Airway peroxidases catalyze nitration of the {beta}2-agonist salbutamol and decrease its pharmacological activity

Abstract

β(2)-agonists are the most effective bronchodilators for the rapid relief of asthma symptoms, but for unclear reasons, their effectiveness may be decreased during severe exacerbations. Because peroxidase activity and nitrogen oxides are increased in the asthmatic airway, we examined whether salbutamol, a clinically important β(2)-agonist, is subject to potentially inactivating nitration. When salbutamol was exposed to myeloperoxidase, eosinophil peroxidase or lactoperoxidase in the presence of hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), both absorption spectroscopy and mass spectrometry indicated formation of a new metabolite with features expected for the nitrated drug. The new metabolites showed an absorption maximum at 410 nm and pK(a) of 6.6 of the phenolic hydroxyl group. In addition to nitrosalbutamol (m/z 285.14), a salbutamol-derived nitrophenol, formed by elimination of the formaldehyde group, was detected (m/z 255.13) by mass spectrometry. It is noteworthy that the latter metabolite was detected in exhaled breath condensates of asthma patients receiving salbutamol but not in unexposed control subjects, indicating the potential for β(2)-agonist nitration to occur in the inflamed airway in vivo. Salbutamol nitration was inhibited in vitro by ascorbate, thiocyanate, and the pharmacological agents methimazole and dapsone. The efficacy of inhibition depended on the nitrating system, with the lactoperoxidase/H(2)O(2)/NO(2)(-) being the most affected. Functionally, nitrated salbutamol showed decreased affinity for β(2)-adrenergic receptors and impaired cAMP synthesis in airway smooth muscle cells compared with the native drug. These results suggest that under inflammatory conditions associated with asthma, phenolic β(2)-agonists may be subject to peroxidase-catalyzed nitration that could potentially diminish their therapeutic efficacy.

Fig. 1.

Structures of salbutamol (1), 6-nitrosalbutamol (2), and salbutamol-derived nitrophenol (3).

Fig. 2.

Interaction of salbutamol with LPO/H₂O₂/NaNO₂ (A and B) and MPO/H₂O₂/NaNO₂ (C and D) systems. A and C, absorption spectra recorded during interaction of salbutamol (20 μM), with H₂O₂ (310 μM), NaNO₂ (300 μM), and LPO (4 nM) (A) or MPO (0.2 U/ml) (C). Spectra were recorded every 30 s. Arrows indicate direction of changes. Absorption peaks at 350 and 276 nm in spectrum a (before H₂O₂ addition) in A and C are from NO₂⁻ and salbutamol, respectively. Insets show time course of absorbance changes at 410 and 314 nm after bolus addition of H₂O₂. B, nitration of salbutamol (100 μM) measured at 410 nm versus [H₂O₂] (●) in the presence of NaNO₂ (250 μM) and LPO (6.5 nM), and versus [NaNO₂] (■) in the presence of H₂O₂ (104 μM) and LPO (10 nM). D, nitration of salbutamol (10 μM) measured at 410 nm versus [H₂O₂] (●) in the presence of NaNO₂ (1000 μM) and versus [NaNO₂] (■) in the presence of H₂O₂ (220 μM) and MPO (0.1 U/ml). All experiments were carried out in phosphate buffer (0.1 M, pH 7.0) containing DTPA (0.2 mM).

Fig. 3.

Absorption spectra of nitrosalbutamol measured at pH 7.9, 7.0, 6.7, 6.3, 5.2, and 4.2 (spectra a-f, respectively). Inset, A₄₁₀ plotted versus pH. The apparent pK_a of 6.6 was determined for the phenolic –OH group in nitrated salbutamol-derived products.

Fig. 4.

A and B, HPLC elution profiles of salbutamol (1, retention time 3.15 min) (A) and two mono-nitrated metabolites of salbutamol (2, retention time 6.19 and 3, retention time 6.75 min) (B). Nitration of salbutamol was carried out using a LPO/H₂O₂/NO₂⁻ nitration system in phosphate buffer, pH 7.0, containing DTPA (0.1 mM). C-E, positive ion electrospray ionization mass spectra of salbutamol 1 (m/z 240.15947, error: 207 ppb, eluted at 3.15 min) (C), nitrosalbutamol 2 (m/z 285.14456, error: 215 ppb, eluted at 6.19 min) (D), and salbutamol-derived nitrophenol 3 (m/z 255.13397, error: 142 ppb, eluted at 6.75 min) (E). F and G, MS/MS spectra and fragmentation patterns of the molecular ions 2 (m/z 285.14) (F) and 3 (m/z 255.13) (G).

Fig. 5.

A and B, HPLC elution profile of molecular ions of m/z 255.13 in breath condensate of asthma patients treated with salbutamol (A; retention time 6.80 min) and from a salbutamol nitrated in vitro (with MPO(LPO)/H₂O₂/NO₂⁻) (B; retention time 6.75 min). C and D, MS/MS spectra of the parent ion of m/z 255.13 in breath condensate (C) and from compound 3 produced by nitration of salbutamol by LPO/H₂O₂/NO₂⁻ in vitro (D). Peaks at m/z 237.17, 199.00, 181.08, and 130.25, originating from the parent ion of m/z 255.13 in D, coincide with peaks in C.

Fig. 6.

Nitration of salbutamol by LPO/H₂O₂/NO₂⁻: effect of ascorbate (A) and thiocyanate (B). A, ascorbate in a concentration-dependent manner causes a lag in net nitration of the drug. Shown are A₄₁₀ versus time traces recorded during interaction of salbutamol (100 μM), with H₂O₂ (4 mM), NaNO₂ (3.5 mM), and LPO (67 nM) in the absence and presence of 55, 88, and 109 μM ascorbate (traces a-d, respectively). B, thiocyanate inhibits nitration of salbutamol (20 μM) by LPO (10 nM), H₂O₂ (312 μM), and NaNO₂ (300 μM). Shown are A₄₁₀ versus time traces recorded in the absence (trace a) and presence of 10, 25, and 50 μM NaSCN (traces b-d, respectively). n = 2.

Fig. 7.

Inhibition by methimazole of salbutamol nitration by LPO/H₂O₂/NO₂⁻ (A) and MPO/H₂O₂/NO₂⁻ (B) systems measured as a change in absorbance at 410 nm. A, salbutamol (100 μM) reacted with LPO (73 nM), NaNO₂ (3.5 mM), and H₂O₂ (4 mM) in pH 7.0 buffer in the absence (trace a) and presence of 27, 55, and 109 μM methimazole (traces b-d, respectively). In traces c and d additional doses of LPO were added at the time indicated by arrows. n = 2. B, salbutamol (50 μM) reacted with MPO (0.2 U/ml), NaNO₂ (5 mM), and H₂O₂ (2.1 mM) in pH 7.0 buffer in the absence (trace a) and presence of 25, 50, and 100 μM methimazole (traces b-d,). Shown are typical kinetic traces.

Fig. 8.

Inhibition by dapsone of salbutamol nitration by LPO/H₂O₂/NO₂⁻ measured as a change in absorbance at 410 nm. Salbutamol (97 μM) reacted with LPO (65 nM), NaNO₂ (3.4 mM), and H₂O₂ (4 mM) in phosphate buffer, pH 7.0, in the absence (trace a) and presence of 2.5, 5, 10, 50, and 100 μM dapsone (traces b-f, respectively). In traces d and e additional doses of LPO were added at the time indicated by arrows. n = 2.

Fig. 9.

Inhibition by dapsone of salbutamol nitration by peroxidase/H₂O₂/NO₂⁻ systems. The extent of nitration is expressed as a change in ΔA₄₁₀ versus control (dapsone omitted) taken as 100% after 10, 30, and 60 min reaction for LPO, MPO, and EPO nitrating systems, respectively. LPO system: [Salbutamol] = 100 μM, [NaNO₂] = 3.4 mM, [H₂O₂] = 4 mM, [LPO] = 65 nM. MPO system: [Salbutamol] = 50 μM, [NaNO₂] = 5 mM, [H₂O₂] = 5 mM, [MPO] = 0.2 U/ml. EPO system: [Salbutamol] = 50 μM, [NaNO₂] = 5 mM, [H₂O₂] = 2 mM, [EPO] = 0.075 U/ml. Reactions were carried out in phosphate buffer, pH 7.0, containing dimethyl sulfoxide (5% v/v) (n = 2).

Fig. 10.

A, ¹²⁵I-CYP displacement from β₂-receptors by intact (○) and nitrated (●) salbutamol. B, cAMP production by smooth muscle cells stimulated by intact (○) and nitrated (●) salbutamol.

Fig. 11.

Proposed mechanisms of the enzymatic nitration of the aromatic moiety of salbutamol. Salbutamol (1) is oxidized to the phenoxyl-type radical (1a), which rearranges to a resonance structure 1b (only one of several resonance forms is shown), in which the unpaired electron is localized in the aromatic ring. The concomitantly generated NO₂^• radicals add to 1b to form nitrated salbutamol, which rearranges to 2 (m/z 285.14). In an alternative pathway, the radical 1a undergoes intramolecular hydrogen atom transfer from the –CH₂OH moiety at C₃ to form a methoxyl-type radical 1c, which after elimination formaldehyde (H₂CO), produces the aromatic radical 1d, which reacts with NO₂^• to form the nitrophenol 3 (m/z = 255.13). In general, the mechanisms of nitration catalyzed by EPO and LPO systems may be similar to that shown in the scheme for MPO. R = CH(OH)CH₂NHC(CH₃)₃.