GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy

Abstract

Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.

Keywords: GABAA receptors; hepatic encephalopathy; hyperammonemia; neurosteroids.

Fig. 1.

Scheme showing the experimental design for hyperammonemic (HA; A) rats and portacaval shunt (PCS; B) rats.

Fig. 2.

Representative patch-clamp current measurements showing GR3027 antagonism of the 3α,21-dihydroxy-5α-pregnan-20-one (THDOC)-enhanced GABA modulation of α1β2γ2L and α5β3γ2L GABA_A receptors and no inhibition of GABA. A: 1 μM GR3027 antagonism of 100 nM THDOC enhanced 30 μM GABA-mediated current response with the α1β2γ2L GABA_A receptor. B: 1 μM GR3027 did not antagonize the 30 μM GABA response of the α1β2γ2L GABA_A receptor. C: concentration response of the GR3027 antagonism of 100 nM THDOC enhanced 30 μM GABA-mediated current response with the α1β2γ2L GABA_A receptor. D: 1 μM GR3027 antagonism of 200 nM THDOC enhanced 0.3 μM GABA-mediated current response with the α5β3γ2L GABA_A receptor. E: 1 μM GR3027 did not antagonize the 0.3 μM GABA response of the α5β3γ2L GABA_A receptor. F: concentration response of the GR3027 antagonism of 200 nM THDOC enhanced 0.3 μM GABA-mediated current response with the α5β3γ2L GABA_A receptors.

Fig. 3.

GR3027 restores motor coordination in beam walking in hyperammonemic and PCS rats. A: control (C V) or hyperammonemic (HA V) rats treated with vehicle and hyperammonemic rats treated with 3 (HA 3), 10 (HA 10), or 20 (HA 20) mg/kg GR3027. B: sham-operated control rats (SM V) or PCS rats treated with vehicle (PCS V) and PCS rats treated with 0.7 (PCS0.7) or 2.5 (PCS2.5) mg/kg GR3027. Values are means ± SE for number of rats indicated under each bar. *Different from control or sham-operated rats: *P < 0.05. Different from hyperammonemic or PCS rats: ^aP < 0.05, ^aaP < 0.01, ^aaaP < 0.001.

Fig. 4.

GR3027 restores spatial memory in the Morris water maze in hyperammonemic and PCS rats. Spatial learning and memory was assessed in control (C V) or hyperammonemic (HA V) rats treated with vehicle and hyperammonemic rats treated with 3 (HA 3), 10 (HA 10), or 20 (HA 20) mg/kg GR3027 (A and B) and in sham-operated control rats (SM V) or PCS rats treated with vehicle (PCS V) and PCS rats treated with 0.7 (PCS 0.7) or 2.5 (PCS 2.5) mg/kg GR3027 (C and D). A and C: escape latencies to reach the platform during learning sessions. B and D: time spent in the correct quadrant during the memory test. Values are means ± SE for the number of rats indicated under each bar. Different from control or sham rats: *P < 0.05. Different from hyperammonemic or PCS rats: ^aP < 0.05.

Fig. 5.

GR3027 restores spatial learning in the radial maze in hyperammonemic and PCS rats. Spatial learning in the radial maze was assessed in control (C V) or hyperammonemic (HA V) rats treated with vehicle or (for hyperammonemic rats) treated with 3 (HA 3), 10 (HA 10) or 20 (HA 20) mg/kg GR3027 (A and B), in sham-operated control rats (SM V) or PCS rats treated with vehicle (PCS V), and in PCS rats treated with 0.7 (PCS 0.7) or 2.5 (PCS 2.5) mg/kg GR3027 (C and D). A and C: working errors during the different sessions. B and D: working errors during days 1 and 2. Values are means ± SE for the number of rats indicated under each bar. Different from control or sham-treated rats: *P < 0.05. Different from hyperammonemic or PCS rats: ^aP < 0.05, ^aaP < 0.01.

Fig. 6.

GR3027 partially restores the circadian rhythm of spontaneous motor activity. Ambulatory counts (A–D) and vertical activity (E–H) were assessed in control (C) and hyperammonemic (HA) rats (A, C, E, G) treated with vehicle (V) or with 20 mg/kg GR3027 and in sham-operated control rats (SMV) or PCS rats (B, D, F, H) treated with vehicle (PCSV) or with 0.7 (PCS0.7) or 2.5 (PCS2.5) mg/kg GR3027. Motor activity during night and day is shown in A, B, E and F and the ratios of activity night to day in C, D, G, and H. Values are means ± SE of 8 rats per group. Different from hyperammonemic or PCS rats: ^aP < 0.05, ^aaP < 0.01. Different from control/sham: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

GR3027 exposures in plasma and in the brain at time for behavioral testing. A and B: in hyperammonemic (A) and PCS (B) rats, the total plasma concentrations of GR3027 are shown in μM. C and D: in hyperammonemic (C) and PCS (D) rats, the unbound brain concentrations of GR3027 are shown in nmol/kg. Note the similar exposures in the different rat models with the doses used, in hyperammonemic rats 3, 10, and 20 mg·kg⁻¹·day⁻¹ and in rats with PCS 0.7 and 2.5 mg·kg⁻¹·day⁻¹. Data are from the end of the study, i.e., after 9 wk of daily treatments with GR3027 in sesame oil given subcutaneously once daily. GAMSA, GABA_A receptor-modulating steroid antagonist.