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. 2017 Oct;33(8):582-590.
doi: 10.1089/jop.2017.0037. Epub 2017 Jul 18.

The In Vivo Effects of the CB1-Positive Allosteric Modulator GAT229 on Intraocular Pressure in Ocular Normotensive and Hypertensive Mice

Elizabeth A Cairns  1 , Anna-Maria Szczesniak  1 , Alex J Straiker  2 , Pushkar M Kulkarni  3 , Roger G Pertwee  4 , Ganesh A Thakur  3 , William H Baldridge  5   6 , Melanie E M Kelly  1   6
Affiliations

The In Vivo Effects of the CB1-Positive Allosteric Modulator GAT229 on Intraocular Pressure in Ocular Normotensive and Hypertensive Mice

Elizabeth A Cairns et al. J Ocul Pharmacol Ther. 2017 Oct.

Abstract

Purpose: Orthosteric cannabinoid receptor 1 (CB1) activation leads to decreases in intraocular pressure (IOP). However, use of orthosteric CB1 agonists chronically has several disadvantages, limiting their usefulness as clinically relevant drugs. Allosteric modulators interact with topographically distinct sites to orthosteric ligands and may be useful to circumvent some of these disadvantages. The purpose of this study was to investigate the effects of the novel CB1-positive allosteric modulator (PAM) GAT229 on IOP.

Methods: IOP was measured using rebound tonometry in anesthetized normotensive C57Bl/6 mice and in a genetic model of ocular hypertension [nose, eyes, ears (nee) mice] before drug administration, and at 1, 6, and 12 h thereafter.

Results: In normotensive mice, topical administration of 5 μL GAT229 alone at either 0.2% or 2% did not reduce IOP. However, a subthreshold dose (0.25%) of the nonselective orthosteric CB1 agonist WIN 55,212-2, when combined with 0.2% GAT229, significantly reduced IOP compared with vehicle at 6 and 12 h. Similarly, combination of subthreshold Δ9-tetrahydrocannabinol (a nonselective orthosteric CB1 agonist; 1 mg/kg) with topical 0.2% GAT229 produced IOP lowering at 6 h. In nee mice, administration of topical 0.2% GAT229 or 10 mg/kg GAT229 alone was sufficient to lower IOP at 6 and 12 h, and 12 h, respectively.

Conclusions: The CB1 PAM GAT229 reduces IOP in ocular hypertensive mice and enhanced CB1-mediated IOP reduction when combined with subthreshold CB1 orthosteric ligands in normotensive mice. Administration of CB1 PAMs may provide a novel approach to reduce IOP with fewer of the disadvantages associated with orthosteric CB1 activation.

Keywords: allosteric modulator; cannabinoid receptor 1; cannabinoids; glaucoma; intraocular pressure.

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Conflict of interest statement

M.E.M.K. is the founder and director of Panag Pharma Inc. Panag develops phytotherapeutics for local and regional treatment of pain and inflammation. E.A.C., A.M.S., P.M.K., R.G.P., G.A.T., and W.H.B. have no existing competing financial interests.

Figures

<b>FIG. 1.</b>
FIG. 1.
The CB1 PAM GAT229 alone does not reduce IOP in normotensive C57Bl/6J mice. (A) The structure of the CB1 PAM GAT229. Given topically at either 0.2% (n = 7, B) or 2% (n = 4, C), GAT229 alone does not reduce IOP in normotensive C57Bl/6 mice 1, 6, or 12 h after administration. CB1, cannabinoid receptor 1; IOP, intraocular pressure; PAM, positive allosteric modulator.
<b>FIG. 2.</b>
FIG. 2.
The IOP-lowering effect of subthreshold WIN is potentiated by the CB1 PAM GAT229 in normotensive C57Bl/6J mice. (A) One hour after administration, but not at 6 or 12 h, 1% WIN significantly decreased IOP compared with contralateral eye receiving vehicle (Tocrisolve; −1.6 ± 0.7 and 0.4 ± 0.6 mmHg from baseline respectively, n = 9). However, a lower dose of WIN (0.25%) administered alone did not significantly decrease IOP compared to contralateral vehicle-treated eyes at any of the time points measured (n = 4), and was therefore considered as a subthreshold dose (B). (C) IOP was significantly lower in eyes receiving 0.2% GAT229 combined with subthreshold WIN (0.25%) at 6 and 12 h after application compared to vehicle (−0.9 ± 0.5 and −0.3 ± 0.9, 0.8 ± 0.8 and 4.2 ± 0.5, and 2.4 ± 1.1 and 5.6 ± 1.2 mmHg from baseline, respectively, n = 5). Paired t-tests, *P < 0.05. CB1, cannabinoid receptor 1; IOP, intraocular pressure; PAM, positive allosteric modulator.
<b>FIG. 3.</b>
FIG. 3.
The IOP-lowering effect of subthreshold Δ9-THC is also potentiated by the PAM GAT229. (A) 5 mg/kg Δ9-THC caused a significant reduction in IOP 1 h after i.p. administration compared to mice receiving i.p. vehicle (P < 0.01, mean difference 1.9 ± 0.6 mmHg, n = 5 mice per group). Administration of 1 mg/kg Δ9-THC (n = 10 mice) did not lower IOP compared with mice receiving vehicle at 1, 6, or 12 h (P > 0.05). (B) In mice receiving i.p. 1 mg/kg Δ9-THC, eyes receiving topical 0.2% GAT229 had significantly larger reduction in baseline IOP at 6 h after administration compared with contralateral eyes receiving topical vehicle (P < 0.05, mean difference 1.7 ± 0.6 mmHg, n = 9). *P < 0.05, **P < 0.01. IOP, intraocular pressure; PAM, positive allosteric modulator; Δ9-THC, Δ9-tetrahydracannabinol.
<b>FIG. 4.</b>
FIG. 4.
The nee mouse has ocular hypertension. Nee mice (nose, eyes, ears) have a mutation in Sh3pxd2b, a podosome adaptor protein, analogous to humans with Frank–Ter Haar syndrome., Compared with WT littermates, nee mice had significantly elevated IOP by p28 (13.9 ± 0.9 and 20.7 ± 2.4 mmHg) that persisted until adulthood (p∼100; 12.1 ± 0.8 and 22.7 ± 1.6 mmHg). T-tests, *P < 0.05, n = 5–19. IOP, intraocular pressure; nee, nose, eyes, ears; WT, wild type.
<b>FIG. 5.</b>
FIG. 5.
The CB1 PAM GAT229 reduces IOP alone in ocular hypertensive nee mice. (A) Six and 12 h following topical administration of 0.2% GAT229, IOP was significantly less compared to baseline than in eyes receiving vehicle (paired t-tests, n = 6, −6.9 ± 2.5 and 0.2 ± 2.5, and −9.9 ± 3.1 and −2.2 ± 1.2 mmHg from baseline, respectively). (B) Twelve hours following i.p. administration of 10 mg/kg GAT229, IOP was significantly reduced compared with nee mice receiving vehicle (t-test, n = 4 mice per group, −11.1 ± 2.2 and −2.4 ± 1.5 mmHg from baseline, respectively). *P < 0.05, **P < 0.01. CB1, cannabinoid receptor 1; IOP, intraocular pressure; PAM, positive allosteric modulator.
<b>FIG. 6.</b>
FIG. 6.
The IOP-lowering effect of subthreshold WIN is not potentiated by GAT229 in nee mice. Unlike normotensive mice (Fig. 2A), administration of 1% WIN did not reduce IOP in nee mice (n = 6; A). Neither subthreshold (0.25%) WIN (n = 4; B) nor the combination of subthreshold WIN and 0.2% GAT229 had a significant effect on IOP (n = 10, C). Paired t-tests. IOP, intraocular pressure.
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