| Lune cancers are the first place in the death of cancers in our Country.Additionally,drug-resistance has been found out and seriously challenged to treat the lung cancer patients.Therefore,innovation of new drug candidates based on new pharmaceutical targets is the clinical merit need.Nicotinamide phosphoribosyltransferase?NAMPT?is a rate limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide?NAD?,which is a crucial cofactor and substrate for a wide range of metabolic enzymes.Efficiently inhibition of NAMPT enzyme activity would causes a direct inhibition of NAD production in cancer cells,leading to loss of ATP and ultimately cause cell death.Two NAMPT inhibitors FK866 and CHS828 had been worked on phase II clinical trials.However,too short half-life and the adverse events after increase of dose even though they potently inhibit NAMPT and/or NAD synthesis in cancer cells.To increase the drug-like properties for NAMPT inhibitors,a series of novel NAMPT inhibitors were designed and synthesized in our early studies.After drug efficacy screening,it was found that a drug candidate J020 exhibited strong inhibitory effect on non-small cell lung cancer?NSCLC?in both in vitro and in vivo experiments.In this study,high performance liquid chromatography tandem mass spectrometry?LC-MS/MS?and Q-trap linear ion traptechnique were used to establish a method for the analysis of J020 in biological samples,including materials from rats,beagles,cynomolgus monkeys and human tissues.In vivo experiments in rats,dogs and monkeys,combined with in vitro cell models,distribution,metabolism,in situ perfusion model,and so on will be used to explore the drug transport,distribution and metabolism properties,including the in vivo-in vitro correlation analysis.The experimental results are the following:1.The oral bioavailability of J020 was 3.9%.?1?From the results of intestinal-hepatic vascular perfusion experiments and the metabolism analysis,it was showed that J020 experiences a considerable first-pass effect on drugs metabolism,in which the content of J020 was decreased by approximately 50%after the first-pass metabolism in liver.?2?From the trans-membrane transport experiment on human colorectal adenocarcinoma cells?caco-2?,it was suggested that J020 was not a good candidate for passive absorption.2.J020 shows its particularity in drug distribution in terms of target organ distribution and remaining half-life.Drug distribution screening results showed:The results of tissue distribution in rats suggest that J020 has a strong affinity to lung.It was rapidly distributed to the lung after intravenous administration.The concentrations of J020 in plasma were decreased rapidly?half-life was 27 min?from 5 min to 120 min after administration,in contrast,the drug contents in the lungs were?0.5-1.2mg/g?much higher than that in other tissues?<0.2mg/g?and blood?<3344 ng/mL?.Therefore,it is obvious that J020 has high affinity toward lung,suggesting that J020 may be much suitable for treatment of lung cancer.3.From drug metabolism and excretion results,it was suggested that the major elimination factor of J020 in rats is its metabolism.?1?The results of metabolic stability for J020 showed that the intrinsic clearance of J020in rats and cynomolgus monkeys liver microsomes was faster?0.44-0.62 mL/min/mg?,but slower in beagle dogs and human liver microsomes?0.08-0.13 mL/min/mg?.?2?Using Q-trap linear ion TRAP technology and LC-MRM-IDA-EPI mode,the metabolites of J020 were identified.Through the comparison of metabolites in vitro between humans and rats,a large number of different metabolites were produced by the reaction of J020 in the rat liver microsomes system,while the content of metabolites in the human liver microsomes was relatively low.Among the metabolites,the hydroxylation metabolite?M1?of methyl benzene was the highest in J020,while the N-oxidation product?M2?of pyridine ring ranks the second,showing the same trend in human and mouse metabolism.?3?The excretion experiment of J020 in rat metabolic cage showed that within 24 hours after administration,the proportion of J020 excreted through kidney was very low?<0.007%?and the proportion of J020 in feces,including bile excretion,was also not more than 1.5%.Meanwhile,metabolites of J020 were detected in urine and feces.This indicates that excretion is not the main elimination pathway of J020.?4?From the drug-drug interaction potential analysis,it was revealed that J020 was not the potent inducer for hPXR-mediated cyp3A4 expression.However,J020 displayed certain inhibitory effect in human P4502D6 enzyme activity(IC50=2.9±0.6?M),suggesting that J020 may cause adverse events,especially for Caucasians who are with high frequency of poorer metabolites in cyp2D6 gene mutation.4.Pharmacokinetics of different species is important for the development of the first-in-human dosage.The results of the screening of pharmacokinetics showed that the pharmacokinetics of J020 in rats and cynomolus monkey were in accordance with the common linear kinetic.The results were the following:?1?The pharmacokinetic results of intravenous injection of four doses for J020 in rats showed a linear relationship between the drug exposure(AUC?0-??)and the dose?R2>0.99?.The pharmacokinetic results of three doses of J020 intravenously injected into cynomolus monkey also showed a linear relationship between the exposure and the dose?R2>0.98?.This indicated that the AUC and peak concentration of J020 were in line with the linear pharmacokinetics.?2?J020 was intravenously injected into rats,beagle dogs and cynomolgus monkeys at a dose of 3 mg/kg,respectively.The pharmacokinetic differences among species were as follows:·The AUC?0-??of J020 was as follows:rat<beagle dog<cynomolgus monkeys?1095 <2374<8785,ng/mL*h?,suggesting that the exposure in large animal like dogs and monkeys?inferred humans?was higher than that in small animals at the same dose.·The half-life of J020 was as follows:rats<beagle dog<cynomolgus monkeys?1.0<4.8<5.6,h?,and the half-life in large animal like dogs and monkeys were 4 or 5times longer than that in rats.The clearance of J020 was changed similarly as half-life:rat>beagle dog>cynomolgus monkeys?3.4>1.3>0.3,L/h/kg?,clearance in large animal like dogs and monkeys was relatively slow.·J020 has a high apparent distribution volume?2.8 L/kg-8.7 L/kg?in the three species,suggesting that J020 is widely distributed throughout the body in different animal species.?3?Prediction of human pharmacokinetic parameters based on allometric amplification model:the allometric amplification equations of half-life,clearance and apparent distribution volume of J020 were as follows:y=1.905x0.498,y=1.717x0.627,y=4.630x1.128,respectively.Using the above equation to predict the pharmacokinetic parameters of human,it is inferred that the possible half-life of 70 kg body weight was 16.6 hours,the clearance rate was 0.352L/h/kg,and the apparent distribution volume was 7.98 L/kg.5.The preliminary safety assessment of J020 showed that the therapeutic window of J020 in rat was not wide.?1?In the acute toxicity test in rats,no death occurred in the 30 mg/kg group during the observation period of 14 days,and the rats were normal.Rats in the 100 mg/kg group began to die on the second day after administration,and all died during the observation period.?2?In the experiment of repeated administration of J020 was 10 mg/kg once two days,no abnormality was observed in rats during the observation period of 14 days.When the dose of J020 was increased to 30 mg/kg,the rats began to die after the administration of the third dose,and all died after the administration of the fifth dose.In a summary,the drug-like property of J020 was assessed through different preclinical evaluation modules and displayed to be a on-going drug candidate.Firstly,J020 possessed a strong inhibitory effect on the new anticancer target NAMPT activity with much better inhibitory potential than that of FK866.Also,J020 demonstrated stronger inhibition on tumor growth in nude mice at a lower dose with the comparison to FK866 and CHS828.Furthermore,the pharmacokinetics of J020 are generally good,including the cynomolgus monkeys'pharmacokinetics.The ADME properties were acceptable for J020.The elimination half-life of J020 in large animal like beagle dogs and cynomolgus monkeysare longer than that of FK866 and CHS828 in animals.The allometric model predicted a half-life of 16.6hours for J020 in humans.Additionally,J020 has a high exposure in lung with a longer half-life,suggesting that J020 may be a candidate for treatment of lung cancers.In addition,J020 still needs an in-depth safety assessment,including the long term toxicity experiments in large animals.Current data suggest that the treatment window of J020is a narrow therapeutic.If J020 is used as an oral drug,the oral bioavailability in large animals needs to be further verified.All these results will provide reference for the follow-up development of J020,a novel anticancer drug,and even for its clinical application and/or multi-target combined therapy.Such developmental experience may be useful for evaluation of innovative other drugs during the drug discovery and development. |
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