The Drug-likeness Evaluation Of N-stearoyltyrosine, A New Endocannabinoid Mimic For Alzherimer’s Disease Therapy

Alzheimer’s disease(AD) is a common degenerative disorder in central nervous system among elderly people, who suffer from gradual deterioration of recognitive ability and the loss of self-care ability. The treatment of AD largely depends on medications, which are merely applicable for early and mid stage symptoms or local blockade of the AD progress with poor therapeutic effect and severe toxicity and side effects. Therefore, it is drastically urged to develop new medicines for attenuating and preventing AD. N-arachidonoylethanolamine(anandamide, AEA) was the first reported endocannabinoid with versatile physiological actions in neuroprotective sectors and effectively counteracting various pathes during the progress of neurodegenerative diseases.N-stearoyltyrosine(Ns Tyr) is an N-fatty-acyl amino acid mimicking AEA. Previously in vivo and in vitro tests of Ns Tyr have shown its neuroprotective effects in certain neural damage models such as oxygen-glucose deprivation(OGD) insult, glutamate-induced insult in primary neural cells, oxidative toxicity in PC12 cells, chronic cerebral hypoperfusion(CCH) and global cerebral ischemia reperfusion(IR) on gerbil jirds, etc in a dose-dependent manner. Thus it is of great necessity to perform drug-likeness tests involving preclinical pharmaceutical tests(optimization in drug synthesis schemes, study on physicochemical properties and stability), pharmacokinetics study and in vitro safety evaluation on Ns Tyr and its salts that are endowed with great potential and promising application scope in AD treatment.In this essay, it is the first time for us to integrally report the synthesis schemes of Ns Tyr and its salts by the hundred grams. We also established in vivo/in vitro determination methodology of Ns Tyr via HPLC, whose excellent profiles as convenient manipulation, high sensitivity and specificity as well as cheap cost in operation and maintenance have been testified through the preclinical trials. Then we performed in vitro evaluation of safety, who was comprised of four parts: cytotoxicity trials, genotoxicity trials, cardiotoxicity trials and reproductive trials. The outcomes of these in vitro trials together with the results of the acute toxicity trial would give us an overall prediction of expected therapeutic window and medication security.In this paper, all determination indexes had been validated and saturated the detection requirements for in vivo and in vitro quantification of Ns Tyr and its salts. As illustrated by the comparison between Ns Tyr and its salts, Ns Tyr-2K demonstrated significant improvement in physicochemical and pharmacokinetic properties. The solubilities were 51.70±0.29 mg/L for Ns Tyr-2K, 92.65±0.52mg/L for Ns Tyr-2Na and 27.62±0.43mg/L for Ns Tyr in aqua. The plasma protein binding ratios were 22.79%±3.26% and 38.46%±4.58% for Ns Tyr and Ns Tyr-2K respectively. The primary pharmacokinetic parameters were(0.75±0.24) h,(0.60±0.17) h in Tmax,(0.11±0.48) μg/m L,(2.40±0.33) μg/m L in Cmax,(1.49±0.96) h,(2.90±0.75) h in T1/2 and(0.13±0.09) μg*h/m L,(3.49±0.61) μg*h/m L in AUC0-72, respectively. Cytotoxicity trial results indicated that Ns Tyr has no obvious toxic effects on proliferation of normal human liver cells like Chang liver cellline with an IC50 at 62 μmol/L in a time-independent manner. The inspection of several organ indexes(weight, organ coefficient, sperm and ovary functionality) in reproductive trial showed no toxic effects on SD rats’ genital system. Results in Ames trial expressed no significant inhibitory or antiseptic effects of Ns Tyr in any tested bacterial strain. Thus without proof of ability to induce mutation in DNA by Ns Tyr, the speculation of genotoxicity could never be projectioned. And finally with no observation of blockade of h ERG potassium channel or prolonged QT interval observed, Ns Tyr was presumed with no apparent cardiotoxicity.To summarize, this paper was to conduct preclinical tests in three major parts as preclinical pharmaceutical tests, pharmacokinetics study and in vitro safety evaluation on Ns Tyr and its salts in order to give theoretical data and exploitative basis for druggability evaluation and establishment of quality control standard.