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Antimicrobial Agents and Chemotherapy, July 2007, p. 2424-2429, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01498-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pharmacokinetics of the Anti-Human Immunodeficiency Virus Agent 1-(β-D-Dioxolane)Thymine in Rhesus Monkeys ^,

Ghazia Asif,^1,3 Selwyn J. Hurwitz,^1,3 Aleksandr Obikhod,^1,3 David Delinsky,^1,3 Janarthanan Narayanasamy,⁴ Chung K. Chu,⁴ Harold M. McClure,² and Raymond F. Schinazi^1,2,3*

Department of Pediatrics,¹ Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, Georgia,² Veterans Affairs Medical Center, Decatur, Georgia 30033,³ College of Pharmacy, University of Georgia, Athens, Georgia⁴

Received 28 November 2006/ Returned for modification 26 March 2007/ Accepted 30 April 2007

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β-D-Dioxolane-thymine (D-DOT) has potent and selective in vitro activity against several clinically important resistant human immunodeficiency virus (HIV) mutants and is in advanced preclinical development. Therefore, the single-dose intravenous and oral pharmacokinetics of D-DOT were studied with three rhesus monkeys. The pharmacokinetic profiles of D-DOT in serum and urine were adequately described by a two-compartment open pharmacokinetic model. D-DOT was rapidly and almost completely absorbed (absorption rate constant = 2.7 h^–1; fraction of oral dose absorbed = 0.82 to 1.06). The average serum beta half-life was 2.16 h. The average central and steady-state volumes of distributions were 0.52 and 1.02 liter/kg of body weight, respectively, and the average systemic and renal clearance values were 0.36 liter/h/kg and 0.18 liter/h/kg. Four or eight percent of administered D-DOT was eliminated in the urine as glucuronide within 8 h after intravenous or oral administration, respectively. D-DOT reached levels in the cerebrospinal fluid in excess of 10 to 20 times the median effective concentration for wild-type HIV and resistant mutants. The potent antiretroviral activity of D-DOT against a lamivudine- and zidovudine-resistant HIV-1 mutant, together with an excellent pharmacokinetic profile for rhesus monkeys, suggest that further development is warranted.

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A racemic mixture of β-dioxolane-thymine [(±)-DOT] was synthesized more than a decade ago and demonstrated a median effective antiviral concentration (EC₅₀) of 20 µM in human immunodeficiency virus type 1 (HIV-1)-infected ATH8 cells and no inhibition of cell growth in uninfected cells up to a 200 µM concentration (32). Later, our group demonstrated more-potent anti-HIV activity (EC₅₀ = 0.3 µM) for the DOT racemate in primary human peripheral blood mononuclear (PBM) cells, which was explained by a lower level of thymidine kinase (TK) needed to phosphorylate (±)-DOT in ATH-8 cells relative to PBM cells (43). The D-enantiomer of DOT (D-DOT) was subsequently found to be more potent than the L-counterpart (EC₅₀ = 0.39 µM versus 4.8 µM, respectively) and did not demonstrate notable cellular toxicity in PBM, Vero, and HepG2 cells or mitochondrial toxicity in HepG2 cells at concentrations greater than 100 µM (14, 16, 26, 40).

The discovery of the antiviral activity of β-D-dioxolane- guanine (DXG) and its prodrug amdoxovir (DAPD) against zidovudine (AZT)- and lamivudine-resistant mutants (19) prompted us to revisit D-DOT to explore the activity against mutant viruses, since D-DOT also contains the dioxolane sugar moiety (see Fig. 1). D-DOT demonstrated potent activity in human PBM cells against lamivudine-resistant (M184V) (EC₅₀ = 0.088 to 0.2 µM), tenofovir-resistant (K65R) (EC₅₀ = 0.21 µM), didanosine-resistant (L74V) (EC₅₀ = 0.33 µM), and zidovudine-resistant (thymidine analog mutations) (EC₅₀ = 0.49 µM) viruses (13, 15, 16). D-DOT-triphosphate also demonstrated activity against wild-type HIV-1 reverse transcriptase and reverse transcriptases of various mutants, including those of thymidine analog mutants and the M184V mutant, in an enzymatic study (27). The activity of dioxolane nucleoside triphosphate against many resistant HIV-1 strains may be due to reduced steric hindrance at the active site and to a specific interaction of the 3'-oxygen atom with nearby enzyme through H bonding (13, 15, 16).

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FIG. 1. Chemical structures of D-DOT (A), D-FDOC (B), DXG (C), β-D-dioxolane-2,6-diaminopurine (DAPD) (D), and APD (E).

D-DOT, like AZT and 3'-deoxy-2',3'-didehydrothymidine (stavudine [D4T]), is also a thymidine analogue that requires cellular TK for activation. The toxicities of AZT and D4T in humans undergoing treatment with these drugs are well known despite their usefulness in drug cocktails (1, 23, 33, 36, 37, 42). Therefore, there is a need to develop TK-dependent nucleoside analogues with limited toxicities that could provide additional treatment options.

The objective of this study was to assess the single-dose pharmacokinetics (PK) of D-DOT in rhesus monkeys given 33.3-mg/kg-of-body-weight intravenous and oral doses and to compare the pharmacokinetics with those of other structurally related antiretroviral nucleoside analogs that were previously administered to rhesus monkeys (7, 8, 10, 11, 12, 24, 28, 30, 34, 39).

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Chemicals. The synthesis of D-DOT (molecular weight = 228.2) (Fig. 1) and the internal standard, AZT, has been described elsewhere (14, 26). The chemical purity of each compound was found to be greater than 98% using high-performance liquid chromatography (HPLC) and spectral analysis. Acetonitrile (HPLC grade) and all other chemicals (analytical grade) were purchased from Fisher Scientific (Fair Lawn, NJ).

Pharmacokinetic studies with rhesus monkeys. Three female rhesus monkeys (Macaca mulatta) weighing from 6.8 to 7.1 kg were used for the pharmacokinetic studies. The animals were maintained at the Yerkes National Primate Research Center at Emory University, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care, in accordance with guidelines established by the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (31) of the National Institutes of Health. Monkeys were administered oral doses of 33.3 mg/kg of D-DOT by gastric intubation in a total volume of 10 ml of water, followed by a further 3 ml of water. After a washout period of at least 4 weeks, these monkeys were given a single dose of 33.3 mg/kg of D-DOT intravenously (i.v.) in 10 ml of pyrogen-free sterile normal saline. The animals were maintained on their backs on a heating pad and covered with a blanket under anesthesia for 4 h after dosing. Anesthesia was performed using a mixture of ketamine HCl (60 mg) and tiletamine HCl plus zolazepam HCl (Telazol; 20 mg) intramuscularly. The animals were monitored for alertness, and additional anesthetics (30 to 60 mg of ketamine HCl) were administered as required. Blood samples were collected through the femoral vein at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after dosing. Cerebrospinal fluid (CSF) samples were collected from treated monkeys at 1 and 2 h after drug administration by a cisternal or lumbar tap using a 22-gauge needle. The monkeys were catheterized for urine collection. Urine samples were collected at 0 to 0.25, 0.25 to 0.5, 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 3, 3 to 4, 4 to 6, and 6 to 8 h after drug administration. Serum, CSF, and urine samples were kept on ice and then frozen at –70°C until used for analyses.

HPLC assays of D-DOT in monkey serum, urine, and CSF samples. HPLC assays were performed using a Hitachi HPLC system (Tokyo, Japan) equipped with a model L-7100 pump, a model L-7400 UV detector, and a model L-7200 autosampler using a Columbus C₁₈ reverse-phase column (4.6 by 250 mm; 5-µm particle diameter; Phenomenex, Torrance, CA). The mobile phase was isocratic for the first 11 min, consisting of 4% acetonitrile and 96% water at a flow rate of 0.7 ml/min. The percent acetonitrile was then changed from 4 to 14% over 11 to 18 min, and the flow rate was varied from 0.7 to 1.0 ml/min. From 18 to 26 min, the percent acetonitrile was varied from 14 to 15%, maintaining a 1.0-ml/min flow rate. The column was equilibrated between 26 to 30 min by 4% acetonitrile at a flow rate of 0.7 ml/min. The retention times of D-DOT and AZT were 19.3 min and 29.3 min, respectively. Peaks were detected at 266 nm by the UV detector.

Preparation of standards and method validation. Standard solutions of D-DOT were prepared in deionized water. Calibration plots for D-DOT were prepared by diluting standard solutions in monkey serum derived from the same monkeys prior to dosing. Concentrations assayed ranging from 0.1 to 100 µg/ml (to convert µg/ml to µM, multiply by 4.4). Standard curves for the analysis of urine and CSF samples were prepared in urine and deionized water, respectively, using concentrations that ranged from 0.1 to 100 µg/ml and 0.1 to 25 µg/ml, respectively. The HPLC method was validated according to the Food and Drug Administration's Guidelines for Industry Bioanalytica Method Validation protocol (41). The intraday accuracy and relative standard deviations of the assay methodologies for serum were evaluated by assaying six samples per concentration level on the same day. For the interday accuracy and precision, samples were analyzed on three separate days. The intraday and interday relative standard deviation was less than 10%, and the intraday and interday accuracy was greater than 90%. The standard curves were linear over concentrations ranging from 0.2 to 100 µg/ml (r² = 0.99). The limit of detection of the assay was 0.1 µg/ml or 0.4 µM (50 ng).

Extraction procedure. For serum samples, 10 µl of AZT (1 mM) was added as an internal standard to a 50- to 100-µl serum sample in a microcentrifuge tube. Acetonitrile (450 µl) was added, followed by thorough vortexing and centrifugation at 9,000 x g for 4 min. The supernatant was evaporated to dryness, reconstituted with water (100 µl), and then vortexed and recentrifuged again at 11,000 x g for 3 min. Supernatant volumes injected onto the HPLC column ranged from 25 to 50 µl. The percent recovery of D-DOT was calculated by comparing the mean peak area for six extracted serum samples with those of the three standard samples with the same amount of nucleoside. Low (1 µg/ml), medium (10 µg/ml), and high (50 µg/ml) concentrations were investigated. The percent extraction recovery was calculated using the formula 100 x A_extracted/A_standard, where A_extracted is the peak area of extracted drug from the biological fluid and A_standard is the peak area of the same amount of the drug without extraction. The extraction recovery of D-DOT was greater than 91%.

For CSF samples, 5 µl of internal standard (AZT; 1 mM) was added to 100 µl of CSF, and then 80 µl of the mixture was injected onto the HPLC column.

DOT and DOT-glucuronide estimation in urine. DOT-glucuronide (G-DOT) was converted into D-DOT in the urine sample by adding 100 µl of 500-U/ml freshly prepared β-glucuronidase, 15 µl acetic acid (0.12 M), and 35 µl of phosphate buffer (40 mM K₂HPO₄, pH 5.8) to 100 µl of urine. D-DOT eliminated unchanged in the same sample was measured by adding 15 µl 0.12 M acetic acid, 100 µl demineralized water, and 35 µl of phosphate buffer to 100 µl of urine. The solutions were mixed and incubated for 12 h at 37°C in a shaker. Ten microliters of 1 mM AZT and 170 µl water were added to 20 µl of incubated β-glucuronidase-treated and untreated urine samples. The samples were injected onto the HPLC column. The G-DOT concentration was calculated as the difference between the D-DOT concentrations in β-glucuronidase-treated and untreated urine (39).

Mass spectrometric analysis of urine sample. A ThermoFinnigan TSQ Ultra ESI quantum triple-quadrupole mass spectrometer (MS/MS) was used to detect G-DOT in the sample. The 5- to 10-fold-diluted and filtered urine sample was injected directly into the MS at a speed of 5 µl/min in the positive field and a collision energy of 30 V. Nitrogen was used as the sheath gas at a pressure of 30 units. The spray voltage and the capillary temperature were 4 kV and 300°C, respectively. A further fragmentation was achieved by a second MS (MS/MS) at a collision energy and pressure of 25 V and 1 mtorr, respectively, and the product ions were scanned for from 70 to 407 (m/z).

Pharmacokinetic analysis. A two-compartment open pharmacokinetic model was simultaneously fitted to the serum concentration profiles of D-DOT derived from each monkey following i.v. and oral dosing (33.3 mg/kg), using a nonlinear regression curve fitting program (WinNonlin, version 5.1.1, 2006; Pharsight Corp., Mountain View, CA). Simultaneous fitting was used to avoid reporting two sets of disposition rate constants for the same animal and to improve estimates of the disposition rate constants k₂₁ and k₁₂, which is otherwise obscured for the oral dose by the kinetics of absorption. This approach assumes that the intercompartment disposition rate constants remain similar once the drug reaches the circulation (20, 21). The adequacy of model fit was assessed by examining the overall dispersion of data over the predicted curves and predicted standard errors of the fitted parameters. A weighting factor of 1/(predicted value)² was used for fitting the serum data in the two-compartmental model (Fig. 2). The accumulation of D-DOT in the urine had not plateaued at 8 h, and urine samples after 8 h were not available, since it is difficult to keep the animals under anesthetic for more than 8 h. Therefore, the calculation of the fraction excreted in the urine and renal clearance (Cl_renal) could not be obtained directly but was derived from a model fitted to the urine excretion data using the pharmacokinetic parameters obtained from the serum data (4). The following equation was used: dX_u/dt = Cl_renal x C_p, where dX_u is the rate of renal accumulation of the compound during the time interval dt and C_p is the predicted concentration of the drug in serum based on the serum data coinciding with the urine collection interval (21). No weighting factor was used for fitting the urine data.

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FIG. 2. Serum concentrations of D-DOT following i.v. (closed symbols) or oral (open symbols) dosing at 33.3 mg/kg. Mean concentrations (± standard deviations [SD]) from two animals () were simultaneously fitted for i.v. and oral data (curves 1 and 2, i.v. and oral predicted, respectively), since the third animal demonstrated a four-times-longer lag time than the other two monkeys.

Other pharmacokinetic parameters listed in Table 1 were calculated using the following standard two-compartment equations. The area under the serum concentration-versus-time curve (AUC) following i.v. dosing was calculated as follows: AUC_i.v. = dose/V x {( – k₂₁)/[( – β)] + (β – k₂₁)/[(β – )β]}, where and β are bioexponentional elimination rate constants from serum and V is the volume of distribution of the central compartment. The AUC after oral dosing was calculated using the equation AUC_oral = F x AUC_i.v., where F is the fraction of oral dose absorbed. The terminal-phase half-life was calculated using the equation t_1/2β = ln(2)/β. Systemic clearance was calculated with the equation Cl = dose/AUC_i.v.; and the mean residence time after i.v. dosing was calculated as MRT_i.v. = [(A/²) + (B/β²)]/[(A/) + (B/β)], where A = dose/V x ( – k₂₁)/( – β) and B = dose/V x (β – k₂₁)/(β – ). The mean residence time extrapolation to infinity after oral dosing was calculated using the equation MRT_oral = MRT_i.v. + 1/K_a, where K_a is the absorption rate constant; and the steady-state distribution volume was calculated with the equation V_ss = Cl x MRT_i.v.; the mean absorption time (MAT) was determined using the equation MAT = 1/K_a (4, 20, 21).

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TABLE 1. Pharmacokinetic parameters of D-DOT (33.3 mg/kg) after simultaneous two-compartment model fits following i.v. and oral administration for three rhesus monkeys (M-1, M-2, and M-3)a

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An open pharmacokinetic model that assumed first-order absorption with two-compartment tissue disposition and a preabsorption lag time was simultaneously fitted to the i.v. and oral serum and urine concentration-versus-time data from each monkey. The model was also fitted to the pooled data from two monkeys that demonstrated similar lag times. The PK parameters are summarized in Table 1 and Fig. 2. D-DOT was rapidly absorbed following oral administration (K_a = 2.7 ± 0.55 h^–1) with peak serum concentrations achieved between 0.25 and 2 h (T_max) after dosing and with the bioavailabilities between 82 and 106%. The peak serum concentrations (C_max) after 33.3-mg/kg oral doses were greater than 100 µM for two monkeys and 89.6 µM for the third. However, the serum concentrations following i.v. administration declined in a biexponential manner, which was not observed following oral doses, presumably due to the concomitant processes of absorption and disposition. The terminal-phase half-life was 2.16 h. The mean volume of distribution of the central compartment was 0.52 liter·kg^–1, and the steady-state volume of distribution was 1.02 liter·kg^–1, suggesting that distribution occurred predominantly into body water.

The average systemic clearance (Cl_systemic) was 0.36 liter·h^–1·kg^–1, and the mean renal clearance (Cl_renal) was 0.18 liter·h^–1·kg^–1. Urine samples were screened by mass spectrometry for the potential presence of G-DOT in monkeys. The appearance of a peak at m/z 405 in positive mode in urine sample by mass spectrometry suggested the presence of G-DOT (molecular weight = 404.05). Further fragmentation of m/z 405 by a second MS gave a major product ion at m/z 229 in positive mode that corresponds to D-DOT. Therefore, levels of G-DOT in the urine were determined indirectly by hydrolyzing the glucuronide to D-DOT by β-glucuronidase in acidic conditions. The average percentage of doses recovered unchanged in urine within 8 h for both oral and i.v. doses were 48% versus 40%. An average of 8 and 4% of D-DOT was eliminated in the form of G-DOT after oral and i.v. dosing, respectively. G-DOT was estimated by an indirect method, using β-glucuronidase to hydrolyze G-DOT into DOT, and therefore may not be precise. The average concentrations of D-DOT in CSF at 1 and 2 h after i.v. dosing were 7.8 and 12.3 µM, respectively, and 3.2 and 4.2 µM, respectively, after oral dosing.

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DXG and its prodrugs, DAPD (amdoxovir or AMDX) and β-D-2-aminopurine dioxolane (APD), and the nucleoside analog β-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine (D-FDOC), like D-DOT, are dioxolane nucleosides but with different base moieties. These nucleosides have well-established selective anti-HIV-1 activities and are in preclinical and clinical development (Fig. 1) (10, 11, 24, 29, 35).

The purpose of this study was to evaluate pharmacokinetics of D-DOT after single-dose (33.3 mg/kg) oral and i.v. administration in rhesus monkeys and compare its PK with those of other dioxolane nucleosides, mentioned above, and the only other two FDA-approved thymidine nucleoside analogs (AZT and D4T). A two-compartment open model was fitted to the serum and urine data (r > 0.99) (Table 1; Fig. 2). D-DOT was almost completely absorbed (F = 95% ± 12%) after oral dosing. In comparison, D-FDOC (24) and DAPD showed lower bioavailabilities (D-FDOC, F = 38%; DAPD, F = 30%), but AZT was also completely absorbed (90 to 100%) after oral dosing in rhesus monkeys (7, 10, 24, 35). The oral dose of 60 mg/kg was used for AZT and D4T with rhesus monkeys (7, 39). Oral bioavailabilities of D4T were 25 to 51% in rhesus monkeys (39), 77 to 83% in cynomolgus monkeys (25), and almost 100% in humans and mice (36, 38). High C_max values were observed with APD, the DXG prodrug, after oral administration (C_max of DXG = 50 to 60 µM within 0.25 to 0.5 h) (11) and D-FDOC (C_max = 34.4 µM) (24), but for D-DOT an even higher C_max value was noted (105 ± 35 µM). However, the time of maximum concentration varied (0.25 to 2 h) due to a longer lag time (1 h) in one animal than the others (0.26 h), suggesting that lag times prior to absorption may vary between individuals. Delayed oral absorption in one of the three monkeys was also reported for β-L-2',3'-dideoxy-5-fluorocytidine (30). The oral-dose AUC and K_a values of D-DOT were also very high (AUC, 349 to 449 µM·h; K_a, 2.08 to 3.15 h^–1), indicating a rapid and greater extent of absorption than that with D-FDOC (AUC, 197 to 227 µM·h; K_a, 0.22 to 0.85 h^–1). The elimination rate from the central compartment (k₁₀) for D-DOT (0.88 h^–1) was higher than that for D-FDOC (0.56 h^–1), with comparable central and higher steady-state volumes of distribution relative to those for D-FDOC (V = 0.53 liter·kg^–1; V_ss = 0.53 liter·kg^–1). However, the higher disposition rate constants for D-DOT (k₁₂ = 2.78 h^–1; k₂₁ = 2.03 h^–1) than for D-FDOC (k₁₂ = 0.58 h^–1; k₂₁ = 0.51 h^–1) are suggestive of rapid equilibration of the drug between the central and peripheral compartments. The average t_1/2β value of D-DOT in rhesus monkeys (2.16 h) was less than that of D-FDOC (2.48 to 5.12 h) but comparable to that of DXG following APD (1.42 to 1.74 h) and DAPD (1.8 to 2.8 h) dosing and higher than that of AZT (0.8 h), D4T (0.80 to 0.87), and several other nucleosides (7, 8, 10, 11, 24, 34). The average systemic and renal clearance values (Cl_systemic, and Cl_renal) for D-DOT were 0.36 and 0.18 liter·h^–1kg^–1, respectively. D-DOT was primarily eliminated unchanged in the urine, but low levels of G-DOT were also detected. The average percentages of administered D-DOT dose recovered unchanged in the urine within 8 h of dosing after oral or i.v. administration were 48 and 40%, respectively, whereas an average of 8 and 4% of the doses were eliminated in the form of glucuronide after oral or i.v. dosing, respectively. However, the glucuronidation of D-DOT is less than that observed for AZT, 3'-fluoro-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, and 3'-azido-2',3'-dideoxy-5-methylcytidine (7, 8, 17, 39) but higher than that for D4T (18, 36, 39). Studies with humans and monkeys have shown that AZT is primarily eliminated as 5'-O-glucuronide (8, 17, 22), and gram quantities were purified from the urine samples (22). D4T has very little or no glucuronidation (18, 38, 39). Therefore, like D4T, D-DOT might be a weaker substrate for uridine-diphospho-glucuronosyl transferase than AZT. A pharmacokinetic study of radiolabeled D4T in monkeys showed less than 50% elimination of administered D4T in 30 days (18), whereas for D-DOT, 44% (i.v.) and 56% (oral) of the total dose was eliminated in the urine within 8 h, suggesting a faster rate of elimination for D-DOT than for D4T. The remaining dose of D-DOT might have been eliminated after 8 h and/or by nonrenal routes. The systemic and renal clearance values (Table 1) also suggest that close to or more than 50% of D-DOT is excreted via renal routes.

Penetration of antiviral drug into the central nervous system is critical in antiviral therapy, since the central nervous system can be a reservoir for HIV-1 (2, 3, 5, 9). The average D-DOT concentrations in CSF at 1 and 2 h after i.v. dosing were 7.8 and 12.3 µM, respectively, corresponding to CSF/serum ratios of 0.09 and 0.18, respectively. The average CSF concentrations of D-DOT at 1 and 2 h post-oral administration were 3.2 and 4.2 µM, corresponding to CSF/serum ratios of 0.05 and 0.06, respectively. The higher CSF/serum ratio at 2 h than at 1 h suggests that D-DOT traverses the blood-brain barrier relatively slowly. The penetration of some antiretroviral agents into the central nervous system is limited due to plasma protein binding, since only the unbound fraction of drug is free to penetrate the blood-brain barrier. However, nucleoside reverse transcriptase inhibitors do not bind significantly to serum proteins (6). Similar CSF penetration kinetics have been reported for APD, dexelvucitabine, and AZT (9, 11, 28). The CSF concentrations of D-DOT were 20- to 40-fold higher than EC₅₀s for wild-type, K65R, L74V, M184V, and AZT-resistant HIV-1 after D-DOT administration by either route (15, 16). The concentration of D-DOT in the serum remained higher than the EC₅₀s against these viruses for at least 12 h after administration.

Taken together, these results indicate that D-DOT has a high C_max value, excellent oral bioavailability, a large AUC, low glucuronidation, and sufficiently high CSF penetration. Therefore, this TK-dependent dioxolane nucleoside with remarkable pharmacokinetic properties that is active against most of the resistant mutants warrants further development for the treatment of HIV-1.

 
This work was supported by NIH grants 5R37-AI-41980 (to R.F.S.), 5R37-AI-25899 and 5R01-AI-32351 (to R.F.S./C.K.C.), RR00165 and 1P30-AI-42847 (to H.M.M.), the Emory Center for AIDS Research, 5P30-AI-50409 (to R.F.S.), and the U.S. Department of Veterans Affairs (to R.F.S.).

In September 2004, RFS Pharma LLC licensed this technology from Emory University and the University of Georgia Research Foundation. R. F. Schinazi is a founder and the director of RFS Pharma LLC. In addition, C. K. Chu and R. F. Schinazi may receive future royalties from products discussed in this paper, including D-DOT, APD, and DAPD.

 
* Corresponding author. Mailing address: Veterans Affairs Medical Center, Medical Research 151H, 1670 Clairmont Road, Decatur, GA 30033. Phone: (404) 728-7711. Fax: (404) 728-7726. E-mail: rschina{at}emory.edu

Published ahead of print on 7 May 2007.

Dedicated to our friend and colleague, Harold M. McClure (1937-2004).

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Antimicrobial Agents and Chemotherapy, July 2007, p. 2424-2429, Vol. 51, No. 7
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