Introduction

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Sampidine, d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] (HI-113),¹ a novel aryl phosphate derivative of stavudine (d4T), is substantially more potent than d4T in inhibiting HIV replication in human peripheral blood mononuclear cells or thymidine kinase-deficient T cells (Venkatachalam et al., 1998; Vig et al., 1998; Uckun and Vig, 2000). HI-113 has been shown to inhibit the replication of HIV-2 and zidovudine (ZVD/AZT)-resistant HIV-1 strains in human peripheral blood mononuclear cells at nanomolar concentrations (Venkatachalam et al., 1998; Vig et al., 1998; Uckun and Vig, 2000). Previous results have also demonstrated that the presence of a single para-bromine group in the phenyl moiety of HI-113 markedly enhances its ability to undergo hydrolysis and thereby produce substantially more of the key metabolite alaninyl-d4T-monophosphate (ala-d4T-MP) (Venkatachalam et al., 1998; Vig et al., 1998; Uckun and Vig, 2000). HI-113 exhibits potent anti-HIV activity in an in vivo SCID mouse model of human acquired immunodeficiency syndrome (F. M. Uckun, T. K. Venkatachalam, D. Maher, and S. Pendergrass, unpublished data). However, the in vivo pharmacokinetics and metabolism of this promising new anti-HIV agent have not yet been characterized. This report details the results of our first study of the in vivo pharmacokinetics and metabolism of HI-113.

	Materials and Methods

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Chemicals. We used HPLC-grade reagents and deionized distilled water. Acetonitrile was purchased from Burdick & Jackson Company (Muskegon, MI). Hydrochloric acid was purchased from Fisher Scientific (Fair Lawn, NJ). Ammonium phosphate and phosphoric acid were purchased from Sigma (St. Louis, MO). The synthetic procedures for the preparation of HI-113, ala-d4T-MP, and d4T (structures shown in Fig. 1) have been previously described in detail (Venkatachalam et al., 1998; Vig et al., 1998; Uckun and Vig, 2000).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Proposed in vivo metabolic pathways of HI-113.

Quantitative HPLC for Detection of HI-113 and Its Metabolites. Each plasma sample (200 µl) was mixed 1:4 with acetone (800 µl) and vortexed for at least 30 s. Following centrifugation, the supernatant was transferred into a clean tube and dried under nitrogen. A 50-µl solution of 50% methanol in 200 mM HCl was used to reconstitute the extraction residue, and 40 µl of the reconstituted sample was subjected to analytical HPLC. The HPLC system used for these studies was a Hewlett Packard (Palo Alto, CA) series 1100 instrument equipped with a quaternary pump, an autosampler, an automatic electronic degasser, an automatic thermostatic column compartment, a diode array detector, and a computer with Chemstation software for data analysis (Chen et al., 1999a,c,e). The analytical column was a Zorbax SB-Phenyl (250 × 4.6 mm; 5 µm; Hewlett Packard) column attached to a guard column (Hewlett Packard). The column was equilibrated prior to data collection. The linear gradient mobile phase (flow rate = 1.0 ml/min) was 100% A/0% B at 0 min, 88% A/12% B at 20 min, and 8% A/92% B at 30 min (A: 10 mM ammonium phosphate buffer, pH 3.7; B: acetonitrile). The detection wavelength was 268 nm. The peak width, response time, and slit were set at >0.03 min, 0.5 s, and 4 nm, respectively.

Stability of HI-113 and ala-d4T-MP in Plasma. Plasma samples were spiked with HI-113 and ala-d4T-MP to yield final concentrations of 250 µM for HI-113 and 100 µM for ala-d4T-MP. Spiked plasma samples were stored at 20°C. At a predetermined time, an aliquot (100 µl) of the spiked plasma sample was extracted by adding 400 µl of acetone to induce the precipitation of proteins.

Stability of HI-113 in Plasma in Presence of Selective Esterase Inhibitors. Plasma samples were preincubated with the esterase inhibitors paraoxon (final concentration = 0.1 mM), physostigmine (final concentration = 0.1 mM), and EDTA (final concentration = 1 M) at 37°C for 30 min. Subsequently, HI-113 was added to yield a final concentration of 250 µM. At a predetermined time, an aliquot (100 µl) of the spiked plasma sample was extracted by adding 400 µl of acetone to induce the precipitation of proteins.

Stability of HI-113 in Murine Liver Homogenates. Freshly obtained livers of female BALB/c mice were homogenized in 1× phosphate-buffered saline (1:1, w/v) by using a Polytron (PT-MR2000) homogenizer (Kinematical AG, Littau, Switzerland). HI-113 was added to the liver homogenate to yield a final concentration of 100 µM and was incubated at 37°C. At a predetermined time, an aliquot (100 µl) of the spiked liver homogenate sample was extracted by adding 400 µl of acetone to induce the precipitation of proteins.

Stability of HI-113 and ala-d4T-MP in Gastric and Intestinal Fluids. The simulated gastric and intestinal fluids that were prepared following the U.S. Pharmacopeia XXII methods were spiked with HI-113 and ala-d4T-MP to yield final concentrations of 100 µM for each compound. The spiked fluids were then placed in a 37°C water bath. At a predetermined time, 100-µl aliquots of the spiked gastric or intestinal fluid were extracted by adding 400 µl of acetone.

Pharmacokinetic Studies in Mice. Female BALB/c mice (6-8 weeks old) (Taconic, Germantown, NY) were housed in a United States Department of Agriculture-accredited animal care facility under standard environmental conditions (12-h light/12-h dark photoperiod, 22 ± 1°C, 60 ± 10% relative humidity). All rodents were housed in microisolator cages (Lab Products, Inc., Maywood, NJ) containing autoclaved bedding. Mice were allowed free access to autoclaved pellet food and tap water throughout the study. All animal studies are approved by the Parker Hughes Institute Animal Care and Use Committee, and all animal care procedures conformed to the principles outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington DC, 1996).

Pharmacokinetic Analysis. Pharmacokinetic modeling and parameter calculations were carried out using the WinNonlin Professional version 3.0 (Pharsight, Inc., Mountain, CA) pharmacokinetics software (Chen et al., 1999b,d,f; Uckun et al., 1999a,b). An appropriate model was chosen on the basis of the lowest sum of weighted squared residuals, the lowest Schwartz Criterion, the lowest Akaike's Information Criterion value, the lowest standard errors of the fitted parameters, and the dispersion of the residuals. F test was also used to discriminate between these hierarchical models (Gabrielsson and Weiner, 1997). The elimination half-life was estimated by linear regression analysis of the terminal phase of the plasma concentration-time profile. The area under the concentration-time curve (AUC) was calculated according to the linear trapezoidal rule between the first sampling time (0 h) and the last sampling time plus C/k, where C is the concentration of the last sampling and k is the elimination rate constant. The systemic clearance (CL) was determined by dividing the dose by the AUC. The clearance of each metabolite was estimated by simultaneous fitting of the concentration versus time curves of the parent drug and metabolites to pharmacokinetic models specified as a system of differential equations (Gabrielsson and Weiner, 1997).

	Results

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Chromatographic Separation of HI-113 and Potential Metabolites of HI-113. We established standard HPLC conditions for simultaneous separation of HI-113 and its metabolites ala-d4T-MP and d4T in plasma. Using the chromatographic separation conditions described under Materials and Methods, the retention times (R_T) measured for HI-113 and its metabolites in spiked samples were 28.7 ± 0.02 min (HI-113A; n = 13; Fig. 2B), 28.9 ± 0.02 min (HI-113B; n = 13; Fig. 2C), 15.3 ± 0.2 min (ala-d4T-MP; n = 30), and 18.5 ± 0.1 min (d4T; n = 30), respectively. At these retention times, no significant interference peaks were observed in the blank plasma samples (Fig. 2, A and B). There was another peak eluting at approximately 27 min (Fig. 2B). The chemical identity of this peak, which may represent another metabolite of HI-113, is currently unknown.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Representative HPLC chromatograms of blank plasma (A), blank plasma spiked with HI-113, d4T, and ala-d4T-MP (B), and plasma samples at 10 min after intravenous injection of 100 mg/kg HI-113 (C).

Stability of HI-113 and ala-d4T-MP. The results shown in Fig. 3A indicate that HI-113 is unstable in plasma. Following incubation with plasma, >95% of HI-113 decomposes within 5 min (Table 1). The decomposition of HI-113 in the plasma samples was complete within 30 min. Hence, immediate extraction of the samples is required after collection to accurately measure the HI-113 levels. In contrast, ala-d4T-MP was stable in the plasma for at least 24 h.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Stability of HI-113 in plasma (A), in liver homogenate (B), in gastric fluid (C), and in intestinal fluid (D). , HI-113; , ala-d4T-MP; , d4T.

Metabolism and Pharmacokinetic Profile of HI-113 Following Intravenous Administration. Following intravenous administration, HI-113 (100 mg/kg) was metabolized to yield HI-113-M1 (R_T = 15.3 min) and HI-113-M2 (R_T = 18.5 min) (Fig. 2C). HI-113-M1 had the same retention time as ala-d4T-MP, whereas HI-113-M2 had the same retention time as d4T (Fig. 2, B and C). The UV spectra of these two metabolites were identical to those of ala-d4T-MP (spectrum match factor representing the degree of similarity between the spectra was 996) and d4T (spectrum match factor = 998), respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Selection of the pharmacokinetic models.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Pharmacokinetic model for describing the disposition of HI-113, ala-d4T-MP, and d4T after i.v. injection of HI-113 (A) and the plasma concentrations of HI-113, ala-d4T-MP, and d4T as a function of time in BALB/c mice following i.v. injection of HI-113 (100 mg/kg, four mice per time point) (B).

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Pharmacokinetic Profile of Ala-d4T-MP Following Intravenous Administration. Following i.v. injection of ala-d4T-MP (75 mg/kg, a dose equimolar to the 100-mg/kg dose of HI-113 discussed above), the concentration versus time curves of its major metabolites ala-d4T-MP and d4T were bi- and monoexponential, respectively. The pharmacokinetic model depicted in Fig. 6A best described the pharmacokinetics of ala-d4T-MP after i.v. administration. According to this model, ala-d4T-MP can either be metabolized to form d4T (CL_m3) or distributed to the extravascular compartment (CL_m1d). D4T derived from ala-d4T-MP is eliminated from the body (CL_me2). When we assumed that both parent ala-d4T-MP and d4T can be eliminated from the body (addition of CL_me1 in Fig. 6A), the predicted curves were not good fits for the observed plasma concentration versus time data. By simultaneous fitting of the parent ala-d4T-MP and d4T plasma concentration values versus time to the described model shown in Fig. 6A, the metabolic clearance of ala-d4T-MP and the formation clearance of d4T (CL_m3) were estimated to be 15.6 ml/min/kg. Ala-d4T-MP was distributed to the extravascular compartment with a CL_m1d of 4.7 ml/min/kg and d4T derived from ala-d4T-MP was eliminated with a relatively high CL_me2 of 88.4 ml/min/kg. The CL_m3 of 15.6 ml/min/kg accounts for 99% of the total systemic clearance (CL = 15.8 ml/min/kg) (Table 2), indicating that most of ala-d4T-MP is biotransformed to form d4T.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Pharmacokinetic model for describing ala-d4T-MP and d4T after i.v. injection of ala-d4T-MP (A) and the plasma concentrations of ala-d4T-MP and d4T as a function of time in BALB/c mice following i.v. injection of ala-d4T-MP (75 mg/kg, five mice per time point) (B).

Pharmacokinetic Profile of d4T Following Intravenous Administration. Following i.v. injection of d4T at a dose level of 40 mg/kg, which is equimolar to the 100-mg/kg dose of HI-113, the plasma concentration of d4T as a function of time can best be described by using a one-compartment model (Fig. 7). The estimated pharmacokinetic parameter values are presented in Table 2. The estimated C_max and AUC values for d4T were 318.9 and 12,173.6 µM · min, respectively. d4T had a short elimination half-life (26.6 min). The systemic clearance of d4T was slow with a CL of only 15.2 ml/min/kg, which is slower than the blood flow to either the kidney or the liver (Davies and Morris, 1993). d4T had a moderately large volume of distribution (V_ss = 581.8 ml/kg), which is approximately equal to the volume of water in the body (Davies and Morris, 1993).

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Plasma concentration of d4T as a function of time in BALB/c mice following i.v. injection of d4T (40 mg/kg, five mice per time point).

Pharmacokinetic Profile of HI-113 Following Oral Administration. The pharmacokinetic behavior of orally administered HI-113 (100 mg/kg) was also examined. Both metabolites (ala-d4T-MP and d4T) were detected, but the concentration of the parent HI-113 was below the detection limit (0.25 µM). The T_max values were 9.3 min for ala-d4T-MP and 45.2 min for d4T. A one-compartment pharmacokinetic model was used to describe the time-dependent concentration changes for ala-d4T-MP and d4T (Fig. 8, A and B). The estimated values for the pharmacokinetic parameters are presented in Table 3. The maximum concentrations (C_max) for ala-d4T-MP and d4T are 15.6 and 29.5 µM, respectively. The elimination half-lives were 56.1 and 102.6 min for ala-d4T-MP and d4T, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Plasma concentrations of ala-d4T-MP (A) and d4T (B) as a function of time in BALB/c mice following oral administration of HI-113 (100 mg/kg, four mice per time point).

	Discussion

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Our results presented herein provide unprecedented evidence that the novel anti-HIV agent HI-113 is quickly metabolized in vivo to form two major metabolites, ala-d4T-MP and d4T after i.v. injection (Fig. 5) as well as after oral administration (Fig. 8). Ala-d4T-MP can also be metabolized further to yield d4T (Figs. 5, 6, and 8).

Stability studies revealed that while HI-113 is readily metabolized in plasma to form ala-d4T-MP, only a small amount of d4T is formed. Furthermore, ala-d4T-MP was found to be stable in plasma. By comparison, a significant amount of d4T was formed after incubation of HI-113 with a liver homogenate (Fig. 3B). These findings are consistent with previous in vitro metabolic studies (Balzarini et al., 1996a,b; Venkatachalam et al., 1998; Saboulard et al., 1999).

Paraoxon, an inhibitor of both cholinesterase and carboxylesterase (Augustinsson, 1961; McCracken et al., 1993; Madhu et al., 1997), significantly prevented the hydrolysis of HI-113 to form ala-d4T-MP and d4T, suggesting that both cholinesterase and carboxylesterase are important for metabolism of HI-113. Physostigmine, an inhibitor of cholinesterase (Augustinsson, 1961; McCracken et al., 1993; Madhu et al., 1997), partially prevented the hydrolysis of HI-113, which further supports the importance of cholinesterase in hydrolysis of HI-113. EDTA, an inhibitor of arylesterase (Augustinsson, 1961; McCracken et al., 1993; Madhu et al., 1997), did not affect the hydrolysis of HI-113, indicating that arylesterase is probably not involved in the hydrolysis of HI-113. However, the importance of the hepatic P450 system in the metabolism of HI-113 is currently unknown.

In the present study, the elimination half-life of d4T following i.v. injection was 26.6 min, which is longer than the reported elimination half-life for d4T (17 min) in mice (Russell et al., 1990). The elimination half-life of intravenously administered d4T was similar to the elimination half-life of d4T derived from ala-d4T-MP (t_1/2 of 26.6 versus 32.4 min). In contrast, the elimination half-life for d4T derived from HI-113 was significantly prolonged (t_1/2 of 116.2 min). Similarly, the elimination half-life for ala-d4T-MP derived from HI-113 was longer than the t_1/2 for ala-d4T-MP administered intravenously (t_1/2 of 138.8 versus 38.9 min).

Orally administered HI-113 also yielded ala-d4T-MP and d4T as the two major metabolites. No parent HI-113 was detectable in the blood after oral administration. This lack of HI-113 in the plasma may be attributed to several factors. First, while HI-113 was stable in gastric fluid and may be absorbed in the stomach, it was quickly hydrolyzed in blood. On the other hand, HI-113 decomposes readily in intestinal fluid to form ala-d4T-MP. This metabolite may be absorbed in the intestine and then further metabolized to yield d4T in the blood. The T_max and t_1/2 values for d4T in mice were longer for orally administered HI-113 (45.2 and 102.6 min, respectively) than for orally administered d4T (5 and 18 min, respectively) (Russell et al., 1990). In comparison with the elimination half-lives measured following i.v. injection, the t_1/2 values for both ala-d4T-MP and d4T were prolonged after oral administration of HI-113.

The exact mechanism for the observed longer elimination half-life of metabolites after administration of the parent compound HI-113 is not clear. Such a phenomenon was observed with other metabolites (Pang and Gillette, 1980; Pang, 1981; Houston and Taylor, 1984; Lin et al., 1984).

There was also a ~10-fold difference in distributional clearance of ala-d4T-MP (47.1 versus 4.7 ml/min/kg) following i.v. injection of HI-113 versus ala-d4T-MP. Since the plasma concentration of ala-d4T-MP after i.v. injection of ala-d4T-MP is ~10-fold higher than that after i.v. injection of HI-113, this distributional process may be nonlinear.

In summary, HI-113 forms two active metabolites with favorable pharmacokinetics after both i.v. as well as oral administration. The intravenous administration of HI-113 results in more prolonged systemic exposure to ala-d4T-MP as well as d4T than the intravenous administration of an equimolar dose of ala-d4T-MP or d4T due to the apparently longer elimination half-lives of HI-113-derived metabolites. Similarly, the oral administration of HI-113 results in prolonged retention of ala-d4T-MP and d4T in the body. Ala-d4T-MP inhibits the replication of the HIV-1 strain HTLVIIIB in human T lymphocytes with an IC₅₀ value of 0.01 µM and a selectivity index of >10,000, whereas D4T inhibits HIV-1 replication with an IC₅₀ value of 0.02 µM and a selectivity index of 100 (F. M. Uckun, unpublished data). Therefore, the potent antiviral activity of HI-113 (IC₅₀ < 0.001 µM; selectivity index >30,000) may be attributed to the activity of both of its metabolites. These results also suggest that HI-113 may serve as a useful prodrug for these active metabolites (Mansuri et al., 1989; Horton et al., 1995; Balzarini et al., 1996a,b; Lea and Faulds, 1996). Furthermore, since i.v. administration of ala-d4T-MP results in formation of d4T, ala-d4T-MP may also find utility as a d4T prodrug.