| Background and ObjectiveLung cancer is one of the most frequently reported malignant diseases in the world currently.Its morbidity and mortality increase rapidly,especially in our country due to past extensive economic development,resulting in air pollution and environmental deterioration,the incidence of lung cancer and mortality showed a linear upward trend.In the current,Lung cancer has became the number one malignant tumor disease that seriously threatens the lives and property of the people.The medical community,based on the different types of cancer cells observed under the microscope,and the size and appearance of the cells based on histology,classify the lung cancer as small cell lung cancer and non-small cell lung cancer.In lung cancer,non-small cell lung cancer accounts for about 84%of the total lung cancer,non-small cell lung cancer usually began to spread in a very early period,and high sensitivity to radiotherapy and chemotherapy,so for non-small cell lung cancer patients should be treated more Chemotherapy rather than surgical treatment.Currently in the clinic,the first-line chemotherapy for patients with advanced non-small cell lung cancer is based on platinum.However,selective EGFR tyrosine kinase inhibitors gefitinib and erlotinib as second and third line treatment of non-small cell lung cancer have also achieved remarkable results.However,drug toxicity and tumor cell resistance greatly limits its clinical application,so it is very necessary to develop new chemotherapeutic drugs for non-small cell lung cancer.Nucleosides are a class of important anticancer drugs,such as 5-fluorouracil and its prodrugs,cytarabine,azacitidine,gemcitabine and so on.It is mainly through the competitive inhibition of DNA synthesis related enzymes to prevent the proliferation of tumor cells,resulting in the death of tumor cells,but in clinical applications also showed some limitations,such as poor lipid solubility of some drugs,short t1/2,low bioavailability,in order to solve these problems,Dr.Wang Zhenyu(Shanghai Medical Institute)focused on Nucleosides which widely used in clinical,Through structural modification and high-throughput screening,FZY-80 was selected as a candidate development compounds.Section 1 Pharmacodynamics study of Novel NucleosideCompound FZY-80Method:1 Anti-tumor activity in vitro:MTT was used to observe the inhibitory effect of FZY-80 on the proliferation of human non-small cell lung cancer cell line A549;2 Anti-tumor activity in vivo:A549 cells were inoculated subcutaneously in Balb/c nude mice.When the tumor grew to about 100 mm3,FZY-80 at doses of 5,10 and 20 mg/kg were orally administrated.The tumor volume was measured and the tumor inhibition rate was calculated.Result:1 The anti-tumor activity in vitro showed that:the compound FZY-80 inhibited the proliferation of A549 cells in a concentration-time dependent manner with an IC50 of 66.9 ?M on A549 cells.The anti-tumor effect of nude mice in vivo showed that:the compound FZY-80 has a good anti-tumor activity and the anti-tumor activity was dose-dependent.2 The inhibitory rates were 25.77%,46.39%and 59.79%,respectively.Gemcitabine,a high-dose and positive drug,has equivalent anti-tumor activity with no more than 10%loss in body weight and no apparently toxicity.Section 2 Anti tumor Mechanism of Novel NucleosideCompound FZY-80Method:1 Flow cytometry was used to detect the effect of different doses of FZY-80 on apoptosis and cycle of tumor cells;2 Western Blot was used to detected P21 protein and Bcl-2 protein.3 Western Blot was used to detected SHH protein and GLI1 protein.Results:1 FZY-80 can induce tumor cell apoptosis,and arrest the cell cycle in GO/G1 phase.With the increase of FZY-80 concentration,the cell apoptosis rate and total apoptosis rate of A549 cells(early apoptosis rate + late apoptosis rate)was significantly higher than C group(P<0.05).2 The results of Western Blot showed that:With the increase of FZY-80 concentration,the expression of P21 in lung cancer cell A549 significantly increased(gray scale increased significantly,P<0.05);With the increase of FZY-80 concentration,Bcl-2 protein expression reduced significantly(gray significantly reduced,P<0.05).FZY-80 upregulates P21 and Bcl-2 proteins in a dose-dependent manner.3 The results of Western Blot showed that:With the increase of FZY-80 concentration,the expression level of SHH in lung cancer cell A549 decreased significantly(P<0.05);With the increase of FZY-80 concentration,the expression of GLI1 Protein expression was significantly reduced(gray significantly reduced,P<0.05).FZY-80 has an inhibitory effect on SHH and GLI1 proteins in a dose-dependent manner.Section 3 Acute Toxicity of Novel Nucleoside CompoundFZY-80 to KM MiceMethod:KM mice were given a single oral dose of 100-2000 mg/kg of compound FZY-80 to observe the animal's state,abnormal reaction and death after administration.The Bliss method was used to calculate LD50,and the acute toxicity symptoms were observed.The cumulative death number and body weight of mice were recorded.Results:The LD50 method were used to test the acute oral toxicity of compound FZY-80.The LD50 of compound FZY-80 was 1053.78 mg/kg and the 95%confidence interval of LD50 was 1000.50-2467.67 mg/kg.Section 4 Long-term Toxicity of Novel Nucleoside CompoundFZY-80 to SD RatsMethod:1 Animal grouping and administration:Take 160 healthy SD rats,male and female.The rats in the low-dose group(group L,20 mg · kg-1 · d-1),middle dose(group M,40 mg · kg-1 · d-1),high-dose group(H group,80 mg · kg-1 · d-1).Six days a week,continuous administration for 3 months(12wk).After stopping taking some animals continue to observe 1 month(4wk),understand the reversibility of toxic reactions and the emergence of delayed toxicity.2 Determination of indicators and time:a daily observation of the general situation,weighing 2 times a week weight,and in the administration of 6wk,12wk and convalescent observation at the end of the hematology,blood biochemistry and pathology.Result:1 General situation:during the administration period and recovery period,except L group 3 rats(LM28,LF40,LM39),M group 1 rat(MF53)due to improper operation of the gavage,the rest of the medication group The general condition of rats was good,no obvious drug-related toxic symptoms were observed,no significant difference compared with the control group.2 Body weight:There was no significant difference in the weight of rats in each treatment group between the two groups(P>0.05).3 Hematology examination:administration of 6wk,12wk when the rats in each administration of leukocytes and the control group were significantly different,compared with the C group(P<0.05),the rest of the test indicators L,M,H group with the same period C group compared to P>0.05.At the end of convalescent period,all the hematological indexes of rats in each administration group had no significant difference compared with those of C group(P>0.05).4 Blood biochemical tests:6wk and 12wk,the rats in each administration of aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase,urea nitrogen,creatinine,total protein were significantly different from the control group,P<0.05 compared with that of group C,and the other test indexes of group L,M and H were P>0.05 compared with group C at the same period.5 Pathological examination:? administration 6wk,H group of hepatocytes loose cytoplasm,a large number of different sizes of vacuoles,see the number of sinusoids in the library of Fufang cells.The remaining organs no significant pathological changes.? administration 12wk:M group of rat liver cells loose cytoplasm,a large number of different sizes of vacuoles,sinusoidal see the number of different library of Fufang cells.H group rats showed focal interstitial nephritis,the junction of the junction of the renal tubular epithelial cells cloudy;hepatocyte cytoplasm loose,there are a large number of different sizes of vacuoles,sinusoidal see the number of different library of cells.The remaining organs no significant pathological changes.? convalescent observation end:no obvious pathological changes in various organs.6 Organ wet weight and viscera coefficient:6wk,12wk administration and convalescence observation end,L,M,H group rats organ weight and viscera coefficient compared with the C group no significant difference(P>0.05).Section 5 Pharmacokinetics of Novel Nucleoside Compound FZY-80 Method:SD rats was used to observed the pharmacokinetics of FZY-80.The rats were intragastrically and intravenously injected with FZY-80(20 mg-kg"1).Blood was collected from the venous plexus at 0,0.25,0.5,1,2,4,6,8,12 and 24 h after administration and anticoagulated with EDTA-K2,centrifuged at 5000 rpm for 5 min.Plasma FZY-80 concentrations were analyzed by liquid chromatography-mass spectrometry(LC-MS/MS).DAS3.0 was used to calculate pharmacokinetic parameters and obtain absolute bioavailability.Results:Pharmacokinetics experiments showed that:The AUC0-t of compound FZY-80(20 mg·kg-1)was 4805.99 ± 956.04 ng·kg-1·d-1 and 7530.00 ± 1360.01 ng·kg-1·d-1,The AUC0-? of compound FZY-80(20 mg·kg-1)was 4986.52 ± 997.30 ng·kg-1·d-1 and 7655.99 ± 1378.08 ng·kg-1·d-1.The absolute bioavailability of compound FZY-80 in rats was 63.82%,indicating that:Compound FZY-80 absorbed better in SD rats after gavage,with a high bioavailability.The peak time(Tmax)of FZY-80(20 mg·kg-11)in SD rats was 2.00 ± 0.38 h,which showed that:The absorption peak was 714.20 ±168.39 ng·kg-1.The elimination half-life(t1/2)was 5.05 ± 1.12 h,indicating that:The retention time in the body is moderate,which is beneficial to exert pharmacological effects.ConclusionThe novel nucleoside compound FZY-80 has obvious anti-tumor activity both in vitro and in vivo.The acute toxicity and long-term toxicity of FZY-80 in mice by oral gavage showed that the toxicity of FZY-80 was low and the absolute bioavailability in rats high degree of elimination of half-life is moderate,with a certain degree of drug development and application value. |
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