Effect of premedications in a murine model of asparaginase hypersensitivity

Abstract

A murine model was developed that recapitulates key features of clinical hypersensitivity to Escherichia coli asparaginase. Sensitized mice developed high levels of anti-asparaginase IgG antibodies and had immediate hypersensitivity reactions to asparaginase upon challenge. Sensitized mice had complete inhibition of plasma asparaginase activity (P = 4.2 × 10(-13)) and elevated levels of mouse mast cell protease 1 (P = 6.1 × 10(-3)) compared with nonsensitized mice. We investigated the influence of pretreatment with triprolidine, cimetidine, the platelet activating factor (PAF) receptor antagonist CV-6209 [2-(2-acetyl-6-methoxy-3,9-dioxo-4,8-dioxa-2,10-diazaoctacos-1-yl)-1-ethyl-pyridinium chloride], or dexamethasone on the severity of asparaginase-induced allergies. Combining triprolidine and CV-6209 was best for mitigating asparaginase-induced hypersensitivity compared with nonpretreated, sensitized mice (P = 1.2 × 10(-5)). However, pretreatment with oral dexamethasone was the only agent capable of mitigating the severity of the hypersensitivity (P = 0.03) and partially restoring asparaginase activity (P = 8.3 × 10(-4)). To rescue asparaginase activity in sensitized mice without requiring dexamethasone, a 5-fold greater dose of asparaginase was needed to restore enzyme activity to a similar concentration as in nonsensitized mice. Our results suggest a role of histamine and PAF in asparaginase-induced allergies and indicate that mast cell-derived proteases released during asparaginase allergy may be a useful marker of clinical hypersensitivity.

Fig. 1.

Schedule for sensitization to E. coli asparaginase and sample collection. (A) Mice received intraperitoneal injections of E. coli asparaginase (10 μg or 2.25 IU per mouse, green arrows) formulated with aluminum hydroxide adjuvant (1 mg, white arrows) on day 0 and day 14 of the immunization schedule to sensitize mice to asparaginase. On day 24, mice were challenged with an intravenous dose of E. coli asparaginase (100 μg or 22.5 IU per mouse, green arrow) to induce hypersensitivity reactions. Samples collected on day 23 (red arrow) were used to assess anti-asparaginase antibody titers before the asparaginase challenge on day 24. For a subset of mice, including those receiving oral dexamethasone, prechallenge samples were available only on day 17 of treatment (red arrow, n = 39). Mice received pretreatment on day 24 (blue arrow) with CV-6209, antihistamine (triprolidine, cimetidine), or a single dose of dexamethasone (i.p.) in an attempt to mitigate the severity of the allergic reaction. (B) These pretreatment agents were administered 5 minutes, 30 minutes, or 2 hours before the asparaginase challenge, respectively. (C) Additional mitigation strategies included pretreatment with multiple doses of dexamethasone (i.p.) on days 0, 14, and 24 of treatment, and pretreatment with continuous oral dexamethasone starting 7 days before, same day, or 7 days after the initial asparaginase immunization dose. Samples collected 2 hours after the challenge on day 24 (black arrow) were used to measure asparaginase activity, antibody titers, and mMCP-1. Data in subsequent figures are color-matched accordingly to indicate the sample used for the laboratory value [black corresponding to day 24 samples (postchallenge dose of asparaginase) and red to day 23 or day 17 samples (prechallenge)].

Fig. 2.

Mice sensitized to E. coli asparaginase developed asparaginase-induced hypersensitivity reactions when challenged. (A) The rectal temperature of sensitized (○) and nonsensitized (□) mice was monitored for 2 hours after intravenous administration of E. coli asparaginase (100 μg) at day 24 (as per Fig. 1A). (B) Compared with nonsensitized controls, sensitized mice with asparaginase-induced allergies experienced a drop in rectal temperature that was quantified by the AUC of the rectal temperature versus time curve (P = 3.3 × 10⁻¹⁰). (C) Sensitized mice had low to no detectable asparaginase activity (P = 4.2 × 10⁻¹³) and (D) developed high anti-asparaginase IgG antibody titers compared with nonsensitized mice (red open circles, P = 1.1 × 10⁻⁷). Sensitized mice showed a drop in antibody levels when comparing the prechallenge and postchallenge samples at day 23 versus day 24 (red versus black open circles, P = 6.7 × 10⁻⁵), suggesting the formation of asparaginase–anti-asparaginase IgG immune complexes. (E) Sensitized mice with asparaginase-induced allergies had elevated levels of mMCP-1 compared with nonsensitized mice in samples collected 2 hours after the challenge (P = 6.1 × 10⁻³).

Fig. 3.

Pretreatment with triprolidine, CV-6209, or dexamethasone (Dex) mitigates the severity of asparaginase-induced allergies. Mice sensitized to E. coli asparaginase were pretreated with triprolidine, cimetidine, the PAF receptor antagonist CV-6209, single-dose dexamethasone, or a combination of the agents as per Fig. 1B; multiple-dose dexamethasone or oral dexamethasone was given as per Fig. 1C. All pretreatment agents except for cimetidine and oral dexamethasone given 7 days after the initial asparaginase immunization dose (at day 0) were able to mitigate the severity of the reaction compared with nonpretreated, sensitized mice (P < 0.03); however, the combination of antihistamine and CV-6209 showed the best ability to reduce the severity of the reaction compared with nonpretreated, sensitized mice (P = 1.2 × 10⁻⁵).

Fig. 4.

Oral dexamethasone given 7 days before asparaginase sensitization can partially restore asparaginase activity. Nonpretreated mice sensitized to asparaginase showed low to no detectable asparaginase activity compared with nonsensitized mice (P = 3.2 × 10⁻¹⁴). Pretreatment with antihistamine, CV-6209, or intraperitoneal dexamethasone (Dex) was not able to rescue asparaginase activity relative to nonpretreated, sensitized mice (P > 0.05). However, mice receiving continuous oral dexamethasone starting 0 or 7 days before asparaginase sensitization had partially restored asparaginase activity compared with nonpretreated, sensitized mice (P = 4.8 × 10⁻² and 8.3 × 10⁻⁴, respectively).

Fig. 5.

Dexamethasone effects the development of anti-asparaginase IgG antibodies. (A) On day 23 [before pretreatment with intraperitoneal single-dose dexamethasone (Dex), triprolidine, cimetidine, or CV-6209], all sensitized mice had elevated anti-asparaginase IgG levels compared with nonsensitized controls (P < 0.04). Mice that received pretreatment with multiple doses of parenteral dexamethasone (with or without triprolidine) on days 0 and 14 before the day 23 blood collection had similar antibody levels as sensitized, nonpretreated mice (P > 0.05). (B) Mice receiving continuous oral dexamethasone starting 0 or 7 days before the asparaginase sensitization had lower measured anti-asparaginase IgG levels compared with sensitized, nonpretreated mice (P = 1.0 × 10⁻³ and 4.1 × 10⁻⁴, respectively) in samples available on day 17 of treatment (before the asparaginase challenge).

Fig. 6.

Elevated levels of mMCP-1 were detected in sensitized mice after asparaginase-induced allergies. Elevated levels of mMCP-1 were detected in all sensitized mice after asparaginase-induced allergies (P < 0.02), except for mice pretreated with a single dose of intraperitoneal dexamethasone (Dex) or a single dose of intraperitoneal dexamethasone in combination with triprolidine.

Fig. 7.

Asparaginase dose adjustments restored activity levels in sensitized mice. Sensitized mice were challenged with 2-, 5-, or 10-fold higher asparaginase doses. (A) The severity of the reaction (drop in rectal temperature) showed a dose-dependent relationship (P < 1.0 × 10⁻³ compared with 1×, sensitized), and mice pretreated with triprolidine and CV-6209 receiving a 5-fold higher dose were able to mitigate the reaction relative to nonpretreated mice (P = 9.8 × 10⁻⁴). (B) Furthermore, dose adjustments ≥5-fold were able to restore asparaginase activity in samples collected 2 hours after the challenge compared with nonsensitized mice (P > 0.05).

Fig. 8.

The severity of asparaginase-induced allergies was correlated with mMCP-1 concentrations. The lower the rectal temperature AUC, the more severe the allergic reaction, and the higher the mMCP-1 concentrations from samples collected after the challenge on day 24 for nonsensitized mice (purple circle) and for sensitized mice receiving 100 μg (gray circle), 200 μg (red circle), 500 μg (green circle), or 1 mg (blue circle) of asparaginase per mouse during the challenge (R² = 0.577, P = 3.0 × 10⁻¹⁶).