Targeting the overproduction of peroxynitrite for the prevention and reversal of paclitaxel-induced neuropathic pain

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain is a major dose-limiting side effect of a large number of antitumoral agents including paclitaxel (Taxol). Thus, CIPN is one of most common causes of dose reduction and discontinuation of what is otherwise a life-saving therapy. Neuropathological changes in spinal cord are linked to CIPN, but the causative mediators and mechanisms remain poorly understood. We report that formation of peroxynitrite (PN) in response to activation of nitric oxide synthases and NADPH oxidase in spinal cord contributes to neuropathological changes through two mechanisms. The first involves modulation of neuroexcitatory and proinflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10 and IL-4) cytokines in favor of the former. The second involves post-translational nitration and modification of glia-derived proteins known to be involved in glutamatergic neurotransmission (astrocyte-restricted glutamate transporters and glutamine synthetase). Targeting PN with PN decomposition catalysts (PNDCs) not only blocked the development of paclitaxel-induced neuropathic pain without interfering with antitumor effects, but also reversed it once established. Herein, we describe our mechanistic study on the role(s) of PN and the prevention of neuropathic pain in rats using known PNDCs (FeTMPyP(5+) and MnTE-2-PyP(5+)). We also demonstrate the prevention of CIPN with our two new orally active PNDCs, SRI6 and SRI110. The improved chemical design of SRI6 and SRI110 also affords selectivity for PN over other reactive oxygen species (such as superoxide). Our findings identify PN as a critical determinant of CIPN, while providing the rationale toward development of superoxide-sparing and "PN-targeted" therapeutics.

Figure 1.

Targeting PN blocks neuropathic pain. A–F, Compared with vehicle (○), administration of paclitaxel (●) led to a time-dependent development of mechano-allodynia and mechano-hyperalgesia that was attenuated by daily subcutaneous injections (D0–D15) of l-NAME (100 mg · kg⁻¹ · d⁻¹; ■, A, B), l-NIL (30 mg · kg⁻¹ · d⁻¹, ▾, A, B), apocynin (100 mg · kg⁻¹ · d⁻¹, ▴, A, B), or FeTMPyP⁵⁺ (C, D) and MnTE-2-PyP⁵⁺ (E, F) at 1, ■; 3, ▾; or 10 mg · kg⁻¹ · d⁻¹, ▴. These drugs did not affect withdrawal thresholds in vehicle groups (A, B: l-NAME, □; l-NIL, ▿; and apocynin, ▵; C, D: FeTMPyP⁵⁺, □; E, F: MnTE-2-PyP⁵⁺, □). Results are expressed as mean ± SD for 6 rats and analyzed by two-tailed, two-way repeated-measures ANOVA with Bonferroni comparisons. *p < 0.001 for paclitaxel versus vehicle and ^†p < 0.001 for paclitaxel versus paclitaxel + experimental drugs.

Figure 2.

Neuropathic pain does not develop upon PNDC discontinuation. When compared with vehicle (○), administration of paclitaxel (●) led to a time-dependent development of mechano-allodynia (A) and mechano-hyperalgesia (B). These events were attenuated by daily subcutaneous injections (D0–D15) of FeTMPyP⁵⁺ (■) and MnTE-2-PyP⁵⁺ (▴) at 10 mg · kg⁻¹ · d⁻¹. Moreover, mechano-allodynia and mechano-hyperalgesia did not develop over an additional 10 d through D25 following discontinuation of FeTMPyP⁵⁺ (■) and MnTE-2-PyP⁵⁺ (▴). Results are expressed as mean ± SD for 6 rats and analyzed by two-tailed, two-way repeated-measures ANOVA with Bonferroni comparisons. *p < 0.001 for paclitaxel versus vehicle and ^†p < 0.01 or ^††p < 0.001 for paclitaxel versus paclitaxel + experimental drugs.

Figure 3.

Neuropathic pain increases nitric oxide synthase and NADPH oxidase activation and PN formation in the spinal dorsal horn. A–E, Compared with vehicle, paclitaxel increased the activity of constitutive/calcium-dependent NOS (black bars) and inducible/calcium-independent NOS (gray bars) (A), and NADPH (B). l-NAME (100 mg · kg⁻¹ · d⁻¹) blocked constitutive/calcium-dependent NOS (A, black bars) activity, whereas l-NAME and l-NIL (30 mg · kg⁻¹ · d⁻¹) inhibited inducible/calcium-independent NOS (A, gray bars) activity. Paclitaxel also increased NADPH oxidase activity, which was attenuated with apocynin (100 mg · kg⁻¹ · d⁻¹, B). As indicated by the formation of NT (red) in fixed frozen spinal cord sections (C–E), PN formation was low in dorsal horn from vehicle-treated animals (C) but substantially increased with paclitaxel treatment (D). This increase was attenuated by MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹; E). C–E are single-plane confocal images. The inset in D is a projected image of an 18 plane Z-stack to illustrate the morphology of a NT-positive cell in the volume of a 10 μm dorsal horn section. Negative controls using normal rabbit serum for NT exhibited only low levels of background fluorescence. Micrographs are taken from superficial layers of dorsal horn of the spinal cord (lumbar enlargement) and are representative of at least 3 images from 3 different animals performed on different days. Enzymatic activities are mean ± SD for 6 rats and analyzed by two-tailed, one-way ANOVA with Dunnett's comparisons to paclitaxel group. *p < 0.001 for paclitaxel versus Veh; ^†p < 0.01 or ^††p < 0.001 for paclitaxel versus paclitaxel + experimental drugs.

Figure 4.

Attenuation of post-translational nitration of MnSOD, GLT-1, and GS by MnTE-2-PyP⁵⁺. A–D, Compared with vehicle, paclitaxel led to post-translational nitration (A) and enzymatic inactivation of mitochondrial MnSOD (B), events blocked by daily subcutaneous injections (D0–D15) of MnTE-2-PyP⁵⁺ (1–10 mg · kg⁻¹ · d⁻¹). Moreover, compared with vehicle, paclitaxel led to significant nitration of the glutamate transporter GLT-1 (C) and GS (D) in spinal cord tissues, events attenuated in rats treated with MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹; C, D). Results are expressed as mean ± SD for 6 rats and analyzed by two-tailed, one-way ANOVA with Dunnett's comparisons to paclitaxel group. *p < 0.05 or **p < 0.001 for paclitaxel versus Veh; ^†p < 0.01 or ^††p < 0.001 for paclitaxel versus paclitaxel + MnTE-2-PyP⁵⁺.

Figure 5.

MnTE-2-PyP⁵⁺ attenuates GFAP, OX-42, and pro-inflammatory cytokine expression and favors anti-inflammatory cytokine production. A–D, Compared with vehicle (Veh), paclitaxel (P) increased GFAP (A), OX-42 (B), TNF-α (C), and IL-1β (D) expression (n = 4), which was significantly attenuated by MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹, i.p.). E, F, Compared with Veh, paclitaxel (P) increased anti-inflammatory IL-10 and IL-4 (E, F, n = 5) and was substantially enhanced by MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹, i.p.). Results are expressed as mean ± SD for (n) rats and analyzed by two-tailed, one-way ANOVA with Dunnett's comparisons to paclitaxel group. *p < 0.05, **p < 0.01, or ***p < 0.001 for paclitaxel versus Veh; ^†p < 0.001 for paclitaxel versus paclitaxel + MnTE-2-PyP⁵⁺.

Figure 6.

Intrathecal (i.th.) delivery of SODm/PNDCs blocks the development of neuropathic pain and modulates spinal cytokine production. A–D, Compared with vehicle (Veh, ○), paclitaxel (P, ●) led to a time-dependent development of mechano-allodynia (A, C) and mechano-hyperalgesia (B, D) that was attenuated by daily i.th. delivery of FeTMPyP⁵⁺ (A, B) or MnTE-2-PyP⁵⁺ (C, D) at 0.1, ■; 0.3, ▾; and 1 nmol/d, ▴). These drugs did not affect withdrawal thresholds in vehicle groups (□, A–D). E, F, Compared with vehicle group, paclitaxel increased TNF-α and IL-1β (E, F) and this was inhibited by MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹, i.th.). G, H, Paclitaxel led to a modest increase in IL-10 and IL-4 (G, H) production compared with vehicle; but was substantially enhanced with daily i.th. delivery of MnTE-2-PyP⁵⁺ (10 mg · kg⁻¹ · d⁻¹). Results are expressed as mean ± SD for 6 rats. Behavioral data were analyzed by two-tailed, two-way repeated-measures ANOVA with Bonferroni comparisons. Data for cytokine production were analyzed by two-tailed, one-way ANOVA with Bonferroni comparisons to paclitaxel group. *p < 0.01 or **p < 0.001 for paclitaxel versus vehicle; ^†p < 0.01 or ^††p < 0.001 for paclitaxel versus paclitaxel + MnTE-2-PyP⁵⁺.

Figure 7.

Systemic or intrathecal delivery of FeTMPyP⁵⁺ or MnTE-2-PyP⁵⁺ reverse established paclitaxel-induced neuropathic pain. A–D, Intraperitoneal (A, B; 10 mg/kg) or intrathecal (C, D; 1 nmol/kg) administration of FeTMPyP⁵⁺ (●) or MnTE-2-PyP⁵⁺ (■) on D16 rapidly reversed mechano-allodynia and mechano-hyperalgesia. FeTMPyP⁵⁺ (○) or MnTE-2-PyP⁵⁺ (□) did not affect baseline mechano-allodynia (A, C) and mechano-hyperalgesia (B, D) values. Results are mean ± SD for n = 6 rats and were analyzed by two-tailed, two-way repeated-measures ANOVA with Bonferroni comparisons to treatment-matched D0 or baseline values. *p < 0.001 for baseline versus D0; ^†p < 0.001 behavior at t versus treatment-matched baseline values.

Figure 8.

Evaluation of SRI6 and SRI110 activities versus MnTM-4-PyP^5+, MnTE-2-PyP⁵⁺ and Ebselen. A, Structures of PNDCs and Ebselen. B, Inhibition of boronic acid oxidation assay. C, SOD-like activity of SRI6 (●) and SRI110 (■) is significantly less than FeTMPyP⁵⁺ (▴); p < 0.001, n = 3 at each concentration. ^aSies and Masumoto, 1997; ^bFerrer-Sueta and Radi, 2009; ^cHunt et al., 1997.

Figure 9.

Oral delivery of “SO-sparing” PNDCs blocks the development of neuropathic pain. A–D, Compared with the vehicle group (○), paclitaxel (●) induced mechano-allodynia (A, C) and mechano-hyperalgesia (B, D) that was blocked by daily (D0–D15) oral delivery (30 mg · kg⁻¹ · d⁻¹) of SRI6 (▾; A, B) or SRI110 (♦; C, D). SRI6 (▿) or SRI110 (♢) alone did not affect withdrawal thresholds in vehicle groups (A–D). E, F, Compared with vehicle, paclitaxel increased TNF-α and IL-1β (E, F) that was significantly attenuated by SRI6 or SRI110 (30 mg · kg⁻¹ · d⁻¹). G, H, Compared with vehicle, paclitaxel increased anti-inflammatory IL-10 and IL-4 (G, H), which was substantially enhanced by SRI6 or SRI110 (30 mg · kg⁻¹ · d⁻¹). Results are expressed as mean ± SD for 5 animals. Behavioral data were analyzed by two-tailed, two-way repeated-measures ANOVA with Bonferroni comparisons. Data for cytokine production were analyzed by two-tailed, one-way ANOVA with Dunnett's comparisons to paclitaxel group. *p < 0.05, **p < 0.01 or ***p < 0.001 for paclitaxel versus vehicle; ^†p < 0.01 or ^††p < 0.001 for paclitaxel versus paclitaxel + SRI6 or SRI110.