Safety and pharmacological characterization of the molecular tweezer CLR01 - a broad-spectrum inhibitor of amyloid proteins' toxicity

Abstract

Background: The "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics.

Methods: Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration.

Results: Examination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "process-specific" mechanism of action.A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h.

Conclusion: Estimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Figure 1

Schematic representation of the interaction between CLR01 and Lys. CLR01 is depicted in blue and Lys in black. The Coulombic attraction between a negatively charged phosphate group at the bridgehead of CLR01 and the positively charged ϵ-NH₃⁺ group of Lys is illustrated in cyan. The binding is stabilized also by hydrophobic interaction between the hydrocarbon side arms of CLR01 and the butylene chain of the Lys.

Figure 2

Impact of CLR01 on tubulin polymerization. Tubulin was allowed to polymerize in the absence or presence of increasing concentrations of CLR01. Perturbation of the polymerization was observed only at 1,000 μM CLR01. The data are an average of three independent experiments and are shown as mean ± SEM.

Figure 3

Liver histopatholologic analysis of mice 24 h following a single IP injection of CLR01. Hepatocytes from A) vehicle-treated, and B) 10-mg/kg-treated mice show moderate amounts of glycogen vacuolation. C) Zone-1 hepatocytes from 100-mg/kg-treated mice show glycogen vacuolation. Zone-2 hepatocytes are normal sized. Zone-3 hepatocytes are pale with granular eosinophilic cytoplasm and some nuclei show pyknosis.

Figure 4

CLR01 plasma concentration following different routes of administration. The graph shows levels of CLR01 in plasma by intravenous (black line) or subcutaneous (blue line) injection at 1 mg/kg, or by oral gavage (red line) at 10 mg/kg over 24 h. Data are given as mean ± SD.

Figure 5

Correction for radioactivity from residual blood in the brain. Comparison of brain perfusion to remove residual blood with subtraction of calculated levels of blood radioactivity at 10 μl of blood per g of brain tissue. Data are given as mean ± SEM. The results were not significantly different and the subtraction method was used in the following experiments.

Figure 6

Blood CLR01 levels in young, middle-aged, and old WT and 3×Tg mice. CLR01 radioactivity levels, measured by scintillation counting, are given as CPM per ml of blood for the six mouse groups between 0.5 - 24 h. At 8 h post administration CLR01 levels drop to 5 - 10% of values observed at 0.5 h. Data are given as mean ± SEM.

Figure 7

Percent brain penetration of CLR01 at 1 h. Percent of CLR01 radioactivity per g of brain was calculated as a function of blood radioactivity levels per ml at 1 h following IV administration of CLR01 ((CPM/g)/(CPM/ml) × 100). Data are given as mean ± SEM. *p < 0.05.

Figure 8

Brain CLR01 levels in young, middle-aged, and old WT and 3×Tg mice. CLR01 radioactivity levels are given per g of brain. Most group × time combinations fall between 10,000 - 20,000 CPM/g (marked with red lines). Double injection studies in aged WT mice show on average double the radioactivity levels of single injection group, 22 m WT. Aged WT mice dosed with 5× the amount of CLR01, show on average 5-times the radioactivity levels of the 1× group, 22 m WT. Data are given as mean ± SEM.

Figure 9

CLR01 dephosphorylation. Molecular structure of theoretical successive CLR01 dephosphorylations at the bridgehead to monophosphate and then to hydroquinone.