Chem. Res. Toxicol. 1998, 11, 91-93
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Detoxification of an Alkylating Drug, N,N-Bis(2-chloroethyl)-p-aminophenylbutyric Acid (Chlorambucil), in Human Gastric Juice and Saliva Jari Hovinen,*,† Raija Silvennoinen,‡ and Juhani Vilpo‡ Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, Wallac Oy, FIN-20101 Turku, Finland, and Departments of Clinical Chemistry and Internal Medicine, Tampere University Hospital and University of Tampere, Medical School, P.O. Box 2000, FIN-33521 Tampere, Finland Received October 30, 1997
N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1) is an orally administered drug widely used in the chemotherapy of chronic lymphocytic leukemia. It is converted in gastric juice into three stable metabolites, which were characterized as N,N-bis(2-hydroxyethyl)p-aminophenylbutyric acid (4), N-(2-hydroxyethyl)-N-[2-(thiocyano)ethyl]-p-aminophenylbutyric acid (5), and N,N-bis[2-(thiocyano)ethyl]-p-aminophenylbutyric acid (6). 4 is the product of chloroambucil hydrolysis, while 5 and 6 are results of the reaction of 1 with saliva-derived thiocyanate ion. The destabilizing effect of low gastric oxonium ion concentration on 1 is also demonstrated.
Introduction N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (1, chlorambucil) (Chart 1) is the front-line treatment of chronic lymphocytic leukemia, which is the most prevalent form of leukemia in Western countries (1). Other indications include Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Waldenstro¨m’s macroglobulinemia, ovarian and breast cancer, some other tumors, and certain autoimmune diseases. Despite chlorambucil’s central role in modern chemotherapy, many details of its pharmacology have remained poorly investigated (2). Although this orally administered drug has been in clinical use since 1961, no data on its reactivity in human gastric juice have been available. We wish to report here our preliminary results on the reactions of chlorambucil in various gastric juices and saliva. It is clearly shown that high gastric pH decreases dramatically the bioavailability of the drug. Also a new metabolic pathway is demonstrated.
Experimental Procedures General. N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (1) was purchased from Sigma. N,N-Bis(2-hydroxyethyl)-paminophenylbutyric acid (4) and N-(2-chloroethyl)-N-(2-hydroxyethyl)-p-aminophenylbutyric acid (3) were synthesized as described in the literature (3, 4). Gastric juices from a healthy voluntary donor were obtained under three different conditions using methods described previously (3). Saliva from 148 different subjects was used as such after centrifugation at 10000g for 10 min. For NMR, all J values are in hertz (Hz). Kinetic Measurements. Reactions were carried out in stoppered tubes at 37 °C ([1] ) 4-9 × 10-4 M). The temperature was adjusted with a water bath and remained constant within 0.1 °C. Aliquots withdrawn at appropriate intervals were cooled with an ice bath and stored at -20 °C. They were melted just prior to HPLC analysis. * Address for correspondence: Wallac Oy, P.O. Box 10, FIN-20101 Turku, Finland. E-mail:
[email protected]. † University of Turku and Wallac Oy. ‡ University of Tampere.
Chart 1
HPLC Analyses. Analyses were performed on a MerckHitachi instrument equipped with a UV detector (λ ) 267 nm), an integrator, and a reversed-phase column (Hypersil C18, 4.6 × 240 mm, particle size 6 µm). Mobile phase: buffer A ) 0.1 M ammonium acetate, buffer B ) 0.1 M ammonium acetate in 50% (v/v) aqueous acetonitrile. Gradient: from 0 to 10 min 100% A, from 10 to 40 min, linear gradient of 100% A to 100% B; flow rate was 1.0 mL min.-1 Isolation of the metabolites (46) was performed on HPLC techniques resembling the above system but using a semipreparative HPLC column (LiChrospher 100 RP18, 10 × 250 mm, particle size 5 µm) and flow rate 3 mL min.-1 Purified products were desalted on HPLC by omitting the buffer salt from the mobile phase and concentrated in vacuo. Their electron spray mass spectra were recorded on a VG ZabSpec-ao TOF instrument, and the spectra were consistent with the proposed structures (see Supporting Information). Their structures were further confirmed on HPLC by spiking with authentic samples synthesized as described below. N,N-Bis[2-(thiocyano)ethyl]-p-aminophenylbutyric Acid, 6. A solution of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (1; 1.0 g, 3.28 mmol) in H2O-MeCN (1:1, v/v; 20 mL) containing potassium thiocyanate (1 M) was heated at reflux for 2 h. The solvent was removed in vacuo. The residue was redissolved in water (20 mL) and extracted with ether. The organic layer was separated, dried (Na2SO4), and concentrated. Purification on silica gel (eluent CH2Cl2-MeOH, 95:5, v/v) yielded the title compound as an oil, which solidified on standing. Mp: 83 °C. 1H NMR (400 MHz, CDCl3): δ 7.18 (2H, d, J ) 8.6, arom), 6.74 (2H, d, J ) 8.6, arom), 3.78 (4H, t, J ) 6.8, 2CH2N), 3.12 (4H, t, J ) 6.8, 2CH2SCN), 2.60 (2H, t, J ) 7.6 CH2COO), 2.37 (2H, t, J ) 7.3, CH2Ph), 1.93 (2H, p, J ) 7.8, CH2CH2CH2). 13C NMR (100.5 MHz, CDCl3): δ 179.4, 143.3, 129.4, 127.9, 114.2, 111.8, 51.5, 33.5, 33.0, 30.7 26.0. IR
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92 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
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Figure 1. HPLC traces of decomposition of chlorambucil (1) in human gastric juice at pH 7.0 induced by a gastric protonic pump antagonist (Losec). For chromatographic conditions, see Experimental Procedures. For reaction conditions, see Table 1. A, t ) 0; B, t ) 1 h, C, t ) 10 h. The large peaks marked with asterisks are present in gastric juice. (KBr/cm-1): 2155 (SCN). Anal. Found: C, 54.56; H, 5.41; N, 11.87; S, 18.27. Calcd for C16H19N3O2S2: C, 54.99; H, 5.48; N, 12.03; S, 18.35. N-(2-Hydroxyethyl)-N-[2-(thiocyano)ethyl]-p-aminophenylbutyric Acid, 5. The title compound was synthesized as desribed above for 6 but using N-(2-hydroxyethyl)-N-(2chloroethyl)-p-aminophenylbutyric acid (3) as the starting material. Purification was performed on silica gel (eluent CH2Cl2MeOH, 9:1, v/v). 1H NMR (400 MHz, CDCl3): δ 7.02 (2H, d, J ) 8.5, arom), 6.74 (2H, d, J ) 8.5, arom), 3.73 (4H, t, J ) 5.4, CH2N), 3.70 (2H, t, J ) 7.3, CH2N), 3.43 (2H, t, J ) 5.2, CH2OH), 3.07 (2H, t, J ) 7.3, CH2SCN), 2.52 (2H, t, J ) 7.6, CH2COOH), 2.29 (2H, t, J ) 7.3, CH2Ph), 1.85 (2H, p, J ) 7.8, CH2CH2CH2). 13C NMR (100.5 MHz, CDCl3): δ 179.2, 145.1, 131.3, 129.6, 114.0, 112.2, 59.9, 54.3, 51.8, 33.8, 33.3, 30.6, 26.4. Anal. Found: C, 57.72; H, 6.58; N, 8.99; S, 10.51. Calcd for C15H20N2O3S: C, 58.42; H, 6.54; N, 9.09; S, 10.40.
Results and Discussion Chlorambucil (1) is given by oral administration, daily dose being usually 4-10 mg. It is adsorbed from the gastrointestinal track, and the peak of plasma concentration is achieved in ca. 1 h (5). 1 is a highly reactive compound that decomposes rapidly in aqueous solutions and forms covalent bonds with a number of nucleophiles (3, 6-8). Keeping this in mind, we investigated its destiny in various human gastric juices in vitro. According to HPLC analysis, 1 is converted into three stable metabolites (Figure 1). They were isolated and characterized on electron spray MS as N,N-bis(2-hydroxyethyl)p-aminophenylbutyric acid (4), N-(2-hydroxyethyl)-N-[2(thiocyano)ethyl]-p-aminophenylbutyric acid (5), and N,Nbis[2-(thiocyano)ethyl]-p-aminophenylbutyric acid (6) (Chart 1). Their structures were further confirmed by HPLC by spiking with authentic samples synthesized via independent route.
Table 1. Half-Lives of Chlorambucil (1) in Various Fluid Matrixes at 37 °C medium
pH
[Cl-], mMa
[SCN-], mMb
t1/2
gastric: pentagastrin normal Losec saliva
1.9 5.5 7.0 7.6
113 91 85 nd
0.09 0.20 2.0 2.1
6 h 53 min 30 min 25 min 19 min
a Potentiometric (3). b Spectrophotometric (14). nd, not determined.
The formation of 4, the product of chlorambucil hydrolysis, is expected. By contrast, the presence of 5 and 6 is quite surprising. There is no information available that thiocyanate would be excerpted by the stomach wall. SCN-, however, is abudantly present in blood (9) and saliva (10-12). The concentration of thiocyanate is dependent on diet and smoking habits. The normal range for thiocyanate concentration for nonsmokers in whole saliva is 0.5-2 mM, while some smokers may have concentrations of up to 6 mM (10-13). Also foods containing cyanide or cyanogenic glucosides increase thiocyanate concentration in saliva. We confirmed that saliva SCN- concentration in 148 subjects was 5.7-0.4 mM when analyzed spectrophotometrically as FeSCN2+ complex (13). The thiocyanate ion concentration in various gastric juice samples varied from 0.1 to 2 mM. Hence, the SCN- in gastric juice, in all likelihood, represents swallowed saliva. Our results also show that high gastric pH and low chloride ion concentration destabilize 1 dramatically (Table 1). While at pH 1.9 the half-life of 1 was almost 7 h, at neural pH the half of the drug was decomposed under 0.5 h. Our observation has several important implications. The role of thiocyanate ion as a critical part in antimi-
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Chem. Res. Toxicol., Vol. 11, No. 2, 1998 93
crobial systems by acting as a substrate for peroxidases has previously been appreciated (12). We demonstrate here that saliva-derived SCN- has an important role in destroying the alkylating capacity of 1. It is very likely that this can be extended to concern other alkylators as well. Furthermore, the novel metabolic fate of 1 described here should at least partially explain the tremendous interindividual differences in the bioavailability of the ingested chlorambucil (2). The role of thiocyanate ion as a pharmacokinetic modulator is under investigation in our laboratories. We demonstrated also that high pH, induced by a gastric protonic pump antagonist (Losec), has a destabilizing effect on chlorambucil. Glugocorticoinds are often given together with 1. The prophylactic or sympthomatic treatment of gastric problems are usually performed by protonic pump or H2-receptor antagonists. Their possible indirect destabilizing effect on chlorambucil shall be taken into account in the clinical practice.
Acknowledgment. This work was supported by the Finnish Society for Cancer Research, Academy of Finland, and the Medical Research Fund of Tampere University Hospital. Supporting Information Available: Electron spray mass spectra of the metabolites (4-6) isolated from a reaction of chlorambucil (1) in human gastric juice (3 pages). Ordering information is given on any current masthead page.
References (1) Faguet, C. B. (1994) Chronic lymphocytic leukemia: An updated review. J. Clin. Oncol. 12, 1974-1990. (2) Lind, M. J., and Ardiet, C. (1993) Pharmacokinetics of alkylating agents. Cancer Surveys 17, 157-188.
(3) Lo¨f, K., Hovinen, J., Reinikainen, P., Vilpo, L. M., Seppa¨la¨, E., and Vilpo, J. A. (1997) Kinetics of chlorambucil in vitro: Effects of fluid matrix, human gastric juice, plasma proteins and red cells. Chem. Biol. Interact. 103, 187-198. (4) Foster, A. B., Jarman, M., Ross, W. C. J., and Tisdale, M. J. (1972) Preparation of N-(2-chloroethyl)-N-(2-hydroxyethyl)arylamines. Possible intermediates to potential carcinolytic agents bearing dissimilar reactive functions. J. Med. Chem. 15, 869-870. (5) Pratt, W. B., Ruddon, R. W., Ensminger, W. D., and Maybaum, J. (1994) The Anticancer Drugs, 2nd ed., Chapter 6, Oxford University Press, New York, NY. (6) Chatterji, D. C., Yager, R. L., and Gallelli, J. F. (1982) Kinetics of chlorambucil hydrolysis using high-pressure liquid chromatography. J. Pharmacol. Sci. 71, 50-54. (7) Cullis, P. M., Green, R. E. and Malone, M. E. (1995) Mechanism and reactions of chlorambucil and chlorambucil-spermidine conjugate. J. Chem. Soc., Perkin Trans. 2 1503-1511. (8) Kundu, G. C., Schullek, J. R., and Wilson, I. R. (1994) The alkylating properties of chlorambucil. Pharmacol. Biochem. Behav. 49, 621-624. (9) Rehak, N. N., Cecco, S. T., Niemela, J. E., and Elin, R. J. (1997) Thiocyanate in smokers interferes with the Nova magnesium ionselective electrode. Clin. Chem. 43, 1596-1600, and references therein. (10) Tenovuo, J. (1985) The peroxidase system in human secretions. In The Lactoperoxidase System. Chemistry and Biological Significance (Pruitt, K. M., and Tenovuo, J. Eds.) pp 101-122, Marcel Dekker Inc., New York, NY. (11) Tenovuo, J. (1989) Nonimmunoglobulin defence factors in human saliva. In Human Saliva: Clinical Chemistry and Microbiology (Tenovuo, J., Ed.) Vol. II, pp 55-91, CRC Press, Inc., Boca Raton, FL. (12) Thomas, E. L., Milligan, T. W., Joyner, R. E., and Jefferson, M. M. (1994) Antibacterial activity of hydrogen peroxide and lactoperoxidase-hydrogen peroxidase-thiocyanate system against oral streptococci. Infect. Immunol. 62, 529-535. (13) Lahti, M., Vilpo, J., and Hovinen, J. Unpublished results. (14) Kolthoff, I. M., Sandell, E. B., Meeham, E. J. and Bruckenstein, S. (1969) Quantitative Chemical Analysis, 4th ed., p 102, The Mackmillan Co., New York.
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