Accelerated Decomposition of 4-Hydroxycyclophosphamide by Human Serum Albumin1

(CANCER RESEARCH 47, 1505-1508, March 15. 1987] Accelerated Decomposition of 4-Hydroxycyclophosphamide by Human Serum Albumin1

Chul-Hoon Kwon, Karen Maddison, Lynn LoCastro, and Richard F. Borch2

Department of Pharmacology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

ABSTRACT Sladek and coworkers have reported a shorter half-life in plasma compared to aqueous buffer for metabolites capable of Cyclophosphamide, a widely used anticancer agent, requires initial generating acrolein (21). We have confirmed this for 4-OHCP metabolic activation to 4-hydroxycyclophosphamide (4-OHCP) to elicit and for its 4-alkylthio-substituted derivative, mafosfamide its activity. The rate of decomposition of <â€¢;v-4-<)IK I' was much faster in plasma than in buffer at pH 7.4. This plasma activity was not affected (ASTA Z 7557), using NMR methods (22). However, the basis by treatment with acid (pH 1.3) or heat ((>(!"( for 30 min). The activity for the increased rate of activation in plasma is not understood. was retained in the macromolecular fraction (>10,000) but not in the The objective of this investigation was to identify the catalytic filtrate. Serum albumin was identified as the catalyst for the elimination species in plasma responsible for this increase and to determine step that generates phosphoramide mustard from aldophosphamide; al the mechanistic details of the process. bumin had no effect on the rate of ring opening of cw-4-OHCP to aldophosphamide. This catalytic activity was dependent on serum albu MATERIALS AND METHODS min concentration and independent of pH over the range of 6.5 to 7.5, in contrast to the buffer-catalyzed reaction. The catalytic rate constants *â€ž, Drugs and Chemicals. c/Ã®-4-Hydroperoxycyclophosphamide was pre (pH 7.4, 37Â°C)for phosphate buffer, human serum albumin, and bovine pared as described elsewhere (4, 6). 3.4-Dihydroxybutyl-yV,/V-bis(2- serum albumin were 1.13, 285, and 83 M~'min~', respectively. Pretreat chloroethyOphosphorodiamidate was prepared as described elsewhere ment of eis-4-OHCP with serum albumin resulted in a time-dependent (4). Sodium phosphate, bovine serum albumin (Fraction V, 98.5% decrease in cytotoxic activity against LI 210 tumor cells in vitro. These albumin), human serum albumin (Fraction V, 98% albumin, essentially data suggest that the albumin-catalyzed reaction of cis-4-OIICP in globulin free), crystalline human serum albumin (>99.5% albumin, plasma represents an important pathway for the transformation of cyclo- globulin free), and human serum albumin solution (99.0% albumin, 1% phosphamide metabolites and further emphasize the importance of con globulin, aseptically prepared) were purchased from Sigma Chemical sidering phosphoramide mustard generated extracellularly versus intra- Co., St. Louis, MO. Other organic reagents and solvents were purchased cellularly and the respective contributions of extracellular and intracel- from Aldrich Chemical Co., Milwaukee, WI. Centricon microconcen- lular phosphoramide mustard to Cyclophosphamide cytotoxicity in vivo. trators, used to separate the M, > 10.000 plasma fraction, were pur chased from Amicon Co., Danvers, MA. Human plasma was obtained from the blood bank. Strong Memorial Hospital, University of Roch INTRODUCTION ester, Rochester, NY, and was frozen at -20Â°Cuntil use. Cyclophosphamide, a widely used antineoplastic and immu- NMR Studies. Unless otherwise noted, NMR studies were carried out as described previously (6). 3IP NMR spectra were recorded on an nosuppressive agent, is of great interest due to its relatively IBM WP-270-SY instrument equipped with an 1BM-VSP multinuclear high oncotoxic specificity (1-3) and its complexity in the acti probe tuned for 109.368 MHz using 10-mm sample tubes, a 5000-H?. vation process (Fig. 1). Cyclophosphamide is a prodrug, and its spectral width, a 10 /'diminution is dimethyl sulfide at 37Â°C.The appropriate buffer (prewarmed to 37Â°C) was then added to give a final c/i-4-OHCP concentration of 30 to 50 subject to general base catalysis (7), and activation may also be catalyzed by 3'-5' exonucleases (8-11). There has been a mM (methanol content, <5% of final volume). A typical buffer mixture consisted of 1.7 ml of 100 mM phosphate buffer and 0.3 ml of D2O. controversy over the identity of the circulating metabolite(s) The appropriate quantity of albumin or separated plasma fraction, that enters cells and ultimately exerts cytotoxic activity. Con prepared from 1.6 ml of plasma, was dissolved in 1.7 ml of 100 mM siderable evidence supports the hypothesis that HCP/Aldo phosphate buffer, and this solution substituted for the above phosphate represents the transport form of CP, and that generation of buffer for comparison studies. A typical plasma mixture consisted of PDA occurs within cells sensitive to this drug (1, 2, 12-16). 1.6 ml of human plasma, 0.17 ml of l M phosphate buffer, and 0.23 ml However, other data suggest that the contribution of extracel of I).() The sample was then introduced into the preequilibrated lular PDA may also be important, especially when the higher spectrometer probe, and spectra were acquired at varying intervals. AUC value for PDA is taken into account (17-20). Time points for each spectrum were assigned at the midpoint of data acquisition. The free induction decay spectra were stored on disk and Received 7/21/86; revised 10/23/86; accepted 12/5/86. subsequently processed by exponential multiplication with 2 Hz of line The costs of publication of this article were defrayed in part by the payment broadening, and relative concentrations of intermediates were deter of page charges. This article must therefore be hereby marked advertisement in mined from the peak heights of their respective phosphorus resonances. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported by Grants CA 34619 and CA 11198 from the Determination of Protein Concentration. Human plasma protein con National Cancer Institute. Department of Health and Human Services, and this centration was measured by using Coomassie Blue G dye-binding assay support is gratefully acknowledged. (23). 2To whom requests for reprints should be addressed. In Vitro Cytotoxic Activity. A soft agar colony-forming assay (24) 1The abbreviations used are: 4-OHCP, 4-hydrooxycyclophosphamide; CP, Cyclophosphamide; Aldo, aldophosphamide; PDA, phosphoramide mustard; was used and modified where necessary. Cultured mouse LI210 cells HSA, human serum albumin; BSA, bovine serum albumin; HCP, hydroxycyclo- were obtained from EG&G Mason Research Institute, Tumor Bank, phosphamide; AUC, area under curve. Worchester, MA. 4-Hydroperoxycyclophosphamide (0.02 mmol, 5.86 1505 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. ALBUMIN-CATALYZED DECOMPOSITION OF ACTIVATED CYCLOPHOSPHAMIDE mg) was treated with 2.2 equivalents of sodium thiosulfate (0.044 mmol, 100 100r 10.92 mg) in 0.2 ml of prewarmed acetate buffer (100 mM, pH 5.25, 37Â°C).AVortex mixer was used to facilitate dissolution. The solution was allowed to stand at 37Â°Cfor 5 min to allow complete reduction to CM-4-OHCP, and Fischer's medium (0.84 ml) (Gibco Lab., Grand Island, NY) was added. This solution was immediately sterilized by passage through a 0.22-nm Millipore filter, and 0.96 ml of 10% aspetically prepared human serum albumin was added. The same vol ume of Fischer's medium was substituted for the albumin solution in the control. This gave a 2-ml final volume of stock solution containing 10 mM 4-OHCP and 48 mg/ml of albumin, which was maintained at 37'C. LI210 cells in exponential growth were suspended in Fischer's medium to give 10-ml volumes at a final density of 2 to 3 x 10s cells/ ml after addition of drug solution. After incubating the stock solutions for 0, 15, 30, and 60 min, appropriate volumes of the drug/albumin or 1O 2O 3O 4O SO X) 20 3O 40 SO drug/Fischer's medium stock solution (5 to 150 ^1) were transferred to TIME (MIN) TIME (MIN) the cell suspensions. The drug-cell suspensions were then incubated for Fig. 2. Reaction of c/s-4-hydroxyc>clophosphamide in phosphate buffer (pH l h at 37'C. The cells were washed 3 times with 3 ml of supplemented 7.4, 100 mM, 37Â°C)(A) and plasma (pH 7.4, 37'C) (B). Points were measured Fischer's medium (containing 10% horse serum) and then resuspended from 31P NMR line intensities; the solid lines represent the best-fit values calculated by the Simplex algorithm. Ã€,c/s-4-OHCP; â€¢,rrans-4-OHCP; â€¢Aldo in 5 ml of fresh medium. A 1-ml portion was used to determine the cell and its hydrate; x. PDA and its products. count (Coulter Counter). From the remainder, a 5-ml suspension of cells was prepared at a density of 10s cells/ml, and between IO2 and Table 1 Rate constants for the elimination of phosphoramide mustard from IO5cells were plated on soft agar and incubated at 37Â°C.Colonies were aldophosphamide at 37'C counted after 10 days. Rate constants are corrected for the contribution from phosphate buffer. All Data Analysis. Rate constants were determined by simultaneous fit linear regression correlation coefficients were >0.98. k,,im, min" of the experimental data points to the values calculated using the Reaction conditions differential Equations A to D. A computer program was written using Phosphate (100 mm, pH 7.4) 0.06 the Simplex algorithm (25) to optimize rate constants kt to kt by Fischer's medium (pH 7.4)Â° 0.04 nonlinear least-squares minimization. Calculated data points were de Plasma (pH 7.5) 0.25 termined every 6 s for the first 10 min, every 30 s for the next 15 min, Plasma ultrafiltrate 0.01 Plasma macromolecules and every min for the remaining time points. Phosphoramide mustard pH7.4 0.23 generation proceeded with good first-order kinetics, so the best-fit pH7.0 0.27 values for A?were determined by linear regression. pH6.5 0.28 pH6.1 0.18 Acid-treated plasma 0.24 Heat-treated plasma (60Â°C,30 min) RESULTS 0.23 Fraction V human serum albumin (98%) (60 0.25 Hydrolysis of 4-OHCP was monitored by 3IP NMR spec- mg/ml) Purified (>99.5%) human serum albumin (60 0.24 troscopy, and the intermediates were quantified by peak inten mg/ml) sity (see "Materials and Methods" for details). Preliminary Bovine serum albumin (70 mg/ml) 0.07 experiments revealed that m-4-OHCP decomposed to PDA " Reaction mixture was 40% Fischer's medium in 30 mM phosphate buffer. (and its products) much faster in plasma than in buffer at pH 7.4 and 37Â°C(Fig. 2). The decomposition rate was unaffected macromolecules (M, > 10,000) in phosphate buffer (100 mM, when plasma was heated at 60Â°Cfor 30 min or acidified to pH pH 7.4, and 37Â°C)was essentially identical to that in whole 1.3 (10% HC1), degassed, and then neutralized to pH 7.4 (10% plasma; however, the buffered plasma filtrate (M, < 10,000) NaOH). Restoration of physiological bicarbonate concentration exhibited a decomposition rate essentially identical to that (25 mM) did not alter the plasma activity. Human plasma was observed in buffer alone. The catalytic activity was independent fractionated by passage through an M, \ 0,000 cutoff filter. The of pH over the range 6.5 to 7.5; at pH 6.1, however, a 30% rate of decomposition of m-4-OHCP in reconstituted plasma decrease in decomposition rate was observed (Table 1). Three separate commercial preparations, containing 98%, 99%, and >99.5% albumin, showed essentially identical catalytic activity, and comparison of plasma and reconstituted albumin solutions at equivalent concentrations indicated that albumin accounted for >90% of the plasma catalytic activity. Having observed that serum albumin catalyzed the decom position of 4-OHCP, we proceeded to investigate its mechanism of action. The kinetic model for the decomposition reaction is shown in Fig. 3 and differential Equations A to D.

dC/dt = -k, C + kiAH (A) dT/dt = k,AH - k,T (B) dAH/dt = k,C + k

AldoHydrate where C, T, AH, and PP are m-4-OHCP, frans-4-OHCP, Aldo Fig. 1. Transformations of cyclophosphamide metabolites. plus its hydrate, and PDA plus its products (aziridinium ion 1506 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. ALBUMIN-CATALYZED DECOMPOSITION OF ACTIVATED CYCLOPHOSPHAMIDE cls-4-OH CP x l *" Aldo and Its Hydrate -, 3 * trans-4-OH CP C k2 AH

PDA and Its Products PP Fig. 3. Kinetic scheme for interconversion of cyclophosphamide metabolites.

25 50 75 100 4-OHCP, UM

Fig. 5. Effect of pretreatment with human serum albumin (48 mg/ml) on the cytotoxic activity of cÃÃ-4-OHCPagainstLI 210 cells in vitro. â€¢.no albumin; Ãœ, O min; O, 15 min; A, 30 min; x. 60 min; â€¢,phosphoramide mustard control. HSA cone Bars, SD (n = 3). Fig. 4. Effect of human serum albumin concentration on the rate constant for ring opening of cii-4-OHCP I I) and on the rate constant for /Ã-elimination(B) of 2 logs of cell kill to 17 UM. The decrease in cytotoxicity PDA from Aldo (pH 7.4 and 37*C). correlates well with the extent of conversion of 4-OHCP to PDA; estimates of conversion based upon decreased cytotoxic and solvolysis products), respectively. The reaction was exam ity are 44% and 84% at 15 and 60 min, respectively, compared ined at 4 different albumin concentrations in phosphate buffer (100 mM, pH 7.4, 37Â°C).Rate constants for ring opening (k,) to predicted conversions of 45% and 91% based upon the rate constants determined above. of m-4-OHCP were determined using the Simplex algorithm (see "Materials and Methods") and were found to be independ DISCUSSION ent of albumin concentration [kt = 0.13 Â±0.01 (SD) min'1; see The basis for CP's relatively high oncotoxic specificity is still Fig. 4]. In contrast, generation of PDA (and its products) was dependent on albumin concentration (Fig. 4) and followed first- not clear, but the initial hydroxylated metabolite 4-OHCP is order kinetics after the pseudoequilibrium was established (10 clearly the pivotal intermediate in both the activation and deactivation processes (Fig. 1). The conversion of 4-OHCP to min). Because equilibration was rapid compared to the elimi nation reaction, AH remained a constant fraction (19%) of the the cytotoxic PDA and acrolein is believed to occur via the 4-OHCP/AH mixture; k-, was readily determined by linear Aldo intermediate, and this general base-catalyzed elimination regression from the plot of \n(PPx â€”PP,) versus time t. The is the rate-limiting step in aqueous buffer under physiological conditions (4-5, 7). The present experiments clearly demon slope from the plot of k<,versus albumin concentration (Fig. 4) gave the catalytic constant Atcal(HSA) for the elimination of strate that plasma albumin catalyzes generation of PDA from PDA from Aldo by albumin (285 vr'rnin"1). Extrapolation to cÃÃ-4-OHCP;thefact that three different albumin preparations zero albumin concentration gave the buffer catalytic constant of varying purity (98 to >99.5%) show equivalent catalytic A:cai(phosphate) = 1.13 M~'min~', in excellent agreement with activity indicates that catalysis by a contaminant is unlikely. the reported rate constant of 1.03 M~'min"' (15).4 BSA was less Serum albumin is the major plasma protein; it is relatively effective as a catalyst than HSA [kc.M(BSA)= 83 M~'min~']. stable to heat and treatment with acid or base, and its main The effect of this albumin-catalyzed reaction on cytotoxicity functions include maintenance of osmotic pressure, fatty acid in vitro was examined by clonogenic assay of LI 210 cells using transport, and sequestration and transport of bilirubin (26). C/5-4-OHCP pretreated with serum albumin (48 mg/ml) for Although numerous drugs bind to serum albumin, it is assumed varying times (Fig. 5). A time-dependent decrease in cytotoxic to lack intrinsic catalytic or enzymatic activity [with the possible activity was observed; the drug concentrations required to pro exception of thioesterase activity (27, 28)]. The detailed mech duce 2 logs of cell kill were 13, 21, 27, and 47 ^M when 4- anism by which albumin catalyzes PDA release remains to be OHCP was pretreated with HSA (60 mg/ml) for 0-, 15-, 30-, elucidated; however, it clearly catalyzes the ^-elimination step and 60-min albumin exposure, respectively. Pretreatment for from Aldo and does not affect the rate of ring opening of 4- 60 min with Fischer's medium in the absence of albumin showed OHCP. This suggests that albumin may bind Aldo or its hydrate a small increase in the drug concentrations required to produce at a site containing a residue that can function as a general base catalyst. Although the 250-fold rate enhancement (HSA versus 4The elimination rate constants reported in this paper are based on the phosphate) is modest for an enzyme-catalyzed reaction, the pH- concentration of aldophosphamide and its hydrate at equilibrium (19% of total metabolites). Comparison with published rate constants that are based upon the rate profile is consistent with macromolecular catalysis with an entire metabolite mixture requires division of the literature values by 0.19. active site functional group having pK0 = 6. Thus albumin 1507 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. ALBUMIN-CATALYZED DECOMPOSITION OF ACTIVATED CYCLOPHOSPHAM1DE

concomitant partitioning of aldophosphamide between irreversible fragmen appears to be functioning as an enzyme with modest general tation and reversible conjugation pathways. J. Med. Chem., 27: 466-485, base catalytic activity. 1984. It is conceivable that albumin might generate PDA via hy 6. Borch, R. F., and Millard, J. The mechanism of activation of 4-hydroxycy- drolysis of the phosphate ester bond. 3,4-Dihydroxybutyl NJV- clophosphamide. J. Med. Chem., 30: in press, 1987. 7. Low, J. E., Borch, R. F., and Sladek, N. E. Further studies on the conversion bis(2-chloroethyl)phosphorodiamidate is a close structural an of 4-hydroxyoxazaphosphorines to reactive mustards and acrolein in inor alogue of Aldo that does not undergo /3-elimination but should ganic buffers. Cancer Res.. 43: 5815-5820, 1983. 8. Voelcker. G., Bielicki. L., and Hohorst, H.-J. Evidence for enzymatic toxi- be equivalent to Aldo as a phosphatase substrate. 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Weber (ed.). Advances in Enzyme their amino acid sequences (26, 27). This suggests that species Regulation, pp. 99-122. Oxford: Pergamon Press, 1985. variability in serum albumin structure may account for some of 12. Domeyer, B E., and Sladek, N. E. Kinetics of cyclophosphamide biotransfor the differences observed in CP metabolite pharmacokinetics mation in vivo. Cancer Res.. 40: 174-180. 1980. 13. Draeger, U., Peter, G., and Hohorst, H.-J. Deactivation of cyclophosphamide and cytotoxicity (16, 18). metabolites by sulfur compounds. Cancer Treat. Rep., 60: 355-359, 1976. The time-dependent decrease in 4-OHCP cytotoxic activity 14. Jardine, I., Fenselau, C., Appier, M., Kan, M-N., Brundrett, R. B., and in vitro when pretreated with serum albumin confirms that, Colvin, M. Simultaneous quantitation by gas chromatography-chemical ion- ization mass spectrometry of cyclophosphamide, phosphoramide mustard, when PDA is generated extracellularly, cytotoxicity is reduced and nornitrogen mustard in the plasma and urine of patients receiving compared to transport of 4-OHCP/Aldo and intracellular PDA cyclophosphamide therapy. Cancer Res., 38:408-415. 1978. 15. Low, J. E., Borch, R. F., and Sladek, N. E. Conversion of 4-hydroperoxycy- generation. This is consistent with the conclusions of Powers clophosphamide and 4-hydroxycyclophosphamide to phosphoramide mus and Sladek that 4-OHCP/Aldo represents the important cir tard and acrolein mediated by bifunctional catalysis. Cancer Res., 42: 830- culating metabolite of CP (16). However, the effects of serum 837, 1982. 16. Powers, J. F., and Sladek. N. E. 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Chul-Hoon Kwon, Karen Maddison, Lynn LoCastro, et al.

Cancer Res 1987;47:1505-1508.

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