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. 2019 Feb 14;20(4):811.
doi: 10.3390/ijms20040811.

Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain

Mei Yee Kwan  1 , Anthony Choo  2 , Taleen Hanania  3 , Afshin Ghavami  4 , Jose Beltran  5 , John Shea  6 , Amidi Barboza  7 , Andrew Hu  8 , Marcie Fowler  9 , Venugopal Rao Neelagiri  10 , Irving Sucholeiki  11
Affiliations

Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain

Mei Yee Kwan et al. Int J Mol Sci. .

Abstract

There is an unmet medical need for the development of non-addicting pain therapeutics with enhanced efficacy and tolerability. The current study examined the effects of AQU-118, an orally active inhibitor of metalloproteinase-2 (MMP-2) and MMP-9, in the spinal nerve ligation (SNL) rat model of neuropathic pain. Mechanical allodynia and the levels of various biomarkers were examined within the dorsal root ganglion (DRG) before and after oral dosing with AQU-118. The rats that received the SNL surgery exhibited significant mechanical allodynia as compared to sham controls. Animals received either vehicle, positive control (gabapentin), or AQU-118. After SNL surgery, the dorsal root ganglion (DRG) of those rats dosed with vehicle had elevated messenger RNA (mRNA) expression levels for MMP-2, IL1-β & IL-6 and elevated protein levels for caspase-3 while exhibiting decreased protein levels for myelin basic protein (MBP) & active IL-β as compared to sham controls. Rats orally dosed with AQU-118 exhibited significantly reduced mechanical allodynia and decreased levels of caspase-3 in the DRG as compared to vehicle controls. Results demonstrate that oral dosing with the dual active, MMP-2/-9 inhibitor, AQU-118, attenuated mechanical allodynia while at the same time significantly reduced the levels of caspase-3 in the DRG.

Keywords: MMP-2; MMP-9; allodynia; caspase-3; dorsal root ganglion; inhibitor; matrix metalloproteinase; neuropathic; pain; spinal nerve ligation.

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Conflict of interest statement

I.S. owns stock in Aquilus Pharmaceuticals, Inc. All other authors declare they have no competing interests.

Figures

Figure A1
Figure A1
Statistical analysis of post-operative von-Frey (VF) baseline paw withdrawal threshold (PWT) of the ipsilateral hind paws. All Chung surgical groups showed a significant decrease in the PWT compared to the sham surgical group. No significant difference in post-operative baseline PWT was detected between the individual Chung surgical groups.
Figure A2
Figure A2
Fisher’s protected least significant difference (PLSD) test of post-operative VF baseline PWT of the ipsilateral hind paws.
Figure A3
Figure A3
Ipsilateral Two-Way RM ANOVA statistical analysis for behavioral portion (presented in Figure 1).
Figure A4
Figure A4
Contralateral Two-Way RM ANOVA statistical analysis for behavioral portion (presented in Figure 2).
Figure 1
Figure 1
Mechanical Response Threshold: Spinal Nerve Ligation (SNL) Chung Model (Chung), Ipsilateral (Ipsi) Paw Withdrawal. Paw withdrawal thresholds following SNL-surgery for ipsilateral hind paws. Data are presented as mean ± SEM. * p < 0.05 vs. (SNL) Chung + Vehicle group on the same day. Post-Surgery Baseline was performed 15 days after SNL-surgery.
Figure 2
Figure 2
Mechanical Response Threshold: Spinal Nerve Ligation (SNL) Chung Model (Chung), Contralateral (Contra) Paw Withdrawal. Contralateral paw withdrawal thresholds following surgery and administration of AQU-118. Data are presented as mean ± SEM.
Figure 3
Figure 3
MMP2 (A) and MMP9 (B) mRNA expression levels in ipsilateral DRG for sham and SNL vehicle groups. MMP-2 mRNA expression levels were found to be significantly elevated after SNL-surgery while MMP-9 mRNA was found to be not significant. Data presented as the average from n = 10 per group ± SEM and analyzed with a two-tailed unpaired t-test (ns, not significant; ****, p < 0.0001).
Figure 4
Figure 4
IL-1β (A) and IL-6 (B) mRNA expression levels in ipsilateral DRG for sham and SNL vehicle groups. Both IL-1β and IL-6 mRNA expression levels were found to be significantly elevated after SNL-surgery in vehicle group compared to sham. Data presented as the average from n = 10 per group ± SEM and analyzed a with two-tailed unpaired t-test. (***, p < 0.001; ****, p < 0.0001).
Figure 5
Figure 5
Correlation between MMP2 and (A) IL-1β mRNA and (B) IL-6 mRNA expression levels in left DRG of SNL & sham Vehicle groups. Pearson correlation coefficients (with 95% confidence) were calculated for sham vehicle (r = 0.7372; two-tailed p value = 0.015) and SNL vehicle (r = 0.8135; two-tailed p value = 0.0042) for MMP2 vs IL-1β mRNA. Similar analysis of MMP2 vs. IL-6 mRNA showed no clear correlation with IL-6 transcript level: sham vehicle (r = 0.4912; two tailed p value = 0.1494) and SNL vehicle (r = 0.1622; two tailed p value = 0.6543).
Figure 6
Figure 6
Comparison of cleaved IL-1β protein level in ipsilateral and contralateral DRG for sham and vehicle groups. Cleaved, active IL-1β protein levels in left (ipsilateral) DRG (A) significantly decreased in the vehicle group (n = 10) compared to the sham (n = 10). (B) No difference between the sham (n = 10) and vehicle group (n = 10) in right (contralateral) DRG. Data presented as the average ± SEM. An unpaired t-test was performed: ns, not significant; **, p < 0.01.
Figure 7
Figure 7
Comparison of Caspase-3 and MBP protein levels in in the ipsilateral DRG for sham and vehicle groups. (A) Caspase-3 protein levels (via western blot) in the left (ipsilateral) DRG significantly increased in the vehicle group compared to the sham. (B) MBP levels (via western blot) significantly decreased for both the upper band (20 kDa) and (C) lower band (15 kDa) in the vehicle group of the left (ipsilateral) DRG as compared to the sham. Protein level is presented normalized to GAPDH and relative to the sham group. Data presented as the average ± SEM. An unpaired t-test was performed: ***, p < 0.001; ****, p < 0.0001.
Figure 8
Figure 8
Comparison of caspase-3 and MBP in the ipsilateral DRG for vehicle and AQU-118 groups. (A) Caspase-3 protein levels in the left (ipsilateral) DRG significantly decreased in the AQU-118 group (n = 20) compared to the vehicle (n = 20). (B) There were no significant changes in MBP levels in both the upper band (20 kDa) and (C) lower band (15 kDa) in AQU-118 group (n = 20) of the left (ipsilateral) DRG as compared to the vehicle group (n = 20). Protein level is presented normalized to GAPDH and relative to the vehicle group. Data presented as the average ± SEM. An unpaired t-test was performed: ns, not significant; *, p < 0.05.
Figure 9
Figure 9
Comparison of MMP2 mRNA expression levels versus caspase-3 in a subset of ipsilateral DRG tissue for both vehicle and AQU-118 groups. Caspase 3 protein and MMP-2 mRNA expression levels were performed in the ipsilateral DRG tissue (via western blot) for both the AQU-118 (n = 10) and vehicle (n = 10) groups and compared to each other. The protein level is presented normalized to GAPDH and relative to the vehicle group (no samples were excluded in analysis). Data presented as the average ± SEM. An unpaired t-test was performed: **, p < 0.01.
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