Study On Myelosuppression And Toxic Mechanism Of An Antitumor Compound, Furnanodiene

ObjectiveTo prepare water-in-oil-in-water multiple emulsions for furanodiene in order to solve difficulties of poor soluble and animal administration, investigate the antitumor activity and preliminary toxicity to S180carcinoma in vivo, and confirm the toxic target organs in mice and the preliminary toxic mechanism. To study the mechanism on furanodiene induced SP2\0apoptosis and its signal transduction pathway, evaluate the pharmacokinetics, tissue distribution of furanodiene multiple emulsions in rats and accumulate in toxic target organs.Methods(1) A Two-step emulsification method was used to prepare the W/O/W multiple emulsions. The prescription and preparation of emulsions were optimized. Both the primary and multiple emulsions were identified through microscope-observation and eosin staining method. The HPLC method was established and used to the quality control of furanodiene multiple emulsions in vitro. S-180sarcoma transplanted mice as animal model and cyclophosphamide as positive control, the tumor growth inhibition rates were measured after intraperitoneal injection and oral at low, middle and high doses. Meanwhile, the weight change, viscera index, blood cell count, serum biochemical indexes and tissue pathological slices were used to evaluate the toxicity.(2) The healthy mice were used as experiment objects with the changes of their weight, viscera index, blood cell count, serum biochemistry and tissue pathological slices as evaluation indexes, the toxic effects and target organs were confirmed after intraperitoneal injection at low, middle and high multiple doses. In order to investigate the preliminary mechanism of furanodiene induced myelosuppression, the changes of apoptosis related protein expression were measured by Western blot technology. Liver microsomes of rats which treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection were prepared by using ultracentrifuge method. The concentration of protein in liver microsome determined by the BCA method and the content of cytochrome P450detected by the Omura and Sato method were used to evaluate the effect of furanodiene on CYP450preliminarily. In order to calculate the enzyme kinetics parameters, a HPLC method for determination of furanodiene in rat liver microsomes was established and the in vitro metabolic rates were studied by incubation with rat liver microsomes. The effect of furanodiene on CYP450subtypes was confirmed by the specific substrate experiments.(3) The plasma concentrations of furanodiene were determined by HPLC-APCI-MS/MS method after intravenous, intraperitoneal injection and oral furanodiene multiple emulsions at low, middle and high single dose in rats, respectively. Pharmacokinetics parameters were calculated by Winnolin software. The cumulative penetration and intestinal residue of furanodiene and its multiple emulsions were determined by the everted gut sacs method. Tissue distribution of furanodiene were studied in rats after oral, intraperitoneal and intravenous injection furanodiene multiple emulsions with single dose. Meanwhile, drug accumulation were monitored in the toxic target organs after oral and intraperitoneal injection furanodiene multiple emulsions with mutable doses, respectively.(4) The growth inhibitions of SP2/0cell cultured in vitro were analyzed by MTT method, than IC50was calculated. Cell morphology changes were observed by using light microscope. Flow cytometry (FCM) was used to study the apoptosis rates, content of DNA and effects of furanodiene on SP2/0cells cycle. The changes of apoptosis related protein expression such as Bax, Bcl-2, Caspase-9, Cyt-c, Fas, Fas-L, Caspase-8and Caspase-3, were measured by Western blot technology. Apoptosis signal transduction pathway was elucidated by the time/dose-effect relationship of protein expression.Results(1) Furanodiene W/O/W multiple emulsions prepared by two-step emulsification method had even particle size, good dispersion and stable quality. The labeled content was99%±2.1%, which fitted to the requirement of formulation quality. Anti-tumor activity results showed that furanodiene could inhibit the growth of S-180sarcoma after intraperitoneal injection at low, middle and high doses and the tumor growth inhibition rates were18.55%±3.5%,19.82%±2.7%and29.82%±3.4%, which showed significant difference compared with multiple emulsions control group (p<0.05). The tumor growth inhibition rate of high dose group was similar to the positive control drug (cyclophosphamide,33.53%±3.5%, p>0.05). There was significant difference only between high dose group (20.76%±4.5%) and multiple emulsions control group after oral at low, middle and high doses (p<0.05), which indicated the poor activity of oral administration group. The survival-extending rate results showed that furanodiene could significantly inhibit the abdominal circumference growth of S-180ascetics’tumor beating mice, but could not prolong the life of mice, which suggested the toxicity lead to death. Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Blood cell count results showed that furanodiene could cause leukopenia (p<0.01). Erythropenia (p<0.05) and thrombocytopenia (p<0.01) occurred in the high dose group. Serum biochemical index results showed that AST level was significantly increased (p<0.01). Tissue pathological slices results suggested that spleen tissue structure was damaged and founded extramedullary hematopoiesis. It indicated that the toxic target organ might be bone marrow hematopoietic system.(2) Blood cell count results showed that furanodiene could cause leukopenia (p<0.01) in healthy mice after intraperitoneal injection at low, middle and high multiple doses. Thrombocytopenia (p<0.05) occurred in the high dose group. Serum biochemical index results showed that GM-CSF level was significantly decreased (p<0.01). TPO level was decreased in the high dose group (p<0.05). ALT, AST, ALP and MDA level compared with blank control group were increased in the high dose group (p<0.05). Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Tissue pathological slices results suggested that cell numbers were decreased in the marrow cavity, spleen was founded extramedullary hematopoiesis and liver also was founded slight extramedullary hematopoiesis. Western blot results showed that furanodiene could increase the ratio of Bax/Bcl-2via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2. The ratio of Bax/Bcl-2was2.22±0.65,5.32±1.56and6.03±2.43, respectively. Compared with blank control group, there was significant difference in low, middle and high dose group (p<0.01) and the dose-effect relationship was good. The clear bands of cleaved caspase-3suggested that furanodiene induced bone marrow apoptosis lead to myelosuppression.Furanodiene could decrease the content of protein and CYP450in rat liver microsome treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection. Compared with blank control group, there was significant difference in the middle and high dose group (p<0.05). Meanwhile, metabolic inhibition of furanodiene was founded in the middle and high dose group. Compared with blank control group, the enzyme kinetics parameters (Km and Vmax) decreased significantly (p<0.05), which showed furanodiene could probably inhibit CYP450. The specific substrate experiments results showed that the metabolic rates, compared with blank control group, S-mephenytoin and phenytoin in rat liver microsomes treated with high multiple doses had significant difference(p<0.01,p<0.05), which indicated the inhibitory effect of furanodiene on CYP2B6and2C9.(3) The concentration-time curve of furanodiene in rats after intravenous injection furanodiene multiple emulsions at a low, middle and high single dose is fitted to a two-compartment model. T1/2α was3.9±2.3,4.3±1.2and4.8±2.1min and T12β was73.2±7.9,64.2±14.7and69.3±12.1min, which showed the rapid distribution and elimination in rats. AUC was83231.34±9784.3,151481.88±14573.1and834672.65±67481.43min-ng-mL"1, respectively. A dose proportionality study indicated that there was good correlation between AUC and dose. The linear equation was y=16845x-8510.6(r=0.987) which showed that the pharmacokinetics of furanodiene in rats was of the linear pharmacokinetic characteristic. The plasma concentration of furanodiene in rats after intraperitoneal injection and oral furanodiene multiple emulsions at a low, middle and high single dose, was too low to calculate the pharmacokinetic parameters. Everted gut sacs experiment results showed that the cumulative penetration of furanodiene group was3456±534,3024±368and2687±286ng and the intestinal residue was8654±1654,7684±957and7342±1256ng in60min at the order of duodenum, jejunum and ileum. The cumulative penetration of furanodiene multiple emulsions group was3456±534,3024±368and2687±286ng and the intestinal residue was2459±625,2861±589and2103±574ng in60min at the order of duodenum, jejunum and ileum. Tissue distribution results showed that furanodiene underwent a rapid and wide distribution in tissues within the time course examined. The highest tissue concentrations after intravenous injection were found in the spleen, followed by liver, heart, lung, kidney small intestine and brain. The highest tissue concentrations after oral or intraperitoneal injection were found in the small intestine, followed by spleen, heart, liver, lung, kidney and brain. Drug accumulation results showed that the drug content in the toxic target organs increased with the extension of administration days. The drug content in spleen was25.3±3.3ng·g-1on the first day and43.1±4.1ng·g-1on the seventh day. The drug content in the bone marrow was1.6±0.4ng·g-1on the first day and5.1‘0.5ng·g-1on the seventh day. The drug content in the thymus was0.9±0.2ng·g-1on the first day and2.5±0.2ng·g-1on the seventh day.(4) MTT results showed that furanodiene could inhibit the growth of SP2/0cell cultured in vitro with a good dose-dependent relationship. The calculated IC50was5.2μng-mL"1. Nuclear morphology characteristic of SP2/0apoptosis cells treated with furanodiene was observed by using light microscope. There were hypodiploid apoptotic peaks pre-G0-G1phase of cell cycle from DNA figure by flow cytometry (FCM). Significant statistics difference (p<0.05) was showed in apoptosis rates of SP2/0cells treated with furanodiene for different time and different concentration by annexin V-FITC method. Western blot results showed that the ratio of Bax/Bcl-2increased form1.83±0.6,8.35±4.3to14.6±5.6with the increase of furanodiene concentration, Cyt-C in the mitochondria decreased form0.7±0.2,0.4±0.3to0.5±0.2, Cyt-C in the cytoplasm increased form2.6±0.7,5.9±1.3to8.3±2.5, Fas increased form1.14±0.42,1.57±0.63to1.73±0.48and Fas-L increased form0.39±0.09,0.58±0.26to0.68±0.32. The ratio of Bax/Bcl-2increased form4.26±1.4,10.99±3.5to14.6±5.6with the extension of furanodiene treated time,Cyt-c in the mitochondria decreased form0.5±0.2,0.3±0.1to0.2±0.1, Cyt-c in the cytoplasm increased form5.3±0.8,6.3±1.7to8.3±2.5, Fas increased form0.69±0.21,0.88±0.24to1.73±0.48and Fas-L increased form0.38±0.14,0.43±0.21to0.68±0.32. All of the results showed that the apoptosis related protein expression had good time-/concentration-dependent relationship. The clear bands of cleaved caspase-9, cleaved caspase-8and cleaved caspase-3suggested that furanodiene induced SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.Conclusions(1) The preparation technology of furanodiene W/O/W multiple emulsions are simple, practical and easily controlled, which can provide guarantee for the study on pharmacodynamics, pharmacokinetics and toxicology. Furanodiene shows strong activity in vivo against S180mice-via intraperitoneal injection, and its main toxic target organs may be bone marrow hematopoietic system and liver.(2) The toxic target organ confirmed in healthy mice is bone marrow hematopoietic system. Toxic mechanism may be that furanodiene can induce apotosis of bone marrow cells via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2and break balance between Bax and Bcl-2. Moreover, furanodiene shows significant thepatotoxicity via inhibitory effect on CYP2B6and2C9.(3) The half-life of furanodiene is short and elimination is rapid. The concentration-time curve in rats after intravenous injection furanodiene multiple emulsions with single dose is fitted to a two-compartment model and the linear pharmacokinetic characteristic. The reason of low concentration after extravascular administration is probably that the transport of emulsion drops may via lymph circulation instead of blood circulation, which can not affect the efficacy and toxicity. Drug accumulation in toxic target organs may be a key reason which plays an important role in the toxic occurrence and development.(4) Furanodiene does not exhibit effect on SP2/0cell cycle; however, it could induce SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.