Orally Bioavailable Prodrugs of ψ-GSH: A Potential Treatment for Alzheimer's Disease

Abstract

Addressing glycation-induced oxidative stress in Alzheimer's disease (AD) is an emerging pharmacotherapeutic strategy. Restoration of the brain glyoxalase enzyme system that neutralizes reactive dicarbonyls is one such approach. Toward this end, we designed, synthesized, and evaluated a γ-glutamyl transpeptidase-resistant glyoxalase substrate, ψ-GSH. Although mechanistically successful, the oral efficacy of ψ-GSH appeared as an area in need of improvement. Herein, we describe our rationale for the creation of prodrugs that mask the labile sulfhydryl group. In vitro and in vivo stability studies identified promising prodrugs that could deliver pharmacologically relevant brain levels of ψ-GSH. When administered orally to a mouse model generated by the intracerebroventricular injection of Aβ1-42, the compounds conferred cognitive benefits. Biochemical and histological examination confirmed their effects on neuroinflammation and oxidative stress. Collectively, we have identified orally efficacious prodrugs of ψ-GSH that are able to restore brain glyoxalase activity and mitigate inflammatory and oxidative pathology associated with AD.

Figure 1

Behavioral assessment and analysis of brain oxidative stress of APP/PS1 mice treated with saline (A/P saline) or oral ψ-GSH (A/P ψ-GSH) and age-matched nontransgenic mice (NTG) for 12 weeks. (A–E) Cognitive assessment was conducted using the Morris water maze test. Performance during the hidden platform training period (four trials per day for 4 consecutive days) was measured by mean escape latency (A) and the path length (B) required to locate the submerged platform. (C, D) Escape latency and the path length results of visible platform trials conducted at the end of the hidden platform training. (E) Retention of memory was assessed by a probe trial 24 h after the hidden platform training. Significant impairment was observed in A/P saline mice compared to the NTG saline group, while oral ψ-GSH displayed only moderate improvement in memory retention (expressed as percent time spent in the area that previously contained the platform). (F–J) Analysis of amyloid burden expressed as soluble (F) and insoluble (G) Aβ1–42 and oxidative stress markers such as protein carbonyls (H), TBARS (I), and reactive oxygen species (ROS, J). Marginal alleviation of oxidative stress and reduction in amyloid burden was observed in the oral ψ-GSH-treated group. Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001).

Figure 2

Chemical structures of ψ-GSH and its prodrugs.

Scheme 1. Synthesis of Prodrugs of ψ-GSH Compounds 1–3 and 5–7

Scheme 2. Synthesis of the ψ-GSH Prodrug 4

Figure 3

(A, B) Conversion of ψ-GSH prodrugs into ψ-GSH examined by incubation with tissue homogenates. The presence of ψ-GSH was detected in the brain (A) and liver (B) incubates of prodrugs 1, 5, and 7. Data are normalized to the highest concentration of ψ-GSH detected in each incubation reaction. No significant differences in the highest ψ-GSH levels were detected among all prodrugs tested. (C, D) In vivo conversion of the lead prodrugs 5 and 7 into ψ-GSH after oral administration in CD1 mice at 250 mg/kg dose in comparison with a therapeutically effective dose of ψ-GSH (500 mg/kg, i.p.). Plasma levels of prodrugs (C) are consistently higher than those achieved by ψ-GSH itself at the time points tested. Prodrug treatment resulted in higher plasma concentrations of ψ-GSH when compared to the levels achieved by i.p. ψ-GSH. A similar increase in the levels of prodrugs and ψ-GSH was also observed in the brain tissues of these mice (D).

Figure 4

Oral ψ-GSH prodrug treatment restored cognitive impairment induced by i.c.v. Aβ1–42. (A) Timeline of the experimental procedure. Three days after pretreatment with the prodrugs, i.c.v. injection of Aβ1–42 was performed and then followed by continued treatment with the prodrugs for an additional 8 days. The T-maze spontaneous alternation test was used to assess cognitive function. (B) Decreased alternation observed in saline-treated Aβ-injected mice was restored to levels comparable to vehicle control mice after prodrug 7 treatment. Treatment with ψ-GSH (i.p), but not oral ψ-GSH, showed significant improvement in alternation. (C) Reciprocal reduction in the number of re-entries was observed in the prodrug-treated mice compared to saline-treated Aβ-injected mice. No spatial bias and motor impairment were evident in any of the treatment groups as assessed by the ratio of arm entries (D) and the total time spent to complete the task (E), respectively. Data are shown as the mean ± SEM. Statistical significance was assessed by a one-way ANOVA with Tukey’s post hoc test (*p  < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). The timeline (A) was created with BioRender.com.

Figure 5

Treatment with the ψ-GSH prodrug 7 reduced reactive astrogliosis and microglial reactivity in i.c.v. Aβ-injected mice. (A) Representative images of reactive astrocytes in the dorsal hippocampus visualized by the GFAP antibody. (B) Quantification of GFAP staining in the dorsal hippocampus of saline and prodrug-treated cohorts. (C) Representative images of activated microglia in the dorsal hippocampus visualized by the Iba1 antibody. (D) Quantification of Iba1 staining in the dorsal hippocampus of the saline and prodrug-treated cohorts. For comparisons between saline, Aβ, and Aβ+ prodrug 7 treated groups, a one-way ANOVA with Tukey’s post hoc test was used for data analysis. (**p < 0.01, ***p < 0.005).

Figure 6

(A, B) Restoration of glyoxalase function was confirmed by quantitation of brain GSH levels (A) and advanced glycation endproducts (AGEs, B). Prodrugs 5 and 7 significantly increased the levels of reduced GSH compared to the Aβ-only group. Consequently, the total AGE content, a surrogate for brain methylglyoxal levels, was significantly reduced. Data are presented as the mean ± SEM, and the groups were compared with a one-way ANOVA with Tukey’s post hoc test (*p < 0.05, **p < 0.01).