Introduction

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Nephrotoxicity is an infrequent but potentially life-threatening complication of high-dose methotrexate (HDMTX) because it can lead to delayed methotrexate (MTX) excretion and a marked enhancement of MTX-induced myelosuppression, mucositis, hepatitis, and dermatitis (Djerassi, 1975; Bleyer, 1978; Frei et al., 1980; Abelson et al., 1983; Stark et al., 1989). We have previously demonstrated that a recombinant form of carboxypeptidase-G₂ (CPDG₂), cloned from Pseudomonas strain Rs-16, which hydrolyzes MTX to 2,4-diamino-N¹⁰-methylpteroic acid (DAMPA) and glutamic acid (Sherwood et al., 1985), rapidly lowers plasma MTX concentrations by more than 98% and provides an alternate route of elimination of MTX in patients with MTX-induced renal dysfunction (Widemann et al., 1995, 1997, 1998).

DAMPA is normally a minor metabolite of MTX, accounting for <5% of the total dose of drug that is excreted in urine (Donehower et al., 1979). DAMPA is presumably formed from MTX that is excreted via the bile into the intestinal tract, hydrolyzed by bacterial carboxypeptidases, and then reabsorbed. It is thought to be an inactive metabolite of MTX because it is not an effective inhibitor of the MTX target enzyme dihydrofolate reductase (Donehower et al., 1979; Widemann et al., 1999). After systemic exposure to CPDG₂, DAMPA plasma concentrations (M) are equivalent to pre-CPDG₂ MTX concentrations (Widemann et al., 1998). DAMPA is approximately 10-fold less water soluble than MTX (aqueous solubility at pH of 7.0: DAMPA 0.85 mg/ml, MTX 9.04 mg/ml), and persistently high concentrations of DAMPA could theoretically lead to further renal toxicity by precipitation in the renal tubules (Donehower et al., 1979). However, in the patients treated for MTX-induced nephrotoxicity with CPDG₂, DAMPA plasma concentrations declined more rapidly than MTX concentrations, which suggests a nonrenal route of elimination for DAMPA (Widemann et al., 1998).

We have characterized the pharmacokinetics and metabolism of i.v.-administered DAMPA in nonhuman primates and studied the metabolic pathways for DAMPA in vitro. We also assessed the cytotoxicity of DAMPA, relative to MTX and the effect of DAMPA on MTX cytotoxicity, in vitro in a human leukemia cell line.

	Materials and Methods

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DAMPA Pharmacokinetics

Animals. Four adult male Rhesus monkeys (Maccaca mulatta) weighing 8.0 to 15.9 kg were used for this study. The animals were fed National Institutes of Health open formula (extruded, nonhuman primate diet) ad libitum and were group housed in accordance with the Guide for the Care and Use of Laboratory Animals, National Research Council (National Academy Press, Washington DC, 1996). Blood samples were drawn through a catheter placed in either the femoral or saphenous vein contralateral to the site of drug infusion.

DAMPA Administration and Plasma Sampling. DAMPA (Sigma Chemical Co., St. Louis, MO) was dissolved in 0.1 N NaOH, the pH was adjusted to 8.5 with HCl, and the drug solution was filter sterilized through a 0.22-µm filter before administration. Animals were hydrated and alkalinized with 20 ml/kg D₅W with 0.5 mEq NaHCO₃/kg over 2 h before DAMPA administration, and they continued to receive i.v. hydration and alkalinization at a rate of 3 ml/kg/h until 10 h after DAMPA administration. Additional NaHCO₃ (0.5-1.0 mEq/kg) was administered if the urine pH was less than 7. DAMPA (200 mg/m²) was administered i.v. over 15 min. Plasma samples were obtained before and at the end of the infusion at 5, 15, 30, 45, 60, and 90 min, and 2, 3, 4, 5, 6, 7, 8, 9, 10, and 24 h after the end of the infusion. Plasma was separated immediately by centrifugation and frozen at 70°C until analysis. Urine was collected from the start of the i.v. hydration until 10 h after the administration of DAMPA. Animals were evaluated for signs of clinical toxicity and monitored with complete blood count and serum electrolytes, blood urea nitrogen, and creatinine twice weekly for 2 weeks after the experiment.

DAMPA Assay. DAMPA was quantified with a previously reported, reverse-phase HPLC method (Widemann et al., 1995, 1997). Briefly, after solid phase extraction with C18 cartridges (Varian, Harbor City, CA), samples were injected onto a 4-µm, C18 Nova-PAK radial compression analytical column (Waters, Milford, MA) with a C18 µm Bondapak guard column (Waters) and eluted isocratically with 80:20 (v/v) 0.1 M sodium phosphate (pH 6.8):methanol at a flow rate of 1.5 ml/min. Eluant was monitored at a wavelength of 303 nm with a 2690 Alliance HPLC separations module with a 996 photodiode array (PDA) detector (Waters). Under these conditions the retention time for DAMPA was approximately 14.5 min. DAMPA metabolites were analyzed with the same assay conditions, and UV spectra were recorded between 200 and 450 nm, with extraction of spectra at 303 nm.

Pharmacokinetic Analysis. A three-compartment open model with first order elimination from the central compartment was fit individually to the plasma DAMPA concentration-time data from the four animals with MLAB (Civilized Software, Bethesda, MD). The model fits were weighted with the MLAB EWT function, which computes a weight vector from estimates of variance. The three-compartment model is described by the following series of differential equations:


where C_c is the concentration of DAMPA in the central compartment at time t, X_p and X_dt are the amounts of drug in the peripheral and deep tissue compartments, k₀ is the drug infusion rate, V_c is the volume of the central compartment, k_cel is the first order elimination rate constant, and k_cp, k_pc, k_cdt, and k_dtc are the intercompartmental first order exchange rate constants. Other pharmacokinetic parameters were derived from the fitted model parameters.

DAMPA Metabolism

Reagents. MTX was obtained from Immunex Corp. (Seattle, WA), CPDG₂ from the Cancer Therapy Evaluation Program of the National Cancer Institute, -glucuronidase from Escherichia coli from Sigma Chemical Co., and young frozen rabbit livers from Pel-Freez (Rogers, AR). 7-Hydroxy-MTX (7-OH-MTX) was kindly provided by Dr. A. Freidouni (Karolinska Institute, Stockholm, Sweden).

Partial Purification of Aldehyde Oxidase (AO). AO, which converts MTX to 7-OH-MTX, was partially purified by a previously described method (Johns et al., 1966; Johns and Loo, 1967). In brief, six white rabbit livers (585 g) were homogenized in water at room temperature. The homogenate was centrifuged, and the supernatant was saved and heated in a 60°C water bath for 10 min. The resulting semisolid gel was centrifuged, and the clear, reddish brown supernatant was decanted into an Erlenmeyer flask. This solution was fractionated with a solution of saturated ammonium sulfate and ammonium hydroxide. The solution became opaque with stirring and was then centrifuged. The pellets were saved, and the supernatant was decanted and stirred with saturated ammoniacal solution again. The solution became opaque with stirring and was centrifuged. The pellets from both fractionations were collected and dissolved in diluted ammoniacal solution. The protein concentration was estimated by UV-spectrophotometry (Waddell and Hill, 1956).

Determination of Specific Activity. The specific activity of the partially purified AO was determined with MTX and DAMPA as substrates. AO hydroxylates MTX and DAMPA at position 7 on the pteridine ring (Johns and Loo, 1967; Valerino et al., 1972). Potassium phosphate buffer (930 µl, 50 µM, pH 7.8) with 0.005% EDTA, 20 µl of partially purified AO, and 50 µl of 1 mM MTX or 1 mM DAMPA (final substrate concentration, 50 µM) was added to a 1.0-cm cuvette. The increase in absorbance at 340 nm was measured for 180 s with an 8452A diode array spectrophotometer (Hewlett Packard, Palo Alto, CA). Measured absorbances were less than 0.8. The amount of 7-OH-MTX formed from MTX was derived with the extinction coefficient 12,000 M¹ cm¹ (Fabre et al., 1986). An extinction coefficient for 7-OH-DAMPA was calculated by incubating 5, 10, 50, 100, 150, and 200 µM DAMPA with AO in 50 µM potassium phosphate buffer, pH 7.8, and determining the increase in absorbance at 340 nm by UV-spectrophotometry once the reaction was complete. We confirmed that the DAMPA was completely converted to 7-OH-DAMPA in these samples by HPLC. The extinction coefficient for 7-OH-DAMPA was then calculated with the Lambert-Beer equation (Segel, 1968).

AO Kinetics with MTX and DAMPA as Substrates. The partially purified AO solution was diluted 25-fold, and various concentrations of MTX and DAMPA were dissolved in 50 µM potassium phosphate buffer, pH 7.8. H-style cuvettes with 2-mm path length were filled with 350 µl of substrate solution combined with 350 µl of dilute AO solution. The rate of production of the 7-OH-metabolites of MTX and DAMPA was measured by monitoring in real-time changes in absorbance at 340 nm. Each reaction was monitored for 180 s, and the first 100 s of the reaction during which the change of absorption was linear was taken to calculate the rate of production of the 7-OH metabolites. The reactions were carried out at 22°C with a Peltier 89090A model (Hewlett Packard). Substrate concentrations in the reaction mixture were 25, 40, 50, 75, 100, 150, 300, and 500 µM, and each substrate concentration was analyzed in five replicates for each compound. The Michaelis-Menten equation was fit to the AO reaction rates at the various substrate concentrations to determine K_m and V_max for MTX and DAMPA with MLAB.

HPLC/Mass Spectroscopy (MS). MS analysis was performed on a Finnigan model TSQ7000 (Finnigan Corporation, San Jose, CA) operated in positive ion electrospray mode. The spray voltage was set at 4.5 kV and the heated capillary was maintained at 240°C. Product ion scans were produced with 1.1 millitorr argon and 13 to 21 eV offset in the collision cell. Extracted samples were introduced into the mass spectrometer after injection onto a phenyl-hexyl, 5 µm, 50- × 2.0-mm HPLC column (Phenomenx Inc., Torrance, CA). The mobile phase contained 1.1 mM ammonium hydroxide and 3.5 mM acetic acid. DAMPA and its metabolites were eluted at a flow rate of 0.25 ml/min with the following gradient: 90:10 (v/v) mobile phase:acetonitrile for the first 2 min to 20:80 (v/v) mobile phase:acetonitrile from minutes 2 to 10. The retention time for DAMPA under these conditions was approximately 9.2 min.

Metabolite Identification. HPLC metabolite fractions from 1) nonhuman primates after DAMPA administration and 2) from two patients treated with CPDG₂ as described in a previously reported study (Widemann et al., 1997) were collected and partially evaporated under a nitrogen stream to remove the methanol in the eluant. The metabolite fractions were applied to 3 ml of C18 solid phase extraction columns (Varian). Columns were washed with 3 ml of 0.1 M sodium phosphate (pH 6.8), and the sample was eluted with 3 × 1 ml of methanol. Extracted samples were centrifuged for 10 min to remove the precipitated phosphate salts, and the supernatant was dried under a nitrogen stream at 38°C. After reconstitution in water, the metabolite fractions were analyzed by HPLC with PDA and MS detection to characterize each metabolite.

DAMPA Cytotoxicity

Reagents. RPMI-1640 with L-glutamate and fetal calf serum was obtained from Mediatech Inc. (Herndon, VA) and sulforhodamine B from Sigma Chemical Co. The Molt-4 T-cell leukemic cell line (American Type Culture Collection, Mannassas, VA) was maintained and grown in RPMI-1640 with L-glutamate and 10% fetal calf serum. The doubling time of Molt-4 cells under these assay conditions was 17 h.

Cytotoxicity Assay. The cytotoxicity of DAMPA was studied in the human leukemia Molt-4 cell line and compared with MTX. The effect of DAMPA on MTX cytotoxicity also was assessed. DAMPA and MTX were dissolved in 0.01 N NaOH, the pH was then adjusted to 7.0 with H₃PO₄, and the solution was filter sterilized through a 0.22-µm filter. Molt-4 cells were plated in a 96-well microplate (Costar, Corning, NY) at a density of 5000 cells/well. After 24 h, when the cells were in logarithmic phase growth, drug or vehicle was added to cells in replicates of three to six wells. The final concentrations of DAMPA alone ranged from 0.00012 to 100 µM; the final concentrations of MTX alone ranged from 0.0001 to 100 µM; and for the combination of the two agents, the MTX concentration ranged from 0.0001 to 10 µM and DAMPA concentration was 100 µM. Control wells had vehicle (0.001 N NaOH, pH adjusted to 7.0 with H₃PO₄) added. After a 48-h drug exposure, cells were stained by use of the sulforhodamine B assay (Skehan et al., 1990). Optical density was measured at wavelengths of 540 and 405 nm (reference) in a Bio-Tek EL 340 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Survival was expressed as a fraction of untreated control. The assay was repeated in three independent experiments.

	Results

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DAMPA Pharmacokinetics

The mean plasma concentration of DAMPA at the end of the 15-min infusion was 51 µM (range 26 to 69 µM). The elimination of DAMPA from plasma was rapid and was well described by the three-compartment model (Fig. 1). DAMPA plasma concentrations fell to <1.0 µM within 1 to 2 h after completion of infusion. The model parameters and the pharmacokinetic parameters derived from the model parameters are listed for each animal in Table 1.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Plasma concentration-time curve of DAMPA in animal RQ293 that received 200 mg/m2 DAMPA i.v. over 15 min. The open symbols represent the measured plasma concentrations and the line represents the three-compartment model fit to the plasma concentration-time data.

View this table: [in this window] [in a new window]   TABLE 1 Pharmacokinetic parameters for DAMPA after a 15-min i.v. infusion of 200 mg/m2 in four nonhuman primates Vc, kcel, kcp, kpc, kcdt, and kdtc are the fitted model parameters for the three-compartment model. Clearance, volume of distribution at steady state (Vdss), and terminal half-life (t1/2) were derived from the model parameters.

The four animals did not experience apparent clinical toxicity attributable to DAMPA. A transient increase in serum creatinine and blood urea nitrogen was documented 4 days after administration of DAMPA in one animal, but returned to baseline levels after additional i.v. fluid hydration. In contrast to the other animals, this animal had received only 6 h of i.v. hydration after DAMPA administration instead of 10 h.

DAMPA Metabolism

Chromatography. In addition to DAMPA, three peaks, which were not present in the plasma before DAMPA administration, were detected in the chromatograms in all animals at the earliest sampling time point. Under the standard assay conditions, DAMPA eluted at approximately 14.5 min and the presumed metabolite peaks (M1, M2, M3) eluted at 16.5, 17.5, and 23.5 min, respectively. M3, the predominant metabolite, peaked at an average of 9 min (range, 0 to 15 min) after the end of the 15-min DAMPA infusion. M3 concentration was estimated with DAMPA standards and UV detection at 303 nm, and the mean peak M3 concentration was 22 µM (range, 15 to 32 µM). M1 and M2 were not quantified because of the lack of baseline separation. To better characterize M1 and M2, the HPLC mobile phase was changed to 83:17 (v/v) 0.1 M sodium phosphate (pH 6.8):methanol at a flow rate of 1.5 ml/min. Under these conditions baseline separation of M1 and M2 was achieved, and the retention times for DAMPA, M1, M2, and M3 were approximately 20, 24.3, 25.7, and 35.1 min, respectively (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Chromatogram of monkey plasma obtained 5 min after the end of a 15-min DAMPA (200 mg/m2) infusion. DAMPA and three additional peaks (M1, M2, M3) were observed at a wavelength of 303 nm. These peaks were not present in the pretreatment plasma sample.

DAMPA metabolism by AO. The protein concentration in the partially purified AO solution was 54 ± 2 mg/ml, and the specific activities with 50 µM MTX and DAMPA as substrates were 8.6 and 21 nmol/min/mg of protein, respectively. The extinction coefficient for 7-OH-DAMPA was calculated to be 10,200 M¹ cm¹.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   AO enzyme kinetics with varying concentrations of MTX (A) and DAMPA (B) as substrates. The symbols represent the data points for five replicate experiments; the solid line represents the fit of the Michaelis-Menten equation. V is the rate of formation of the 7-hydroxylated products 7-OH-MTX and 7-OH-DAMPA.

Metabolite Identification. M1 was identified as hydroxy-DAMPA (OH-DAMPA). The HPLC retention time and UV spectrum (Fig. 4A) for M1 were identical with those of the product of incubating DAMPA with AO and incubating 7-OH-MTX with CPDG₂. M1 from nonhuman primates was also identical with a metabolite from the plasma of patients who had received CPDG₂ for HDMTX-induced renal dysfunction. M1 was confirmed to be OH-DAMPA by MS. The positive ion single quad scan of M1 and 7-OH-DAMPA standard yielded a protonated molecular ion at 342 Da. The product ion spectrum of M1 and 7-OH-DAMPA contained the diagnostic ion at m/z 191 Da.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   A, UV spectra obtained with PDA detector for M1 metabolite (confirmed to be OH-DAMPA by mass spectral analysis) from plasma of nonhuman primates (NHP) and a patient who had received CPDG2 for delayed MTX excretion, resulting from MTX-induced nephrotoxicity, and products of the incubation of DAMPA with AO and 7-OH-MTX with CPDG2. B, M2 metabolite (confirmed to be DAMPA-glc by mass spectral analysis) from plasma of NHP and a patient who had received CPDG2 as a methotrexate rescue agent. C, M3 metabolite (confirmed to be OH-DAMPA-glc by mass spectral analysis) from plasma of NHP and a patient who had received CPDG2 as a methotrexate rescue agent.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   MS product ion scans of DAMPA (A) (parent ion m/z 326 at 23 eV); OH-DAMPA (parent ion m/z 342 at 23 eV) (B); DAMPA-glc (parent ion m/z 502 at 23 eV) (C); and OH-DAMPA-glc (D) (parent ion m/z 518 at 13 eV).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Proposed metabolic pathways of MTX in the presence of AO and CPDG2: MTX is converted to 7-OH-MTX by AO (a), and hydrolyzed to DAMPA by CPDG2 (b). OH-DAMPA results from the oxidation of DAMPA by AO (c) and from hydrolysis of 7-OH-MTX by CPDG2 (d). Glucuronyl transferase (GT) converts DAMPA (e) and OH-DAMPA to glucuronidated products (f).

DAMPA Cytotoxicity

DAMPA alone was not cytotoxic to Molt-4 human leukemia cells at concentrations up to 100 µM, and DAMPA did not significantly alter the cytotoxicity profile of MTX (Fig. 7). The IC₅₀ value for MTX and MTX in the presence of 100 µM DAMPA was 0.038 ± 0.02 and 0.043 ± 0.012 µM, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Survival of Molt-4 cells as a fraction of untreated control after incubation with 0.0001 to 100 µM MTX (), 0.0001 to 100 µM MTX in the presence of 100 µM DAMPA (), and 0.00012 to 100 µM DAMPA (). Points and error bars represent the mean and standard deviation of three experiments with three to six replicates per experiment. Solid lines represent the curve fit with a four-parameter model.

	Discussion

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DAMPA is normally a minor metabolite of MTX (Donehower et al., 1979) but when CPDG₂ is used as a rescue agent for patients with delayed MTX excretion resulting from HDMTX-induced renal failure, plasma concentrations of DAMPA, which is the product of CPDG₂-catalyzed hydrolysis of MTX, are equivalent to pre-CPDG₂ MTX concentrations. The more rapid elimination of DAMPA than MTX in those patients with renal dysfunction who received CPDG₂ as a rescue agent suggested that there is a nonrenal route of elimination for DAMPA. Nonrenal elimination would be advantageous in the setting of HDMTX-induced renal dysfunction because DAMPA is approximately 10-fold less water soluble than MTX and if excreted renally, it could precipitate in renal tubules and lead to further deterioration of the already compromised renal function in these patients. Therefore, an understanding of the pharmacokinetics and metabolism of DAMPA is of importance for the use of CPDG₂ as a rescue agent in patients with HDMTX-induced renal dysfunction.

After i.v. administration to nonhuman primates with normal renal function, DAMPA is rapidly eliminated. By 1 to 2 h after the end of the 15-min DAMPA infusion, plasma concentrations were <5% of the peak (end of infusion) plasma concentration. The terminal half-life of DAMPA in nonhuman primates was 51 min, which is somewhat shorter than the 62-min terminal half-life of MTX (100 mg/m² as i.v. bolus) in the same species.

Renal excretion of unchanged DAMPA accounted for 46% of the administered dose over the 10 h after the i.v. infusion. Based on the terminal half-life of 51 min, more than 99% of the DAMPA dose should have been eliminated by 10 h, suggesting that approximately 50% of the drug was metabolized. Under our HPLC conditions three metabolites were identified in monkey plasma and urine and these metabolites coincided with metabolites observed in patients who had received CPDG₂ as a rescue agent. Based on the retention time in HPLC assays, the UV spectra, a comparison to the products of in vitro incubation of DAMPA with AO and 7-OH-MTX with CPDG₂, the effect of -glucuronidase on the metabolites, and MS of each metabolite, we identified the metabolites as OH-DAMPA, DAMPA-glc, and OH-DAMPA-glc. The identical HPLC retention times and UV spectra of the products of in vitro incubation of 7-OH-MTX + CPDG₂, DAMPA + AO, and OH-DAMPA-glc + -glucuronidase support that OH-DAMPA and OH-DAMPA-glc are hydroxylated in position 7 of the pteridine ring. OH-DAMPA-glc was the predominant metabolite in plasma of nonhuman primates but accounted for only a small fraction of the total dose excreted in urine. Absolute quantification of metabolites in plasma and urine was not possible because of the lack of pure standards, and fecal excretion of DAMPA and its metabolites was not studied.

In rabbits, rhesus monkeys, and humans, MTX is converted to 7-OH-MTX by AO (Johns and Loo, 1967; Jacobs et al., 1976, 1977). In addition, in mice, DAMPA is also a substrate for AO, yielding 7-OH-DAMPA (Valerino et al., 1972). In vitro enzyme incubation studies with rabbit liver AO confirmed that DAMPA is a substrate for AO, and the affinity of the enzyme for DAMPA (apparent k_m = 131 µM) was similar to MTX (apparent k_m = 142 µM). Saturation of DAMPA by AO in vitro occurs at concentrations exceeding 250 µM, which is substantially higher than clinically observed plasma DAMPA concentrations in most patients who receive CPDG₂ as a rescue agent.

DAMPA has been presumed to be an inactive MTX metabolite based on a lesser degree of dihydrofolate reductase inhibition, and cytotoxicity studies in murine leukemic cells (Kessel, 1969; Valerino et al., 1972; Chaykovsky et al., 1974; Adamson et al., 1992). Our in vitro cytotoxicity studies of DAMPA and MTX demonstrated that DAMPA alone also was not cytotoxic in a human leukemia cell line at concentrations of up to 100 µM and that DAMPA did not significantly alter the cytotoxicity of MTX. DAMPA is only 3.9% as effective an inhibitor of dihydrofolate reductase as MTX (Widemann et al., 1999). The IC₅₀ of DAMPA, however, is more than 4 logs greater than the IC₅₀ of MTX, suggesting that DAMPA may not gain intracellular access as readily as MTX. Decreased intracellular uptake of DAMPA compared with MTX has been reported in murine leukemia cells (Kessel, 1969). Our studies further support that DAMPA is an inactive metabolite of MTX. The lack of cytotoxic activity of DAMPA supports the use of CPDG₂ as a rescue agent for patients with HDMTX-induced renal dysfunction, who may have plasma DAMPA concentrations exceeding 100 µM after treatment with CPDG₂.

In conclusion, in nonhuman primates and humans metabolism is a major route of DAMPA elimination. Metabolism underlies the more rapid elimination of DAMPA compared with MTX in patients with MTX-induced renal dysfunction after administration of CPDG₂.