Predictors of C0/dose were CYP3A5*1, hematocrit, age, CYP3A4*22, use of concomitant CYP3A4 inhibitor or inducer, ALT, estimated glomerular filtration rate, tacrolimus formulation (once vs twice daily), ABCB1 diplotype and time after transplantation. The effect of ABCB1 diplotype was small but strongly accentuated in CYP3A4*22 carriers and non-existent in CYP3A5 expressors. ABCC2 diplotype had a limited effect on C0/dose that was only statistically significant in CYP3A5 non-expressors.

High Intrapatient Variability of Tacrolimus Concentrations Predicts Accelerated Progression of Chronic Histologic Lesions in Renal Recipients.

High intrapatient variability (IPV) of tacrolimus concentrations is increasingly recognized as a predictor of poor outcome in solid organ recipients. How it relates to evolution of histology has not been explored. We analyzed tacrolimus IPV using the coefficient of variability (CV) from months 6-12 after transplantation in a cohort of 220 renal recipients for whom paired protocol biopsies at 3 mo and 2 years were available. Recipients in the highest CV tertile had an increased risk of moderate to severe fibrosis and tubular atrophy by 2 years compared with the low-IPV tertile (odds ratio [OR] 2.47, 95% confidence interval [CI] 1.09-5.60, p = 0.031; and OR 2.40, 95% CI 1.03-5.60, p = 0.043, respectively). Other predictors were donor age, severity of chronic lesions at 3 mo, and presence of borderline or subclinical rejection at 3 mo. Chronicity score increased significantly more in the high CV tertile group than in the middle and low tertiles (mean increase 1.97 ± 2.03 vs. 1.18 ± 2.44 and 1.12 ± 1.80, respectively; p < 0.05). CV did not predict evolution of renal function, which did not deteriorate within the 2-year follow-up period. These results indicate that high IPV is related to accelerated progression of chronic histologic lesions before any evidence of renal dysfunction.

Verapamil hepatic clearance in four preclinical rat models: towards activity-based scaling.

The current study was designed to cross-validate rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and the isolated perfused rat liver (IPRL) model against in vivo pharmacokinetic data, using verapamil as a model drug. Michaelis-Menten constants (Km), for the metabolic disappearance kinetics of verapamil in RLM and SRH (freshly isolated and cryopreserved), were determined and corrected for non-specific binding. The 'unbound' Km determined with RLM (2.8 µM) was divided by the 'unbound' Km determined with fresh and cryopreserved SRH (3.9 µM and 2.1 µM, respectively) to calculate the ratio of intracellular to extracellular unbound concentration (Kpu,u). Kpu,u was significantly different between freshly isolated (0.71) and cryopreserved (1.31) SRH, but intracellular capacity for verapamil metabolism was maintained after cryopreservation (200 vs. 191 µl/min/million cells). Direct comparison of intrinsic clearance values (Clint) in RLM versus SRH, yielded an activity-based scaling factor (SF) of 0.28-0.30 mg microsomal protein/million cells (MPPMC). Merging the IPRL-derived Clint with the MPPMC and SRH data, resulted in scaling factors for MPPGL (80 and 43 mg microsomal protein/g liver) and HPGL (269 and 153 million cells/g liver), respectively. Likewise, the hepatic blood flow (61 ml/min/kg b.wt) was calculated using IPRL Clint and the in vivo Cl. The scaling factors determined here are consistent with previously reported CYP450-content based scaling factors. Overall, the results show that integrated interpretation of data obtained with multiple preclinical tools (i.e. RLM, SRH, IPRL) can contribute to more reliable estimates for scaling factors and ultimately to improved in vivo clearance predictions based on in vitro experimentation.

Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1.

The effects of human immunodeficiency virus (HIV) protease inhibitors (PI) on the accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in organic anion transporting polypeptide (OATP)-1B1 and -1B3-expressing Chinese hamster ovary (CHO) cells. In addition, interaction studies in Caco-2 monolayers, known only to express the OATP2B1 isoform, were conducted using the established OATP substrate estrone 3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers. CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 microl min(-1) mg(-1) protein in OATP1B1 and OATP1B3-transfected cells, respectively. K(i)-values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and OATP1B3-expressing cells. Lopinavir was the most potent inhibitor (K(i) = 0.5-1.4 microM) of OATP1B-mediated CGamF accumulation compared with atazanavir, darunavir, ritonavir, and saquinavir (K(i) between 1.4 and 3.3 microM). Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir, and ritonavir showing substantial effects. In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver, and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine.

P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes.

Recently, sandwich-cultured (SC) rat hepatocytes have been used as an in vitro model to assess biliary excretion of drugs and xenobiotics. The purpose of the present study was to validate the use of SC rat hepatocytes for the in vitro assessment of P-glycoprotein (P-gp)-mediated biliary drug excretion. The specific and fluorescent P-gp substrate rhodamine 123 (Rh123) and the P-gp substrate digoxin were selected as model compounds. Rh123 and digoxin accumulation and Rh123 efflux under standard and Ca(2+)-free conditions were quantified in SC rat hepatocytes to determine substrate secretion into canalicular networks in vitro. The major role of P-gp in the biliary excretion of these compounds was confirmed by inhibition experiments with the potent P-gp inhibitor GF120918. Hepatocyte culture conditions, including media type and time in culture, significantly affected Rh123 biliary excretion. P-gp expression, as assessed by Western blot, was increased with culture time. Dexamethasone (an in vivo inducer of P-gp) concentrations ranging from 0.01 to 1 microM in the cell culture medium did not influence P-gp expression or Rh123 biliary excretion. Rh123 and digoxin biliary clearance values, predicted from SC rat hepatocyte data, were consistent with values reported in vivo and in isolated perfused rat liver studies. In conclusion, the results of this study demonstrate the utility of SC rat hepatocytes as an in vitro model to study and predict the biliary excretion of P-gp substrates.

Strategies for absorption screening in drug discovery and development.

This review gives an overview of the current approaches to evaluate drug absorption potential in the different phases of drug discovery and development. Methods discussed include in silico models, artificial membranes as absorption models, in vitro models such as the Ussing chamber and Caco-2 monolayers, in situ rat intestinal perfusion and in vivo absorption studies. In silico models such as iDEA can help optimizing chemical synthesis since the fraction absorbed (Fa) can be predicted based on structural characteristics only. A more accurate prediction of Fa can be obtained by feeding the iDEA model with Caco-2 permeability data and solubility data at various pH's. Permeability experiments with artificial membranes such as the filter-IAM technology are high-throughput and offer the possibility to group compounds according to a low and a high permeability. Highly permeable compounds, however, need to be further evaluated in Caco-2 cells, since artificial membranes lack active transport systems and efflux mechanisms such as P-glycoprotein (PgP). Caco-2 and other "intestinal-like" cell lines (MDCK, TC-7, HT29-MTX, 2/4/A1) permit to perform mechanistic studies and identify drug-drug interactions at the level of PgP. The everted sac and Ussing chamber techniques are more advanced models in the sense that they can provide additional information with respect to intestinal metabolism. In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism, however, it is time consuming, and therefore not suited for screening purposes. Finally, in vivo absorption in animals can be estimated from bioavailability studies (ratio of the plasma AUC after oral and i.v. administration). The role of the liver in affecting bioavailability can be evaluated by portal vein sampling experiments in dogs.

Increased absorption of the antiviral ester prodrug tenofovir disoproxil in rat ileum by inhibiting its intestinal metabolism.

Previous studies have shown that strawberry extract increases the transepithelial transport of tenofovir disoproxil, an esterase-sensitive prodrug of the antiviral compound tenofovir (formerly PMPA), across Caco-2 monolayers. This increase in transport was at least partially due to inhibition of its intestinal metabolism. To further study the feasibility of this absorption enhancing strategy, the influence of various concentrations of strawberry extract (0-2%) on the intestinal absorption of tenofovir disoproxil (100 microM) was assessed using an in situ perfusion model with immediate blood sampling from the mesenteric vein, a model closer to the in vivo situation than the in vitro Caco-2 system. Inclusion of strawberry extract (1%) resulted in a 7-fold increase in the appearance of tenofovir equivalents. The metabolism of tenofovir disoproxil in the intestinal perfusate was significantly lower in the presence of strawberry extract (1%), showing that the metabolism of tenofovir disoproxil is reduced by the flavoring extract.

In vitro, ex vivo, and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil.

Caco-2 monolayers (in vitro), rat intestinal sheets mounted in modified Ussing Chambers (ex vivo), and in situ intestinal perfusion of rat ileum were used as models to determine and compare the absorption characteristics of the antiviral agent 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir) and its bis(pivaloyloxymethyl)-ester prodrug [bis(POM)-PMEA, adefovir dipivoxil]. Although metabolism of adefovir dipivoxil was more pronounced in the ex vivo and in situ models than in the Caco-2 system, the transport of 'total adefovir' [= adefovir dipivoxil and its metabolites mono(POM)-PMEA and adefovir] was comparable in the three models. Compared with transport of the parent compound (adefovir), use of adefovir dipivoxil resulted in a significant increase in transport of total adefovir in the in vitro ( approximately 100-fold) and the in situ ( approximately 10-fold) models; in contrast, the ex vivo method failed to demonstrate a remarkable transport enhancement when using the ester prodrug. Similar to the results obtained in the Caco-2 model, the inclusion of the P-glycoprotein inhibitor verapamil resulted in transport enhancement during in situ perfusion of rat ileum with adefovir dipivoxil; however, no effect of verapamil could be observed in the ex vivo model. The results of this study confirm the utility of both the in vitro and in situ models to assess intestinal transport and metabolism of adefovir dipivoxil. The ex vivo model appeared to be less appropriate because of its inability to discriminate transport following administration of adefovir or adefovir dipivoxil and because of the absence of an effect of verapamil on transport when using adefovir dipivoxil.

Hydration changes implicated in the remarkable temperature-dependent membrane permeation of cyclosporin A.

Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.

Evaluation of the potential of ion pair formation to improve the oral absorption of two potent antiviral compounds, AMD3100 and PMPA.

9-(2-phosphonyomethoxypropyl)adenine (PMPA) and AMD3100 are highly potent and selective antiretroviral agents. Since PMPA is negatively charged and AMD3100 positively charged at physiological pH, their transepithelial transport and their potential for oral drug delivery is very low. In this study, ion pair formation was evaluated as a possible strategy to enhance transepithelial transport of PMPA and AMD3100. Positively charged counter ions such as t-hexyl-, t-heptyl-, t-octylammonium bromide and dodecyl-, tetradecyl-, hexadecyltrimethylammonium bromide were used to form ion pairs with PMPA, while sodium taurodeoxycholate (in vitro experiments) and sodium taurocholate (in vivo experiments) were used as counter ions for AMD3100. The effect of counter ions on transepithelial transport of PMPA (1 mM) and AMD3100 (1 mM) was investigated by measuring the flux across Caco-2 monolayers. An enhancement in drug transport could be observed at a concentration of 2 mM of hexadecyltrimethylammonium bromide (counter ion for PMPA) and 10 mM of sodium taurodeoxycholate (counter ion for AMD3100), but at the concentrations used, the absorption enhancing effect could be attributed to a reduction of the integrity of the monolayers. When AMD3100 transport was tested at a concentration of 200 microM, no flux was observed, even in the presence of relatively high concentrations of counter ion (20 times the concentration of AMD3100). Results obtained from partitioning studies of the drugs in the presence or absence of counter ion revealed that competition by other ions was responsible for the absence of an effect: when pure water was used as the aqueous phase, a reduction up to 24.4+/-1.4% and 17.0+/-1.3% of the initial aqueous concentration was observed for PMPA and AMD3100, respectively; however, as soon as other ions were present in the aqueous phase, the effect of the counter ion was diminished (25-50 mOsm) or completely abolished (270-305 mOsm). The absorption enhancing effect of counter ions was also studied in vivo: pharmacokinetic studies in rabbits showed that the oral bioavailability of AMD3100 in the presence of 4 equivalents of taurocholic acid remained very low and was only 3.2-fold better (i.e. 3.6%) in comparison to pure AMD3100. In view of the results obtained in the Caco-2 system, this absorption enhancement can be attributed to an effect on monolayer integrity rather than to the potential to form ion pairs. We can conclude that the formation of ion pairs may not be very efficient as a strategy to enhance transepithelial transport of charged hydrophilic compounds, as competition by other ions may abolish the beneficial effect of counter ions.

Inhibition of intestinal metabolism of the antiviral ester prodrug bis(POC)-PMPA by nature-identical fruit extracts as a strategy to enhance its oral absorption: an in vitro study.

PURPOSE: To explore the usefulness of fruit extracts as enhancers of the oral absorption of esterase-sensitive prodrugs. METHODS: Inhibition of esterase-mediated degradation by nature-identical fruit extracts was evaluated using 1) p-nitrophenylacetate (model substrate for esterase-activity) in rat intestinal homogenates and 2) bis(isopropyloxycarbonyloxymethyl)-(R)-9-[(2-phosphonomethoxy++ +)propyl]adenine [bis(POC)-PMPA] (esterase-sensitive prodrug of the antiviral agent PMPA) in Caco-2 cell homogenates and in intestinal homogenates from rat, pig and man. Subsequently, transport of the ester prodrug was studied across Caco-2 monolayers in the presence or absence of fruit extracts. RESULTS: In homogenates from rat ileum, the esterase activity could be reduced significantly by the inclusion of fruit extracts (1%): the initial enzymatic degradation of p-nitrophenylacetate was inhibited by 77% (strawberry), 16% (passion fruit) and 57% (banana). A similar inhibition of bis(POC)-PMPA metabolism by fruit extracts was observed in intestinal homogenates from several species and in homogenates from Caco-2 cells. Transport of total PMPA across Caco-2 monolayers was enhanced 3-fold by co-incubation with strawberry extract (1%). The fraction of intact prodrug appearing in the acceptor compartment increased from virtually zero to 67%. CONCLUSIONS: The results suggest that co-incubation with nature-identical fruit extracts might be useful as a strategy to enhance the transepithelial transport of esterase-sensitive prodrugs through inhibition of intracellular metabolism of the prodrug.

Carrier mechanisms involved in the transepithelial transport of bis(POM)-PMEA and its metabolites across Caco-2 monolayers.

PURPOSE: To investigate the role of carrier mechanisms in: [1] the polarized transport of the bis(pivaloyloxymethyl)- [bis(POM)-] ester prodrug of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] and [2] the directional secretion of its metabolites. METHODS: Caco-2 monolayers were used to study the modulation effect of carriers on the transport of bis(POM)-PMEA and the efflux of intracellularly formed metabolites mono(POM)-PMEA and PMEA from the cells. The interaction of bis(POM)-PMEA and its metabolites with the efflux mechanisms present in Caco-2 monolayers was investigated by testing the effect of various concentrations of verapamil (30, 100, 300, microns) or indomethacin (10-500 microns) on transport and efflux. RESULTS: Polarity in transport of bis(POM)-PMEA (50 micron) across Caco-2 monolayers was noted: transport of total PMEA [=bis(POM)-PMEA, mono(POM)-PMEA and PMEA] was significantly higher in basolateral (BL) to apical (AP) direction (14.5 +/- 0.4%) than transport in the opposite (AP to BL) direction (1.7 +/- 0.2%). This difference was reduced in a concentration dependent way when verapamil (0-100 microns) was included in both AP and BL incubation media. After loading the cells with bis(POM)-PMEA (100 micron) for 1 hr, studies on efflux of PMEA and mono(POM)-PMEA from the Caco-2 monolayers over a 3 hr period, revealed that both metabolites were preferentially secreted towards the AP compartment. Efflux of PMEA toward AP and BL compartments amounted to 14.6 +/- 1.1% and 5.3 +/- 0.4, respectively, of the initial intracellular amount of total PMEA, while efflux of mono(POM)-PMEA towards AP and BL compartments was limited to 2.3 +/- 0.1% and 0.5 +/- 0.1%, respectively. When 10 micron indomethacin was included in the AP incubation medium, efflux of PMEA was decreased to 7.8 +/- 0.3% and 3.3 +/- 0.3% towards the AP and BL compartments, respectively. The decrease in efflux by indomethacin was concentration-dependent up to 100 micron. Transepithelial transport of total PMEA was also reduced in the presence of 30 micron indomethacin, as reflected in smaller concentrations of PMEA and mono(POM)-PMEA in the acceptor compartment, irrespective of the transport direction. CONCLUSIONS: The data obtained in this study suggest that bis(POM)-PMEA is substrate for a P -glycoprotein-like carrier mechanism in Caco-2 monolayers, while its metabolites mono(POM)-PMEA and PMEA are transported by a non-P-glycoprotein efflux protein.

Antiretroviral efficacy and pharmacokinetics of oral bis(isopropyloxycarbonyloxymethyl)-9-(2-phosphonylmethoxypropyl)adenine in mice.

To overcome the low oral bioavailability of the highly potent and selective antiretroviral agent (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), a new lipophilic ester derivative, i.e., the bis(isopropyloxycarbonyloxymethyl)-ester [bis(POC)-PMPA], was prepared. The usefulness of bis(POC)-PMPA as an oral prodrug for PMPA was investigated in the intestinal mucosa Caco-2 cell monolayer model. The total transport of bis(POC)-PMPA was 2.7%, whereas it was less than 0.1% for PMPA. Bis(POC)-PMPA was considerably metabolized inside the epithelial cells, since the majority of the compound was recovered after transport in the form of the monoester metabolite [mono(POC)-PMPA]. In contrast, bis(POC)-PMPA was relatively resistant to degradation at the luminal side of the Caco-2 cells. Pharmacokinetic studies with mice showed that the oral bioavailability of bis(POC)-PMPA (calculated from the curves of the concentration of free PMPA in plasma) was 20%. Neither bis(POC)-PMPA nor mono(POC)-PMPA could be recovered in plasma, suggesting the efficient release of the active drug PMPA after oral administration of bis(POC)-PMPA. Severe combined immunodeficient (SCID) mice infected with Moloney murine sarcoma virus (MSV) and treated orally with bis(POC)-PMPA for 5 or 10 days (dosages, 50, 100, or 200 mg of PMPA equivalent per kg of body weight per day) showed a significant delay in MSV-induced tumor appearance and tumor-associated death. The antiviral efficacy of oral bis(POC)-PMPA was related to the dosage and treatment period and was not significantly different from that of subcutaneous PMPA given at an equivalent dose. The favorable pharmacokinetic profile, marked antiviral efficacy, and low toxicity make bis(POC)-PMPA an attractive oral prodrug of PMPA that should be further pursued in clinical studies with patients infected with human immunodeficiency virus or hepatitis B virus.

Comparison of the disposition of ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] in Caco-2 monolayers.

PURPOSE: To evaluate the potential of several bis-ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) to enhance the oral absorption of PMEA. METHODS: Caco-2 monolayers were used to estimate intestinal transport and metabolism of the bis(pivaloyloxymethyl)-ester [bis(POM)-] and a series of bis(S-acyl-2-thioethyl)-esters [bis(SATE)-] of PMEA. An LC-MS method was used for the identification of unknown metabolites which were formed from the SATE-esters. RESULTS: During transport across Caco-2 monolayers, all esters were extensively degraded as could be concluded from the appearance of the mono-ester and free PMEA in apical as well as basolateral compartments. Incubation of SATE-esters with the monolayers resulted in the formation of two additional metabolites, which were identified as 2-thioethyl-PMEA and its dimerisation product. All ester prodrugs resulted in enhanced transepithelial transport of total PMEA (i.e. the bis-esters and their corresponding metabolites, including PMEA), but significant differences could be observed between the various esters. Transport of total PMEA ranged from 0.4 +/- 0.1% for the bis[S(methyl) ATE]-ester to 15.3 +/- 0.9% for the more lipophilic bis[S(phenyl)ATE]-PMEA. A relationship between total transport of the esters and their lipophilicity (as estimated by their octanol/water partition coefficient) was established (r2 = 0.87). Incubation of prodrug esters with homogenates from Caco-2 cells showed large differences in susceptibility of the compounds to esterases, the half-lives of the bis-esters varying from 4.3 +/- 0.3 min for the bis[S(phenyl)ATE]-PMEA to 41.5 +/- 0.8 min for its methyl analogue. In addition, intracellularly formed PMEA was observed to be further converted by the cells to the diphosphorylated PMEA (PMEApp). CONCLUSIONS: Several SATE-esters of PMEA can be considered as potential alternatives to bis(POM)-PMEA, due to enhanced epithelial transport, sufficient chemical and enzymatic stability and adequate release of PMEA. Toxicological studies as well as in vivo experiments are required in order to further explore the potential of those SATE-esters as prodrugs for oral delivery of PMEA.

Transport, uptake, and metabolism of the bis(pivaloyloxymethyl)-ester prodrug of 9-(2-phosphonylmethoxyethyl)adenine in an in vitro cell culture system of the intestinal mucosa (Caco-2).

PURPOSE: To evaluate intestinal transport, uptake and metabolism characteristics of the bis(pivaloyloxymethyl)-ester [bis(POM)-ester] of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA]. METHODS: Intestinal transport, uptake and metabolism of bis(POM)-PMEA were studied using an in vitro cell culture system of the intestinal mucosa (Caco-2 monolayers). Concentrations of bis(POM)-PMEA and its metabolites mono(POM)-PMEA and PMEA were determined using a reversed-phase HPLC method. Enzymatic stability of bis(POM)-PMEA was evaluated by incubation with purified liver carboxylesterase, homogenates of Caco-2 cells and scraped pig small intestinal mucosa. RESULTS: The use of bis(POM)-PMEA as a prodrug of PMEA resulted in a significant increase in transport of total PMEA [bis(POM)-PMEA, mono(POM)-PMEA and PMEA] across Caco-2 monolayers. While transepithelial transport of PMEA (500 microM) was lower than 0.1% during a 3 hr incubation period, transport of total PMEA after addition of bis(POM)-PMEA (100 microM) amounted to 8.8% over the same incubation period. Only 23% of the amount transported appeared as intact bis-ester at the basolateral side, while 33% of this amount was free PMEA and 44% was mono(POM)-PMEA, suggesting susceptibility of the prodrug to chemical and enzymatic degradation. Uptake studies revealed that only negligible amounts of bis(POM)-PMEA (< 0.2%) were present inside the cells. Very high intracellular concentrations of PMEA were found (approximately 1.2 mM, after a 3 hr incubation with 50 microM bis(POM)-PMEA), which suggests that PMEA was trapped inside the cells probably due to its negative charge. This explains that efflux of PMEA was relatively slow (25% of the intracellular amount in 3 hr). Enzymatic degradation of the prodrug by carboxylesterase was confirmed by incubation of bis(POM)-PMEA with purified enzyme (Km = 87 microM and Vmax = 9.5 microM/min). Incubation of bis(POM)-PMEA (10 microM) with cell homogenate of Caco-2 monolayers and pig small intestinal mucosa produced similar degradation profiles. CONCLUSIONS: The use of the bis(POM)-prodrug significantly enhances the intestinal permeability of PMEA. Intracellular trapping of PMEA in the intestinal mucosa may result in slow release of PMEA to the circulation after oral administration of bis(POM)-PMEA.