Establishment Of A Methodology For The Detection Of A Novel HDAC6 Inhibitor And Preliminary Study Of Its Pharmacokinetics And Safety In Rats

ObjectiveAlzheimer’s disease（AD）is the most common form of dementia.The pathogenesis of this disease is attributed to the extracellular accumulation of β-amyloid protein（Aβ）plaques in the cortical and peripheral regions of the brain,as well as the intracellular neurofibrillary tangles formed by hyperphosphorylated Tau proteins.With the aggravation of aging,it is predicted that by 2050,1 out of every 85 individuals worldwide will suffer from AD,resulting in an increasingly heavy burden on healthcare.Although three innovative drugs for Alzheimer’s disease（Lecanemab,Aducanumab,and GV-971）have been approved for marketing in the past three years,they can only temporarily alleviate disease symptoms.Currently,there is no medication that has been proven to cure or halt the progression of AD.Therefore,it is necessary to continue searching for novel mechanisms and drug targets that drive disease progression,as well as to develop innovative drugs.W5(C₁₉H₁₆ClN₃O₂S)is a newly synthesized HDAC6 inhibitor with high selectivity.Previous in vitro studies have shown that W5 can inhibit Aβ aggregation,promote neuronal growth,and target metal homeostasis imbalance,suggesting a potential multi-target approach for anti-AD.In order to further investigate the absorption,distribution,and metabolism of W5 in animal bodies,this study aims to establish a fast and efficient HPLC method to detect the content of W5 in biological samples.According to this method,a preliminary study of W5 pharmacokinetics was carried out in rats to obtain the analysis results of relevant PK parameters and the penetration ability of the drug’s blood-brain barrier.It provides a reliable basis for subsequent application in clinical research.Furthermore,a preliminary study was conducted to investigate the safety of long-term administration of W5 in rats,laying the foundation for further validation of the drug’s pharmacokinetic effects.Methods1.Establishment of W5 biological sample analysis methodUsing an ultraviolet-visible spectrophotometer,a full wavelength scan ranging from 200 nm to 1100 nm was performed with methanol as the reference solution to determine the detection wavelength of W5.The chromatographic conditions for the analysis of compound W5 were determined through the optimization of the chromatographic column,the type and proportion of the mobile phase,and the flow rate.Explore the extraction recovery rates of different extraction solvents and redissolved solvents in blank brain tissue and plasma,and determine the pre-treatment method with good extraction efficiency and specificity.Finally,the established method was validated through parameters such as specificity,linearity,quantitative limit,lowest quantitative limit,detection limit,precision,accuracy,extraction recovery rate,and stability.2.Preliminary study of pharmacokinetics of W5 in ratsIn this study,rats were subjected to gavage using 100 mg/kg of W5 solution.Brain tissues were separated,and blood was collected from the abdominal aorta at 5,15,30,45,60,120,1 80,240,and 360 minutes following administration.At each time point,three rats were used as parallel samples.27 rat brain tissue and plasma samples were processed according to a predetermined bioprocessing method for biological samples.Analysis was carried out by injecting the sample into the HPLC system after filtration through an organic membrane filter with a pore size of 0.22 μm.Based on the obtained data of drug concentration and time,the drug-concentration time curve is plotted.The DAS 2.0 pharmacokinetic analysis software was utilized to calculate the primary PK parameters using a non-compartmental model and statistical moment method.3.Safety study of W5 in ratsThirty SD rats were randomly divided into three groups:the control group,the low-dose W5 group,and the high-dose W5 group.After one week of adaptive feeding,the administration group was given compound W5 at doses of 15 mg/（kg·bw）/day and 30 mg/（kg·bw）/day,while the control group was administered the same amount of W5 solvent.The experiment lasted 8 weeks.Throughout the entire research period,we observed the clinical symptoms of rats on a daily basis,including their fur,pupils,behavior,and posture.Additionally,we recorded the weight of the rats once a week.After the end of the medication cycle,the final weight of the rats was recorded.Brain,liver,kidney samples from sacrificed rats were collected to calculate relative organ weights.The serum obtained after processing was used for biochemical serum analysis and testing of oxidative stress markers.In rats,liver and kidney function-related indicators such as albumin（ALB）,blood urea nitrogen（BUN）,creatinine（CREA）,aspartate aminotransferase（AST）,alkaline phosphatase（ALP）,and alanine aminotransferase（ALT）are measured using a fully automated biochemical analyzer according to its analysis program.According to the results of the preliminary experiment,different concentrations of serum were selected to determine the activity of superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）,as well as the contents of glutathione（GSH）and malondialdehyde（MDA）using a microplate reader.Results1.Establishment of W5 biological sample analysis method and methodology validationThe chromatographic conditions for detecting W5 were determined as follows:highperformance liquid chromatography;detector:diode array detector;detection wavelength:251 nm;chromatographic column:SHIMADZU C18（250 mm×4.6 mm,5 μm）;mobile phase:organic phase（methanol/acetonitrile=1/1）:aqueous phase（0.05%H₃PO₄+1.5%THF）=70:30;flow rate:1 mL/min;column temperature:40℃;injection volume:10 μL;and sample collection time of 24 min.Determine the pre-processing method for biological samples as follows:take 0.1 g of brain tissue sample and add it to acetonitrile extraction solvent in a volume ratio of 1:5.Homogenize the mixture using a homogenizer（7 m/s,15 s per cycle,3 cycles）,and then centrifuge it at a speed of 10000 rpm for 10 min.Take 100 μL of plasma sample and add it to ethyl acetate extraction solvent in a volume ratio of 1:6.After vortexing for 1 min,centrifuge it at a speed of 10000 rpm for 10 min.Then,transfer the supernatant to a new centrifuge tube for nitrogen blowdown treatment.Finally,reconstitute the sample with mobile phase and filter it through a 0.22 μm membrane before measuring the content of W5.After methodological verification,the established HPLC method showed good linearity within the concentration range of 0.06-0.70μg/mL in both brain tissues and plasma,with determination coefficients all greater than 0.99.The relative standard deviations of intra-day and inter-day precision measurements were both less than 13%,with deviations within ± 7%.The extraction recovery rates for brain tissue samples ranged from 95%to 108%,while those for plasma samples ranged from 98%to 103%.The method demonstrated reliable stability under short-term,long-term,repeated freeze-thaw,and sample preparation conditions.The method is simple,fast,highly sensitive,and highly selective,meeting the requirements of biological sample analysis.2.Pharmacokinetic study results of W5 in ratsThe area under the concentration-time curve（AUC）of the drug in brain tissue and plasma were 2.014 mg/L^*h and 1.361 mg/L^*h,respectively.The mean residence time（MRT）（0-t）was 5.240 h and 3.734 h,while the elimination half-life was 3.576 h and 2.351 h,respectively.The time to reach maximum concentration（Tmax）was 1.0 h and 0.5 h,respectively.The clearance rate was 49657878.686 L/h/kg and 73.449 L/h/kg,while the apparent volume of distribution was 256265803.558 L/kg and 249.206 L/kg,respectively.The maximum blood drug concentration was 0.523 mg/L and 0.413 mg/L,respectively.3.Safety study of W5 in ratsDuring an 8-week experiment,all groups of rats consumed normal food and water,had normal pupillary changes,and exhibited no abnormal behavior.There was no significant difference in the average weight change,as well as the absolute and relative weight of the brain,liver and kidney between the W5 administration group and the control group（P>0.05）.With regards to the activity of ALT,AST,ALP and the content of ALB,BUN,and CREA in rat serum,there was no significant difference between the W5 administration group and the control group（P>0.05）.There was no significant change in the levels of T-SOD,CuZn-SOD,CAT,GSH-Px,GSH,and MDA in the serum of rats after receiving W5（P>0.05）.Conclusions1.This study established a fast,simple,and reliable HPLC method with high sensitivity and selectivity,which can accurately determine the content of W5 in biological samples.2.Preliminary pharmacokinetic studies of W5 in rat models have shown that it can penetrate the blood-brain barrier and has a short half-life of elimination in the bloodstream.These findings serve as a fundamental basis for subsequent pharmacokinetic studies of W5 in other animal models and in human subjects.3.W5 has demonstrated good safety in aspects such as growth and development,liver and kidney function,and oxidative stress levels in rats,laying the foundation for further research and application.