Toxicologic Characteristics And Toxicity Mechanisms Of DATR, A New Investigational Antitumor Drug

According to the report of WHO,there was the cancer that 760 thousand people were died of,which as much as 13 percent,in all of the total deaden 58,000,000 in 2005.A few days ago, a research paper was publicated by Centro International de Investigationes sobre et Cancer(CIIC),which said that based on the present invasion trend of cancer,there would be 50%more cancer incidence happened all of the world in 2020,and then the total number of cancer patients would be increased 15 million a year.In the last several decades,many methods of cancer therapy emerged,including operation, the radiation therapy,the chemotherapeutics,the hormonal therapy,the gene therapy and the immunotherapy.The chemotherapeutics is a method that administrading antitumor chemical drugs to treated cancer patients.These drugs could distribute quickly after the administration. Either the local tumor or the distant metastatic tumor could be killed,so chemotherapeutics is a kind of whole body therapy.The effectiveness of chemotherapeutics depends on tumor type and severity.Under most circumstance,chemotherapeutics was combined with operation or radiation therapy,though sometimes it is the only choice to some special cancers.So many chemotherapeutic drugs were sort:alkylating agent,antimetabolite,antibiotics,vegetable drug, hormone and endocrine secretion drug.They were also classified:the cell cycle specific agent and the cell cycle non-specificity agent.The shortages of chemotherapeutics are quite visible, such as the large adverse reaction,drug tolerlance and so on.Tumor necrosis factor related apoptosis-inducing ligand(TRAIL),which called Apo2 ligand(Apo2L),was a new discovered TNF superfamiliy member after TNF and FasL.Recent years,TRAIL has been considered to be a promising antitumor drug,because of its apoptosis selectivity of tumor cells but not normal cells.There were several versions of TRAIL till now, all of them anthropogenicly lack some amid acid of the N-terminal comprared with wild TRAIL,as TRAIL 95～281,TRAIL114～281 and so on.DATR(recombinant solube human tumor necrosis factor-related apoptosis-inducing ligand mutant,rsh TRAIL mutant),another TRAIL version,was studied by Chengdu Diao Pharmaceutical Group Co.,Ltd(Chengdu, China).It has been proved that compared with wild TRAIL,DATR has the same spatial structure and physico-chemical property,including isoionic point and dissolubility.But there was a little elevation in bioactivity.Toxicology,pharmacology and molecular biology related methods were used in our study. We administrated DATR,which were protein product of artificial TRAIL gene,in healthy adult mouse,rats and crab-eating monkeys,to observe the acute and cronic toxicology of DATR and the reciprocity of trauma.Furthermore,to observe the interaction and possible adverse effect of DATR combinately administrated with some clinical antitumor drugs.Oxaliplatin,polyene paclitaxel and irinotecan were selected and administrated respectely combined with DATR.All of our work was to preliminarily assess the toxicologic characteristics of DATR and to provide rationale and pre-clinical experiment provements for the clinical application of DATR.【PURPOSE】:Our purpose was to prelimilarily assess the toxicologic characteristics and mechanism of the toxicology of DATR.【METHODS and CONSEQUENCES】:1.Acute toxicity and reciprocity of DATR in mice,rats and crab-eating monkeysTo observe the acute toxicity in mice,rats and monkeys,all the animals were iv DATR once.Furthermore,another group of mice were ip DATR.The consequences showed that DATR might affect the central nervous system,respiratory apparatus,nerve muscle and autonomic nerve of mice and rats.The LD₅₀ of iv DATR in mice and rats were 1018.0 mg/kg and 262.0 mg/kg respectively,and the 95%confidence interval were 901.3～1149.7 mg/kg and 238.8～287.5 mg/kg singly.The LD₅₀ of ip DATR in mice was1432.1mg/kg and the 95%confidence interval was 1237.9～1656.8mg/kg.Four crab-eating monkeys were once iv DATR respectively,the dosages ranged from 90.0mg/kg to 303.8mg/kg.The results showed that NOAEL(no observed adverse effect level) was 90.0 mg/kg,the minimum toxic reaction dosage was 135.0mg/kg and the sever toxic reaction dosage was more than 202.5mg/kg.Declined activities,less spirit and a little foodintake were observed in the experiment.Beside these,RBC,Hb and Hct were detected to be declined,and ALT,AST,Tbill,Dbill,BUN,Cr and LDH were detected to be increased.The target organs of toxic reaction might be hematological system,alimentary system and uropoiesis system.2.long-term toxicity and reciprocity of DATR in rats and crab-eating monkeysRats were arranged into four different dosage groups,and monkeys were arranged into five different dosage groups including a TRAIL positive group.All the animals were iv DATR every morning for 7 consecutive days,which were interrupted with 7 withdrawing days.There were 4 separated administrating weeks in all.The purpose was to investigate the long-term toxicity and its dependability with doses.The results in crab-eating monkeys diaplayed all the monkeys' foodintake in every medication administration teams ranged from 10～90mg/kg declined,especially the monkeys in 90mg/kg DATR group and positive controls 90mg/kg TRAILgroup.Pathological investigate demonstrated nodosity granuloma or confluence inflammatory necrosis focus in the liver parenchyma of(?)27(10mg/kg),(?)29(30mg/kg),(?)11(90mg/kg) and(?)31(TRAIL 90mg/kg) at the end of administration period.These indicated DATR might educe toxic action on liver.In the renal of(?)27(10mg/kg) and(?)29(30mg/kg),the endothelial cell of proximal convoluted tubule exhibit hydropic degeneration,even found isolated or multiple cellular inflammatory granulomatosis locus.These manifested DATR may have renal toxicity in crab-eating monkeys. Furthermore,DATR may also influence hematological system,because it was found increasing of DBIL and TBIL in blood biochemical investigation and decreasing of RBC,HGB,Hct and MCV in hemology investigation.The results in rats,whose dosages ranged form 20 to 180mg/kg,diaplayed the safety dose of 20mg/kg and toxic dose of 60mg/kg.The main toxic reactions were foodintake and bodyweigh stepped down,the increasing of ALT,AST,Tch,BUN,Cr,the decreasing of RBC and Hct,hydropic degeneration in definite aera of renal parenchyma cells and some bone marrow toxicity.The target organs of toxic reaction might be alimentary system,uropoietic system and hematological system.3.TUNEL investigate the apoptosis of liver in crab-eating monkeys and of renal in ratsFor DATR inhibit tumor growth through inducing tumor cell apoptosis,we designed TUNEL experiment to investigate the apoptosis of liver cell in crab-eating mongkeys and renal cells in rats to further study the mechanisum of hepatotoxicity and nephrotoxicity aroused by DATR.The results showed there were no significant differences of apoptosis cell numbers between dosage groups and controls.Compared with controls,the apoptosis liver cells of monkeys in DATR10mg/kg group and 30mg/kg group had no obvious differences.But liver cells of monkeys in DATR 90mg/kg group significantly apoptosed.The liver cells of monkeys in TRAIL 90mg/kg had no statistical difference,but the apoptosis numbers of liver cells in each monkeys of this group were eaqual or exceeded the ones in DATR 90mg/kg group.Perhaps just because of the only one extremely severe apoptosis exist,it did not caused statistical significance.So we believed the apoptosis condition in liver of TRAIL 90mg/kg group monkeys were even much severe than the condition in liver ofDATR 90mg/kg group monkeys. It confirmed that DATR induced apoptosis in normal liver hepatocellulars,which caused hepatotoxicity in crab-eating monkeys,while the nephrotoxicity in SD rats were independence with DATR induced apoptosis.4.DATR was respectively combinatory administrated with oxaliPiatin,polyene paclitaxel and irinotecan to observe the possible toxic reaction and histopathological changesWe designed this experiment to observe toxic reaction when DATR combine administrated with some clinical used antitumor drugs,further to provide information and accordance for clinical applications and compatibility.DATR were arranged at different dosage level combining with oxaliPlatin,polyenepaclitaxel and irinotecan respectively.Adverse effects and its relationship with dosage were surveyed.No obvious different adverse effect were seen while DATR was combing administrated with oxaliPlatin,polyenepaclitaxel and irinotecan respectively,and no new toxic reactions were observed.When DATR was combined with the three anti-tumor drug,the influences in hematological system,urinary system,and immune system should be monitored.【CONCLUSIONS】:As a new antitumor drug,DATR's toxic effect mainly appeared in hematological system, alimentary system,such as liver,and urinary system,such as renal.The hepatoxicity aroused by DATR in crab-eating monkeys might because normal liver cells apoptosis induced by DATR, while nephrotoxicity was not the same reason,perhaps due to bone marrow depression of DATR.No obvious different adverse effect were seen while DATR was combing administrated with oxaliPlatin,polyenepaclitaxel and irinotecan respectively,and no new toxic reactions were observed.