Universidad Católica Santo Toribio de. Mogrovejo Integrantes Calderón Dávila Ana Elisa Pinglo Chapiama Wendy Narro Julca Miguel Angel. ley (Pickett) Burwell, and Mary Johnston. (Burwell) Butler; and papers, . Virginia Historical Society, P.O. Box , Richmond, VA Full Name and. DER. ADMINISTRATIVO; DER. CONSTITUCIONAL; Asistencia legal; Defensoría del Pueblo. Justia Legal Resources. Find a Lawyer.
|Published (Last):||10 January 2009|
|PDF File Size:||18.55 Mb|
|ePub File Size:||1.31 Mb|
|Price:||Free* [*Free Regsitration Required]|
The activation of FLT3 is initiated by the binding of the FLT3 ligand, which is expressed by stromal cells, to the receptor. Two major types of FLT3 mutations have been identified: These mutations confer an adverse prognostic influence with chemotherapy failure and relapse [ 5 — 8 ]. Therefore, many researchers and pharmaceutical companies have tried to find FLT3 inhibitors as potential therapeutic agents of AML. Several clinical candidates targeting FLT3 have been reported including lestaurtinib [ 11 ], tandutinib [ 12 ], sorafenib [ 13 ], KW [ 14 ], and quizartinib [ 15 ].
Among them, lestaurtinib is indolocarbazole derivative and well known multi-targeted tyrosine kinase inhibitors. Therefore, the development of new potent and selective FLT3 kinase inhibitors is needed at the present time.
Our group previously reported that indirubin analogues potently inhibit FLT3 kinase [ 21 ]. After further development of the indirubin derivatives and their inhibitory activity, we identified a novel FLT3 inhibitor, LDD, through a kinase inhibitory assay of synthesized compounds, which significantly inhibited the growth of an AML cells.
The ability of LDD to suppress tumor cell growth in vivo and in vitro makes it a promising candidate to treat AML patients as well as to possibly treat other types of cancers also. We previously reported that a series of 5-substituted indirubin derivatives are potent FLT3 inhibitors [ 21 ], which effectively inhibited the growth of acute myeloid leukemic cells. While the indirubins had a potent kinase inhibitory activity, their poor solubility in water caused some physiological problems.
To address the solubility problems of these indirubin derivatives, in this study, we designed and synthesized new analogues with hydrophilic functional groups on the molecules. Several indirubin analogues were synthesized, and their structure activity relationship was investigated Supplementary Table 1. The IC 50 s against other kinase activities were also measured Table 1.
cinemararro by Angel Narro on Prezi
In vitro activity of LDD against select kinases. MV cells are leukemic cells with a FLT3 kinase mutation. MV cell growth and survival are known to be dependent on the FLT3 activity [ 22 ]. The cytotoxicity by LDD was measured and shown in Table 2. Immortalized T lymphocytes Jurkat cells, prostate cancer PC-3 cells, breast cancer MCF-7 cells, and erythroleukemia K cells were also subjected to the cytotoxicity assay. Anti-proliferative activities of LDD against various cancer cell lines.
The calculated CI value at GI 50 was 0. Doxorubicin and LDD were treated at a ratio of The CI value was calculated with the CompuSyn software. Apoptotic cell death by Oey was measured with annexin V staining.
The apoptotic cell population increased in a dose-dependent manner shown in Figure 2B. The LDD treatment induced a significant change 2321 the cell lwy populations. The sub-G1 population indicating 32221 dead cell population increased by the LDD treatment from 4.
The results in Figure 3 are consistent with the cell growth suppression Table 32221 and apoptosis of the MV cells Figure 2.
Cells were then subjected to cell cycle analyses using a flow cytometer. G 1 phase, M2: Cyclin D1 levels were detected by western blot. The ability of the LDD compound to inhibit the downstream signal transduction pathway was assessed by western blot analysis.
MV cells were treated with LDD for 4 h at the indicated concentration. The pharmacokinetic property of LDD was investigated. The relevant pharmacokinetic parameters are listed in Table 3. This result is consistent with the result of the parallel artificial membrane permeability PAMPA assay. Additionally, the non-renal clearance of LDD was the main route of elimination due to the negligible contribution of the CL R to CL, and the urinary excretion was 0.
Nevertheless, the F of LDD was low, at 1. Due to the low bioavailability, the intravenous route of administration was used for the in vivo xenograft study. Pharmacokinetic study of LDD Blood samples were collected at the indicated time points after the injection. AUC, area under the curve; A e 0—24 htotal amount excreted in h urine; BW, body weight; CL time-averaged total body clearance; CL NR, time-averaged nonrenal clearance; GI 24 htotal amount recovered from the 32221 gastrointestinal tract including its contents and feces at 24 h; MRT, mean residence time; V ss, apparent volume of distribution at steady state.
From the metabolite information acquired from the pharmacokinetic experiments, the major metabolite LDD is expected to contribute to the anti-tumor effects of LDD As shown in Figure 23212the tumor sizes in the LDD group were dramatically smaller than those of the control group. There was no significant difference in body weight between the groups during the administration period Supplementary Figure 1.
In vivo antitumor efficacy of LDD The tumor size was measured, and the tumor volumes were calculated A. At day 21 after drug administration, mice were sacrificed, and the tumor 23212 measured 2221.
In this study, the indirubin derivative LDD compound was characterized as a FLT3 inhibitor with anti-leukemic activity. Based on the pharmacokinetic profile of LDD Figure 5an in vivo xenograft study was performed. The tumor volume and weight were dramatically suppressed by LDD Figure 6 indicating the potential of LDD as an antileukemic agent.
The combination effect of a cytotoxic drug and targeted agent should be evaluated because antagonism may exist between the two drugs. The combination index, CI, was measured using the principle based on Chou et al. As mechanisms lye the anti-leukemic effects, apoptotic cell death Figure 2 and cell cycle arrest Figure 3 by the LDD treatment was investigated in this study. Other potential mechanisms such as differentiation and cellular senescence were also explored.
Discovery of a FLT3 inhibitor LDD1937 as an anti-leukemic agent for acute myeloid leukemia
Differentiation of leukemic cells was evaluated with Wright-Giemsa staining. However, differentiated cells were not observed in LDDtreated cells data not shown. The pharmacokinetic properties of LDD were 32221 Table 3. Based on the low F value 1. The pharmacokinetic profile of the 2322 LDD was measured ely is shown in Table 3. These favorable pharmacokinetic properties may contribute to the effective anti-tumor activity in vivo. Its indication is newly diagnosed AML that is FLT3 positive, in combination with standard cytarabine and daunorubicin induction and cytarabine consolidation.
Monotherapy of midostaurin for induction therapy is not an approved indication. The oral route of leyy of 50 mg twice daily with food is recommended. In terms of clinical application, pulmonary toxicity and interaction with CYP3A4 inhibitor and inducer are major disadvantages of this drug.
Midostaurin is a derivative of staurosporine, a pan-kinase inhibitor. Improvement of the kinase selectivity, overcoming adverse effects especially pulmonary toxicity, and the removal of the drug interaction mediated by CYP3A4 will result in a better drug than that of midostaurin. Many approaches for developing novel therapies for AML are ongoing, such as antibodies against CD33, epigenetic targets, and T cell immunotherapy [ 27 ].
FLT3 targeting is still a promising approach to overcome the treatment failure of AML despite the insufficient clinical results from recent trials. Experiences from FLT3 inhibitor clinical trials have lwy, and the follow-up analysis of the clinical data suggests that more effective FLT inhibitors are still required [ 28 ].
Here, we presented the LDD compound which has great potency in vitro and in vivo for antileukemic activity. The IC 50 was calculated with nonlinear regression using Prism version 5.
Cells were treated with each compound alone and a combination of two compounds. Cell viability was assessed as described above, and the combination index CI was calculated with the CompuSyn software version 1.
The synthetic procedures for all the compounds are available in the supporting information Supplementary Figures 2—5. Ten microliters of the total volume of the kinase reaction were added to the wells of lwy well assay ,ey.
The kinase reactions were incubated for 90 min. The signal was measured on an EnVision multi-label reader. The data were analyzed with the BD Accuri C6 software.
After labelling, the cells were washed twice with rinse buffer. Cell cycle distribution was analyzed by flow cytometry with an Accuri C6 flow cytometer. The antibodies used were as follows: Goat anti-rabbit IgG ; 1;5, and anti-mouse IgG ; 1: All mice were provided food and water ad libitum and were then maintained during this study.
A blood sample of approximately 0. The turbo ion-spray interface was operated at an ion capillary voltage of 3. Separation was done on a reverse-phase C 18 column BEH, 1. After measuring the concentrations of the LDD and LDD, standard methods were used to calculate the pharmacokinetic parameters using a non-compartmental analysis WinNonlin 2.
The drug or the control PBS was administered daily for a duration of 21 days. Tumor sizes were measured twice a week for 21 days, and the tumor volumes were calculated with the following formula: After 21 days, the mice were sacrificed, and the tumor weights were measured. FMS-like receptor tyrosine kinase-3; GI Imprinting Control Region; IC Participated in research design: Choi YH and Chin ; Performed data analysis: Characterization of the protein encoded by the flt3 flk2 receptor-like 232221 kinase gene.
The role of FLT3 in haematopoietic malignancies. Internal tandem duplication lry the flt3 gene found in acute myeloid leukemia.