Study On The Early Toxicity And Its Mechanism Of Antitumor Compound MC004

It is widely known that new drug research and development (R&D) is a process of high risk, large expense and long term. During the drug preclinical research period, toxicity is one of the determinants of R&D. For this reason, it is important to give up compounds that are unsuitable for further R&D so as to lower the investment risk and save the cost. How to find the potential toxicity of drug candidates in order to speed up the R&D process, to reduce the cost and to increase the success rate has become an important direction of the toxicology study. In recent years, with the prolonged survival of tumor patients, the long–term adverse effects of antineoplastic drugs have become a focus of relevant studies. Moreover, the reproductive cells are easily affected by the antineoplastic as they proliferate very quickly. As the antineoplastic kills tumor cells, it can also cause massive deaths of spermatogenic cells and consequently damages spermatogenesis, or even induces infertility. So the reproductive toxicity of xenobiotics has been attracting more attention over the years. MC004, a water-soluble pro-drug of triptolide with broad antitumor spectrum and high antitumor performance, is synthesized from the diterpene triepoxide, triptolide. Since MC004 is still in early stage of research and development, systematic study of MC004 on its general toxicity, toxicity target organ and toxicity functioning mechanism hasn't been launched and there are few data available to evaluate its development perspective. However, as a synthesized compound of triptolide, its reproductive toxicity should be duly concerned. In this study, the short-term toxicity screening and optimization of the discovery toxicology technologies and the sub-acute toxicity test have been applied together to evaluate the early toxicity of the antitumor compound MC004. In this sub-acute toxicity study, Wistar rats were administrated every other day. These two series of toxicological experiments on MC004 were conducted in order to investigate the toxicity characteristics and its toxic target organs. Furthermore, the mechanism of the reproductive toxicity of MC004 was studied as well in order to provide toxicological foundation for its further development as a novel antineoplastic.The screening and optimization technologies were used in the study of early toxicology. In the general toxicity test, the LD50 of MC004 to female mice examined by the up and down acute toxicity test was 2.0 mg/kg(1.92～2.42 mg/kg). About 4 hours after the contamination, toxic symptoms appeared and death usually occurred between 24 to 60 hours after the contamination. The cell toxicity of MC004 to HepG2 and HK-2 was detected by MTT and LDH leakage test, MC004 inhibited the proliferation of both cell lines in a dose-dependent manner and increased the leakage of LDH, damaged the cytomembrane and increased the permeability, also in a dose- and time-dependent manner. The IC50 values of MC004 to HepG2 and HK-2 were respectively 0.81μM and 0.042μM for a 48 h continuous exposure. In the genetic toxicity test, the results of Ames test showed no mutagenicity to TA97 and TA102 Salmonella typhimurium with or without the S9 metabolic system at the concentration of 1.0, 10.0, 100.0, 500.0, 1000.0 and 2000.0μg in each dish. In the vitro reproductive toxicity test, midbrain cells from 13-day old rat embryos were executed to detect the teratogenicity. And the results revealed that MC004 could inhibit both proliferation and differentiation of midbrain cells, and decrease proliferation and colony formation of nerve cells in a dose-dependent manner. The 50% inhibition concentration of differentiation (ICD50) and proliferation (ICP50) of midbrain cells to MC004 were 0.0624μg/ml and 0.1963μg/ml, the ratio of ICP50 to ICD50 was 3.1, indicating that MC004 might have patient teratogenetic effect in vitro.In the vivo dosing study, male Wistar rats were randomly divided into four groups with 12 rats in each group. The rats of control group were given intravenous injections with normal saline and the exposure groups were administered respectively at doses of 0.25,0.50 and 0.75 mg·kg-1 MC004. All rats were injected once in two days for continuous 28 days. There were no significant toxicity manifestations, but the body weight of rats treated with MC004 increased slowly, especially the high-dose group, until the 19th day after first dosing, the body weight in 0.75 mg/kg was significantly low compared with that of the control group. The hematology and the plasma biochemistry parameters were examined. It was found that the levels of RBC, HGB and HCT in all MC004-treated groups decreased significantly in a dose-dependent manner, RDW increased in a dose-dependent manner, the number of WBC was significantly reduced in 0.75 mg/kg group, indicating that MC004 might have toxic effects on hematological system. There was no obvious difference in the plasma levels of indicating hepatic function, such as ALT, AST, ALP and TBIL, and there was no significant finding between the control and the exposure groups in the liver coefficient and liver histopathology. There were no changes in the routine uronoscopy parameters, in kidney histopathology and in the plasma biochemistry levels of indicating renal function, such as CREA and BUN. However, the weight of kidney and the kidney coefficient decreased in a dose-dependent manner, only the high-dose group decreased compared with the control group. So no sign of hepatotoxicity and nephtotoxicity was found with the dose of 0.75 mg/kg in the subacute toxicity test. In the reproductive toxicity test, effects of MC004 on reproductive organs, reproductive cells and enzymes activity of male rats in the testes were detected. Additionally, the effects of MC004 on fertility of the healthy and mature female rats after the males were contaminated were examined. The results are as follows: Firstly, Both the absolute weight and the organ/body ratios for testis, epididymis and epididymal cauda were dose-dependently decreased, and there were significantly decreases compared with the control group. Histopathological examination found that there were atrophy and degeneration of seminiferous tubules, irregular arrangement of spermatogenic cells, remarkable loss of spermatogenic cells; absence of sperms and secretions in the epididymis. Secondly, the sperm number and the sperm density were significantly decreased by treatment, the motility of the sperms, the percentage of the motility sperms and the rapid percentage of the sperms dose-dependently decreased, and the parameters indicating sperm motility including VAP, VSL, VCL dose-dependently decreased, ALH and BCF obviously decreased in both the mid- and high-dose group, STR and LIN significantly decreased in the high-dose group, while the abnormal sperm rate increased compared with that of the control group and the deformity rates of the low-, mid-,and high-dose group were respectively 18.9%, 23.1% and 21.8%. Most of the abnormal sperms were head absent, and then were polymorph, fold tail, absent hook sperms and a few of short tail or double head/tail sperms. Thirdly, the results of enzymes activity in the testes of rats showed that the activity of LDH, LDH-x, ACP and SDH in all treated groups dose-dependently decreased and (highly) significantly decreased compared with the control group, and the activity of SDH was reduced and had highly significant difference compared with the control group, indicating that MC004 could significantly reduce the spermatogenic energy, interfere with the capacitation and affect the spermatogenesis. Fourthly, after the tenth dosing, one normal female without MC004 treatment was placed into the cage of each male overnight for mating. The results showed that the body weights with MC004 treatment increased slowly, especially the high-dose group. The pregnant rats were euthanized and laparohysterectomized on gestational day 15. The results showed that there was a dose-dependent decrease in the weight of uterus, placenta, ovary, the mean number of implants, the mean number of living fetuses, and a dose-dependent increase in the mean percentage of preimplantation and the mean percentage of postimplantation. The results above indicated that MC004 could affect the pregnancy and development of female rats indirectly by interfering with the fertility of male rats, and cause significant early-embryonic development toxicity.In the study of MC004 on reproductive toxicity mechanism, MC004 could cause the detached germ cells of male rats in sertoli cell and germ cell(S-G) co-culture model increased in a dose- and time-dependent manner. The IC50 values of MC004 for S-G and Sertoli were 0.18μg/ml and 1.84μg/ml respectively, and the former is around 10 times bigger than the latter. The results indicated that MC004 damaged the germ cell first, and the tolerance of sertoli cell to MC004 is a little stronger than the germ cell. Besides, MC004 could dose-dependently increase the leakage of LDH in sertoli cells, indicating that MC004 could increase the permeability and damage the cytomembrane of sertoli cells. Male Wistar rats were injected intravenously with MC004 at 0.25 mg/kg for 1 or 3 days and euthanized after 24 h. The results showed that there were significant decreases in the weight of testes and the viscera coefficient after 3 doses. TUNEL staining of apoptotic cells showed that the percentage of apoptotic-positive cells increased between the single dosing and the three-time dosing group, both obviously increased compared with the control group. Primary spermatocytes and second spermatocytes were affected selectively in the MC004 treated groups, while the spermatogonia were mainly affected in the control group, indicating that the effect of MC004 induced apoptosis is cell-specific.In conclusion, MC004 has obvious reproductive toxicity to male rats and its major toxic target organs are testis. There are atrophy and degeneration of seminiferous tubules, irregular arrangement of spermatogenic cells, and remarkable loss of spermatogenic cells, at the dose of 0.25 mg/kg. MC004 can decrease the enzymes activity in the testes including LDH-x, ACP and SDH; MC004 can affect the spermatogenesis by interfering with the spermatogenic energy and the capacitation, and result in decrease in the number of sperms, motility and increase in abnormal sperms. Besides, MC004 can affect the pregnancy and development of female rats indirectly by interfering with the fertility of male rats, and cause significant early-embryonic development toxicity. Further, MC004 can damage the interaction between the sertoli and the germ cell by causing the detached germ cell to increase. These results might be the major reason for causing the atrophy of testes and epididymis, the decreased number of sperm and the decreased quality of sperms, while the sperm energy dysmetabolism and the abnormal spermatogenic cell apoptosis may be the main mechanism interfering with the interaction between the sertoli and germ cell. However, as an antitumor compound, we must consider its pharmacodynamics dose, the pharmacokinetics index and its suitable clinical patients before evaluating the development perspective of MC004.