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. 2024 Aug 26:15:1451634.
doi: 10.3389/fphar.2024.1451634. eCollection 2024.

The deuterated pyrazoloquinolinone targeting α6 subunit-containing GABAA receptor as novel candidate for inhibition of trigeminovascular system activation: implication for migraine therapy

Affiliations

The deuterated pyrazoloquinolinone targeting α6 subunit-containing GABAA receptor as novel candidate for inhibition of trigeminovascular system activation: implication for migraine therapy

Pi-Chuan Fan et al. Front Pharmacol. .

Abstract

Introduction: The α6 subunit-containing GABAA receptors (α6GABAARs) are highly expressed in the trigeminal ganglia (TG), the sensory hub of the trigeminovascular system (TGVS). Hypo-GABAergic transmission in the TG was reported to contribute to migraine-related behavioral and histopathological phenotypes. Previously, we found that Compound 6, an α6GABAAR-selective positive allosteric modulator (PAM), significantly alleviated TGVS activation-induced peripheral and central sensitization in a capsaicin-induced migraine-mimicking model.

Methods: Here, we tested whether the deuterated analogues of Compound 6, namely DK-1-56-1 and RV-I-29, known to have longer half-lives than the parent compound, can exert a similar therapeutic effect in the same model. The activation of TGVS was triggered by intra-cisternal (i.c.) instillation of capsaicin in male Wistar rats. Centrally, i.c. capsaicin increased the quantity of c-Fos-immunoreactive (c-Fos-ir) neurons in the trigeminal cervical complex (TCC). Peripherally, it increased the calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG, and caused CGRP release, leading to CGRP depletion in the dura mater.

Results: DK-I-56-1 and RV-I-29, administered intraperitoneally (i.p.), significantly ameliorated the TCC neuronal activation, TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin, with DK-I-56-1 demonstrating better efficacy. The therapeutic effects of 3 mg/kg DK-I-56-1 are comparable to that of 30 mg/kg topiramate. Notably, i.p. administered furosemide, a blood-brain-barrier impermeable α6GABAAR-selective antagonist, prevented the effects of DK-I-56-1 and RV-I-29. Lastly, orally administered DK-I-56-1 has a similar pharmacological effect.

Discussion: These results suggest that DK-I-56-1 is a promising candidate for novel migraine pharmacotherapy, through positively modulating TG α6GABAARs to inhibit TGVS activation, with relatively favourable pharmacokinetic properties.

Keywords: GABA; positive allosteric modulator; pyrazoloquinolinone; trigeminal ganglia; trigeminovascular system; α6GABAAR.

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Conflict of interest statement

A patent application has been jointly filed by P-CF, JC, and L-CC for the chemical structures and therapeutic applications of α6GABAAR-modulating PQ compounds, but the patent right is co-owned by the inventors’ institutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

FIGURE 1
FIGURE 1
Chemical structures of α6GABAAR-selective positive allosteric modulators (PAMs) used in this study. Note that DK-I-56-1 and RV-I-29 are deuterated analogues of Compound 6.
SCHEME 1
SCHEME 1
The timeline of the experimental procedure.
FIGURE 2
FIGURE 2
Effects of DK-I-56-1 (i.p.) on capsaicin-induced neuronal activation and anti-α6GABAAR effects of furosemide in the trigeminal cervical complex (TCC), trigeminal ganglia (TG) and dura mater. Immunohistograms (A-i) and the total number of activated neurons (A-ii), i.e. c-Fos-immunoreactive (c-Fos-ir) neurons, in the TCC of rats having received intracisternal (i.c.) instillation of 10 nmol capsaicin in the group pretreated with i.p. injection of DK-I-56-1 at 1 (D1), 3 (D3) or 10 (D10) mg/kg, as well as with 3 mg/kg DK with 20 mg/kg furosemide (i.p.) (D3+Furosemide). The sham group received ic. instillation of the vehicle instead of capsaicin (Sham). A close-up image (inset) of c-Fos-containing TCC neurons (A-i) is shown in each treatment group. Note that DK-I-56-1 at 1, 3 or 10 mg/kg significantly attenuates capsaicin-induced TCC neuronal activation. The inhibitory effect of DK-I-56-1 in the TCC was significantly reversed by furosemide, a highly selective allosteric inhibitor for α6GABAARs. (B-i) DK-I-56-1 at 1, 3 or 10 mg/kg significantly prevents capsaicin-induced CGRP-ir in TG. The capsaicin-induced CGRP-ir inhibited by DK-I-56-1 at 3 mg/kg is significantly reversed by furosemide (B-ii). (C-i) DK-I-56-1 at 3 or 10, but not 1, mg/kg significantly suppressed capsaicin-induced depletion of dura CGRP-ir. The capsaicin-induced CGRP depletion inhibited by DK at 3 mg/kg is significantly reversed by furosemide (C-ii). Scale bar: (A-i) 500 um (a); 100 um (b). (B-i) 500 um (a and b); 250 um (c). (C-i) 1,000 um (a); 400 um (b). 100 um (c). CC: central canal; Sp5C: spinal trigeminal nucleus caudalis. In each rat, total CGRP-ir fluorescence in 3 TG sections was measured. Shown is the average CGRP-ir fluorescence of all tested rats. The length of CGRP-ir nerve fibers (arrowhead), stained by immunohistochemistry, in the dura mater was quantified by ImageJ. The total length of CGRP-ir fibers (in pixel) in six fixed comparable areas of the dura mater in each rat was collected. Filled arrowhead: CGRP-ir nerve fiber; Empty arrowhead: dural vessel. Data are presented as median and interquartile ranges. The n number shown in parentheses is the number of tested rats. *p < 0.0125 x i, vs. the Vehicle (Capsaicin) group; # p < 0.01 x i, vs. the Sham group; i: the rank of p value (Supplementary Tables S1, S2). (Kruskal–Wallis test followed by post hoc Mann-Whitney U test with Benjamin-Hochberg correction). @ p < 0.05 by Mann-Whitney U test.
FIGURE 3
FIGURE 3
Effects of RV-I-29 (i.p.) on capsaicin-induced neuronal activation and anti-α6GABAAR effects of furosemide in the trigemino-cervical complex (TCC), trigeminal ganglia (TG) and dura mater. Immunohistograms (A-i) and (A-ii) c-Fos-immunoreactive (c-Fos-ir) neurons in the TCC of rats having received intracisternal (i.c.) instillation of 10 nmol capsaicin in the group pretreated with i.p. injection of RV-I-29 at 1 (R1), 3 (R3) or 10 (R10) mg/kg, as well as with 3 mg/kg RV plus 20 mg/kg furosemide (i.p.) (R3+Furosemide). The sham group received i.c. instillation of the vehicle instead of capsaicin (Sham). A close-up image (inset) of c-Fos-containing TCC neurons (A-i) is shown in each treatment group. Note that RV-I-29 at 3 or 10, but not 1 mg/kg significantly attenuates capsaicin-induced TCC neuronal activation. The inhibitory effect of RV in the TCC was significantly, but not fully, reversed by furosemide. (B-i) RV-I-29 at 3 or 10, but not 1 mg/kg significantly prevents capsaicin-induced CGRP-ir in TG (B-ii). The capsaicin-induced CGRP-ir inhibited by RV-I-29 at 3 mg/kg is partially reversed by furosemide. (C-i) RV-I-29 at 10, but not one or 3, mg/kg significantly suppressed capsaicin-induced depletion of dura CGRP-ir (C-ii). Scale bar, data presentation and statistical analyses are the same as in Figure 2. Note that the immunohistograms of Cap and Sham groups are the same as in Figure 2.
FIGURE 4
FIGURE 4
Comparison in effects of Compound 6, RV-I-29 and DK-I-56-1 (i.p.) on capsaicin-induced neuronal activation as well as topiramate in the trigemino-cervical complex (TCC), trigeminal ganglia (TG) and dura mater. (A) c-Fos-immunoreactive (c-Fos-ir) neurons in the TCC of rats having received intracisternal (i.c.) instillation of 10 nmol capsaicin in the group pretreated with i.p. injection of compound 6 (yellow bars), RV-I-29 (orange bars) or DK-I-56-1 (green bars) at 1, 3 or 10 mg/kg, as well as topiramate, a known clinically effective anti-migraine drug, at 30 mg/kg (brown bars). The sham group received i.c. instillation of the vehicle instead of capsaicin (Sham). Note that only DK-I-56-1, but not Compound 6 or RV-I-29 at 1 mg/kg significantly attenuates capsaicin-induced TCC neuronal activation. Although all the three drugs at 3 and 10 mg/kg significantly attenuates capsaicin-induced TCC neuronal activation, the effectiveness is borderline different at 3 mg/kg (p = 0.051) and significantly different at 10 mg/kg (p = 0.006) among the three drugs. DK at 10 mg/kg is significantly better than compound 6 (p = 0.004) or RV (p = 0.009). DK-I-56-1 at 3 mg/kg is better than compound 6 (p = 0.014). (B) In TG, only DK-I-56-1, but not Compound 6 or RV-I-29, at 1 mg/kg significantly attenuates capsaicin-induced CGRP-ir. The effectiveness is significantly different at 1 mg/kg (p = 0.026) among the three drugs. DK at 1 mg/kg is significantly better than compound 6 (p = 0.004). (C) In dura mater, the three drugs at 1 mg/kg do not significantly suppress capsaicin-induced depletion of dura CGRP-ir. Both Compound 6 and DK-I-56, but not RV-I-29 at 3 mg/kg, significantly attenuates capsaicin-induced CGRP-ir. All of them are effective at 10 mg/kg. The effectiveness is significantly different at 3 mg/kg (p = 0.026) among the three drugs. Compound 6 and DK-I-56 are better than RV-I-29 at 3 mg/kg (p = 0.011 and 0.043, respectively). Data presentation and statistical analyses are the same as in Figure 3. Differences among groups at the same dosage were compared using the Kruskal–Wallis test followed by post hoc Mann-Whitney U test. @ p < 0.05 and @@ p < 0.01 by Mann-Whitney U test.
FIGURE 5
FIGURE 5
Effects of DK-I-56-1 (p.o.) on capsaicin-induced neuronal activation in the trigemino-cervical complex (TCC), trigeminal ganglia (TG) and dura mater. Immunohistograms (A-i) and the total number of activated neurons (A-ii), i.e. c-Fos-immunoreactive (c-Fos-ir) neurons, in the TCC of rats having received intracisternal (i.c.) instillation of 10 nmol capsaicin in the group pretreated with p.o. administration of DK-I-56-1 at 1 (OD1), 3 (OD3) or 10 (OD10) mg/kg. The sham group received ic. instillation of the vehicle instead of capsaicin (Sham). A close-up image (inset) of c-Fos-containing TCC neurons (A-i) is shown in each treatment group. Note that oral treatment of DK-I-56-1 at 10 mg/kg significantly attenuates capsaicin-induced TCC neuronal activation. (B) DK-I-56-1 at 3 or 10 mg/kg significantly prevents capsaicin-induced CGRP-ir in TG. (C-i) DK-I-56-1 at 10, but not one or 3, mg/kg significantly suppressed capsaicin-induced depletion of dura CGRP-ir. (C-ii). Scale bar, data presentation and statistical analyses are the same as in Figure 2. *p < 0.0167 x i, vs. the Vehicle (Capsaicin) group; # p < 0.0125 x i, vs. the Sham group; i: the rank of p value (Supplementary Table S3). (Kruskal–Wallis test followed by post hoc Mann-Whitney U test with Benjamin-Hochberg correction). @ p < 0.05 by Mann-Whitney U test.

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