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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on May 18, 2005; DOI: 10.1124/jpet.104.082412

0022-3565/05/3142-883-890$20.00
JPET 314:883-890, 2005
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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Amino Acid Ester Prodrugs of 2-Bromo-5,6-dichloro-1-({beta}-D-ribofuranosyl)benzimidazole Enhance Metabolic Stability in Vitro and in Vivo

Philip L. Lorenzi1, Christopher P. Landowski, Xueqin Song, Katherine Z. Borysko, Julie M. Breitenbach, Jae Seung Kim, John M. Hilfinger, Leroy B. Townsend, John C. Drach, and Gordon L. Amidon

Departments of Pharmaceutical Sciences (P.L.L., C.P.L., X.S., G.L.A.) and Medicinal Chemistry (L.B.T., J.C.D.), College of Pharmacy, and Department of Biologic and Materials Sciences (K.Z.B., J.M.B., J.C.D.), School of Dentistry, University of Michigan, Ann Arbor, Michigan; and Therapeutic Systems Research Laboratories, Inc. (J.S.K., J.M.H.), Ann Arbor, Michigan

Received December 18, 2004; accepted May 16, 2005.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
2-Bromo-5,6-dichloro-1-({beta}-D-ribofuranosyl)benzimidazole (BDCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV), but it lacks clinical utility due to rapid in vivo metabolism. We hypothesized that amino acid ester prodrugs of BDCRB may enhance both in vitro potency and systemic exposure of BDCRB through evasion of BDCRB-metabolizing enzymes. To this end, eight different amino acid prodrugs of BDCRB were tested for N-glycosidic bond stability, ester bond stability, Caco-2 cell uptake, antiviral activity, and cytotoxicity. The prodrugs were resistant to metabolism by BDCRB-metabolizing enzymes, and ester bond cleavage was rate-limiting in metabolite formation from prodrug. Thus, BDCRB metabolism could be controlled by the selection of promoiety. In HCMV plaque-formation assays, L-Asp-BDCRB exhibited 3-fold greater selectivity than BDCRB for inhibition of HCMV replication. This potent and selective antiviral activity in addition to favorable stability profile made L-Asp-BDCRB an excellent candidate for in vivo assessment and pharmacokinetic comparison with BDCRB. In addition to rapid absorption and sufficient prodrug activation after oral administration to mice, L-Asp-BDCRB exhibited a 5-fold greater half-life than BDCRB. Furthermore, the sum of area under the concentration-time profile (AUC)BDCRB and AUCprodrug after L-Asp-BDCRB administration was roughly 3-fold greater than AUCBDCRB after BDCRB administration, suggesting that a reservoir of prodrug was delivered in addition to parent drug. Overall, these findings demonstrate that amino acid prodrugs of BDCRB exhibit evasion of metabolizing enzymes (i.e., bioevasion) in vitro and provide a modular approach for translating this in vitro stability into enhanced in vivo delivery of BDCRB.

Benzimidazole D-ribonucleosides such as BDCRB were identified as potent inhibitors of HCMV replication over a decade ago (Townsend and Drach, 1992Go; Townsend et al., 1995Go). In addition to excellent potency, BDCRB exhibited low toxicity to uninfected cells due to its viral-specific mechanism of action (Krosky et al., 1998Go; Underwood et al., 1998Go; Scheffczik et al., 2002Go; Scholz et al., 2003Go). Despite these desirable attributes, the clinical development of BDCRB was restricted due to rapid N-glycosidic bond cleavage after oral and intravenous administration to rats and monkeys (Good et al., 1994Go). The enzymes 8-oxoguanine DNA glycosylase (OGG1), and N-methylpurine DNA glycosylase (MPG), were recently implicated in cleavage of the BDCRB glycosidic bond (P. L. Lorenzi, C. P. Landowski, X. Song, L. B. Townsend, J. C. Drach, and G. L. Amidon, manuscript submitted for publication). Thus, the main limitation to use of BDCRB in HCMV therapy seems to be metabolism by these and possibly additional enzymes.

Rapid drug inactivation prevents clinical utility or increases the requisite dosing frequency for numerous antiviral and anticancer nucleoside drugs (Ensminger et al., 1978Go; Ensminger and Gyves, 1983Go; Cooney et al., 1987Go; Carson et al., 1988Go; Hartman et al., 1990Go; Good et al., 1994Go). The evasion of drug-metabolizing enzymes, or bioevasion, is a strategy that could potentially overcome such problems. Conceptually the exact opposite of drug-targeting, bioevasion, is achieved by designing drug molecules to not be substrates for enzymes. Although numerous chemical modification strategies have been used to evade drug metabolism, ester prodrugs have consistently demonstrated enhanced metabolic stability (Birnie et al., 1963Go; Gangwar et al., 1996Go; Pauletti et al., 1996Go; Pauletti et al., 1997Go; Bak et al., 1999Go; Gudmundsson et al., 1999Go; Wang et al., 1999Go; Song and Siahaan, 2002Go; Siccardi et al., 2003Go, 2004Go). Ester prodrugs of floxuridine, for example, demonstrated reduced affinity for metabolizing enzymes and a corresponding bioavailability increase in humans (Mukherjee et al., 1963Go). Furthermore, ester prodrugs can be activated to the parent drug through ester bond hydrolysis, which is catalyzed by esterases. Our group has identified one such prodrug-activating enzyme, biphenyl hydrolase-like protein (Kim et al., 2003Go), that activates amino acid ester prodrugs of acyclovir, ganciclovir, floxuridine, zidovudine, BDCRB, and gemcitabine (Kim et al., 2004Go). These studies led to the utilization of amino acids as promoieties in the search for bioevasive prodrugs with enhanced metabolic stability (Vig et al., 2003Go; Landowski et al., 2005Go) and suggested that amino acid ester prodrugs of BDCRB may confer stability and facilitate modulation of BDCRB N-glycosidic bond metabolism.

In a previous report, we described the synthesis of a variety of amino acid ester prodrugs of BDCRB and their potential as substrates of the intestinal oligopeptide transporter PEPT1 (Song et al., 2005Go). In this report, we describe the evaluation of the ester and N-glycosidic bond stabilities of these BDCRB prodrugs in a variety of biologically relevant media. We also describe the antiviral activity of these compounds evaluated in vitro in a HCMV-infected cell system. Finally, the disposition of BDCRB after oral administration of BDCRB and a promising prodrug candidate, L-Asp-BDCRB, were also determined in mice. The combined results of these studies suggest that amino acid ester prodrugs of BDCRB can evade N-glycosidic bond-metabolizing enzymes, enhance the in vitro efficacy of BDCRB without increasing toxicity, and enhance the delivery of BDCRB in vivo.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. BDCRB and its aglycone were synthesized in the laboratory of Dr. Leroy B. Townsend. Amino acid ester prodrugs of BDCRB were synthesized and obtained as TFA salts as described previously (Song et al., 2005Go). The chemical structures of BDCRB, its aglycone, and the amino acid ester prodrugs of BDCRB are shown in Fig. 1. Human OGG1 (hOGG1) and murine MPG (mMPG) enzymes were purchased from Trevigen (Gaithersburg, MD). All other chemicals were of analytical grade or better. Stock drug solutions were prepared at 100 mM in dimethyl sulfoxide.



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  		Fig. 1. Chemical structures of BDCRB, BDCRB aglycone, and amino acid ester prodrugs of BDCRB.

 
Metabolism by OGG1 and MPG. Three independent metabolism time-course reactions were conducted in triplicate in the presence and absence of selected enzymes as follows. Briefly, the reaction buffer for hOGG1 reactions contained 1 mM dithiothreitol, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 0.1 mg/ml bovine serum albumin, and the reaction buffer for mMPG reactions contained 1 mM dithiotreitol, 100 mM KCl, 1 mM EDTA, and 10 mM HEPES, pH 7.4. Reactions were initiated by the addition of drug solutions to reaction buffer either with or without enzyme at 37°C, where final drug and enzyme concentrations in the reaction mixture were 200 µM and 3.0 µg/ml, respectively. Aliquots taken at 0, 2, 5, 10, 15, and 20 min were quenched with ice-cold methanol, and BDCRB aglycone was quantitated by HPLC to assess N-glycosidic bond cleavage. Triplicate aglycone values from each independent experiment were averaged, and the resulting initial rate of aglycone formation was determined as described under "Data Analysis". The data are reported as mean ± S.D. determined for the three independent reactions.

Metabolism by Mouse Intestine and Liver Homogenates. Relative N-glycosidic and ester bond cleavage rates were determined in mouse tissue homogenates. A 25-g female mouse was euthanized with sodium pentobarbital, and liver and duodenum pieces were removed and immediately placed on ice in buffer C (10 mM HEPES, 25 mM KCl, and 5 mM MgCl2, pH 7.4). Tissues were decanted and washed several times with buffer C to remove blood. For liver homogenate preparation, liver was then minced into small chunks with a razor blade and added to a tube containing buffer C and 0.5% Triton X-100. The tissue was homogenized with a Tissue Tearor for 10 s at speed 1, and the resulting homogenate kept on ice. For intestinal homogenate, the mucosal layer of the intestinal tissue was scraped into a tube containing buffer C and 0.5% Triton X-100. Tubes containing liver and intestinal tissue with detergent were incubated at room temperature for 8 to 10 min and then pipetted to lyse the cells and intracellular organelles. The homogenates were inspected with a phase contrast microscope after treatment with trypan blue stain to confirm minimal 90% cell breakage. The homogenized intestinal and liver suspensions were centrifuged at 600g and 4°C for 5 min to pellet debris, and the supernatant was used in metabolism reactions. Total protein was determined with the DC protein microplate assay (Bio-Rad, Hercules, CA) using a bovine serum albumin standard. As described above for the pure enzymes, three independent stability studies were conducted in triplicate at a protein concentration of 200 µg/ml in buffer C. N-Glycosidic bond cleavage was assessed by rate of aglycone formation, and ester bond cleavage was assessed by rate of parent drug formation.

Caco-2 Cell Uptake. To examine the relative uptake of the BDCRB prodrugs with respect to the parent drug, Caco-2 cell (ATCC HTB37, passage no. 76) (American Type Culture Collection, Manassas, VA) uptake experiments were performed by administering 0.4 mM drug or prodrug to triplicate wells. Cells were plated onto six-well plates at a density of 16,000 cells/cm2 and grown to 80 to 90% confluence, at which point the experiment was initiated by adding 0.4 mM drug prepared in pH 6 Hanks' balanced salt solution to each well at 37°C. Supernatant was aspirated at 5, 10, 20, 30, 60, and 120 min, and cells were immediately washed three times with ice-cold uptake buffer. Cells were then combined with 500 µl of 0.5% Triton X-100 and shaken with a tilting shaker for 30 min to achieve complete lysis, which was confirmed by visual inspection under phase contrast microscope. A 100-µl aliquot of ice-cold acetonitrile was introduced to each well and vigorously mixed by pipetting to precipitate protein and extract drug. Ten microliters of homogenate protein was saved for protein quantitation with the Bio-Rad DC microassay procedure, and the remaining homogenate was transferred into wells of a GF/B filter plate (Whatman, Clifton, NJ). The filter plate was centrifuged at 400g for 1 min, and the filtrate was transferred to HPLC tubes and stored at -80°C until HPLC analysis. Data are reported as the mean of triplicate determinations.

Antiviral Activity and Cytotoxicity. Antiviral activity and cytotoxicity were determined as described previously (Turk et al., 1987Go). Briefly, primary human foreskin fibroblast (HFF) cells were grown in monolayer cultures with minimal essential medium containing either Hanks' or Earle's salts supplemented with 10% calf serum or 10% fetal bovine serum. Cells were passaged at 1:2 to 1:4 dilutions using 0.05% trypsin plus 0.02% EDTA in HEPES-buffered saline. Plaque-purified isolate P0 of the Towne strain of HCMV was kindly provided by Dr. Mark F. Stinski (Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA). Stock HCMV was prepared by infecting HFF cells at a multiplicity of infection of <0.01. Plaque-forming units per cell were determined as detailed previously (Turk et al., 1987Go). Virus titers were determined by using monolayer cultures of HFF cells for HCMV as described previously (Prichard et al., 1990Go). HCMV plaque reduction assay was performed by infecting HFF cells in 24-well cluster dishes with approximately 100 plaque-forming units of HCMV per well (Turk et al., 1987Go). After virus adsorption, compounds dissolved in growth medium were added to duplicate wells at four to eight selected concentrations. After incubation at 37°C for 7 days, the cell sheets were fixed and stained with crystal violet, and microscopic plaques were enumerated. Drug efficacy, determined as the 50% inhibitory concentration (IC50), was defined as the concentration required to decrease the growth rate to 50% of the untreated control cells. Growth rate was calculated from the slope of a semilogarithmic plot of cell number against time for the treated culture as a percentage of the control.

To determine cytotoxicity, the effects of the compounds during two population doublings of KB cells were determined by staining the cells with crystal violet and spectrophotometrically quantitating the dye that eluted from stained cells. Briefly, 96-well cluster dishes were seeded with KB cells at 3000 to 5000 cells per well. After incubation overnight at 37°C, the test compounds were added in triplicate at six to eight concentrations. The plates were incubated at 37°C for 48 h, rinsed, fixed with 95% ethanol, and stained with 0.1% crystal violet. Acidified ethanol was added, and the plates were read at 570 nm. Cytotoxicity, determined as the 50% cytotoxic concentration (CC50), was defined as the concentration required to decrease the growth rate to 50% of the untreated control cells.

Dose-response relationships were examined by linear regression of the percentage of inhibition of parameters derived in the preceding sections against log drug concentrations. IC50 concentrations were then calculated from the regression lines.

HPLC. Drug and metabolite concentrations were determined by conversion of peak areas using standard curves on a Waters HPLC system (Waters, Milford, MA). The system consisted of two Waters 515 pumps with pump control module, a Waters 717plus auto-sampler, a Waters 996 photodiode array detector, a Waters Xterra C18 reversed phase column (5 µm, 4.6 x 250 mm) equipped with guard column, and computer with Waters Empower software. The aqueous mobile phase (solvent A) was 10 mM ammonium acetate, pH 8.2, with triethylamine, and the organic mobile phase (solvent B) was acetonitrile.

Elution conditions for BDCRB, L- and D-Val-BDCRB, and the aglycone were isocratic using a 58:42 mixture (solvent A:B) with detection at 298.9 nm. The retention time was 6 min for BDCRB and 11 min for its aglycone. L-Val-BDCRB, D-Val-BDCRB prodrugs eluted at 8 and 9 min, respectively. L- and D-Asp-BDCRB were eluted with a 62:32 mixture (solvent A:B) under isocratic conditions, which yielded retention times of 3.5, 6.7 and 13.5 min for prodrug, parent drug and aglycone, respectively. L-Ile-BDCRB and D-Ile-BDCRB were eluted isocratically with a 70:30 mixture (solvent A:B), which yielded retention times of 14 and 14.4 min for the prodrugs and 4.5 and 7.8 min for the parent drug and its aglycone. In all cases, the injection volume and flow rate were 50 µl and 1.0 ml/min.

Standard solutions of prodrugs, BDCRB, and aglycone were prepared by serial dilution of known concentrations in the same buffers and quenching solutions used for the reactions, and standard curves were analyzed with the appropriate HPLC method. The limit of detection was defined as 3 times the S.D. of three independent determinations of 12.5 µM standards. The limit of detection for BDCRB and its aglycone were 1.0 and 1.8 µM, respectively.

In Vivo Pharmacokinetics. BDCRB and L-Asp-BDCRB suspensions were prepared at 10 mg/kg in 100 µl of 0.5% carboxymethylcellulose for oral administration to C57BL/6 8-week-old female mice (n = 2). Solutions were briefly sonicated before administration. Since the anesthetic action of ketamine at the recommended dose (100 mg/kg) was prolonged after oral dosing of our drugs (P. Lorenzi, unpublished data), C57BL/6 8-week-old female mice were anesthetized using an i.m. 50 mg/kg ketamine dose in the hind leg. This lower ketamine dose facilitated oral gavage without completely anesthetizing the animals. All animal experiments were performed in accordance with institutional guidelines and were approved by the University Committee on Use and Care of Animals, University of Michigan. Ten-microliter serial blood samples were obtained through the tail vein at 5, 15, and 30 min and 1, 2, 3, 4, 5 and 6 h following BDCRB administration and at 5, 15, 30 and 45 min and 1, 2, 3, 4, 5, 6, 7, and 8 h for L-Asp-BDCRB. Mice were periodically sedated with 10 µl of additional ketamine as needed. All samples were placed into Microfuge tubes containing 90 µl of ethyl acetate and the internal standard, which was the aglycone of BDCRB's 2-chloro homolog, TCRB (1 µM final). Samples were pipetted vigorously and then centrifuged at 6000g for 5 min at 4°C to spin down protein precipitate. The ethyl acetate layers were transferred into fresh tubes, and the extraction from blood was repeated with an additional 100 µl of ethyl acetate and combined with the first extract. Samples were evaporated to dryness and stored at -80°C until reconstitution and analysis by LC/MS/MS.

LC/MS/MS. BDCRB prodrugs BDCRB, BDCRB aglycone, and the internal standard TCRB aglycone were quantitated using an LC/MS/MS system composed of a Micromass Quattro II with MassLynx version 1.4 software and an HP1100 LC system (Hewlett Packard, Palo Alto, CA). Samples were separated with a C18 column (2.1 x 50 mm) and 1:1 (v/v) acetonitrile/water mobile phase containing 0.5% formic acid. The mass spectrometer was interfaced with the LC via electrospray source and was operated in positive ion mode for the mass analysis. The injection volume and flow rate were 10 µl and 0.2 ml/min.

The precursor/daughter ion transitions were monitored using multiple reaction monitoring mode at m/z 398.9 -> m/z 266.9 for BDCRB, m/z 266.9 -> m/z 266.9 for BDCRB aglycone, m/z 222.4 -> m/z 222.4 for TCRB aglycone, and m/z 513.9 -> m/z 266.9 for L-Asp-BDCRB.

Calibration curves were constructed by weighted (1/x) least square regression of peak area versus concentrations of the calibration standards. The LOQ for L-Asp-BDCRB, BDCRB, and the aglycone in LC/MS/MS analyses was approximately 10 nM.

Data Analysis. Initial metabolite appearance rates, v0, were determined from the initial slope of the line formed by plotting aglycone amount as a function of time. Data from pure enzyme and tissue homogenate reactions are reported as mean ± S.D. from three independent experiments with data from each experiment determined in triplicate. Statistical significance was assessed by two-tailed unpaired t-tests with GraphPad Prism 4.02.

Blood concentration-time profiles were generated with normalization of the concentration data to internal standards and correction for extraction efficiencies, which were complete for BDCRB and its aglycone and 20% for L-Asp-BDCRB. In addition, data obtained after L-Asp-BDCRB administration were dose-normalized for comparison to data generated from BDCRB administration by multiplying with the factor 1.576 —the mole ratio of BDCRB to that of the TFA salt of L-Asp-BDCRB (BDCRB molecular weight, 398.04; L-Asp-BDCRB-TFA salt, molecular weight 627.16; both dosed at 10 mg/kg). Maximum plasma concentrations (Cmax) and the time to reach Cmax (Tmax) were obtained from these profiles. The area under the concentration-time profile from 0 h to Tlast (AUClast) was calculated using the trapezoidal rule. Individual estimates of the elimination rate constant (k) were obtained by log-linear regression of the terminal portions of the plasma concentration time curves. Half-life (t1/2) was calculated from the elimination rate using the equation t1/2 = 0.693/k. The AUC from 0 to infinity (AUCinf) was calculated as AUClast + Clast/k.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Metabolism by OGG1 and MPG. hOGG1 and mMPG, two enzymes previously identified to cleave the N-glycosidic bond of BDCRB, were initially investigated for their ability to metabolize BDCRB prodrugs. The results shown in Fig. 2 indicate that in marked contrast to BDCRB itself, BDCRB prodrugs were not substrates of these two enzymes.



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  		Fig. 2. BDCRB and prodrugs as substrates for DNA repair enzymes. N-Glycosidic bond cleavage by hOGG1 and mMPG. Data represent initial rate of aglycone appearance after addition of 200 µM prodrug to 3.0 µg/ml enzyme ({blacksquare}) or to reaction buffer lacking enzyme ({square}). Data are expressed as mean ± S.D. of triplicate assays from three independent experiments.

 
Metabolism by Mouse Intestine and Liver Homogenates. The results of stability studies in mouse intestine and mouse liver homogenates, surrogate systems for the organs, are shown in Figs. 3 and 4. N-Glycosidic bond cleavage rates (Fig. 3) were roughly 2- to 3-fold greater in liver homogenate than intestinal homogenate, and ester bond cleavage rates (Fig. 4) were roughly 2-fold greater in liver homogenate as well. In addition, prodrug ester bond cleavage rates in each homogenate were 4- to 10-fold greater than the corresponding N-glycosidic bond cleavage rates. Furthermore, with the exception of L-Lys and L-Pro, prodrug N-glycosidic bond cleavage was 40- to 100-fold slower than that of BDCRB.



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  		Fig. 3. N-Glycosidic bond cleavage in mouse tissue homogenates. Metabolism of BDCRB and prodrugs in mouse intestine and liver homogenates. Data represent initial rate of aglycone appearance after addition of 200 µM drug or prodrug to 200 µg/ml protein in reaction buffer ({blacksquare}) or to reaction buffer alone ({square}). Data are expressed as mean ± S.D. of triplicate assays from three independent experiments.

 


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  		Fig. 4. Ester bond cleavage in mouse tissue homogenates. Prodrug activation in mouse intestine and liver homogenates. Data represent initial rate of BDCRB appearance after addition of 200 µM prodrug to 200 µg/ml protein in reaction buffer ({blacksquare}) or to reaction buffer alone ({square}). Data are expressed as mean ± S.D. of triplicate assays from three independent experiments.

 

The enzymatic activation of several prodrugs seemed to be solubility-limited. In particular, L-Tyr(Et), L-Tyr(Me), L-Phe(p-Cl), D-Phe, and L-Phe prodrugs precipitated in the reaction mixture. Thus, these five prodrugs were excluded and not considered for further testing. Figure 4 illustrates the following stability trend: D-Ile** > L-Ile** > D-Asp ≥ D-Val** > L-Asp*** > L-Val* > L-Lys*** > L-Pro (*p < 0.05, **p < 0.01, and ***p < 0.001, where the symbol refers to comparison with the next compound in the series).

Caco-2 Cell Uptake. Relative BDCRB and prodrug uptake rates assessed at 0.4 mM are shown in Fig. 5. All prodrugs exhibited slower uptake than BDCRB, and uptake rate decreased as follows: BDCRB > L-Ile > D-Ile > L-Val > D-Val > L-Pro > L-Asp > D-Asp > L-Lys. Furthermore, no statistical difference was observed between the uptake of L- and D-prodrugs.



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  		Fig. 5. BDCRB and prodrug uptake by Caco-2 cells. Initial uptake rates after administration of 0.4 mM BDCRB or prodrug to Caco-2 cells grown to confluence in six-well plates. Data are reported as mean ± S.D. from triplicate wells.

 
Antiviral Activity and Cytotoxicity. The results of antiviral activity and cytotoxicity assays with BDCRB and its prodrugs are summarized in Table 1. Most compounds exhibited efficacy (IC50) similar to the parent drug except for isoleucine-containing prodrugs, which were approximately 40-fold less efficacious than BDCRB. Prodrugs with lysine and isoleucine promoieties were slightly more toxic than the parent drug with CC50 values ranging from 60 to 80 µM, whereas all other prodrugs exhibited toxicities similar to that of BDCRB (~100 µM). L-Asp-BDCRB was the least cytotoxic with a CC50 of 110 µM. The reasonably high antiviral activity and low toxicity of L-Asp-BDCRB coupled with its high ester and glycosidic bond stability in various biological media suggest that L-Asp-BDCRB might be an attractive candidate for in vivo evaluation.


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  		TABLE 1 Antiviral activity and cytotoxicity of BDCRB and prodrugs HCMV plaque reduction assay was performed by infecting HFF cells with HCMV as indicated in the text. Four to eight drug concentrations were used to calculate IC50 by plotting the percentage of plaque reduction in the presence versus absence of drug as a function of drug concentration. Cytotoxicity was evaluated in KB cells with six to eight drug concentrations added in triplicate. Plates were incubated at 37°C for 48 h, rinsed, fixed with 95% ethanol, stained with 0.1% crystal violet, and read at 570 nm.

 

In Vivo Pharmacokinetics. The disposition of BDCRB and L-Asp-BDCRB after oral administration to mice was assessed to determine whether a prodrug that exhibited "bioevasive" properties in vitro could potentially improve BDCRB delivery in vivo. Whole blood concentration-time profiles of BDCRB and its aglycone after oral administration of BDCRB and those of prodrug, BDCRB, and aglycone after oral administration of L-Asp-BDCRB are shown in Fig. 6. The BDCRB profile after L-Asp-BDCRB prodrug dosing was found to be similar to the BDCRB profile obtained after BDCRB oral dosing. Table 2 shows a comparison of pharmacokinetic parameters determined from these in vivo studies. It is seen from Table 2 that peak whole blood concentration, Cmax, and AUC for BDCRB normalized for dose are roughly 1.5-fold higher after L-Asp-BDCRB oral administration compared with BDCRB administration. Time to peak BDCRB concentration, Tmax, however, increased from 30 min after BDCRB oral dosing to 45 to 60 min with L-Asp-BDCRB dosing. Dose normalized Cmax and AUC for aglycone were 1.7- to 2-fold higher with L-Asp-BDCRB dosing compared with BDCRB oral administration. Tmax values for aglycone were somewhat shorter after L-Asp-BDCRB administration compared with BDCRB administration.



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  		Fig. 6. Mean whole blood concentration-time profiles. L-Asp-BDCRB ({circ}), BDCRB ({blacksquare}), and aglycone ({square}) concentrations after oral administration of 10 mg/kg BDCRB or 10 mg/kg L-Asp-BDCRB. Data are reported as mean of two determinations, and data obtained after L-Asp-BDCRB administration are dose-normalized by the mole ratio of BDCRB to L-Asp-BDCRB.

 

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  		TABLE 2 Pharmacokinetic parameters determined after BDCRB and prodrug administration to mice After intragastric oral administration to mice, blood was serially sampled over 6 h after BDCRB dosing and 8 h after L-Asp-BDCRB dosing. Samples were extracted with ethyl acetate and dried before reconstitution in mobile phase and LC/MS/MS analysis. Parameters obtained for L-Asp-BDCRB administration were obtained from dose-normalized concentration profiles as indicated in the text. Data for two mice (n = 2) are presented.

 

With L-Asp-BDCRB oral dosing, an additional contribution from AUCprodrug roughly equivalent to AUCBDCRB was obtained. The sum of dose-normalized AUCprodrug and dose-normalized AUCBDCRB after L-Asp-BDCRB dosing was roughly 3- to 3.4-fold greater than AUCBDCRB after BDCRB dosing. Interestingly, the half-life of L-Asp-BDCRB was 5-fold greater than that of BDCRB, which was approximately 1 h after both prodrug and parent drug dosing. The aglycone half-life after L-Asp-BDCRB administration was nearly 3-fold greater than with BDCRB administration. Last, the substantial (~50%) contribution to AUCinf relative to AUClast of L-Asp-BDCRB indicates that a longer time period is required for L-Asp-BDCRB absorption to be accurately determined.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
BDCRB is a potent antiviral agent whose development was halted as a result of low bioavailability attributed to its rapid in vivo N-glycosidic bond metabolism to the inactive aglycone in animal models. In this report, we evaluated the potential of amino acid ester prodrugs of BDCRB to evade N-glycosidic bond metabolism to identify suitable candidate prodrugs for further testing in vivo. Although BDCRB was metabolized by the DNA repair enzymes hOGG1 and mMPG, its amino acid ester prodrugs were not substrates of these enzymes (Fig. 2). These results suggested that amino acid ester prodrugs of BDCRB may evade metabolizing enzymes encountered in first-pass organs and other tissues. The N-glycosidic bond stability of BDCRB and its prodrugs was therefore examined in vitro in surrogate systems for first-pass organs. Mouse tissues were selected due to higher MPG and OGG1 expression compared with humans and since other animal models lack this high expression (Chakravarti et al., 1991Go). The parallel trends observed for N-glycosidic bond stability in mouse tissue homogenates (Fig. 3) and in pure enzyme systems (Fig. 2) support the possible role of these DNA glycosylase enzymes in glycosidic bond cleavage in vivo. Furthermore, the observation that amino acid ester prodrugs of BDCRB exhibited an N-glycosidic bond stability enhancement in vitro suggested that such metabolic bioevasion by the prodrugs might enhance the half-life and systemic exposure of BDCRB in vivo.

Together with the above-mentioned findings, the observation that high ester bond stability (Fig. 4) conferred greater N-glycosidic bond stability to prodrugs (Fig. 3) suggests that prodrug ester bond cleavage must take place before N-glycosidic bond cleavage can occur. Significantly slower ester bond cleavage rates compared with N-glycosidic bond cleavage rates also indicate that the former determines overall N-glycosidic bond stability of the prodrug. Thus, BDCRB metabolism could be controlled by choice of the amino acid promoiety. This amino acid prodrug strategy therefore provides a modular approach to enhancing the delivery of BDCRB and essentially serves as a "controlled release" strategy. Since prodrug activation or "release" to the parent BDCRB, however, is not the sole parameter that affects in vivo pharmacokinetics and efficacy, the relative uptake of BDCRB and prodrugs was evaluated in Caco-2 cells, a well established model of the human intestine, to predict in vivo absorption.

The lack of stereochemical preferences in uptake of prodrugs containing L- and D-promoieties in Caco-2 cells suggested that uptake was passive. Furthermore, the observation that charged aspartic acid and lysine prodrugs exhibited negligible uptake compared with more hydrophobic prodrugs and BDCRB itself (Fig. 5) suggested that uptake was likely a function of lipophilicity.

Although uptake was very low for charged prodrugs, assessment of antiviral activity and cytotoxicity suggested that L-Asp-BDCRB may be a candidate for in vivo testing. Amino acid promoieties are generally believed to impart minimal toxicity after prodrug activation since they are endogenous, and cytotoxicity measurements (Table 1) were consistent with this hypothesis, with the exception of lysine- and isole-ucine-containing compounds. Surprisingly, L-Asp-BDCRB exhibited the highest CC50/IC50, a measure of selectivity. Coupled with its moderate ester bond stability, these findings therefore suggested that L-Asp-BDCRB would be a good candidate for assessment of in vivo pharmacokinetics.

Preliminary pharmacokinetic studies were conducted to assess the disposition of BDCRB after oral administration of BDCRB or L-Asp-BDCRB to mice. Resulting BDCRB profiles were consistent with previous studies in rats (Good et al., 1994Go). Dose-normalized BDCRB and aglycone concentrations after L-Asp-BDCRB oral dosing were higher than those obtained after BDCRB dosing, suggesting substantial systemic activation of the prodrug. These studies also suggest that it may be possible to enhance oral bioavailability of BDCRB via administration of L-Asp-BDCRB.

The total mass at the first sampling time point, 5 min, and all subsequent time points after L-Asp-BDCRB administration was higher than the total mass measured at those times after BDCRB administration (Fig. 6). This observation suggests that the L-Asp-BDCRB prodrug is efficiently transported across mouse intestine followed by sequential degradation to the parent and then to the aglycone. Greater Tmax,BDCRB values after L-Asp-BDCRB compared with BDCRB administration suggest delayed systemic activation of the prodrug to BDCRB. Furthermore, the accompanying 3-fold greater half-life of aglycone suggests saturation of aglycone elimination.

The combined presence of prodrug and BDCRB in systemic circulation after L-Asp-BDCRB administration suggests transport of L-Asp-BDCRB across mouse intestine, perhaps by carrier-mediated mechanisms involving amino acid transporters (Yang et al., 2001Go; Hatanaka et al., 2004Go). Such a phenomenon could also explain the observed antiviral efficacy of L-Asp-BDCRB (Table 1). Although the uptake data in Caco-2 cells (Fig. 5) may seem to be inconsistent with such a contention, it is very possible that transporter expression profiles in Caco-2 cells are dramatically different in mouse intestine and in HCMV-infected cells.

L-Asp-BDCRB exhibited several pharmacokinetic enhancements over BDCRB in vivo. First, the 5-fold greater half-life of L-Asp-BDCRB suggested efficient in vivo bioevasion. Second, the higher Cmax,BDCRB and AUCBDCRB after L-Asp-BDCRB compared with that from BDCRB administration suggests 1) significant prodrug activation over a 6- to 8-h period, and 2) the potential for higher therapeutic effect and higher relative oral bioavailability. In this regard, it is noteworthy that Good et al. (1994Go) reported a mean oral bioavailability of around 50% after BDCRB administration in rats. Considering the rather high similarity of BDCRB profiles and pharmacokinetic parameters between our study in mice and that reported by Good et al. (1994Go) in rats, and coupled with the observation that the sum of dose-normalized AUCprodrug and AUCBDCRB after L-Asp-BDCRB dosing was roughly 3- to 3.4-fold greater than AUCBDCRB after BDCRB dosing, it is tempting to suggest the potential of obtaining relatively high oral bioavailabilities after L-Asp-BDCRB dosing. More extensive studies with a greater number of animals would be required to determine whether such a possibility can be fully realized.

In summary, amino acid prodrugs of BDCRB exhibited successful evasion of metabolizing enzymes, i.e., bioevasion. This strategy provided a flexible approach for translating in vitro bioevasion into enhanced BDCRB delivery in vivo. The observation of more potent and selective antiviral activity by L-Asp-BDCRB compared with BDCRB suggests that the enhanced delivery of this prodrug in vivo could translate into enhanced in vivo efficacy, but further testing will be necessary to determine whether this is true.


   Acknowledgements
 
We thank Dr. Chandrasekharan Ramachandran for critical evaluation of the manuscript; Hiro Tsume and Scott Ocheltree for teaching animal techniques; Chi Ho Ngan for assistance with the enzyme kinetics experiments; Dr. John D. Williams for synthesizing the BDCRB aglycone and for helpful discussions; Dr. Lynda S. Welage for unrestricted use of her HPLC system; and Jack Hinkley for helpful discussions and providing additional reagents.


   Footnotes
 
This work was performed in the laboratories of G.L.A. and J.C.D. (University of Michigan College of Pharmacy and School of Dentistry) and was made possible by National Institutes of Health Grants R01-GM37188 and P01-AI46390. P.L.L. was supported by Training Grant GM07767 from the National Institute of General Medical Sciences. The contents are solely the responsibility of the authors and do not necessarily represent the official views of National Institute of General Medical Sciences.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.082412.

ABBREVIATIONS: BDCRB, 2-bromo-5,6-dichloro-1-({beta}-D-ribofuranosyl)benzimidazole; HCMV, human cytomegalovirus; OGG1, 8-oxoguanine DNA glycosylase; MPG, N-methylpurine DNA glycosylase; TCRB, 2,5,6-trichloro-1-({beta}-D-ribofuranosyl)benzimidazole; TFA, trifluoroacetic acid; hOGG1, human 8-oxoguanine DNA glycosylase; HPLC, high-performance liquid chromatography; HFF, human foreskin fibroblast; LC/MS/MS, liquid chromatography/tandem mass spectrometry.

1 Current address: Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD. Back

Address correspondence to: Dr. Gordon L. Amidon, Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109-1065. E-mail: glamidon{at}umich.edu


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