1032
Chem. Res. Toxicol. 1997, 10, 1032-1036
A Proposed Mechanism for p-Aminoclonidine Allergenicity Based on Its Relative Oxidative Lability Charles D. Thompson,† Prakash R. Vachaspati,† Stanley P. Kolis,† Pamela H. Gulden,† Michael E. Garst,‡ Anne Wiese,‡ Stephen A. Munk,‡ W. Dean Harman,† and Timothy L. Macdonald*,† Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virginia 22901, and Allergan, Inc., Irvine, California Received May 7, 1997X
p-Aminoclonidine (apraclonidine) is a selective R2 adrenergic agonist used to reduce intraocular pressure in the treatment of glaucoma. Use of apraclonidine is frequently associated with severe local allergic effects which warrant discontinuation of the drug in affected patients. We have assessed the oxidative lability of apraclonidine relative to a panel of adrenergic agonists and/or known allergens: amodiaquine, epinephrine, clonidine, and brimonidine. These compounds were compared by their electrochemical potentials as well as their oxidative lability in the presence of several oxidative enzyme systems (i.e., horseradish peroxidase, lactoperoxidase, myeloperoxidase, and diamine oxidase). The half-lives for enzymatic oxidation of these compounds were found to parallel the electrochemical oxidation potentials in the order: amodiaquine ∼ epinephrine < apraclonidine 1.70 >1.70
120 >120
3.0 ( 0.6 120 >120
6.7 ( 0.2 5.0 ( 0.3 20 ( 1.2 >120 >120
120 >120
a The anodic peak potentials (E ) and the half-lives for metabolism by HRP/H O , LPO, MPO/Cl-, and DAO for apraclonidine, p,a 2 2 amodiaquine, epinephrine, brimonidine, and clonidine.
8.3 Hz, Cys CH-CH2-S), 3.29 (d, d, 1H, J ) 14.3, 4.4 Hz. Cys CH-CH2-S), 3.59 (s, 2H, Gly NH-CH2-CO2H), 3.66 (m, 1H, γ-Glu CH), 3.70 (b, s, 4H, -NH-CH2-CH2-NH-), 4.28 (d, d, 1H, J ) 8.3, 4.4 Hz, Cys CH-CH2-S), 6.91 (s, 1H, Ph). The peak at δ ) 3.59 ppm, assigned to the R protons of the glycine residue, was found to shift in a concentration dependent manner.
Results The anodic peak potentials and the half-lives for oxidation by horseradish peroxidase, lactoperoxidase, myeloperoxidase, and diamine oxidase for apraclonidine, epinephrine, amodiaquine, brimonidine, and clonidine are compiled in Table 1. Under the conditions employed for our electrochemical studies, amodiaquine (Ep,a ) 0.42 V), epinephrine (Ep,a ) 0.65 V), and apraclonidine (Ep,a ) 1.00 V) were oxidized, while brimonidine and clonidine proved to be stable (Ep,a > 1.70 V; the potential at which the buffering medium was oxidized). None of the potentials appeared to be reversible under the criteria outlined in the Materials and Methods. The half-lives for oxidation of these compounds by enzymatic methods using a standardized set of conditions for oxidation were in the order: amodiaquine ≈ epinephrine < apraclonidine , clonidine ≈ brimonidine, which correlates to the electrochemically determined oxidation potentials. MPO in the absence of chloride ions failed to oxidize any of the five drugs. The experiments in which H2O2 was used were terminated at 120 min due to the destruction of H2O2 under the experimental conditions. Oxygen-mediated DAO oxidation led to the oxidation of amodiaquine and epinephrine only. Biotransformation of the R2 agonist apraclonidine by the enzymatic oxidative systems resulted in the formation of a yellow polymeric material. Analogous polymeric species were noted in the HRP-mediated oxidation of acetaminophen (17). In the presence of GSH, oxidation of apraclonidine with HRP/H2O2 exhibited a longer halflife, presumably due to competitive oxidation of GSH by HRP, and resulted in the formation of apraclonidineGSH adducts, in addition to polymer. The apraclonidine-GSH adducts were analyzed by HPLC-MS. The spectrum obtained by summing all of the scans collected during HPLC elution of the HRPapraclonidine-GSH incubation is depicted in Figure 1A. This spectrum has peaks for oxidized glutathione (m/z ) 613, m/2z ) 307), a glutathione-apraclonidine adduct (adduct A: m/z ) 550, m/2z ) 276), and an adduct corresponding to glutathione-apraclonidine minus a chloride (adduct B: m/z ) 516, m/2z ) 259). The structures of these adducts are shown in Chart 2. These adducts appear capable of a second round of oxidation followed by conjugation. However, we did not observe any bis-glutathione adducts under the experimental conditions employed in these studies. The total ion currents for adducts A and B indicate that the ratio of A/B is ≈5/1. The molecular ion peak for adduct A (m/z
Figure 1. (A) Mass spectrum obtained by summing all of the scans collected during HPLC elution of the HRP/H2O2-catalyzed oxidation of apraclonidine in the presence of GSH. (B) CAD spectrum of adduct A. (C) CAD spectrum of adduct B.
Chart 2. Structures of the Two Apraclonidine-GSH Adducts
) 550) has associated isotope peaks at m/z ) 552 and 554 as expected for the presence of two chlorine atoms, whereas the molecular ion peak for adduct B (m/z ) 516) has a single isotope peak at m/z ) 518 due to the one remaining chlorine atom. CAD spectra for adduct A and B are presented in Figure 1B,C, respectively. The absence of isotope peaks in the CAD spectra is due to the selection by the first quadrupole of only the m/z of the molecular ions for the adducts (i.e., m/z ) 550 for adduct A and m/z ) 516 for adduct B). The assigned
Oxidative Reactivity of p-Aminoclonidine
Chem. Res. Toxicol., Vol. 10, No. 9, 1997 1035
Scheme 1. Mechanisms of Oxidative Bioactivation to Reactive Intermediates for Amodiaquine (A) and Epinephrine (B) and Proposed Mechanism for Oxidative Bioactivation of Apraclonidine to a Reactive Species (C)
structures of the apraclonidine-glutathione conjugates are consistent with the mass spectral data and the 1H NMR spectrum obtained for purified adduct A (see the Materials and Methods section).
Discussion The long term ocular use of apraclonidine is associated with local allergic reactions that are sufficiently severe to require the discontinuation of therapy. This behavior is similar to that reported for epinephrine, a nonselective adrenergic receptor agonist, in which 80% of long term users were unable to continue therapy (18). The severity of the local reactions to apraclonidine and epinephrine has limited the utility of these agents in glaucoma therapy. A hypothesis for the allergenicity of these agents has been advanced by Butler et al. (8), who postulate that “adrenergic agents may reduce the volume of conjunctival cells, thereby producing a widening of the inter-cellular spaces through which potential allergens may reach the subepithelial tissues to produce the described local reactions.” This hypothesis implicates all adrenergic agents to be ocular allergens and has significant ramifications for the therapeutic utility of this class of agents. We propose an alternate hypothesis that recognizes the oxidative sensitivities of apraclonidine and epinephrine
and their potential bioactivation to reactive intermediates. The subsequent covalent binding of the reactive intermediate to tissue macromolecules produces antigens capable of initiating the allergic reactions. The facile oxidation of apraclonidine by the nonspecific oxidative enzyme systems HRP, MPO/Cl-, and LPO (Table 1) and the formation of GSH adducts derived from the bioactivated species support this mechanistic proposal underlying the observed allergy. Our initial studies were done with hydrogen peroxide-supported HRP (9), because of its widespread use as a nonspecific, one-electron oxidant in analogous studies of the oxidative bioactivation of foreign and endogenous compounds (17, 19-21). The studies of LPO, MPO, and DAO, which are enzymes of mammalian origin unlike HRP, may be more relevant for understanding the bioactivation of these drugs in humans. For example, the oxidation of xenobiotics to reactive species by MPO has been studied extensively as a model for mammalian bioactivative processes, since MPO appears to be a prominent xenobiotic-metabolizing enzyme in human peripheral blood leukocytes (22). Table 1 summarizes the data for oxidation of amodiaquine, epinephrine, apraclonidine, clonidine, and brimonidine by HRP, LPO, MPO/Cl- and DAO. The data demonstrate the oxidative lability of amodiaquine, epinephrine, and apraclonidine to the various oxidative
1036 Chem. Res. Toxicol., Vol. 10, No. 9, 1997
enzyme systems. Only brimonidine and clonidine proved stable to all of the enzymatic oxidation conditions employed in this study. Significantly, these two agents have been reported to have relatively low allergenic potential (23, 24). In general, the half-lives for enzymatic oxidation of these compounds were found to parallel the electrochemical oxidation potentials in the order: amodiaquine ∼ epinephrine < apraclonidine , clonidine ∼ brimonidine. Oxidative bioactivation to a reactive intermediate followed by covalent binding to biological macromolecules is a common mechanism for many known allergens and protoxins (10-14, 25). For example, the bioactivation of amodiaquine (Scheme 1A) to toxic and allergic species has been demonstrated to involve bioactivation-conjugation with thiol moieties on proteins (13). The oxidation of catecholamines including epinephrine is established to proceed via orthoquinone intermediates (14) to produce dopaquinone congeners which may polymerize to neuromelanin or bind to tissue macromolecules (Scheme 1B). We propose an analogous series of transformations for apraclonidine illustrated by the formation of the GSH adducts in Scheme 1C. Although we have employed nonspecific oxidases as models to address the potential for metabolism of apraclonidine to reactive species, it is possible that oxidation of apraclonidine may occur through both nonspecific and specific enzyme-catalyzed mechanisms in the eye. The eye is endowed with many specific enzyme systems (26) such as cytochrome P450 and cyclooxygenase for the metabolism of various foreign substances. Transformation of apraclonidine by these enzymes may also lead to the putative bis-iminoquinone reactive intermediate. These enzymes, therefore, may play an important role in the development of apraclonidine allergenicity in vivo. Our data support the hypothesis that the allergenicity of some adrenergic agonists correlates with their oxidative lability. We believe it is this potential for bioactivation to reactive species which may undergo protein conjugation and not a general adrenergic agonism effect that underlies the observed allergenicities. Thus, the development of oxidatively stable R2 adrenergic agonists may provide nonallergenic compounds for the treatment of glaucoma.
Acknowledgment. The authors thank Dr. Donald F. Hunt of the University of Virginia for making the LC/ MS analysis possible. Additionally, the authors would like to acknowledge the support of the NIH through Grant NS 64378 and through a Cell and Molecular Pharmacology Traineeship (C.D.T., T32GM07055).
References (1) Mittag, T. W. (1996) Adrenergic and dopaminergic drugs in glaucoma. The Glaucomas, (Ritch, R., Shields, M. B., Krupin, T., Eds) pp 1409-1424, 2nd edition, Mosby Publishing, Missouri. (2) Gharagozoloo, N. Z., Relf, S. J., and Brubaker, R. F. (1988) Aqueous flow is reduced by alpha-adrenergic agonist apraclonidine hydrochloride. Ophthalmology 95, 1217-1220. (3) Hurvitz, L. M., Kaufman, P. L., Robin, A. L., Weinreb, R. N., Crawford, K., and Shaw, B. (1991) New developments in the drug treatment of glaucoma. Drugs 41, 514-532.
Thompson et al. (4) Robin, A. L., Pollack, I. P., and deFaller, J. M. (1987) Effects of topical ALO 2145 (p-aminoclonidine hydrochloride) on the acute intraocular pressure rise after argon laser iridotomy. Arch. Ophthalmol. 105, 1208-1211. (5) Wiles, S. B., MacKenzie, D., and Ide, C. H. (1991) Control of intraocular pressure with apraclonidine hydrochloride after cataract extraction. Am. J. Ophthalmol. 111, 184-188. (6) Krawitz, P. L., and Podos, S. M. (1990) Use of apraclonidine in the treatment of acute angle closure glaucoma. Arch. Ophthalmol. 108, 1208-1209. (7) Robin, A. L. (1988) Short term effects of unilateral 1% apraclonidine therapy. Arch. Ophthalmol. 106, 912-915. (8) Butler, P., Mannschreck, M., Lin, S., Hwang, I., and Alvarado, J. (1995) Clinical experience with the long term use of 1% apraclonidine. Arch. Ophthalmol. 113, 293-296. (9) Thompson, C. D., Macdonald, T. L., Garst, M. E., Wiese, A., and Munk, S. A. (1997) Mechanisms of adrenergic agonist-induced allergy: bioactivation and antigen formation. Exp. Eye Res. 64, 767-773. (10) Basketter, D., Dooms-Goossens, A., Karlberg, A. T., and Lepoittevin, J. P. (1995) The chemistry of contact allergy: why is a molecule allergenic? Contact Dermatitis 32, 65-73. (11) Riley, R. J., and Leeder, J. S. (1995) In vitro analysis of metabolic predisposition to drug hypersensitivity reactions. Clin. Exp. Immunol. 99, 1-6. (12) Nelson, S. D. (1982) Metabolic activation and drug toxicity. J. Med. Chem. 25, 753-765. (13) Harrison, A. C., Kitteringham, N. R., Clarke, J. B., and Park, B. K. (1992) The mechanism of bioactivation and antigen formation of amodiaquine in the rat. Biochem. Pharmacol. 43, 1421-1430. (14) O’Brien, P. J. (1991) Molecular mechanism of quinone cytotoxicity. Chem.-Biol. Interact. 80, 1-41. (15) Van Gelder, B. F., and Slater, E. C. (1962) The extinction coefficient of cytochrome c. Biochem. Biophys. Acta 58, 593-595. (16) Thompson, C. D., Gulden, P. H., and Macdonald, T. L. (1997) Identification of modified atropaldehyde mercapturic acids in rat and human urine after felbamate administration. Chem. Res. Toxicol. 10, 457-462. (17) Potter, D. W., Miller, D. W., and Hinson, J. A. (1986) Horseradish peroxidase-catalyzed oxidation of acetaminophen to intermediates that form polymers or conjugate with gluthathione. Mol. Pharmacol. 29, 155-162. (18) Becker, B., and Morton, W. R. (1966) Topical epinephrine in glaucoma suspects. Am. J. Ophthalmology 62, 272-277. (19) Samokyszyn, V. M., Freeman, J. P., Maddipati, K. R., and Lloyd, R. V. (1995) Peroxidase-catalyzed oxidation of pentachlorophenol. Chem. Res. Toxicol. 8, 349-355. (20) Ross, D., Larsson, R., Andersson, B., Nilsson, U., Lindquist, T., Lindeke, B., and Moldeus, P. (1985) The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase and the fate of glutathione during such oxidations. Biochem. Pharmacol. 34, 343-351. (21) Josephy, P. D., Eling, T. E., and Mason, R. P. (1983) Oxidation of p-aminophenol catalyzed by horseradish peroxidase and prostaglandin synthase. Mol. Pharmacol. 23, 461-466. (22) Hofstra, A. H., and Uetrecht, J. P. (1993) Myeloperoxidasemediated activation of xenobiotics by human leukocytes. Toxicology 82, 221-242. (23) Ritch, R., Liebmann, J. M., Greenfield, D. S., and Lama, P. (1997) Alpha-agonist allergy: Is there cross reactivity between apraclonidine and brimonidine? Inv. Ophthalmol. Vis. Sci. 38, S559. (24) Scheper, R. J., von Blomberg, B. M. E., De Groot, J., Goeptar, A. R., Lang, M., Oostendorp, R. A. J., Bruynzeel, D. P., and Van Tol, R. G. L. (1990) Low allergenicity of clonidine impedes studies of sensitization mechanisms in guinea pig models. Contact Dermatitis 23, 81-89. (25) Orton, T. C., and Lowery, C. (1981) Practolol metabolism III. Irreversible binding of [14C]practolol metabolite(s) to mammalian liver microsomes. J. Pharmacol. Exp. Ther. 219, 207-212. (26) Kumar, G. N. (1994) Drug metabolizing enzyme systems in the eye. In Ocular Therapeutics and Drug Delivery (Reddy, I. K., Ed.) pp 149-167, Technomic Publishing Co., Pennsylvannia.
TX9700735
