Abstract
The widely used anticancer prodrug cyclophosphamide (CPA) is activated in liver by a 4-hydroxylation reaction primarily catalyzed by cytochrome P-4502B and P-4502C enzymes. An alternative metabolic pathway involves CPA N-dechloroethylation to yield chloroacetaldehyde (CA), a P-4503A-catalyzed deactivation/neurotoxication reaction. The in vivo modulation of these alternative, competing pathways of P-450 metabolism was investigated in pharmacokinetic studies carried out in the rat model. Peak plasma concentrations (Cmax) for 4-OH-CPA and CA were increased by 3- to 4-fold, and apparent plasma half-lives of both metabolites were correspondingly shortened in rats pretreated with phenobarbital (PB), an inducer of P-4502B and P-4503A enzymes. However, PB had no net impact on the extent of drug activation or its partitioning between these alternative metabolic pathways, as judged from AUC values (area-under-the-plasma concentration × time curve) for 4-OH-CPA and CA. The P-4503A inhibitor troleandomycin (TAO) decreased plasma Cmax and AUC of CA (80–85% decrease) without changing theCmax or AUC of 4-OH-CPA in uninduced rats. In PB-induced rats, TAO decreased AUCCA by 73%, whereas it increased AUC4-OH-CPA by 93%. TAO thus selectively suppresses CPA N-dechloroethylation, thereby increasing the availability of drug for P-450 activation via 4-hydroxylation. By contrast, dexamethasone, a P-4503A inducer and antiemetic widely used in patients with cancer, stimulated large, undesirable increases in theCmax and AUC of CA (8- and 4-fold, respectively) while reducing the AUC of the 4-hydroxylation pathway by ∼60%. Tumor excision/in vitro colony formation and tumor growth delay assays using an in vivo 9L gliosarcoma solid tumor model revealed that TAO suppression of CPA N-dechloroethylation could be achieved without compromising the antitumor effect of CPA. The combination of PB with TAO did not, however, enhance the antitumor activity of CPA, despite the ∼2-fold increase in AUC4-OH-CPA, suggesting that other PB-inducible activities, such as aldehyde dehydrogenase, may counter this increase through enhanced deactivation of the 4-hydroxy metabolite. Together, these studies demonstrate that the P-4503A inhibitor TAO can be used to effectively modulate CPA metabolism and pharmacokinetics in vivo in a manner that decreases the formation of toxic metabolites that do not contribute to antitumor activity.
Cytochrome P-450 enzymes, expressed at high levels in liver, catalyze the oxidative metabolism of numerous drugs and other foreign chemicals (Guengerich, 1997), including antitumor drugs such as cyclophosphamide (CPA) (LeBlanc and Waxman, 1989). More than 20 distinct forms of liver P-450, each with a unique substrate specificity, have been characterized from livers of several mammalian species, including humans (Nelson et al., 1996). Striking interindividual differences in the levels of expression of specific P-450s are observed in many species, including humans (Shimada et al., 1994; George et al., 1996), and these interindividual differences contribute to the differential toxic responses and therapeutic effects that can result from the use of cancer chemotherapeutic drugs, many of which are subject to P-450 metabolism (LeBlanc and Waxman, 1989; Kivisto et al., 1995).
CPA and its isomer, ifosfamide (IFA), are anticancer alkylating agent prodrugs that require hepatic P-450-catalyzed metabolism to exhibit cytotoxic activity (Sladek, 1988; Fleming, 1997). Studies carried out in humans as well as in the rat model have established that CPA can be metabolized by liver P-450 enzymes along two alternative pathways (Fig. 1): 1) CPA 4-hydroxylation yields 4-hydroperoxy-CPA (4-OH-CPA), which ultimately decomposes to yield phosphoramide mustard, a DNA-alkylating, cytotoxic metabolite, and 2) CPA N-dechloroethylation yields dechloroethyl-CPA and chloroacetaldehyde (CA), which are formed in equimolar amounts. Unlike CPA, dechloroethyl-CPA does not yield DNA-crosslinking metabolites and is devoid of antitumor activity, whereas its byproduct CA is associated with various host toxicities, including neurotoxicity and urinary tract toxicity (Springate et al., 1997; Carlson et al., 1998). AlthoughN-dechloroethylation is generally viewed as a minor metabolic pathway for CPA compared with IFA, substantial variation in the extent to which CPA is metabolized byN-dechloroethylation has been reported for individual patients with cancer (Boddy et al., 1992; Yule et al., 1995; Busse et al., 1997). Secondary metabolic pathways include further oxidation of the 4-OH-CPA rearrangement product aldophosphamide by aldehyde dehydrogenase to yield carboxyphosphamide, an inactive, nontoxic CPA metabolite (Radin et al., 1991; Sladek, 1993). In the case of IFA, which is subject to corresponding pathways of metabolism,N-dechloroethylation is a major route of biotransformation, with up to 50% of drug metabolized by this undesirable drug inactivation/toxication pathway (Fleming, 1997).
CPA 4-hydroxylation and CPA N-dechloroethylation are catalyzed by distinct subsets of liver P-450 enzymes (Yu and Waxman, 1996). P-4502B enzymes, which are strongly inducible in rat liver by phenobarbital (PB) pretreatment (Waxman and Azaroff, 1992), and P-4502C enzymes, which are constitutively expressed in livers of uninduced adult rats, are the major hepatic P-450 catalysts of CPA 4-hydroxylation in the rat model (Clarke and Waxman, 1989). In contrast, up to 95% of CPA N-dechloroethylation is catalyzed by P-4503A enzymes in both uninduced and drug-induced rat liver microsomes (Yu and Waxman, 1996). This differential role of individual liver P-450 enzymes in the catalysis of CPA 4-hydroxylation versus N-dechloroethylation suggests the possibility of using P-450 form-selective inducers and inhibitors to modulate the balance between these two alternative metabolic routes. Studies using isolated rat liver microsomes suggest that PB induction of liver P-450 enzymes may be ineffective with respect to modulation of CPA metabolism because PB pretreatment increases liver microsomal CPA 4-hydroxylation and CPA N-dechloroethylation to similar extents (Yu and Waxman, 1996). By contrast, the P-4503A-selective inhibitor troleandomycin (TAO) (Chang et al., 1994) selectively blocks CPAN-dechloroethylation in both uninduced and PB- or dexamethasone (DEX)-induced rat liver microsomes (Yu and Waxman, 1996), suggesting that TAO might be a useful in vivo modulator for suppression of the undesirable N-dechloroethylation pathway while increasing net metabolism of CPA via the therapeutically productive 4-hydroxylation pathway. Pharmacokinetic studies are required, however, to test this hypothesis and to evaluate the therapeutic impact of any such modulation strategy in vivo. The present rat model pharmacokinetic study was therefore carried out 1) to determine whether P-450-catalyzed CPA N-dechloroethylation can be suppressed in vivo using the P-450 form-selective inhibitor TAO and 2) to establish whether this modulation can be effected without compromising the therapeutic activity of CPA, as judged by using in vivo tumor excision and tumor growth delay assays.
Experimental Procedures
Materials.
CPA and IFA were obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD). 4-hydroperoxy-CPA was a gift from Dr. J. Pohl (ASTA Pharma, Bielefeld, Germany). TAO was obtained from Pfizer, Inc. (Brooklyn, NY). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI). Fischer 344 rats (males, 150–250 g; females, 105–130 g) were purchased from Taconic, Inc. (Germantown, NY). Silastic tubing and PE50 tubing were purchased from Baxter Scientific (Boston, MA). Syringes, needles and syringe filters were purchased from VWR, Inc. (Boston, MA).
Rat Jugular Vein Cannulation.
Jugular vein cannulation followed an established protocol (Raff and Fagin, 1984) with modifications. Rats were anesthetized with a combination of ketamine (95 mg/kg) and xylazine (12 mg/kg). The skin on the right side of the midline of the neck was pinched with a straight forceps, and a small incision (1 cm long, maximum) was made ∼0.5 cm above the bone from the right foreleg using a sharp gold-handled scissors. The jugular vein was exposed after gently teasing apart the fascia overlying the jugular vein using two pairs of toothed forceps. The anterior end of the jugular vein was tied using a piece of 3-0 silk to prevent bleeding from the head region, and then a small snip in the vein was made using iridectomy scissors. A cannula consisting of Silastic tubing (inside of the vein) connected with PE50 tubing (outside of the vein) was filled with sterile 0.9% saline containing 20 U heparin/ml and then inserted into the vein ∼1.3 cm toward the heart, corresponding to the point where the PE50 and Silastic tubing were joined. Blood should be easily withdrawn through the cannula if it is functional at this step. The cannula was tied to the vein by a piece of 3-0 silk. The PE50 tubing was exteriorized at the back of the neck to prevent the rat from pulling out the cannula. A hollow rod (trocar) was inserted through an incision in the skin of the rat’s midscapular region. The syringe attached to the tubing was removed, and the tubing was quickly threaded through the trocar. Once threaded, the trocar was removed from the rat, and the syringe was reattached to the tubing. The wound was closed by stitching.
To secure the cannula, a Rodent Infusion Set (Lomir Inc., Quebec, Canada) consisting of a rodent jacket attached at back with a 40-cm length of stainless steel spring coil was used. The rat was placed in the jacket while it was still anesthetized, and the cannula was threaded through a spring coil that extended through the cage top and was locked in place by a rodent swivel, tether connector, and swivel retainer. The end of the catheter was bent and sealed with a small piece of polyethylene tubing. This Rodent Infusion Set protected the cannula from breaking or being disturbed by the rat. It also allowed the rat to move freely and access food and water, and it facilitated blood sampling during the pharmacokinetic experiments. Pharmacokinetic studies were generally performed 3 days after surgery, at which time the animal’s weight gain had resumed.
Pharmacokinetic Experiments.
Rats given CPA alone or in combination with P-450 modulators (PB, TAO, PB plus TAO, or DEX) were treated as follows: 1) PB, 80 mg/kg in 0.9% saline i.p. × 4 daily injections, followed by CPA administration ∼24 h after the last dose of PB; 2) TAO at 500 mg/kg i.p. administered 2 h before CPA administration and prepared as described below; 3) PB plus TAO cotreatment: PB at 80 mg/kg/day × 4, as above, and TAO at 500 mg/kg i.p. either 24 or 2 h before CPA administration (no difference was observed between these two regimens; data not shown); and 4) DEX at 50 mg/kg in 0.5 ml corn oil i.p. × 3 daily injections, followed by CPA treatment ∼24 h after the last injection. TAO used for these experiments was prepared fresh daily according to Arlotto et al. (1987) with modifications: TAO was suspended in saline, and 1 M HCl was then added at ∼1:1 molar ratio until TAO completely dissolved. The sample was then diluted in 0.9% saline to a final required volume of ∼0.9 ml, giving ∼0.1 M HCl, and the pH was adjusted to pH 4 with 1 M NaOH. The final TAO solution was filtered through a 0.45-μm sterile syringe filter. CPA or IFA (100 mg/kg in 0.5 ml of 0.9% saline and filtered through a 0.22-μm sterile syringe filter) was given i.v. through the jugular vein catheter, which was then flushed with 0.5 ml of 0.9% saline. Blood samples (0.5 ml each) were withdrawn via the cannula just before CPA injection and then 4, 10, 20, 30, 40, 60, 90, 120, and 180 min after CPA administration. The fluid removed at each sampling was replaced by 0.4 ml of 0.9% saline plus 0.1 ml of heparin (20 U/ml). The 4-min time point was not taken in the case of female rats due to their lower body size, which limited the total blood volume that could be withdrawn during a pharmacokinetic experiment. Consequently, AUC (area-under-the-curve, plasma × concentration) and Cmax values for the female rats adjusted to take this into account could be up to ∼10% and ∼15% higher than the values calculated from the available plasma concentration-time curve data for both 4-OH-CPA and CA, respectively; these values were estimated by extrapolation from the semilog concentration-time plot and the Tmax(4 and 10 min for 4-OH-CPA and CA, respectively) data obtained from male Fischer 344 rats. Pharmacokinetic analysis of IFA metabolites was carried out in essentially the same manner, as summarized elsewhere (Brain et al., 1998).
Processing of Blood Samples.
Blood samples were withdrawn via the jugular vein cannula and immediately mixed with 20 μl of heparin (100 U/ml). An aliquot (260 μl) of heparinized blood, for analysis of 4-OH-CPA or 4-OH-IFA, was immediately mixed with 2.6 μl of 0.5 M semicarbazide to stabilize the initial 4-hydroxy metabolite (Clarke and Waxman, 1989), followed by centrifugation at 16,000g for 4 min. The separated plasma (100 μl) could be frozen at −80°C for at least 1 week without change in 4-OH-CPA levels, as determined by fluorimetric analysis (see below). A second aliquot (208 μl) of heparinized blood, for determination of CA, was immediately mixed by vortexing with 2.1 μl of 100 mM formaldehyde to stabilize CA (Kaijser et al., 1993), followed by the addition of 25.2 μl of ice-cold 70% perchloric acid to precipitate protein. After centrifugation at 16,000g for 4 min, CA in the supernatant (140 μl) was immediately derivatized to 2-aminothiazole by heating with 21 μl of 100 mM thiourea at 90°C for 1 h. The supernatant after perchloric acid precipitation from the t = 0-min time point was divided into two equal aliquots. One aliquot was used for determination of the background plasma CA level. The 5 μl of 0.5 μM CA standard was added to the second aliquot, which was used to determine the percent recovery of CA. Both samples were then derivatized as described above. Derivatized samples could be stored at −80°C for up to 2 weeks before analysis without any change in metabolite levels.
Quantification of 4-OH-CPA and 4-OH-IFA.
A fluorimetric assay (Alarcon, 1968; Masurel et al., 1990) with modifications (Chang et al., 1993) was used to quantify 4-OH-CPA and 4-OH-IFA and their aldophosphamide tautomers free of interfering substances (Voelcker et al., 1979). Briefly, this assay involves trapping the primary 4-hydroxy/aldophosphamide metabolite with semicarbazide, acidic decomposition to acrolein, and condensation with 3-aminophenol to yield 7-hydroxyquinoline, a fluorescent compound. Frozen plasma samples were thawed on ice, and protein was precipitated by the sequential addition of 40 μl of 5.5% (w/v) zinc sulfate, 40 μl of saturated barium hydroxide, and 20 μl of 0.01 M hydrochloric acid. After centrifugation at 16,000g for 15 min, 125 μl of the supernatant was added to 67 μl of a solution containing 6 mg of 3-aminophenol and 6 mg of hydroxylamine hydrochloride dissolved in 1 ml of 1 M hydrochloric acid (freshly prepared). The samples were heated at 90°C for 30 min in the dark and then allowed to cool to room temperature. Water was added to reach the minimum volume of a fluorometer cell (0.6 ml in this study) before measurement of fluorescence (excitation at 350 nm and emission at 515 nm; Shimadzu Spectrofluorophotometer RF-1501). The recovery of 4-OH-CPA in plasma (60 ± 3%, mean ± S.D.) was determined by adding 4-hydroperoxy-CPA (0–200 μM) standard to plasma obtained from untreated rats, followed by derivatization, as done for the pharmacokinetic samples. A similar recovery was obtained for 4-OH-IFA (Brain et al., 1998). A standard curve was generated from 4-hydroperoxy-CPA (0–200 μM) dissolved in a 0.1 M potasium phosphate/0.1 mM EDTA (pH 7.4) buffer followed by the same derivatization as described above (r2= ∼0.99). The detection limit of this method was 1 μM (0.1 nmol of 4-hydroperoxy-CPA in 100 μl of plasma).
Quantification of CA.
Frozen samples collected from the pharmacokinetic experiments were warmed to room temperature. Sample clean-up on a Varian Associates (Harbor City, CA) Bond Elut SCX (cation exchange) column and HPLC analysis after derivatization with thiourea to yield 2-aminothiazole were as detailed previously (Yu and Waxman, 1996) . The recovery of CA in plasma was 68 ± 18.5% (mean ± S.D., n = 20), and the detection limit was ∼28 pmol of CA in 140 μl of deproteinized plasma (0.2 μM).
Data Analysis.
The plasma concentrations (Cpl) of 4-OH-CPA (or 4-OH-IFA) and CA were plotted as a function of time. Because theCpl of both 4-OH-CA (or 4-OH-IFA) and CA declined monoexponentially when plotted on a semilog concentration-time graph, the pharmacokinetics of these metabolites were approximated by a one-compartment model. The apparent first order elimination rate constant (ke) was obtained by linear regression analysis of ln(Cpl) versus time afterTmax (correlation coefficientr2 > 0.9 in all cases). The elimination rate constant was used to calculate the apparentT1/2 of 4-OH-CPA and CA from (T1/2) = ln 2/ke. Because 4-OH-CPA and CA are both metabolites of the parent drug, T1/2 values determined in this manner are apparent T1/2values, which depend both on the rate of metabolite formation and on the intrinsic rate of metabolite elimination. AUC values were calculated for each metabolite concentration-time profile by numerical intergration using the linear trapezoidal method. The AUC extrapolated from the last pharmacokinetic time point t to infinity was estimated as the last measured Cpldivided by the elimination rate constant: AUC(t–∞) =Cplt/ke. Total AUC values reported in this study were calculated from AUC = AUC(0–t) + AUC(t–∞).Cmax was determined as the peak plasma concentration observed, and Tmax was the time at which Cmax was reached. Statistical analysis based on unpaired, two-tailed Student’st test (assuming unequal variances) were carried out on data obtained from the different rat groups using StatWork (Cricket Software, Inc.) or Excel 5.0.
Tumor Excision Assay.
9L gliosarcoma cells (Barker et al., 1973) were implanted s.c. in adult male rats on both thighs, as described previously (Chen and Waxman, 1995). CPA treatment at 100 mg/kg in 0.9% saline i.p. was given 9 to 12 days after tumor implantation, at which time the tumor size was ∼100 to 125 mm2 in area. Rats were randomized and divided into four treatment groups: uninduced controls (untreated, UT), uninduced controls treated with CPA, rats induced with PB for 4 days before CPA treatment, and PB-induced rats given TAO 2 h before CPA. In a separate series of experiments, rats were divided into three treatment groups: control (TAO vehicle plus CPA vehicle), CPA alone (with TAO vehicle), and CPA treatment with TAO given 2 h before CPA. PB induction and TAO treatment were as described inPharmacokinetic Experiments. All rats were sacrificed 24 h after CPA treatment, and tumor excision assays were performed as described elsewhere (Chen et al., 1997), except that Dulbecco’s modified Eagle’s medium (GIBCO BRL, Grand Island, NY) was used in place of α-minimal essential medium, and the cells were seeded onto 6-well tissue culture plates at densities ranging from 100 to 5000 cells/well for untreated controls (colony efficiency ∼7%) and from 10,000 to 100,000 cells/well for CPA-treated samples.
Tumor Growth Delay Experiments.
9L gliosarcoma cells were implanted s.c. on each thigh of adult male rats at 2 × 106 cells/injection site per rat. Drug treatment was initiated 11 days after tumor implantation, at which time the mean tumor size of each group was 110 to 140 mm2 in area (n = 6 tumors/group). Rats were randomized into four treatment groups: saline, TAO at 500 mg/kg i.p. with saline (CPA vehicle), CPA at 100 mg/kg i.p. with TAO vehicle, and CPA plus TAO in combination, with TAO given 2 h before CPA, as described above. Tumor measurements (lengths and widths) were taken twice a week using a vernier caliper, and tumor size was determined by the area method (Tomayko and Reynolds, 1989). Animals were sacrificed when the mean tumor area of the control group reached ∼1000 to 1200 mm2.
Results
CPA and IFA Pharmacokinetics in Uninduced Male and Female Rats.
Plasma concentration-time profiles for 4-OH-CPA and CA after i. v. administration of CPA were compared in adult male and female rats (Fig. 2, A and F). PlasmaCmax values for 4-OH-CPA and CA were 77 and 17 μM, respectively, in male rats. In female rats, theCmax values for both metabolites were significantly lower: 43 μM for 4-OH-CPA and 6.3 μM for CA (Table1). The lower initial rates of CPA metabolism seen in the females may reflect the absence of two male-specific P-450 enzymes, which, respectively, contribute a substantial fraction of liver microsomal CPA 4-hydroxylation (P-4502C11) (Clarke and Waxman, 1989) and CPAN-dechloroethylation (P-4503A2) (Yu and Waxman, 1996). In contrast to the Cmax values, the AUC values of 4-OH-CPA were similar for male and female rats (3860 and 3230 μM · min, respectively), whereas the AUC of CA was ∼2-fold higher in male rats than in female rats (1250 and 620 μM · min, respectively) (Table 1). The apparent T1/2of 4-OH-CPA and CA were both shorter in the males (28 versus 41 min for 4-OH-CPA and 46 versus 60 min for CA), but these differences did not reach statistical significance (Table 1). Because the CPA metaboliteT1/2 measured in this study in large part reflect the rates of metabolite formation (see Discussion), these findings are consistent with in vitro studies showing that male liver microsomes metabolize CPA along both the 4-hydroxylation and theN-dechloroethylation pathways at higher rates than microsomes from female rats (Weber and Waxman, 1993; Yu and Waxman, 1996).
In comparison with CPA, IFA pharmacokinetics were associated with much higher Cmax and AUC values for CA formation. This effect was seen both in male rats, as described elsewhere (Brain et al., 1998), and in females (Fig.3; speckled bars versus hatched bars). In males, ∼25% of total CPA metabolism occurred via theN-dechloroethylation pathway, as judged from AUCCA values (also see Table2, below), whereas with IFA, theN-dechloroethylation pathway increased to ∼70%. In the case of the females, the extent of N-dechloroethylation was somewhat lower: 16% for CPA and ∼55% for IFA (Fig. 3B).
Effect of PB Induction on CPA Metabolism and Pharmacokinetics.
PB is an inducer of P-450 enzymes 2B1 and 2B2 and, to a lesser extent, several other hepatic P-450 enzymes (Waxman and Azaroff, 1992). Compared with uninduced rats, PB pretreatment significantly increased mean plasma Cmax values of 4-OH-CPA (3.1-fold increase) (Table 1; Fig. 2, C versus A; note difference in scale of y-axis). CPA 4-hydroxylation is primarily catalyzed by P-4502B1 and P-4502B2 in PB-induced rat in liver microsomes (Clarke and Waxman, 1989). However, PB also increased the rate of CPAN-dechloroethylation, as indicated by the 4.5-fold increase in Cmax of CA (Table 1; Fig.4A). This PB-induced increase is primarily due to P-4503A enzymes, which are PB inducible (Cooper et al., 1993), and are the dominant catalysts of this microsomal activity (Yu and Waxman, 1996). The rapid metabolism of CPA in PB-pretreated rats resulted in very short apparent T1/2for both 4-OH-CPA (T1/2 = 9.2 min) and CA (T1/2 = 16 min) (Table 1) and in a lowTmax value (≤4 min for both metabolites).
The short T1/2 of 4-OH-CPA seen in PB-induced rats is essentially identical to the intrinsicT1/2 of 4-OH-CPA (T1/2 = 8.1 min), measured by administering preformed 4-OH-CPA to rats (Hong et al., 1991). Thus, in contrast to UT rats, the rate of formation of 4-OH-CPA from CPA in PB-induced rats is not rate determining in its overall disposition. Similarly, the shortT1/2 (CA) seen in PB-induced rats (T1/2 = 16 min; compare with otherT1/2, CA, values shown in Table 1) may largely reflect the intrinsic plasma T1/2of this metabolite.
As a consequence of the high Cmax at a low Tmax and the short apparentT1/2 for 4-OH-CPA in PB-induced rats, there was no net increase in the AUC of 4-OH-CPA after PB pretreatment (Table1; Fig. 4B). The overall induction by PB of both CPA metabolic pathways is consistent with in vitro studies, in which hepatic microsomal activities for 4-hydroxylation and N-dechloroethylation of CPA were increased to similar extents by PB pretreatment (Yu and Waxman, 1996).
TAO Suppression of N-Dechloroethylation Pathway.
TAO is a P-4503A-specific inhibitor (Chang et al., 1994). The treatment of rats with TAO 2 h before CPA injection reduced the rate and the extent of CPA N-dechloroethylation significantly, as shown by the decrease in both plasmaCmax (6-fold decrease) (Fig. 2, B versus A; Fig. 4A) and AUC values for CA (4.8-fold decrease) (Fig. 4B) in TAO-treated rats (Table 1). This finding is consistent with our earlier in vitro finding that P-4503A is the dominant catalyst of liver microsomal CPA N-dechloroethylation (Yu and Waxman, 1996). By contrast, TAO did not have a significant effect on the plasmaCmax and AUC of 4-OH-CPA compared with untreated controls (Fig. 2, A versus B). Thus, TAO inhibition of CPAN-dechloroethylation leads to an increase in the overall fraction of drug metabolites formed via the 4-hydroxylation pathway. The Tmax values for 4-OH-CPA and CA in TAO-treated rats (10 and 20 min, respectively) were somewhat longer those than in UT rats (4 and 10 min, respectively). The apparentT1/2 of 4-OH-CPA was increased from 28 min in control rats to 40 min in TAO-treated rats, whereas the apparentT1/2 of CA was not significantly changed (46 min in UT rats versus 52 min in TAO rats) (Table 1). Overall, these pharmacokinetic results are consistent with earlier in vitro findings that TAO can inhibit liver microsomal P-4503A-mediated CPAN-dechloroethylation in a selective manner (Yu and Waxman, 1996), that is, without affecting CPA 4-hydroxylation, which is primarily catalyzed by P-4502C enzymes in adult male rats (Clarke and Waxman, 1989).
Effect of Combination of PB and TAO.
The treatment of PB-induced rats with TAO before CPA injection maintained the high plasma Cmax 4-OH-CPA, whereas theN-dechloroethylation pathway was suppressed by ∼85% (cf., plasma Cmax of CA decreased from 77 to 12.1 μM) (Fig. 2, C versus D; Fig. 4A). Although the apparentT1/2 of 4-OH-CPA (17 min) was still shorter than that in untreated controls (T1/2 = 28 min), the AUC of 4-OH-CPA was significantly increased to 6980 μM · min, which was the highest value among all the treatments examined (Table 1; Fig. 4B). On the other hand, the PB plus TAO combination substantially reduced the AUC of CA to 550 μM · min, corresponding to only ∼8% of the AUC value for 4-OH-CPA in these same animals. Thus, although PB increased the rates of CPA 4-hydroxylation and N-dechloroethylation, TAO selectively suppressed P-4503A-catalyzed CPA N-dechloroethylation.
Major Increase in CPA N-Dechloroethylation Pathway after DEX Treatment.
P-4503A enzymes have been implicated as major catalysts of CPA N-dechloroethylation, both in the rat (Yu and Waxman, 1996) and in human liver (Bohnenstengel et al., 1996; Ren et al., 1997). Because DEX is a strong inducer of P-4503A enzymes in many species (Cooper et al., 1993; Kocarek et al., 1995), we investigated the impact of DEX induction on CPA pharmacokinetics. DEX pretreatment increased the Cmax of CA by ∼8.5-fold, to 144 μM (Figs. 2E and 4A), while significantly increasing AUCCA 4-fold, from 1250 to 5160 μM · min (Fig. 4B; Table 1). The apparentT1/2 of CA was correspondingly decreased, from 46 to 21 min, consistent with the more rapid conversion of CPA to CA indicated by the Cmax data (see below). DEX also had a major impact on metabolism of CPA via the 4-hydroxylation pathway, which was suppressed in the DEX-induced rats. This effect was most striking for AUC4-OH-CPA, which was decreased by ∼56%, from 3860 to 1680 μM · min (Fig.4B; Table 1). Thus, a substantially greater fraction of CPA is metabolized via the therapeutically nonproductiveN-dechloroethylation pathway in DEX-induced rat liver.
Modulation of CPA Metabolism: Comparison of In Vivo versus In Vitro Analysis.
The initial rate of formation of a metabolite is more difficult to quantify in vivo than in in vitro microsomal assays due to the fact that the plasma concentration of a metabolite at any given point in time reflects the balance between its rate of formation and its rate of elimination. However, for a metabolite that is generated rapidly, its initial plasma concentration is essentially proportional to its rate of formation because metabolite elimination is less of a factor at this early stage. Based on this assumption, the initial rate of CPA 4-hydroxylation taken as a percent of the initial rate of total P-450 metabolic activity (4-hydroxylation plusN-dechloroethylation) can be estimated from the initial plasma concentration of 4-OH-CPA (taken at t = 4 min after drug administration in our analysis) divided by the sum of the plasma concentrations of 4-OH-CPA and CA at the same time point, as shown in the middle column of Table 2. These values are compared with the initial rates of CPA 4-hydroxylation (specific enzyme activities) divided by the initial rates of 4-hydroxylation plusN-dechloroethylation, assayed in vitro in rat liver microsomes, in the first column of Table 2. This ratio, which corresponds to the partition ratio for CPA 4-hydroxylation versus CPAN-dechloroethylation in microsomal incubations, corresponds fairly well to the partition ratio for 4-OH-CPA formation based on initial rates of 4-OH-CPA formation determined in vivo for each of the P-450 inducer and inhibitor treatments studied. Moreover, the overall partitioning of P-450 metabolites between the 4-hydroxylation andN-dechloroethylation pathways, calculated in a similar manner based on AUC values [AUC4-OH-CPA divided by (AUC4-OH-CPA + AUCCA); Table 2, last column], also is in good agreement with the predictions of the in vitro liver microsomal data. This suggests that the exposure to these CPA metabolites in vivo is primarily governed by their rates of formation, rather than their elimination rates, and that this exposure can be predicted based on liver microsomal P-450 enzyme activities.
Together, the findings presented in Table 2 suggest the following conclusions: 1) TAO substantially enhances the extent to which CPA is metabolized by P-450 via the therapeutically useful 4-hydroxylation pathway, both in uninduced and in PB-induced rats (percent 4-hydroxylation in vivo increased to 93–98% of total P-450 metabolism versus 74–84% in uninduced rats and 65–76% in PB-induced rats); 2) PB induction does not increase, but rather may decrease somewhat, the extent of CPA 4-hydroxylation in vivo (percent 4-hydroxylation: 65–76% in PB group versus 74–84% in UT group); and 3) DEX markedly suppresses the extent of 4-hydroxylation in vivo, down to 24% to 28% of total P-450 metabolism (Table 2).
P-450 Modulation of N-Dechloroethylation without Loss of CPA Antitumor Activity.
The impact of TAO pretreatment on the antitumor activity of CPA was examined to investigate whether TAO compromises the therapeutic activity of CPA under conditions in which it effectively suppresses CPA N-dechloroethylation. These experiments used the rat gliosarcoma tumor model 9L (Barker et al., 1973), which grows in Fischer 344 rats and is responsive to CPA. A tumor excision assay was used to quantify antitumor activity accumulating in vivo over a 24-h period after CPA treatment, as determined using an in vitro colony formation assay. Adult male rats were implanted with 9L tumors by s.c. injection and then were induced with PB over a 4-day period, beginning on day 8 after tumor implantation. On day 12, rats were given TAO or vehicle control, as indicated, followed by CPA given as a single i.p. injection at 100 mg/kg 2 h later. For these experiments, PB and TAO were given using the same doses and schedules used in the pharmacokinetic studies. Tumors were excised from the rats 24 h after CPA treatment and then were dispersed to give single-cell suspensions that were plated at various densities on tissue culture dishes. The number of surviving tumor cells was then determined by a colony formation assay. Figure5A shows that CPA alone reduced the 9L tumor cell survival fraction by ∼20-fold, from a relative value of 1 in the control group to 0.05 ± 0.037 (p < .05 for UT versus CPA) after CPA treatment. In rats pretreated with PB or with PB plus TAO, CPA reduced the survival fraction to 0.095 ± 0.081 and 0.097 ± 0.10, respectively (p > .05 for CPA versus CPA plus PB and p > .05 for CPA versus CPA plus PB plus TAO; n = 6–11 tumors/group). In a separate set of experiments, carried out in uninduced rats, CPA alone decreased tumor cell survival to 0.014 ± 0.011 of control (p < .05 for UT versus CPA), whereas CPA plus TAO decreased tumor cell survival to 0.009 ± 0.006 (p> .05 for CPA versus CPA plus TAO; n = 6 tumors/group) (Fig. 5B). Thus, TAO does not interfere with the antitumor effect of CPA.
To evaluate the longer-term impact of TAO pretreatment on CPA antitumor activity in vivo, a tumor growth delay assay was carried out. As shown in Fig. 6, treatment of the 9L tumors with CPA effected a delay in tumor growth of ∼7 to 10 days compared with saline-treated control rats or rats treated with TAO in the absence of CPA. Moreover, the same degree of tumor growth delay was achieved in rats given CPA in combination with TAO under conditions in which it effectively suppresses CPA N-dechloroethylation. Thus, TAO can effectively suppress the N-dechloroethylation pathway without loss of antitumor effect.
Discussion
The present study, carried out in the rat model, demonstrates that several commonly used liver P-450-inducing agents and P-450 inhibitors can have a major impact on CPA metabolism and pharmacokinetics, and in some cases, they may be used to modulate the balance between the two alternative, competing pathways for P-450-catalyzed CPA metabolism in vivo. In particular, the P-4503A inhibitor TAO was found to selectively suppress CPA N-dechloroethylation, greatly decreasing formation of the neurotoxic and therapeutically inactive metabolite CA. The effective modulation of the pharmacokinetic profiles of CPA using TAO is consistent with our earlier prediction, based on in vitro rat hepatic microsomal studies, that the two alternative CPA metabolic pathways are catalyzed by distinct subsets of liver P-450 enzymes (Yu and Waxman, 1996). A similar situation occurs in human liver, where the CPA N-dechloroethylation pathway is dominated by CYP3A4 (Z. Huang, P. Roy and D. J. Waxman, unpublished results), whereas CPA 4-hydroxylation is catalyzed by several P-450s, including CYP2B6, CYP3A4, and multiple CYP2C enzymes (Chang et al., 1993). Although CPAN-dechloroethylation is generally much less extensive than the corresponding N-dechloroethylation pathway in patients with cancer who are given IFA (Fleming, 1997), several recent studies show that dechloroethyl-CPA is a significant CPA metabolite in cancer patients, albeit with striking interindividual differences (Boddy et al., 1992; Yule et al., 1995; Busse et al., 1997). Accordingly, the present rat model studies raise the possibility that TAO or perhaps other CYP3A-selective inhibitors (Bourrie et al., 1996) may have use in patients that exhibit more extensive CPAN-dechloroethylation in an effort to suppressN-dechloroethylation while increasing the net extent of CPA metabolism via 4-hydroxylation. By contrast, in the case of IFA, there is greater overlap between the subsets of P-450 enzymes catalyzing 4-hydroxylation and N-dechloroethylation (Yu and Waxman, 1996), making it more difficult to alter the balance between these two pathways using P-450 modulators (Brain et al., 1998). However, given the successful and potentially beneficial modulation of CPA pharmacokinetics through the use of TAO, alone or in combination with PB, that is described here, it may be useful to further explore suitable modulation strategies with IFA, where metabolism along theN-dechloroethylation/drug toxification pathway is more extensive, with detrimental clinical effects (Kurowski and Wagner, 1993; Boddy et al., 1996; Brain et al., 1998).
The factors that dictate the major interindividual variation in CPAN-dechloroethylation in individual patients with cancer (Boddy et al., 1992; Yule et al., 1995; Busse et al., 1997) are not presently known but likely include the patient’s recent history of exposure to liver CYP3A inducers, which in the case of human hepatocytes includes drugs such as DEX, rifampin, and PB (Maurel, 1996), in addition to IFA and CPA (Chang et al., 1997). In this regard, the present pharmacokinetic studies demonstrate that P-4503A inducers such as DEX can have a dramatic impact on the metabolic partitioning of CPA between the 4-hydroxylation and N-dechloroethylation pathways in the rat model. Enhanced clearance of CPA has been observed after DEX treatment of pediatric patients with cancer (Yule et al., 1996); however, whether this leads to an increase in the rate or the extent of CPA N-dechloroethylation is still uncertain (Yule et al., 1995). Human CYP3A enzymes, which are known to be responsive to inducers such as DEX (Watkins et al., 1985; Maurel, 1996), do carry out the major fraction of CPA N-dechloroethylation in human liver microsomes (Z. Huang, P. Roy and D. J. Waxman, unpublished results), suggesting that this could be the case. Accordingly, it seems warranted to investigate whether CYP3A inducers, including steroids and other antiemetics commonly administered to patients with cancer (Levitt et al., 1993), have a significant impact on CPAN-dechloroethylation and CA formation. If this is found to be the case, then exposure of patients with cancer who are undergoing chemotherapy to P-4503A inhibitors or, more generally, to drugs and other chemicals that serve as P-450 inducers or inhibitors (Guengerich, 1997) could contribute to the clinically significant interindividual differences in CPA metabolism and pharmacokinetics (Ayash et al., 1992;Yule et al., 1996).
4-OH-CPA is the primary activated metabolite of CPA, and it ultimately decomposes via aldophosphamide to yield the DNA-alkylating metabolite phosphoramide mustard. Consequently, it is expected that the antitumor effect of CPA will be proportional to the plasma AUC of 4-OH-CPA (Sladek, 1988). This hypothesis is supported by the present finding that although the rate of CPA activation, and hence theCmax of 4-OH-CPA, was greatly increased by PB pretreatment, the plasma AUC of 4-OH-CPA was essentially unchanged by PB pretreatment, and correspondingly, there was no significant difference in the antitumor effect of CPA measured in a tumor excision assay, with and without PB pretreatment. Earlier studies in rats and mice also reported no improvement in the antitumor activity of CPA when PB was used as a liver P-450 inducer (Alberts and van Daalen Wetters, 1976; Hipkens et al., 1981). It is surprising, however, that the combined modulator regimen of PB plus TAO also did not increase the antitumor activity of CPA, as quantified in a tumor excision assay, despite the fact that TAO increased by almost 2-fold the plasma AUC of 4-OH-CPA compared with PB alone. Preliminary rat tumor growth delay studies using PB plus TAO also indicated no increase in CPA antitumor activity compared with PB alone (data not shown). The apparent inconsistency between the increase in plasma AUC of 4-OH-CPA in PB plus TAO-treated rats versus the absence of an increase in the antitumor activity of CPA may in part relate to the induction by PB of liver aldehyde dehydrogenase (Hipkens et al., 1981; Sessink et al., 1996), which deactivates 4-OH-CPA by oxidation of aldophosphamide (the tautomeric form of 4-OH-CPA and an immediate precursor of phosphoramide mustard) (Fig. 1) to form the therapeutically inactive carboxyphosphamide (Sladek, 1993). Indeed, carboxyphosphamide produced from CPA is increased >8-fold in PB-induced rat liver microsomes in the presence of NAD plus glutathione (designed to mimic in vivo conditions) compared with uninduced rat liver microsomes (Sessink et al., 1996). Therefore, the net amount of phosphoramide mustard generated intratumorally may not be increased in rats given PB or PB plus TAO. It may be useful to examine other known inducers of P-450-catalyzed CPA activation (Clarke and Waxman, 1989; Yu and Waxman, 1996) to determine whether they can be used to enhance hepatic CPA activation without parallel induction of aldehyde dehydrogenase.
A second explanation for the absence of improved CPA antitumor activity after PB plus TAO treatment may relate to the impact of PB induction on the Cmax andT1/2 of 4-OH-CPA. 4-OH-CPA/aldophosphamide can serve as an important “transport form” of phosphoramide mustard due to its higher stability and greater lipophilicity compared with phosphoramide mustard, enabling the activated drug to more readily enter cells (Sladek, 1988). In this regard, 4-OH-CPA has been proposed to have greater therapeutic activity when present in blood at a relatively low concentration but for longer times compared with a brief exposure to high drug levels (Voelcker et al., 1984). Thus, for a given AUC of 4-OH-CPA, constant level pharmacokinetics (i.e., lowCmax but longT1/2 ) confers higher therapeutic activity than bolus pharmacokinetics (Voelcker et al., 1984). This observation, which is not typical of direct-acting anticancer alkylating agents, may relate to blood flow and/or membrane-limited penetration of 4-OH-CPA into tumor cells, which frequently have a disproportionately lower or more irregular blood supply than normal tissue (Netti et al., 1996;Brown and Giaccia, 1998). Consequently, a large portion of 4-OH-CPA may be excreted from the body or may decompose to phosphoramide mustard before it can reach tumor cells, particularly in situations in which the apparent T1/2 of 4-OH-CPA may be very short, as is presently shown to occur in PB-induced rats. In this regard, it is interesting to note that in the case of 4-OH-CPA (and also for CA), the rate of metabolite formation appears to be the primary determinant of the metabolite’s overall apparent plasmaT1/2 because in all cases, the metabolite’s apparent T1/2 is decreased by P-450 inducer treatments that increase the rate of metabolite formation, as measured from Cmaxvalues at t = 4 min after drug administration. Finally, the suggestion that circulating 4-OH-CPA may not efficiently penetrate solid tumors is supported by recent studies showing a dramatic increase in CPA antitumor activity when CPA-activating P-450 enzymes are expressed in tumors in the context of a prodrug activation-based cancer gene therapy strategy (Chen and Waxman, 1995; Chen et al., 1997).
The antitumor activity of CPA is influenced by multiple biochemical factors and enzymatic steps, some of which are amenable to pharmacological modulation, as demonstrated by the present rat model pharmacokinetic study for CPA and in a previous study for IFA (Brain et al., 1998). Further studies are required for a full understanding of the extent to which P-450 inhibitors and inducers that are administered in the clinic in combination with CPA affect drug metabolism and clinical pharmacokinetics in patients with cancer. The potential clinical use of P-450 modulation approaches such as the suppression by TAO of CPA N-dechloroethylation described in the present rat model study may also warrant further investigation.
Acknowledgments
We thank Dr. Eric Widmaier (Boston University) for sharing his expertise with the jugular vein catheterization procedure.
Footnotes
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Send reprint requests to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington St., Boston, MA 02215. E-mail: djw{at}bio.bu.edu
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↵1 This work was supported in part by National Institutes of Health Grant CA49248 (D.J.W.). K.G. and P.D. contributed equally to this study as part of their Masters dissertation research for the Royal Danish School of Pharmacy, Copenhagen. E.G.C.B. received fellowship support from L’Association Pour la Recherche sur le Cancer.
- Abbreviations:
- P-450 or CYP
- cytochrome P-450
- CPA
- cyclophosphamide
- 4-OH-CPA
- 4-hydroxy-cyclophosphamide
- CA
- chloroacetaldehyde
- IFA
- ifosfamide
- 4-OH-IFA
- 4-hydroxy-ifosfamide
- PB
- phenobarbital
- TAO
- troleandomycin
- DEX
- dexamethasone
- UT
- untreated (uninduced) rat treatment group
- AUC
- area-under-the-curve, plasma X concentration
- Cmax
- peak plasma concentration
- Tmax
- time ofCmax
- Cpl
- plasma concentration
- T1/2
- apparent half-life, which reflects both the rate of metabolite formation and the intrinsic rate of metabolite elimination
- Received June 11, 1998.
- Accepted September 29, 1998.
- The American Society for Pharmacology and Experimental Therapeutics