Asistencia Mujer B - ADITAMENTO DE SERVICIOS DE ASISTENCIA

ADITAMENTO DE SERVICIOS DE ASISTENCIA – MUJER B

3.2.1.1. Asistencia Mujer B

To test weather the inhibition of the enzymatic activity of LSD1 in a cell-free assay could be translated in pharmacological effects in cells, active compounds were tested in six AML cellular models and namely HL-60, MV4-11, KASUMI, U937, OCI-AML3 and THP-1 cell lines. These AML lines are representative of three AML subtypes according to the FAB classification, which ranks AMLs based on their differentiation status, being M1 the least and M6 the most differentiated class.289

Table 4.2: FAB classification of the AML cell lines used.

M2 M4 M5

KASUMI1 OCI-AML3 MV4-11

HL-60 THP-1

U937

After incubation with the LSD1 inhibitors, cell survival was quantified using CellTiter- Glo®_{, a luminescent reagent that detects the presence of metabolically active cells by} measuring the quantity of adenosine triphosphate (ATP) in the system (Figure 4.7).

Figure 4.7: CellTiter-Glo®_{luminescence.}

The luciferase contained in Ultra-GloTM_{uses the luciferin as substrate in the presence of ATP, releasing} bioluminescence. Adapted from the manufacturer manual.290

The first set of experiments was performed to estimate the anti-proliferative activity of TCP in the AML cell lines and define the appropriate experimental conditions (concentrations and course of treatment) to evaluate the activities of the novel TCP- derived analogues. To do so, cells were treated with five concentrations (1 µM, 3 µM, 10 µM, 30 µM and 100 µM) and cultured for 48 h and 72 h (Figure 4.8).

Figure 4.8: Effects of TCP on the proliferation of AMLs (48 h and 72 h).

Survival (RLU) was normalised to pre-treatment levels (untreated cells). Statistical significance was determined with two-way ANOVA and corrected for multiple comparisons using Dunnett’s test. Data are shown as means ± STD (n=5); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

TCP treatments revealed to hinder the viability of KASUMI, HL-60, MV4-11 and OCI- AML3 cells at 3-10 µM, while U937 and THP-1 were not significantly affected. Generally, marked effects were registered following 72 h exposure.

The anti-proliferative activities of 4.10 and 4.11, which demonstrated exceptional abilities in suppressing LSD1 enzymatically, were next evaluated. The compounds were first tested at two concentrations (1 µM and 100 nM). As the enzymatic inhibition of such compounds increased by 50-fold compared to TCP, cellular activity was predicted to fall in this range of concentrations. Viable numbers were measured after 24 h, 48 h, 72 h and 120 h with CellTiter-Glo® (Figure 4.9 and 4.10).

Figure 4.9: Effects of TCP analogue 4.10 (100 nM and 1 µM; 24 h, 48 h, 72 h and 120 h) on the proliferation of AMLs.

Survival (RLU) was normalised to pre-treatment levels for each cell line. Statistical significance was determined with one-way ANOVA and corrected for multiple comparisons using Dunnett’s test. Data are shown as means ± STD (n=5); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4.10: Effects of TCP analogue 4.11 (100 nM and 1 µM; 24 h, 48 h, 72 h and 120 h) on the proliferation of AMLs.

This preliminary evaluation confirmed the enhanced anti-proliferative activity of the novel compounds compared to the scaffold TCP. The effects exerted by 4.10 and 4.11 resulted similar. A substantial drop in cell proliferation was generated after prolonged exposures (48-120 h) and the most significant effects were detected in HL-60, THP-1, KASUMI and MV4-11 cells, whereby survival was significantly decreased at 1 µM. Interestingly, after short-time incubation (24 h and 48 h) an increment in cell proliferation was observed, mostly evident in OCI-AML3. The registered data are in agreement with the increased expression of LSD1 in poorly differentiated AMLs reported in the literature. LSD1 is greatly expressed in cells ranked in the FAB-M1 subtype84 and HL-60 and KASUMI belong to the FAB-M2 subtype, also characterised by poor differentiated blasts. A high reduction in cell viability was also reported for

MV4-11 cells, ranked in the M5 subtype and characterised in contrast, by cells with a more differentiated status.

After assessment of 4.10 and 4.11, the anti-leukaemic activity of the remaining compounds, belonging to the new library of TCP derivatives, was examined in similar experimental conditions. Although the most evident decrease in cell proliferation was recorded at 120 h, prolonged incubation can lead to cell stress and depletion of medium nutrients. For these reasons, the effects of 4.12-4.25 were evaluated following 72 h treatment and at the previously reported concentrations (1 µM and 100 nM, Figures 4.11-4.21). As compounds 4.26 and 4.27 have been synthesised later on to further extend the library, these were not included in the first tests.

Figure 4.11: Effects of TCP analogue 4.12 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.12: Effects of TCP analogue 4.13 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.13: Effects of TCP analogue 4.14 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Survival (RLU) is normalised to pre-treatment levels for each cell line. Statistical significance was determined with one-way ANOVA and corrected for multiple comparisons using Dunnett’s test. Data are shown as means ± STD (n=5); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.14: Effects of TCP analogue 4.15 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.15: Effects of TCP analogue 4.16 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.16: Effects of TCP analogue 4.17 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.17: Effects of TCP analogue 4.18 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.18: Effects of TCP analogue 4.21 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.19: Effects of TCP analogue 4.23 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.20: Effects of TCP analogue 4.24 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

Figure 4.21: Effects of TCP analogue 4.12 (100 nM and 1 µM, 72 h) on the proliferation of AMLs.

With the exception of compound 4.21, which was devoid of activity, all the tested compounds caused a significant decrease of AMLs viability at the selected concentrations. In accordance with the data obtained with the previous evaluations of TCP, 4.10 and 4.11 the cell lines resulting most sensitive to anti-LSD1 treatments were KASUMI, HL-60, THP-1 and MV4-11 cells.

To further characterise the active new compounds, IC50s were determined by exposing AMLs to a broader range of concentrations (10 µ M, 3 µM, 1 µM, 0.3 µM, 0.1 µM, 0.03 µM, 0.01 µM, 0.003 µM and 0.001 µM) and the survival rates measured after 72 h incubation.

Figure 4.22: Dose-response curves showing the effects of 4.10 on AMLs proliferation (72 h).

The X-axis is in logarithm of concentration (M, Molar); Y-axis is the % of RLU (relative luminescence unit) compared to 100% activity (vehicle control, DMSO). Data are shown as means ± STD (n=5).

Figure 4.23: Dose-response curves showing the effects of 4.11 on AMLs proliferation (72 h).

The X-axis is in logarithm of concentration (M, Molar); Y-axis is the % of RLU (relative luminescence unit) compared to 100% activity (vehicle control, DMSO). Data are shown as means ± STD (n=5).

Figure 4.24: Dose-response curves showing the effects of novel TCP-derivatives on AMLs proliferation (72 h).

The X-axis is in logarithm of concentration (M, Molar); Y-axis is the % of RLU (relative luminescence unit) compared to 100% activity (vehicle control, DMSO). Data are shown as means ± STD (n=5).

Figure 4.25: Dose-response curves showing the effects of novel TCP-derivatives on AMLs proliferation (72 h).

The X-axis is in logarithm of concentration (M, Molar), Y-axis is the % of RLU (relative luminescence unit) compared to 100% activity (vehicle control, DMSO). Data are shown as means ± STD (n=5).

Table 4.3: In vitro anti-proliferative results of novel TCP-analogues onAMLs.

Values are reported in µM ± STD (n=5).

Results revealed that after 72 h treatment, the new TCP derivatives were able to arrest cell proliferation and particularly, 4.10 and 4.11 were active in the entire panel of analysed AMLs. Interestingly, the dose-response curves revealed that none of the new analogues produced a 100% inhibition of cells proliferation.

IC50 (µM ± STD, n=5)

Compound

KASUMI U937 HL-60 OCI-AML3 THP-1 MV4-11 TCP 32±1.8 > 100 84±15.0 89±13.0 81±3.9 63±8.4 4.10 0.03±0.03 1.6±0.09 1.7±0.14 1.8±0.08 0.1±0.08 0.18±0.07 4.11 0.7±0.03 1.2±0.02 0.09±0.01 0.06±0.04 0.2±0.02 0.19±0.4 4.14 0.4±0.01 0.6±0.01 0.6±0.01 1.0±0.10 0.1 ± 0.08 4.15 0.4±0.27 0.6±0.08 1.2±0.01 4.16 0.3±0.01 0.1±0.02 4.17 0.06±0.008 0.4±0.1 0.2±0.07 4.18 0.02±0.001 0.2±0.02 4.23 2.3±0.4 0.6±0.3 4.25 0.2 ± 0.07 4.26 0.1±0.02 0.2±0.01 4.27 0.3±0.01

In document COMPROMISO DE LA ASEGURADORA (página 98-109)