General toxicity assessment of the novel aldose reductase inhibitor cemtirestat.

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b] indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 "Inactive" outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic ß-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 µM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies.

Artichoke leaf extract, as AKR1B1 inhibitor, decreases sorbitol level in the rat eye lenses under high glucose conditions ex vivo.

In the previous study, the artichoke leaf extract showed effective inhibition of AKR1B1, the first enzyme of polyol pathway, which reduces high level of glucose to osmotically active sorbitol, important for development of chronic diabetic complications. In the present study, the effect of artichoke leaf extract and of several participating phenols (caffeic acid, chlorogenic acid, quinic acid, and luteolin) was tested on sorbitol level in rat lenses exposed to high glucose ex vivo, on cytotoxicity as well as on oxidative stress in C2C12 muscle cell line induced by high glucose in vitro. The concentration of sorbitol was determined by enzymatic analysis, the cytotoxicity was provided by WST-1 test and intracellular content of reactive oxygen species was determined by fluorescence of 2'-7'-dichlorofluorescein probe. The extract and the compounds tested showed significant protection against toxic effects of high concentration of glucose in both models. On balance, the artichoke leaf extract thus represents a prospective preventive agent of development of chronic diabetic complications, probably due to phenols content, concerning preclinical and clinical studies.

Structure optimization of tetrahydropyridoindole-based aldose reductase inhibitors improved their efficacy and selectivity.

In our previous study, tetrahydropyridoindoles carboxymethylated in position 8 were identified as aldose reductase (ALR2) inhibitors with mild efficacy and selectivity yet with significant antioxidant activity. In the present study we proceeded with optimization of the tetrahydropyridoindole scaffold by shifting the carboxymethyl pharmacophore from position 8 to position 5, with the aim to improve the biological activity. Commercial databases were screened for the presence of tetrahydropyridoindoles carboxymethylated in position 5 and an experimental set of eight compounds was created. Mild inhibition characterized by IC50 in micromolar range was recorded for compound 8 with the isopropyl substituent at the piperidine nitrogen (position 2). This alkylated tertiary nitrogen is characterized by a rather high basicity (pKa ∼ 10.4) with complete protonization at physiological pH. On the other hand, ALR2 inhibition activity of the low basicity derivatives 3-7 with an acyl substituted nitrogen in position 2 (pKa ∼ -1 to -3) was characterized with IC50 values in low and medium nanomolar region. Docking into the binding site of human recombinant enzyme AKR1B1 performed for 3 revealed an interaction network responsible for the high affinity and selectivity. In ex vivo experiment, sorbitol accumulation in isolated rat eye lenses was significantly inhibited by 3 in the presence of high glucose, starting at a concentration as low as 0.1 µM. Moreover, in streptozotocin-induced diabetic rats, compound 3 administered intragastrically (i.g., 50 mg/kg/day) for five consecutive days significantly inhibited sorbitol accumulation in red blood cells and the sciatic nerve. Molecular obesity indices predicted along with water solubility point an excellent "lead-likeness" of compound 3, with prospects of further structure optimizations.

Enhancing activity and selectivity in a series of pyrrol-1-yl-1-hydroxypyrazole-based aldose reductase inhibitors: The case of trifluoroacetylation.

Aldose reductase (ALR2) has been the target of therapeutic intervention for over 40 years; first, for its role in long-term diabetic complications and more recently as a key mediator in inflammation and cancer. However, efforts to prepare small-molecule aldose reductase inhibitors (ARIs) have mostly yielded carboxylic acids with rather poor pharmacokinetics. To address this limitation, the 1-hydroxypyrazole moiety has been previously established as a bioisostere of acetic acid in a group of aroyl-substituted pyrrolyl derivatives. In the present work, optimization of this new class of ARIs was achieved by the addition of a trifluoroacetyl group on the pyrrole ring. Eight novel compounds were synthesized and tested for their inhibitory activity towards ALR2 and selectivity against aldehyde reductase (ALR1). All compounds proved potent and selective inhibitors of ALR2 (IC50/ALR2 = 0.043-0.242 µΜ, Selectivity index = 190-858), whilst retaining a favorable physicochemical profile. The most active (4g) and selective (4d) compounds were further evaluated for their ability to inhibit sorbitol formation in rat lenses ex vivo and to exhibit substrate-specific inhibition.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

Antioxidant action of 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid, an efficient aldose reductase inhibitor, in a 1,1'-diphenyl-2-picrylhydrazyl assay and in the cellular system of isolated erythrocytes exposed to tert-butyl hydroperoxide.

OBJECTIVES: The subject of this study was 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (compound 1), an efficient aldose reductase inhibitor of high selectivity. The antioxidant action of 1 was investigated in greater detail by employing a 1,1'-diphenyl-2-picrylhydrazyl (DPPH) test and in the system of isolated rat erythrocytes. METHODS: First, the compound was subjected to the DPPH test. Second, the overall antioxidant action of the compound was studied in the cellular system of isolated rat erythrocytes oxidatively stressed by free radicals derived from the lipophilic tert-butyl hydroperoxide. The uptake kinetics of 1 was studied and osmotic fragility of the erythrocytes was evaluated. RESULTS: The DPPH test revealed significant antiradical activity of 1. One molecule of 1 was found to quench 1.48 ± 0.06 DPPH radicals. In the system of isolated erythrocytes, the compound was readily taken up by the cells followed by their protection against free radical-initiated hemolysis. Osmotic fragility of the erythrocytes was not affected by 1. CONCLUSIONS: The results demonstrated the ability of 1 to scavenge DPPH and to protect intact erythrocytes against oxidative damage induced by peroxyl radicals. By affecting both the polyol pathway and oxidative stress, the compound represents an example of a promising agent for multi-target pharmacology of diabetic complications.