Protective Effect Of DS-1226 On Cyclophosphamide-induced Bone Marrow Suppression In Mice

Dammarane Sapogenins (DS-1226) is an extract derived from Asian Ginseng by an alkaline hydrolysis of total ginsenosides and purification process, which showed that DS-1226 was effective to chemotherapy associated myelosuppression after it was tested in clinical trial. To investigate the potential protective effect of DS-1226 on chemotherapy associated myelosuppression, this study was primarily focusing on cyclophosphamide-induced myelosuppression in mice with respect to hematopoiesis and immunomodulation. The research data will provide for investigational new drug (IND) for bone marrow suppression protection.Establishment of a mouse model of cyclophosphamide-induced myelosuppressionBALB/c mice were intraperitoneally (ip) injected with a dose of 100,150,200 and 250 mg/kg of cyclophosphamide (CTX) respectively to induce myelosuppression. Analysis of peripheral blood cells was demonstrated that WBC was found a significant decrease at doses of 200 mg/kg and 250 mg/kg cyclophosphamide ip compared to that of doses of 100 mg/kg and 150 mg/kg. While a dose of 250 mg/kg cyclophosphamide caused a significant decrease of mice body weight with transient viable condition. It suggested that a dose of 200 mg/kg cyclophosphamide ip was optimal for myelosuppression mice model. Three other murine models using BALB/c, C57BL/6J and Kunming mice intraperitoneally (ip) injected with a dose of 200 mg/kg cyclophosphamide were also tested. Suppression of WBC counts was increased at D5 in C57BL/6J and Kunming mice models. While WBC counts were increased on D7 in BALB/c mice model and WBC suppression was consistently decreased. It was found that BALB/c mouse is the best option for myelosuppression mice model.Hematopoietic effect of DS-1226 on cyclophosphamide-induced myelo-suppression in miceThe general condition, peripheral blood cell counts, bone marrow histology, bone marrow smear were examined. It is showed that DS-1226 orally administered at doses of 37.5,75,150 mg/kg could improve the general condition, augment peripheral blood counts (take D3 as example, three doses of DS-1226 could augment peripheral WBC counts from 2.18×109/L to3.67×109/L,3.82×109/L and 3.81×109/L respectively), and increase the ratio of bone marrow nucleated and non nucleated cells, promote hematopoiesis in bone marrow at the high dose on D3 treatment, and decrease rate of polychromatic erythrocytic micronucleus formation induced by cyclophosphamide on D7 in a dose dependent manner. The hematology data demonstrated that oral intake of DS-1226 has potent hematopoietic effect on cyclophosphamide-induced myelosuppression in mice. Moreover, hematopoietic effect of intraperitoneally administered DS-1226 on cyclophosphamide-induced myelosuppression in mice was observed. It is showed that DS-1226 intraperitoneally administered at doses of 10,20, 40 mg/kg could reverse decreased peripheral blood cell counts, however, no significant improvement was found in bone marrow histological examination.Immunomodulatory effect of DS-1226 on cyclophosphamide-induced myelo-suppression in miceMouse spleen, thymus index and lymphocyte transformation rate as well as serum IL-2 and IL-6 levels were measured. It is showed that DS-1226 orally administered at doses of 37.5,75,150 mg/kg could increase mouse spleen, thymus index, ConA stimulated lymphocyte transformation rate, and serum IL-2 level at 3 days treatment; while increase mouse spleen index, LPS stimulated lymphocyte transformation rate, and serum IL-6 levels at 7 days treatment. These findings indicated that DS-1226 could stimulate immune function. Studies also showed that DS-1226 intraperitoneally administered could increase mouse thymus index and ConA stimulated lymphocyte transformation rate at 3 days treatment; while increase mouse spleen index, at 7 days treatment.Acute toxicity study of DS-1226 CapsuleThe oral LD50 of DS-1226 capsule in mice was not determined; it revealed that DS-1226 is to be very low toxic. Acute toxicity study in mice showed that the oral maximum tolerated dose (MTD) of DS-1226 is 8 g/kg, no adverse effects were observed at the MTD. It suggests that DS-1226 is considerably safe and non-toxic for oral intake.In conclusion, DS-1226 helps mitigate cyclophosphamide-induced myelo-suppression by promoting hematopoiesis and enhancing immune function. The enhanced immune response maybe related to modulation of serum IL-2 and IL-6 levels. Preliminary data will support investigational new drug (IND) for bone marrow suppression protection.