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Preparation and In Vivo Evaluation of a Water-Soluble Prodrug for 2R--Tocotrienol and as a Two-Step Prodrug for 2,7,8-Trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC) in Rat

Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan (N.A., J.T., K.M., R.H., K.F., M.Y., To.F., Y.K.); and Department of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (Ta.F., A.H., K.I.)

(Received December 21, 2006; accepted May 23, 2007)

	Abstract

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2R--Tocotrienol (-T3) is currently receiving attention because it has beneficial effects not observed with -tocopherol. To achieve the effective delivery of -T3, we synthesized three kinds of ester derivatives of -T3 and evaluated their use as hydrophilic prodrugs for -T3 in vitro and in vivo. 2R--Tocotrienyl N,N-dimethylamino-acetate hydrochloride (compound 3) was a solid compound, with high solubility and stability in water, and was converted to -T3 by esterases in rat and human liver. Intravenous administration of 3 in rats led to a rapid increase in the plasma, liver, heart, and kidney levels of -T3. The bioavailability (plasma level) after intravenous administration was 82.5 ± 13.4% and 100 ± 11.3% for 3 and -T3 in surfactant, respectively, and the availability in liver was 213 ± 47.6% and 100 ± 4.8% for 3 and -T3 in surfactant, respectively. Furthermore, the systemic availability of 2,7,8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC), a metabolite of -T3, was 78.6% for compound 3, 47.1% for -T3 in surfactant, and 100% for racemic -CEHC. Based on these results, we identified compound 3 as the most promising water-soluble prodrug of -T3 and two-step prodrug of S--CEHC.

Recent studies have shown that -T3 is metabolized to 2,7,8-trimethyl-S2-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC) in rat (Hattori et al., 2000) and human (Lodge et al., 2001). S--CEHC was originally discovered as a natriuretic factor and named LLU- by Wechter et al. (1996). S--CEHC was later found to be a major metabolite of 2R--tocopherol (Murray et al., 1997). Furthermore, S--CEHC has been shown to inhibit the generation of prostaglandin E₂, an important mediator synthesized during inflammation via the cyclooxygenase-2-catalyzed oxidation of arachidonic acid (Jiang et al., 2000; Grammas et al., 2004). Thus, -T3 and its metabolite, S--CEHC, are expected to be useful therapeutic agents.

Therapeutic formulation of -T3 is difficult because it is a highly viscous oil, nearly insoluble in water, and readily oxidized by atmospheric oxygen. These physicochemical properties of -T3 limit its therapeutic application and complicate its administration. Like -T3, S--CEHC is readily oxidized, and, furthermore, its rapid elimination causes it to have a very low bioavailability (Murray et al., 1997; Hattori et al., 2001). It is expected that the effective delivery of -T3 will achieve sufficient levels of S--CEHC. When administered in the form of an oil solution or an oil emulsion, tocotrienols have poor bioavailability, regardless of the route of administration (e.g., parenteral, oral, or topical) (Yap et al., 2003). To solubilize a lipophilic compound in water, a large amount of surfactant, for example, polyoxyethylene hydrogenated castor oil, or a self-emulsifying drug delivery system is needed. The use of surfactants is, however, undesirable for a parenteral dosage because it generally causes toxic reactions such as anaphylaxis.

Formation of water-soluble ester prodrugs has long been considered an efficient way to improve the aqueous solubility of poorly soluble drugs that contain a hydroxyl group. The most commonly used esters for forming prodrugs are those containing ionizable groups such as dicarboxylic acid hemiester. The phenolic functional group in -T3 can be easily esterified, and some of the ester derivatives are expected to provide the desired improvements in water solubility and stability to oxidation. An ideal prodrug should exhibit sufficient aqueous solubility and should be rapidly converted into the parent drug in vivo. We previously observed that aminoalkylcarboxylic acid esters of another vitamin E family member, -tocopherol, which, like -T3, contains a C5-desmethyl chroman ring but a different hydrocarbon tail (phytyl) at the C2 position, have good water solubility and a high susceptibility to esterase-catalyzed hydrolysis in rat and human liver. In addition, the esters could increase plasma and liver availability of -tocopherol after i.v. administration in rats (Takata et al., 2002). The ester of -tocopherol can reduce photo-inflammation in mouse skin and prevents UV-induced skin pigmentation when applied topically (Kuwabara et al., 2006; Yoshida et al., 2006). In the current studies, to overcome the problems of administering -T3 and S--CEHC, we prepared several aminoalkylcarboxylic acid esters of -T3 and examined their effectiveness as two-step prodrugs of S--CEHC when administered in an aqueous formulation.

	Materials and Methods

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Instruments. Melting point measurements were made using a BY-1 micro melting point apparatus (Yazawa, Tokyo, Japan) and were uncorrected. Microanalyses and measurements of ¹H NMR and mass spectroscopy (MS) were carried out at the Central Microanalytical Department of Pharmaceutical Sciences, Fukuoka University. The ¹H NMR spectra were determined at 500 MHz in CDCl₃ using a JEOL JNM-A500 spectrometer (JEOL, Tokyo, Japan). The chemical shifts are expressed in (ppm) using tetramethylsilane as the internal standard. Fast atom bombardment mass spectra were obtained using a JEOL JMS-HX110 spectrometer.

Chemicals. Tocotrienol-rich fraction of palm oil and racemic -CEHC were kindly supplied by Eisai Co., Ltd. (Tokyo, Japan). Eserine (physostigmine hemisulfate) was purchased from Sigma Chemical Co. (St. Louis, MO). N-tert-Butyloxycarbonyl (N-t-Boc) aminoacetic acid and N-t-Boc-N-methylaminoacetic acid were purchased from Peptide Institute, Inc. (Osaka, Japan). N, N-Dimethylaminoacetic acid hydrochloride, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole, and triphenylphosphine were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Polyoxyethylene hydrogenated castor oil was purchased from Nikko Chemical (Tokyo, Japan). All other chemicals were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). Male Sprague-Dawley rats (7 weeks old), Sprague-Dawley rat liver microsomes, and human liver microsomes were purchased from Charles River Japan Inc. (Kanagawa, Japan). Human plasma was obtained from healthy volunteers (aged 21-22 years). All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University.

Purification of 2R--Tocotrienol from the Tocotrienol-Rich Fraction of Palm Oil. Tocotrienol-rich fraction of palm oil was fractionated by flash chromatography and normal phase HPLC, and the -T3 was isolated without delay by solvent evaporation in vacuo and then stored under argon at -30°C. A 1.2-g sample of tocotrienol-rich fraction of palm oil was separated by flash chromatography on a 40 x 150-mm column (Biotage, Charlottesville, VA) containing Wakogel LP40 (particle size 20-40 µm; Wako Pure Chemical Co.) and elution with a gradient of 97:3 to 80:20 (v/v) n-hexane/isopropyl ether. The major fraction containing -T3 (0.6 g) was further separated by HPLC using a Wakosil-II 5SIL-100 normal-phase column (10 x 300 mm; Wako Pure Chemical Co.) and elution with 97.5:2.5 (v/v) n-hexane/ethyl acetate to afford 2R--tocotrienol (-T3): colorless oil; MS m/z 410 (M⁺). ¹H NMR d (CDCl₃): 6.36 (1H, s, 5-H), 5.10 (3H, m, 3', 7', 11'-H), 4.15 (1H, s, 6-OH), 2.68 (2H, m, 4-H₂) 2.13-1.95 (16H, m, including 2.13 [3H, s, 7-CH₃]), 2.11 [3H, s, 8-CH₃]), 1.79-1.52 (16H, m, including 1.68 [3H, s], 1.59 [6H, s], 1.58 [3H, s]), 1.26 (3H, s, 2-CH₃). Anal. calculated for C₂₈H₄₂O₂: C, 81.90; H, 10.31. Found: C, 81.77; H, 10.37.

Synthesis of the -T3 Esters. General procedure for primary and secondary aminoacetic acid esters of -T3 (compounds 1 and 2). A mixture of 2.5 mmol of -T3, 2.7 mmol of N-t-Boc-aminoacetic acid, and 2.7 mmol of dicyclohexylcarbodiimide in 20 ml of dry pyridine was stirred at room temperature for 24 h. After evaporation in vacuo, the residue was triturated with 50 ml of isopropyl ether, and the dicyclohexylurea was removed by filtration. The filtrate was evaporated in vacuo, and the trituration was repeated twice. The N-t-Boc-aminoacetic acid ester of -T3 was isolated by flash chromatography (Biotage) on a column containing Wakogel LP40 and elution with 97.5:2.5 (v/v) n-hexane/isopropyl ether. The acetone solution of the N-t-Boc-aminoacetic acid ester was added to 4 N HCl in dioxane, and the mixture was stirred for 30 min. The solvent was evaporated in vacuo, and the residue was recrystallized from acetone to give the hydrochloride salt of 1 and 2.

2R--Tocotrienyl glycinate hydrochloride (1). White solid; yield 61%; melting point 195°C-198°C; MS m/z, 468 (M - HCl + H⁺). ¹H NMR (CDCl₃): -tocotrienyl moiety 6.61 (1H, s, 5-H), 5.10 (3H, m), 2.60 (2H, m, 4-H₂), 2.16-1.92 (16H, m, including 2.02 [3H, s, 7-CH₃], 1.92 [3H, s, 8-CH₃]), 1.69-1.49 (16H, m, including 1.67 [3H, s], 1.58 [6H, s], 1.55 [3H, s]), 1.24 [3H, s, 2-CH₃]), glycine moiety 8.67 (2H, s, NH₂), 4.04 (2H, s, -NCH₂CO-). Anal. calculated for C₃₀H₄₅NO₃HCl + 1.5H₂O: C, 67.84; H, 9.30; N, 2.64. Found: C, 67.91; H, 9.06; N, 2.60.

2R--Tocotrienyl N-methylglycinate hydrochloride (2). White solid; yield 64%; melting point 130°C-132°C; MS m/z, 482 (M - HCl + H⁺). ¹H NMR (CDCl₃): -tocotrienyl moiety 6.66 (1H, s, 5-H), 5.11 (3H, m), 2.66 (2H, m, 4-H₂), 2.10-1.95 (16H, m, including 2.08 [3H, s, 7-CH₃], 2.01 [3H, s, 8-CH₃]), 1.80-1.52 [16H, m, including 1.67 [3H, s], 1.59 [6H, s], 1.56 [3H, s]), 1.24 [3H, s, 2-CH₃], N-methylglycine moiety 10.01 (1H, s, NH), 4.04 (2H, s, -NCH₂CO-), 2.81 (3H, s, CH₃N-). Anal. calculated for C₃₁H₄₇NO₃HCl + 1.3H₂O: C, 68.75; H, 9.42; N, 2.59. Found: C, 68.74; H, 9.17; N, 2.65.

2R--Tocotrienyl N, N-dimethylglycinate hydrochloride (3). To a dry pyridine solution of -T3 (2.5 mmol), 2.7 mmol of N,N-dimethylaminoacetic acid hydrochloride and 2.7 mmol of dicyclohexylcarbodiimide were added. The reaction mixture was stirred at room temperature for 20 h, and the dicyclohexylurea formed was removed by filtration. After the solvent was evaporated, the residue was treated with 100 ml of water and made alkaline by adding sodium bicarbonate. The solution was then extracted three times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated. The residue was separated by flash chromatography (Biotage) using a column packed with Wakogel LP40 and elution with 8:2 (v/v) n-hexane/ethyl acetate. Because the free base of the ester is very unstable (i.e., hydrolyzed) at high concentrations, the corresponding fractions were collected directly in isopropyl ether containing 3% HCl in dioxane, and the precipitate was collected and recrystallized from acetone to give the hydrochloride salt of 3: white solid; yield 83%; melting point 160°C-161°C; MS m/z, 496 (M - HCl + H⁺). ¹H NMR (CDCl₃): -tocotrienyl moiety 6.63 (1H, s, 5-H), 5.10 (3H, m, 3', 7',11'-H), 2.72 (2H, m, 4-H₂), 2.13-195 (16H, m, including 2.12 [3H, s, 7-CH₃], 2.02 [3H, s, 8-CH₃]), 1.81-1.59 (16H, m, including 1.68 [3H, s], 1.60 [6H, s], 1.59 [3H, s]), 1.28 (3H, s, 2-CH₃). N,N-dimethylglycine moiety 4.21 (2H, s, NCH₂CO), 3.09 (6H, s, (CH₃)₂N). Anal. calculated for C₃₂H₄₉NO₃HCl: C, 72.20; H, 9.47; N, 2.63. Found: C, 72.03; H, 9.30; N, 2.53.

2R--Tocotrienyl acid succinate (4). To a dry dichloromethane containing -T3 (2.5 mmol), 2.7 mmol of succinic anhydride and dimethylaminopyridine were added. The reaction mixture was stirred at room temperature for 20 h, and the solvent was evaporated in vacuo. The residue was treated with 100 ml of water and acidified with 1 N HCl. The solution was then extracted three times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated. The residue was fractionated by flash chromatography (Biotage) on a column containing Wakogel LP40, 60 A (particle size 20-40 µm; Wako Pure Chemical Co.) and elution with 8:2 (v/v) n-hexane/ethyl acetate (8:2, v/v): colorless oil; MS m/z, 510 (M⁺). ¹H NMR (CDCl₃): 6.56 (1H, s, 5-H), 5.10 (3H, m), 2.70 (2H, m, 4-H₂), 2.13-1.95 (16H, m, including 2.10 [3H, s, 7-CH₃], 2.01 [3H, s, 8-CH₃]), 1.79-1.56 (16H, m, including 1.67 [3H, s], 1.60 [6H, s], 159 [3H, s]), 1.26 (3H, s, 2-CH₃), succinyl moiety 2.88 (2H, t), 2.80 (2H, t). Anal. calculated for C₃₂H₄₆O₅: C, 75.26; H, 9.08. Found: C, 75.02; H, 9.13.

Water Solubility. The aqueous solubility of 1 to 4 was determined by adding 50 µmol of each compound to 1 ml of water in amber vials maintained at 25 ± 0.1°C in a constant-temperature water bath. The vials were shaken for 24 h, and the contents were filtered through Columngard-LCR4 membranes (0.5-µm pore size; Nihon Millipore Kogyo K.K., Yonezawa, Japan). The ester concentrations in the filtrates were determined by the HPLC method described below. The adherence of the esters to the filter was ignored. HPLC indicated that, during the entire procedure, less than 0.5% of -T3 was formed from the esters.

Analysis of Hydrolysis. The hydrolysis of the esters was studied at 37°C in isotonic phosphate buffer (pH 7.4) containing rat plasma, rat liver microsomes, human plasma, or human liver microsomes. Stock solutions of esters 1 to 4 were made in 5% (v/v) aqueous methanol. The enzymatic reactions were initiated by adding 50 µl of stock solution of esters (final concentration 0.1-0.8 mM) and 50 µl of isotonic phosphate buffer to amber test tubes containing 900 µl of preheated reaction medium containing rat or human plasma (90% final concentration) or rat or human liver microsomes in isotonic phosphate buffer (pH 7.4) (final concentration 0.11 mg protein/ml). The solutions were incubated at 37°C, and at appropriate times, 100-µl samples of the reaction were removed and added to 350 µl of ethanol. The ethanol-diluted samples were vortexed for 2 min and centrifuged at 1000g for 5 min. A 50-µl sample of the supernatant was analyzed by HPLC, and the initial hydrolytic rate (moles of -T3 formed per liter of reaction) was calculated from the initial slope of the plot of -T3 generated versus time. HPLC detected hydrolysis of 4 but not of 1 to 3 using rat plasma, rat liver microsomes, human plasma, or human liver microsomes. Because significant hydrolysis of 4 in isotonic phosphate buffer (pH 7.4) was observed during the experiment, the differential rate of parent drug regeneration in reaction medium and chemical hydrolysis of 4 in isotonic phosphate buffer (pH 7.4) was used as the initial hydrolytic rate. In phosphate buffer, the apparent first-order rate constants for hydrolysis were obtained by linear regression analysis of the natural logarithm of concentration versus time (r > 0.97).

The effects of eserine on the hydrolysis of the esters in the liver microsome were examined as described above except that 50 µl of eserine aqueous solution (final concentration 0-2.5 mM) was added in place of isotonic phosphate buffer (pH 7.4) to the liver microsome solution at the beginning of the experiment.

HPLC Analysis of -T3 and Compounds 1 to 4. The Shimadzu HPLC system (Shimadzu, Kyoto, Japan) used in this study consisted of an LC6A pump, an SIL 9A autosample injector, an SPD-10AV UV detector, and an RF-540 spectrofluorophotometer equipped with a 12-µl liquid chromatography flow cell and C-R7A peak integrators. The eluent was monitored spectrophotometrically at 283 nm and spectrofluorometrically at 325 nm with excitation of 298 nm. Compounds -T3 and 1 to 4 were analyzed using a CAPCELL PAK C18 UG120 reversed-phase column (4.6 x 150 mm; Shiseido, Tokyo, Japan) with a mobile phase of 7:3 (v/v) methanol/acetonitrile delivered at 0.7 ml/min. The compounds were quantified using linear calibration curves of the peak area versus the concentration of the compound.

Disposition of the Prodrug and -T3 after i.v. Administration in Rats. We first performed a preliminary study of the dose effect of the selected prodrug (compound 3) on the plasma disposition of -T3 in rats. Compound 3 was dissolved in distilled water containing 15% (v/v) propylene glycol, and -T3 was solubilized with water containing 10% (w/v) polyoxyethylene hydrogenated castor oil and 15% (v/v) propylene glycol (-T3-H). The solution of racemic -CEHC was solubilized in saline solution containing 33% (v/v) polyethylene glycol. Before experiments, male Sprague-Dawley rats were fasted for 16 h but allowed access to water ad libitum. The drugs were administered via the left femoral vein, which was exposed by means of a small incision made under light ether anesthesia. Rats were injected with 5, 10, and 25 mg/kg 3 or the equivalent of -T3 in a volume of 0.1 ml/100 g body weight. After 0.25, 0.5, 1, 2, 4, 8, and 24 h, 300-µl blood samples were taken from the external jugular vein using heparinized syringes. The plasma was isolated by centrifugation, after which 100-µl samples of the plasma were added to 350 µl of ethanol, vortexed for 2 min, and centrifuged at 1000g for 5 min. The supernatant was analyzed by HPLC as described above (see HPLC Analysis of -T3 and Compounds 1 to 41).

On the basis of the initial experiment, we next treated rats with 3 and -T3-H at 25 mg/kg Eq of -T3. Under ether anesthesia, at 0.25, 0.5, 1, 2, 4, 8, and 24 h, 4.5 ml of blood was taken from the abdominal artery using a syringe containing 0.5 ml of 3.2% sodium citrate, and the liver, kidneys, and heart were removed. The plasma was immediately separated by centrifugation at 5°C and stored at -80°C until HPLC analysis. The tissues were homogenized with 3 volumes of 1.15% KCl containing 1 mM eserine using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) and stored at -80°C until used for HPLC. The plasma and homogenized tissue samples (100 µl) were added to 350 µl of ethanol, vortexed for 2 min, and centrifuged at 1000g for 5 min. The supernatant layer was determined by the HPLC method described above.

Plasma Disposition of S--CEHC in Rats. Rats were treated and administered drugs (3, -T3, and racemic -CEHC) as described above. After 0.25, 0.5, 1, 2, 4, 8, and 24 h, 300-µl blood samples were taken from the external jugular vein using heparinized syringes. The plasma was immediately separated by centrifugation at 5°C and stored at -80°C until HPLC analysis. Plasma samples (50 µl) were examined for the level of S--CEHC as described below.

HPLC Analysis for S--CEHC. S--CEHC levels in rat plasma were assessed according to our previously reported method, which allows separate determination of the S- and R-enantiomers of -CEHC (Hattori et al., 2001). The HPLC system consisted of an L-7100 pump (Hitachi, Tokyo, Japan) and two PU610-10 pumps (GL Sciences, Tokyo, Japan), two L-7480 fluorescence detectors (Hitachi), two 807-IT integrators (Jasco, Tokyo, Japan), and two HV-992-01 six-port valves (Jasco). In brief, -CEHC in rat plasma (50 µl) was derivatized with 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole, acetylated with acetyl chloride, and deproteinized with 4:1 (v/v) CH₃CN/ethanol. After purification with an Empore C₁₈ cartridge (GL Sciences), the -CEHC derivative was injected into a column-switching HPLC system (Hattori et al., 2001), with the following three columns: TSKgel SuperPhenyl (100 x 4.6 mm; Tosoh, Tokyo, Japan), TSKgel ODS-80Ts (250 x 4.6 mm; Tosoh), and CHIRALCEL OD-RH (150 x 4.6 mm; Daicel Co. Ltd., Tokyo, Japan) connected through two six-port valves equipped with a trapping column. The fraction including the -CEHC derivative separated on the phenyl column was introduced to the ODS column and separated again on the chiral column. The mobile phase compositions and flow rates, respectively, were 650:350:1 (v/v/v) H₂O/CH₃CN/trifluoroacetic acid and 0.8 ml/min for the phenyl column, 400:600:1 (v/v/v) H₂O/CH₃CN/trifluoroacetic acid and 0.3 ml/min for the ODS column, and 95:5 (v/v) CH₃OH/CH₃CN and 0.3 ml/min for the chiral column. Eluted compounds were detected spectrofluorometrically at 560 nm with excitation at 450 nm.

Pharmacokinetic Analysis. The plasma and liver concentration versus time curve was analyzed using independent and statistical moment methods (Yamaoka et al., 1978, 1981). Both the maximum concentration (C_max) and its corresponding time (T_max) were directly obtained from the observed data. The systemic and tissue availabilities for -T3 after i.v. administration of 3 relative to -T3-H administration was determined from the ratio of the area under the concentration-time curve (AUC) of -T3 based on eqs. 1 and 2, respectively. The selective advantage value (Eriksson and Tozer, 1987) for -T3 in the liver was calculated using eq. 3. The systemic availability for S--CEHC after i.v. administration of 3 relative to racemic -CEHC administration was determined from the ratio of AUC of S--CEHC based on eq. 4.

(1)

(2)

where AUC^Plasma_{-T3, prodrug}, AUC^Plasma_-T3,-T3-H, AUC^tissue_{-T3, prodrug}, and AUC^tissue_{-T3, -T3-H} are the AUC values for -T3 in the plasma and tissues after the administration of 3 and -T3-H, respectively; AUC^Plasma_{prodrug, prodrug} is the AUC value for 3; D_prodrug, D_-T3-H, and D_{racemic -CEHC} are the doses of 3, -T3-H, and racemic -CEHC, respectively; and AUC^Plasma_{S--CEHC, prodrug} and AUC^Plasma_{S--CEHC, racemic -CEHC} are the AUC values for increased S--CEHC in the plasma after the administration of 3 and racemic -CEHC, respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Chemical structures of -T3, S--CEHC, and 2R--tocopherol ester derivatives.

	Results

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Hydrolysis of the R--Tocotrienyl Esters in Rat Plasma, Rat Liver Microsomes, Human Plasma, and Human Liver Microsomes. To develop a useful prodrug, the linkage between the parent drug and the promoiety should be stable in formulations but rapidly cleaved in vivo. The most successful prodrug of -T3 should be converted to the parent drug by enzyme(s) encountered after administration. We therefore examined the kinetics for the hydrolysis of esters 1 to 3 and the acid succinate ester 4 to -T3 in isotonic phosphate buffer (pH 7.4), rat plasma, rat liver microsomes, human plasma, and human liver microsomes at 37°C and pH 7.4. Compared with compound 4, all of the aminoalkylcarboxylic acid esters were highly stable. Compound 3 was the only ester to show modest stability in isotonic phosphate buffer (Table 1). The stability of this series of esters decreased in the following order: tertiary amine 3 » primary amine 1 » secondary amine 2 > succinyl hemiester 4. HPLC analysis revealed that the hydrolysis of the esters to -T3 was enhanced by the rat liver microsomes, rat plasma, and human liver microsomes but not by human plasma.

View this table: [in this window] [in a new window]   TABLE 1 Kinetic parameters for the hydrolysis of the aminoalkylcarboxylic acid esters and acid succinate ester of 2R--tocotrienol in vitro at pH 7.4 and 37°C

The kinetics of the hydrolysis fit the Michaelis-Menten model (Lineweaver and Burk, 1934), indicating that the process was mediated by an enzyme. Representative Lineweaver-Burk plots for the hydrolysis by the rat and human liver microsomes are shown in Figs. 2 and 3. The V_max and K_m values, as determined from linear regression analysis of 1/velocity versus 1/concentration plots, are shown in Table 1.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Representative Lineweaver-Burk plots of initial rates for the hydrolysis of the -T3 ester derivatives by rat liver microsomes at pH 7.4 and 37°C. Open squares, 1; closed squares, 2; open circles, 3; closed circles, 4. Each point represents the mean ± standard deviation from three experiments. The lines represent the least-square regression lines (r > 0.98).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Representative Lineweaver-Burk plots of initial rates for the hydrolysis of the -T3 ester derivatives in human liver microsomes at pH 7.4 and 37°C. Open squares, 1; closed squares, 2; open circles, 3; closed circles, 4. Each point represents the mean ± standard deviation from three experiments. The lines represent least-square regression lines (r > 0.98).

View larger version (13K): [in this window] [in a new window]   FIG. 4. Effect of eserine on the enzymatic hydrolysis of compound 3 by rat and human liver. Closed circles, rat liver microsomes; open circles, human liver microsomes. The initial concentration of 3 was 0.4 mM. Each point represents the mean ± standard deviation from three experiments.

Disposition of the Prodrug in Rats. Compound 3 was examined for potential parenteral use because of its high water solubility, stability in solution, and effective conversion to -T3. We initially examined the effect of 3 on the plasma disposition of -T3 when injected i.v. into rats at a dose range of 5 to 25 mg/kg Eq of -T3 (Fig. 5). We found a rapid and dose-dependent appearance of -T3 in plasma after administration of 3, indicating that -T3 was rapidly generated from 3 in vivo and that the process did not saturate in the dose range tested. Based on these findings, we carried out further studies using a dose of 25 mg/kg Eq of -T3.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Mean plasma concentration of -T3 after the i.v. administration of 3 in rats. The doses are 25 mg/kg (closed circles), 10 mg/kg (open squares), and 5 mg/kg (closed triangles) equivalent for -T3. Each point represents the mean ± standard deviation of three rats.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Mean concentration of intrinsic 3 and -T3 after the i.v. administration of 3 and -T3-H in the rats. Open triangles, intrinsic 3 after administration of 3; closed circle, -T3 after administration of 3; open circles, -T3 after administration of -T3-H. Each point represents the mean ± standard deviation of three rats. The dose was 25 mg/kg equivalent for -T3.

View this table: [in this window] [in a new window]   TABLE 2 Pharmacokinetic parameters in plasma and liver after the i.v. administration of -T3-H and 3 in rats Values are the mean and S.D. of three rats at a dose of 25 mg/kg equivalent for -T3.

View this table: [in this window] [in a new window]   TABLE 3 Pharmacokinetic parameters in kidney and heart after the i.v. administration of -T3-H and 3 in rats Values are the mean and S.D. of three rats at a dose of 25 mg/kg equivalent for -T3.

The liver and plasma distribution values of -T3 and intrinsic 3 at 0.25 h after administration of 3 were 31.4 ± 5.7% (-T3 in liver), 16.5 ± 4.3% (intrinsic 3 in liver), 4.0 ± 0.6% (-T3 in plasma), and 8.8 ± 3.4% (intrinsic 3 in plasma) of the initial dose. At this time, the distribution of -T3 after administration of -T3-H was 24.5 ± 0.7% (-T3 in liver) and 8.9 ± 2.2% (-T3 in plasma) of the initial dose. The rapid and liver-specific uptake of intrinsic 3 along with the rapid and specific appearance of -T3 in the liver clearly indicated that the generation of -T3 occurred mainly in the liver. It appeared that these characteristics of 3 might allow the liver-specific delivery of -T3. In fact, a remarkable liver-specific delivery of -T3 was observed after the administration of 3 as indicated by selective advantages of 4.1 and 1.0 for 3 and -T3-H, respectively. After the administration of 3, we did not observe the specific delivery of -T3 into kidney and heart, but the availabilities of -T3 were relatively high (74.2% and 78.5%, respectively).

Plasma Disposition of S--CEHC after i.v. Administration of the Prodrug in Rats. Because S--CEHC contains the same chromanol structure as -T3, it is also readily oxidized by atmospheric oxygen. S--CEHC has a 20-fold more potent natriuretic activity than R--CEHC (Murray et al., 1997) but is rapidly eliminated after i.v. administration (Hattori et al., 2001). These characteristics limit the therapeutic application of S--CEHC. It has been reported that -T3 is efficiently converted to S--CEHC without epimerization at chroman C2 (Hattori et al., 2001). Therefore, it seems that the use of the prodrug of -T3 as a two-step prodrug of S--CEHC should overcome the problems of delivering S--CEHC.

To examine the ability of 3 to act as a two-step prodrug for S--CEHC, rats were injected i.v. with 3 or a racemic mixture of -CEHC, and the plasma disposition of S--CEHC was examined. HPLC analysis detected only S--CEHC in the plasma during the course of the experiment (Fig. 7). This agreed with previous results examining the prodrug of -Toc (Takata et al., 2002). Figure 8 shows the time course of appearance of S--CEHC in the plasma after administration of 3, -T3-H, and racemic -CEHC. The pharmacokinetic parameters for S--CEHC are summarized in Table 4. After the i.v. administration of 3, the plasma level of S--CEHC rapidly increased, and the maximum accumulation was achieved 1 h after administration. In addition, the mean residence time (MRT) for S--CEHC was 7-fold longer than for racemic -CEHC. The relative systemic availabilities for S--CEHC (F) after administration of 3, -T3-H, and racemic -CEHC were 78.6%, 47.1%, and 100%, respectively.

View larger version (12K): [in this window] [in a new window]   FIG. 7. Representative chromatograms from enantiomeric separation of fluorescent -CEHC derivative. A, racemic -CEHC standard. The first and second eluted enantiomers were S- and R-form, respectively (Hattori et al., 2001). B, rat plasma sample 1 h after i.v. administration of 3.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Mean plasma concentration of S--CEHC in rats after the i.v. administration of 3 and -T3-H. Closed circles, 3; open circles, -T3-H. Each point represents the mean ± S.D. of four rats. The doses are 25 mg/kg equivalent for -T3.

View this table: [in this window] [in a new window]   TABLE 4 Pharmacokinetic parameters for S--CEHC after the i.v. administration of -T3-H and 3 in rats -CEHC was not soluble in distilled water for injection; thus, -CEHC was solubilized with water containing 33% polyethylene glycol and used for i.v. administration. The low water solubility of -CEHC compelled us to adopt a low dose of it for the disposition study.

	Discussion

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Four esters of -T3 were cleaved at different rates, and all of the aminoalkylcarboxylic acid esters (1-3) were hydrolyzed more rapidly than succinyl hemiester 4. The obtained results clearly demonstrated that the conversion of the aminoalkylcarboxylic acid esters and the succinic acid ester are catalyzed by enzyme(s) located in rat liver, rat plasma, and human liver, and that esters 1 to 3 are better substrates for the enzyme(s) than 4. Furthermore, although the rat liver microsomes were diluted more than the plasma (final concentrations 0.87% and 90%, respectively), the catalytic efficiency (V_max/K_m) value was higher in the liver preparation, suggesting that the esters are more effectively hydrolyzed in rat liver than in rat plasma. The data in Table 1 also reveal that the reactivity of the liver enzyme(s) toward the aminoalkylcarboxylic acid esters depends mainly on the structure of the amino functionality on the promoiety. Specifically, the cleavage increased in the following order: tertiary amine 3 = secondary amine 2 > primary amine 1. A higher rate was due to an increased V_max, indicating a higher enzymatic capacity.

For therapeutic application of the esters as prodrugs, they must be readily cleaved by a human enzyme. Human liver microsomes (Table 1) but not human plasma increased the rate of their hydrolysis. In addition, all of the aminoalkylcarboxylic acid esters 1 to 3 were hydrolyzed more rapidly than succinyl hemiester 4 by the human liver microsomes, and the reactivity for the aminoalkylcarboxylic acid esters increased in the following order: tertiary amine 3 > secondary amine 2 > primary amine 1.

Previously, we observed that the N,N-dimethylaminoacetic acid ester of -tocopherol can act as water-soluble prodrug of -tocopherol (Takata et al., 2002). The number and position of methyl substituents in the chroman ring of -T3 and -tocopherol are the same, but the hydrocarbon side chains are different: -T3 has an unsaturated hydrocarbon chain, and -tocopherol has a saturated hydrocarbon chain. Comparison of the structures of the N,N-dimethylaminoacetic acid esters of -T3 and -tocopherol reveal important insights into their different dispositions. In agreement with a previous report (Takata et al., 2002), we found that the T_max and MRT for -T3 in plasma and liver (Table 2) following i.v. administration of the esters were relatively short compared with those for -tocopherol. Thus, the isoprenyl side chain of -T3 is a crucial determinant of disposition characteristics. Notably, the unsaturated side chain of -tocotrienol, which is the same as that of -T3, appears to allow for more effective penetration and better distribution in cell membranes than -tocopherol, which has the same saturated side chain as -tocopherol (Suzuki et al., 1993). These findings suggest that differences in the side chain contribute to the observed differences in the disposition of -T3 and -tocopherol.

The animal disposition studies clearly indicated that 3 is a good candidate for the parenteral prodrug of -T3. Delivery of -T3 by i.v. administration of 3 can achieve a rapid and accurate onset and could alter several prospective biological activities of -T3 not observed with -tocopherol, for example, anticancer and tumor-suppressing activities. The oral bioavailability of -T3 was very low and was increased by food intake (Yap et al., 2001). The dissolution rate of poorly water-soluble compounds in the gastrointestinal tract is generally considered as the great variable factor for the oral bioavailability. The dissolution rate of these compounds is extremely influenced by the diet and the flow of bile secretion; thus, the oral bioavailability is low and its variation becomes large. Therefore, it can be expected that the water-soluble prodrug of -T3 might also be effective if delivered orally.

Although the systemic availability of -T3 after administration of 3 was lower than that of -T3-H, the liver availability of -T3 was higher in the former case. Therefore, the high systemic availability of S--CEHC appears to be related to extensive delivery of -T3 into the liver after administration of 3. Although the mechanism for the delivery of S--CEHC by administration of 3 cannot be confirmed, the selective appearance of S--CEHC suggests that 3 was first converted to -T3 and then metabolized to S--CEHC. In fact, -T3 is known to be metabolized to -CEHC by CYP3A4 and CYP4F2 (Sontag and Parker, 2002; Parker et al., 2004; Zhou et al., 2004). Thus, when 3 is taken together with drugs that are principally metabolized by these cytochrome P450 enzymes, the disposition kinetics of S--CEHC after administration of 3 may be altered because of competitive inhibition.

In conclusion, the hydrochloride salt of the N,N-dimethylaminoacetic acid ester of -T3 displayed a high melting point, sufficient water solubility, high stability in solution, and high susceptibility to enzymatic hydrolysis by rat and human liver enzymes. Because the aim of the present prodrug development was to overcome problems of crystallization and stability in solution, ester derivatives are desirable prodrugs of -T3. Animal experiments supported the idea that an ester of -T3 could be useful as a two-step prodrug of S--CEHC when administered i.v. The prodrug may also avoid the toxicity of the solubilizing agent HCO-60. The ability to effectively deliver -T3 and S--CEHC as a prodrug should promote interest in their therapeutic use.

	Footnotes

 
doi:10.1124/dmd.106.014365.

ABBREVIATIONS: -T3, R--tocotrienol; S--CEHC, 2,7,8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman; R--CEHC, 2,7,8-trimethyl-2R-(ß-carboxyethyl)-6-hydroxychroman; MS, mass spectroscopy; N-t-boc, N-t-butyloxycarbonyl; -T3-H, -T3 solubilized with water containing 10% (w/v) polyoxyethylene hydrogenated castor oil and 15% (v/v) propylene glycol; AUC, area under the concentration-time curve; MRT, mean residence time; C_max, maximum concentration; T_max, time at the maximum concentration.

¹ Current affiliation: Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.

² Current affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.

Address correspondence to: Jiro Takata, Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan-ku, Fukuoka 814-0180, Japan. E-mail: jtakata{at}fukuoka-u.ac.jp

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