Dual HDAC/BRD4 Inhibitors Relieves Neuropathic Pain by Attenuating Inflammatory Response in Microglia After Spared Nerve Injury

Abstract

Despite the effort on developing new treatments, therapy for neuropathic pain is still a clinical challenge and combination therapy regimes of two or more drugs are often needed to improve efficacy. Accumulating evidence shows an altered expression and activity of histone acetylation enzymes in chronic pain conditions and restoration of these aberrant epigenetic modifications promotes pain-relieving activity. Recent studies showed a synergistic activity in neuropathic pain models by combination of histone deacetylases (HDACs) and bromodomain and extra-terminal domain (BET) inhibitors. On these premises, the present study investigated the pharmacological profile of new dual HDAC/BRD4 inhibitors, named SUM52 and SUM35, in the spared nerve injury (SNI) model in mice as innovative strategy to simultaneously inhibit HDACs and BETs. Intranasal administration of SUM52 and SUM35 attenuated thermal and mechanical hypersensitivity in the absence of locomotor side effects. Both dual inhibitors showed a preferential interaction with BRD4-BD2 domain, and SUM52 resulted the most active compound. SUM52 reduced microglia-mediated spinal neuroinflammation in spinal cord sections of SNI mice as showed by reduction of IBA1 immunostaining, inducible nitric oxide synthase (iNOS) expression, p65 nuclear factor-κB (NF-κB) and p38 MAPK over-phosphorylation. A robust decrease of the spinal proinflammatory cytokines content (IL-6, IL-1ß) was also observed after SUM52 treatment. Present results, showing the pain-relieving activity of HDAC/BRD4 dual inhibitors, indicate that the simultaneous modulation of BET and HDAC activity by a single molecule acting as multi-target agent might represent a promise for neuropathic pain relief.

Keywords: BET; Cytokines; HDAC; Microglia; NF-kB; Neuropathic pain.

Fig. 1

Effect of dual HDAC/BRD4 inhibitors on acute thermal pain. A Antinociceptive profile of SUM52 against an acute thermal stimulus (hot plate test). B Time course study showing that SUM52 100 μM increased the pain threshold 90 min after the intranasal administration. C and D SUM35 (5–500 μM) did not produce any significant antinociceptive effect. E Normalizing data to the control group, SUM52 100 μM produced a significant analgesic effect compared to SUM35 100 μM treated mice (C) (n = 8 per group). F Chemical structure of SUM35 and SUM52

Fig. 2

SM52 relieves pain hypersensitivity in SNI mice (n = 8 per group). Dose–response curve of SUM52 (10–100 µM) showed thermal (A) and mechanical (C) antiallodynic activity in the ipsilateral side (ipsi) of SNI mice compared to before treatment values. Comparison of the effect of SUM52 in the contra and ipsilateral side showed the absence of any thermal (B) or mechanical (D) analgesic effect in the contralateral side

Fig. 3

SUM35 attenuated pain hypersensitivity in SNI mice (n = 8 per group). Dose–response curve of SUM52 (10–200 µM) showed thermal (A) and mechanical (C) antiallodynic activity in the ipsilateral side (ipsi) of SNI mice compared to before treatment values. Comparison of the effect of SUM52 in the contra and ipsilateral side showed the absence of any thermal (B) or mechanical (D) analgesic effect in the contralateral side

Fig. 4

Time-course comparison of the antiallodynic activity of dual inhibitors. Comparison of thermal (A) and mechanical (C) antiallodynic activity of effective dose of SUM35 and SUM52 normalized to the CTRL group in SNI mice detected on day 7 (d7) and 14 (d14) post-surgery. Comparison of the time-course curves for the highest effective dose of the dual inhibitors against thermal (B) and mechanical (D) hypersensitivity. Lack of locomotor impairment by SUM52. At the highest effective dose, SUM52 did not alter motor coordination (E), spontaneous motility or exploratory activity (F) in comparison with vehicle-treated CTRL SNI mice. n = 8 per group

Fig. 5

Time-course comparison of the antiallodynic activity of epigenetic modulators. Comparison of thermal (A) and mechanical (D) antiallodynic activity of SAHA, iBET762 and their combination showing potentiating effects in SNI mice. Comparison between SUM52 and SAHA + iBET762 combination against thermal (B) and mechanical (C) allodynia showed comparable efficacy between treatments. Comparison between SUM35 and SAHA + iBET762 combination against thermal (E) and mechanical (F) allodynia showed a lower antiallodynic activity by the dual inhibitor (n = 8 per group)

Fig. 6

Effect of dual inhibitors on HDAC1 and BRD4 protein. A Inhibition curves for SUM35 and SUM52 on BRD4-BD1 in comparison with ( +)-JQ1 (SUM35 IC₅₀ = 1722 nM; SUM52 IC₅₀ = 758 nM). B Inhibition curves for SUM35 and SUM52 on BRD4-BD2 in comparison with ( +)-JQ1 (SUM35 IC₅₀ = 118 nM; SUM52 IC₅₀ = 10.5 nM). C Partial inhibition of HDAC1 enzymatic activity by SUM35 and SUM52 in comparison with SAHA. Microphotographs (D) and quantification analysis (E) of intrinsic fluorescence of SUM52 detected in spinal cord samples of SNI mice after i.n. delivery compared to vehicle-treated control mice. ***P < 0.001 vs CTRL (F) SNI procedure increased the expression of spinal BRD4 in the ipsilateral side on day 7 (d7) and 14 (d14) post-surgery that returned to basal values after SUM52 treatment. G Spinal cord samples from SNI mice showed an increased expression of HDAC1 protein in the ipsilateral side (SNI) on d7 and d14 that was completely prevented by SUM52 administration (SUM52 SNI). Dashed lines represent the protein expression value from SNI contra. *P < 0.05, **P < 0.01 vs contra; §P < 0.05, §§P < 0.01 vs vehicle-treated SNI ipsi (SNI)

Fig. 7

Effect of SUM52 on SNI-induced microglia activation and neuroinflammation. A Intrinsic fluorescence of SUM52 was detected in IBA1 positive microglial cells of SNI mice (merged images). Scale bar = 20 µm. B Immunofluorescence images of lumbar spinal cord of SNI mice (scale bar 100 µm) with the quantification analysis that showed the reduction of IBA1 immunostaining by SUM52. Dashed lines represent IBA1 immunostaining value from SNI contra. **P < 0.01, *** P < 0.001 vs contra; P < 0.05 vs vehicle-treated SNI ipsi (SNI). Spinal cord samples from SNI mice, collected on day 7 (d7) and 14 (d14) post-surgery, showed an increase of the proinflammatory microglia marker iNOS (C), increased phosphorylation of the proinflammatory transcription factor p65 (D) and p38 MAPK (E), increased levels of proinflammatory cytokines IL-6 (F) and IL-1ß (G) in the ipsilateral side that were reduced up to basal levels by SUM52. Dashed lines represent the protein expression value from SNI contra. *P < 0.05, **P < 0.01 vs contra; §P < 0.05, §§P < 0.01 vs vehicle-treated SNI ipsi (SNI)