Introduction

Abstract Introduction Results & Discussion References

New fluorine-containing ADM¹ analogs have been synthesized to increase antineoplastic activity and to decrease cardiodtoxicity of ADM. For example, the Research Laboratories of Dong-A Pharmaceutical Company (Yongin, South Korea) recently developed DA-125, which is currently being evaluated in phase II clinical trials. DA-125 is a -alanine derivative of M1 and is a water-soluble prodrug of M1 (fig. 1). In the preceding papers, simultaneous HPLC analysis of DA-125 and its metabolites (M1-M4) in plasma, urine, and tissue homogenates (1); stability in plasma, blood partitioning, and pharmacokinetics of DA-125 or M1 in rats (2); pharmacokinetics of DA-125 in beagle dogs (3); nonlinear metabolism of DA-125 in rats (4); and phase I clinical trials of DA-125 (unpublished data) have been reported. DA-125 was rapidly hydrolyzed to form M1 by losing the -alanine moiety; the disappearance half-lives of DA-125 in plasma from humans, dogs, rats, and mice were 0.54, 0.54, 1.72, and 1.97 min, respectively (2). M1 was metabolized to M2 and M3, then both M2 and M3 were further metabolized to M4 (fig. 1) in mice (4), rats (2, 4), dogs (3), and human subjects (unpublished data). M1 was reduced to M2 by the aldo-keto reductase and transformed to M3 by losing the sugar moiety (fig. 1). M4 was formed from M2 by losing the sugar moiety and from M3 by the aldo-keto reductase (fig. 1). M1 was the only active metabolite among M1-M4 and had a high affinity for the lungs of mice (4), rats (4), and dogs (3) after iv administration of DA-125. In acute tests on DA-125 in rats and mice, the values of both LD₁₀ and LD₅₀ after intravenous administration of the drug to both animal types were 4-5 times higher than those of ADM. DA-125 was found to have higher in vitro and in vivo cytocidal activities, and lower cardiotoxicity and hematotoxicity than ADM (5).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Structures of DA-125, M1, M2, M3, M4, and fluorescein (internal standard), and metabolic pathways of DA-125.

In many other studies, SHRs (6-12) and DOCA-salt rats (10-14) have been used as the animal models for human primary (essential) and secondary hypertension, respectively. The 8-hr urinary excretion of furosemide per gram of kidney was significantly lower, the nonrenal clearance of furosemide was significantly faster, and the 8-hr urine output and urinary excretion of sodium and chloride per gram of kidney were significantly lower after an iv administration of furosemide to 16-week-old SHRs than to their age-matched control Wistar rats. However, these phenomena were not observed between 16-week-old DOCA-salt rats and their age-matched control Sprague-Dawley rats (11). The mean fraction of an iv dose of acetaminophen excreted in 24-hr urine as the acetaminophen-sulfate and the acetaminophen-glucuronide were significantly greater and smaller, respectively, in 16-week-old SHRs than in their age-matched control Wistar rats. However, there was no significant difference in either the sulfotransferase or the glucuronyltransferase activities toward acetaminophen between 16-week-old SHRs and their age-matched control Wistar rats (12). The pharmacokinetics and pharmacodynamics of an iv bumetanide were not significantly different between 16-week-old SHRs and their age-matched control Wistar rats, and 16-week-old DOCA-salt rats and their age-matched control Sprague-Dawley rats, respectively (10).

The purpose of the present study was to investigate whether any difference observed in the pharmacokinetics of DA-125 was caused by either the hereditary characteristics of SHRs (between 6-week-old and 16-week-old SHRs) or the hypertension state itself (between 16-week-old SHRs and 16-week-old DOCA-salt-induced hypertensive rats). In the present study, the pharmacokinetics of M1-M4 were evaluated after iv administration of DA-125 (20 mg/kg body weight) to 16-week-old SHRs after chronic exposure to hypertension (15) and to their age-matched control normotensive Kyoto-Wistar rats. Similar studies were also performed in 16-week-old DOCA-salt rats and their age-matched control Sprague-Dawley rats, and in 6-week-old SHRs [corresponding to the early phase of the development of hypertension, at which time blood pressure remains within the normotensive range (15)] and their age-matched control Kyoto-Wistar rats. Some factorssuch as activities and amount of the aldo-keto reductase in the liver, tissue distribution of M2 and M4, and renal and biliary excretion of M2 and M4 (before and after incubation with -glucuronidase)were also investigated to discover why plasma concentrations and resultant AUCT values of both M2 and M4 increased significantly in both the SHRs and the DOCA-salt rats at 16 weeks of age, when compared with those of their age-matched control rats.

Materials and Methods

DA-125 and M1-M4 were kindly donated by Research Laboratories of Dong-A Pharmaceutical Company. Fluorescein, internal standard for the HPLC assay, DOCA, NADPH, and -glucuronidase (Helix pomatia type 2, -glucuronidase activity of 97,400 units/ml and sulfatase activity of 1,000-5,000 units/ml) were products of Sigma Chemical Company (St. Louis, MO). Other chemicals were of reagent grade or HPLC grade and used without further purification.

Male SHRs at 5 or 15 weeks of age and their age-matched control Kyoto-Wistar rats, and Sprague-Dawley rats at 12 weeks of age were purchased from Charles River Company (Atsugi, Japan). At 6 and 16 weeks of age, systolic blood pressure of SHRs and their control Kyoto-Wistar rats were measured using tail cuff plethysmography (Narcotrace 40, NBS, Houston, TX). The 16-week-old SHRs with systolic blood pressure higher than 170 mm Hg, and their control Kyoto-Wistar rats with systolic blood pressure lower than 120 mm Hg, were used for the study. The 6-week-old SHRs and their control Kyoto-Wistar rats, all having systolic blood pressure lower than 110 mm Hg, were used for the study. Sprague-Dawley rats were randomly divided into two groups: the DOCA-salt rats and their control rats. DOCA-salt rats received a subcutaneous injection of 12.5 mg/kg body weight of DOCA (5 mg/ml) dissolved in cotton seed oil every 3 days and 1% NaCl as drinking water ad libitum during 12-16 weeks of age. The control Sprague-Dawley rats were given a subcutaneous injection of the same volume of cotton seed oil every 3 days and tap water ad libitum during 12-16 weeks of age. Systolic blood pressure was similarly determined at 16 weeks of age. DOCA-salt rats with systolic blood pressure higher than 150 mm Hg and their control Sprague-Dawley rats with systolic blood pressure lower than 110 mm Hg were used for the study.

Intravenous Study. In the early morning at the end of 6 or 16 weeks (after overnight fasting with tap water ad libitum), the carotid artery and the jugular vein were catheterized with polyethylene tubing (Clay Adams, Parsippany, NJ) under light ether anesthesia. Both cannulae were exteriorized to the dorsal side of the neck, wherein each cannula terminated with the long Silastic tubing (Dow Corning, Midland, MI). The two Silastic tubings were covered with a wire to allow free movement of the rat. Each rat was housed individually in a rat metabolic cage (Daejong Scientific, Seoul, South Korea) and allowed 4-5 hr to recover from the anesthesia before the study. They were not restrained at any time during the study. DA-125 (dissolved in 1 mM lactic acid at pH 4.0/normal saline-injectable solution), 20 mg/kg body weight, was administered by an iv infusion in 1 min via the jugular vein (total injection volume was ~1.5 ml) of each of the SHRs (N = 7), Kyoto-Wistar rats (N = 9), DOCA-salt rats (N = 8), and Sprague-Dawley rats (N = 13) at 16 weeks of age, and SHRs (N = 5) and Kyoto-Wistar rats (N = 6) at 6 weeks of age. Blood samples (0.25 ml) were collected via the carotid artery before (to serve as a control) and at 1 (at the end of the infusion), 5, 15, 30, 45, 60, 90, 120, 240, 360, 480, and 600 min after iv administration of DA-125. Heparinized 0.9% NaCl-injectable solution (15 units/ml), 0.25 ml, was used to flush the cannula after each blood sampling to prevent blood clotting. Blood samples were centrifuged immediately to minimize the "blood storage effect" (2) of plasma concentrations of M1-M4; and 100 µl of each plasma was stored at 20°C until the HPLC analysis of M1-M4 (1). At 24 hr, a large volume of blood was collected through the carotid artery, and each rat was killed by cervical dislocation. At the same time, the metabolic cage was rinsed with 20 ml of distilled water. This, along with the washings of the cut bladder, was combined with 24-hr urine. After measuring the exact volume of the combined 24-hr urine, an aliquot of the combined 24-hr urine was collected and frozen before HPLC analysis of M1-M4 (1).

Determination of Aldo-Keto Reductase Activities in Rat Liver. Aldo-keto reductase activities were determined on 105,000g cytoplasmic extracts of the liver of SHRs, Kyoto-Wistar rats, DOCA-salt rats, and Sprague-Dawley rats (N = 5, each) at 16 weeks of age by the reported method (16). Each rat liver was perfused with 0.25 M sucrose, and ~1 g of liver was homogenized (Ultra-Turrax) in 5 ml of 0.1 M potassium phosphate buffer (pH 7.4). After centrifugation for 10 min at 12,000g (Beckman L-80, Palo Alto, CA), the supernatant was further centrifuged for 60 min at 105,000g. The supernatant was diluted 5 times with 0.02 M potassium phosphate buffer (pH 7.4) containing 1 mM of NADPH in a total volume of 0.5 ml. Enzyme kinetics for the 105,000g liver extracts were determined at DA-125 concentrations of 0.05, 0.1, 0.2, 0.4, and 0.8 mM. Reaction mixtures were incubated for 15 min in a water-bath shaker kept at 37°C and at a rate of 50 opm. Reactions were stopped by mixing 0.5 ml of 0.1 N HCl into the reaction mixture, and measured the concentration of M1-M4 using HPLC (1). V_max and K_m were determined using the Lineweaver-Burk plot. The amount of proteins in the 105,000g liver supernatant was determined by the procedure of Lowry et al. (17).

HPLC Analysis of M1-M4. M1-M4 in the biological samples were analyzed by the reported HPLC method (1). A 0.1-ml volume of 0.1 N HCl, 0.1 ml of internal standard (3 µg/ml fluorescein dissolved in distilled water), 0.1 ml of methanol, and 1 ml of ethyl acetate were added to 0.1 ml of the biological sample. After extracting and evaporating the organic layer, a 0.1-ml volume of Britton-Robinson buffer:methanol (1:2, v/v) was added to reconstitute the residue. After vortex sonication and centrifugation, 80 µl of the supernatant was injected directly onto the reversed-phase column. The mobile phase, 1% acetic acid:isopropyl alcohol:methanol (70:20:10, v/v) was run at a flow rate of 1.5 ml, and the column effluent was monitored by fluorescence detector with an excitation wavelength of 488 nm and an emission wavelength of 556 nm. Retention times for M1, M2, M3, M4, and internal standard were 4.8, 3.6, 9.8, 6.2, and 7.8 min, respectively. Detection limits for M1-M4 in both plasma and urine were 50 ng/ml. The mean within-day and between-day coefficients of variation for M1-M4 in both plasma and urine were lower than 6.06%.

Pharmacokinetic Analysis. AUCT values of M1-M4 were calculated by the trapezoidal rule-extrapolation method (18); this method used the logarithmic trapezoidal rule (19) for calculation of the area during the declining plasma-level phase and the linear trapezoidal rule for the rising plasma-level phase. Values of CL of M1 and CL_R of M1-M4 were estimated by dividing the dose of M1 (calculated from the dose of DA-125) by the AUCT value of M1, and the amount of M1-M4 excreted in 24 hr urine by the AUCT values of M1-M4, respectively. Because the AUCT values of M1-M4 were estimated up to 8 or 10 hr, the values of CL and CL_R would be somewhat overestimated.

Statistical Analysis. Levels of statistical significance were assessed using the t test between two means for unpaired data. Significant differences were judged as p < 0.05. All results were expressed as mean ± SD.

	Results and Discussion

Abstract Introduction Results & Discussion References

It has been reported (2) from our laboratory that the pharmacokinetic parameters of M1 were comparable between an iv administration of DA-125 and M1 to rats, and the in vitro degradation half-life of DA-125 to M1 in rat plasma was 1.72 min, indicating that DA-125 is rapidly transformed to M1 after an iv administration of DA-125 to rats. Therefore, estimation of the pharmacokinetic parameters of M1 after an iv dose of DA-125 in the present rats did not seem to be any different from the values after an iv dose of M1. DA-125 is a water-soluble prodrug of M1, and only M1 has antineoplastic activity among M1-M4. Plasma concentrations of DA-125 decayed rapidly, with a mean terminal half-life of 1.64 min after iv administration of DA-125 (20 mg/kg body weight) to five rats (2). Therefore, concentrations of DA-125 were not measured in the present rat study.

Mean arterial plasma concentration-time profiles of M1-M4 after an iv administration of DA-125 (20 mg/kg body weight) to 16-week-old SHRs (N = 7) and their age-matched control Kyoto-Wistar rats (N = 9) are shown in fig. 2, and the relevant pharmacokinetic parameters are listed in table 1. After an iv administration of DA-125, each plasma concentration of M1, M2, and M4 seemed to decline in a polyexponential fashion and almost "constant" plasma concentrations of M1, M2, and M4 were maintained from 1 to 2 hr and from 8 to 10 hr for both groups of rats (fig. 2). This could be due to the continuous formation of M2 from M1 and M4 from M3, respectively; because the mean terminal half-lives of M2 and M4 were 38.8 and 43.9 min, respectively, when 5 mg/kg body weight of each of the M2 and M4 were administered intravenously to rats (4). It was found (4) that M4 was formed mainly from M3, and a negligible amount of M4 was formed from M2 when 5 mg/kg body weight of each of the M2 and M3 were injected intravenously to rats. In the present rat study, M3 was only detected for up to 5 and 30 min in the plasma of 16-week-old SHRs and their control Kyoto-Wistar rats, respectively (fig. 2), due to its rapid and "almost" complete conversion to M4 (4). AUC values of M4 were comparable between an iv administration of M3 and M4 (5 mg/kg body weight) to rats, indicating that M3 is "almost" completely metabolized to M4 (4).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Mean arterial plasma concentration-time profiles of M1, M2, M3, and M4 after iv administration of DA-125 (20 mg/kg body weight) to 16-week-old SHRs (N = 7, ) and their age-matched control Kyoto-Wistar rats (N = 9, ). Bars represent SD. *p < 0.05; **p < 0.01; ***p < 0.001.

It is of interest to note that most of the plasma concentrations of M2 and M4 in the 16-week-old SHRs were significantly higher than in their control rats (fig. 2), and this resulted in significantly greater AUCT values for M2 (68.9 vs. 29.3 µg-min/ml) and M4 (53.4 vs. 33.4 µg-min/ml) in the SHRs (table 1). M2 was the main metabolite excreted in the 24-hr urine among M1-M4; mean percentages of an iv dose excreted in 24-hr urine as M2 (expressed in terms of DA-125) by 16-week-old SHRs and their control rats were 4.89% and 4.40%, respectively. Corresponding values for M1, M3, and M4 were almost negligible (table 1).

To determine whether the increase in plasma concentrations and the resultant AUCT values of both M2 and M4 in 16-week-old SHRs, compared with their control rats, is due to the heredity of the SHRs or other factors (such as hypertension state itself), DA-125 was also intravenously administered to 6-week-old SHRsat which time the blood pressure remains within the normotensive rangeand their control rats. Mean arterial plasma concentration-time profiles of M1-M4 after an iv administration of DA-125 (20 mg/kg body weight) to 6-week-old SHRs and their control rats are shown in fig. 3; relevant pharmacokinetic parameters are listed in table 2. Note that the plasma concentrations of M1-M4 (except for a few concentrations) and their pharmacokinetic parameters (except for the CL value of M1, and the AUCT and CL values of M3) in 6-week-old SHRs and their control rats were not significantly different. The aforementioned data indicated that significantly increased plasma concentrations and the resultant AUCT values of both M2 and M4 in 16-week-old SHRs was not due to the hereditary characteristics of the SHRs. Similar results were also reported with furosemide (11) and acetaminophen (12). M2 was also the main metabolite excreted in 24-hr urine among M1-M4 in both rat groups (table 2).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Mean arterial plasma concentration-time profiles of M1, M2, M3, and M4 after iv administration of DA-125 (20 mg/kg body weight) to 6-week-old SHRs (N = 5, ) and their age-matched control Kyoto-Wistar rats (N = 6, ). Bars represent SD. *p < 0.05; **p < 0.01.

To confirm the significantly increased plasma concentrations and the resultant AUCT values of both M2 and M4 in 16-week-old SHRs is due to the hypertension state itself; DA-125 was also intravenously administered to 16-week-old DOCA-salt rats and their control rats. Mean arterial plasma concentration-time profiles of M1, M2, and M4 after an iv administration of DA-125 (20 mg/kg body weight) to 16-week-old DOCA-salt rats and their control rats are shown in fig. 4, and the relevant pharmacokinetic parameters are listed in table 3. Because M3 was only detected in plasma up to 15 min in both rat groups, it was thus not included in fig. 4. Interesting results were obtained from 16-week-old DOCA-salt rats (fig. 4 and table 3); results were similar to those obtained from 16-week-old SHRs (fig. 2 and table 1). Plasma concentrations of both M2 and M4 in 16-week-old DOCA-salt rats were significantly higher than in their control rats, thus resulting in a significant increase in AUCT values of M2 (130 vs. 60.8 µg-min/ml) and M4 (92.0 vs. 31.5 µg-min/ml) in DOCA-salt rats. Previous data indicated that significantly increased plasma concentrations and resultant AUCT values of both M2 and M4 after an iv administration of DA-125 to 16-week-old SHRs, compared with their age-matched control Kyoto-Wistar rats, was due to the hypertension state itself and not due to any hereditary characteristics of the SHRs. Again, M2 was the main metabolite excreted in 24-hr urine among M1-M4 in both groups of rats (table 3).

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Mean arterial plasma concentration-time profiles of M1, M2, and M4 after iv administration of DA-125 (20 mg/kg body weight) to 16-week-old DOCA-salt rats (N = 8, ) and their age-matched control Sprague-Dawley rats (N = 13, ). Bars represent SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Some factors could be considered to explain the significantly increased plasma concentrations and the resultant AUCT values of both M2 and M4 in both 16-week-old SHRs (fig. 2 and table 1) and DOCA-salt rats (fig. 4 and table 3). First, the increased aldo-keto reductase activities in the liver (and hence increased metabolism of M2 from M1 and M4 from M3 by the enzyme) of both 16-week-old SHRs and DOCA-salt rats could be a factor. However, this factor was ruled out, because the aldo-keto reductase activities were very similar between 16-week-old SHRs and their control rats, and similar results were also obtained between 16-week-old DOCA-salt rats and their control rats (table 4). However, the increased amount of aldo-keto reductase (and hence increased metabolism of M2 from M1 and M4 from M3) in the liver of both 16-week-old SHRs and DOCA-salt rats could contribute to this phenomenon. Although, protein contents in the liver were not significantly different between 16-week-old SHRs and their control rats (8.60 ± 0.307 vs. 8.67 ± 0.735 mg protein/ml in the liver supernatant), the liver weight increased significantly (10) in the 16-week-old SHRs than those in age-matched control rats (3.03 ± 0.191 vs. 2.76 ± 0.238% of body weight, p < 0.01). Although liver weights were similar between 16-week-old DOCA-salt rats and their control rats (10), the contents of protein in the liver increased significantly in the 16-week-old DOCA-salt rats (9.24 ± 0.739 vs. 7.76 ± 0.871 mg protein/ml liver supernatant, p < 0.05). Because the contribution of CL_R to total body CL of M1 in the present (table 1) and previous (4) rat studies, and M3 in rats (4) was negligible, formation of M2 (from M1) and M4 (from M3) by the aldo-keto reductase in the liver may contribute considerably to the increased plasma concentrations; the resultant AUCT values of both M2 and M4 after an iv administration of DA-125 to both SHRs and DOCA-salt rats that were 16 weeks of age. Second, tissue distribution changes of both M2 and M4 could be another factor. However, this factor was remote, because the tissue distribution of both M2 and M4 was generally comparable between 16-week-old SHRs and their control rats (table 5), and between 16-week-old DOCA-salt rats and their control rats (table 6), except a few organs. Third, the decreased urinary excretion of M2 and M4 could also be a factor. However, this factor was again ruled out because the 24-hr urinary excretion of both M2 and M4 before and after incubation with -glucuronidase was not significantly different between 16-week-old SHRs and their control rats, and similar results were also obtained between 16-week-old DOCA-salt rats and their control rats (table 7). Moreover, the contribution of CL_R to a total body CL of both M2 and M4 was negligible after an iv administration of DA-125 to Sprague-Dawley rats (4). Finally, the decreased biliary excretion of M2 and/or M4 could contribute to the higher plasma concentrations and the resultant AUCT values of M2 and/or M4 in 16-week-old SHRs and the DOCA-salt rats. The 8-hr biliary excretion of M2 before and after incubation with -glucuronidase decreased significantly in 16-week-old SHRs, compared with those in their control rats, and similar results were also reported in 16-week-old DOCA-salt rats (table 8). However, 8-hr biliary excretion of M4 before and after incubation with -glucuronidase was not significantly different between 16-week-old SHRs and their control rats, and similar results were also obtained between 16-week-old DOCA-salt rats and their control rats (table 8). Glucuronide formation of M1-M4 in the 8-hr bile samples after 1-min iv administration of DA-125 (2.5 mg/kg) to 3 male and 2 female beagle dogs was almost negligible (3). However, the value was considerable in the present rat studies (table 8). This could be due to species difference, and similar results were also reported with clofibric acid (21).

In conclusion, the significantly higher plasma concentrations and the resultant AUCT values of M2 in both 16-week-old SHRs and DOCA-salt rats than those in their age-matched control rats were due to significantly decreased biliary excretion of M2 and possibly due to the increased amount of aldo-keto reductase in the liver. However, increase in the two previously described pharmacokinetic parameters in the case of M4 was possibly due solely to the increased amount of aldo-keto reductase in the liver.