Combination drug therapy
A technology of compounds and combination products, applied in drug combinations, antineoplastic drugs, pharmaceutical formulations, etc.
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Embodiment 1
[0290] Antiproliferative effects of compound A and compound B in prostate cancer cells
[0291] LNCaP prostate cancer cells are androgen receptor positive and depend on androgen for cell growth. Cells were grown in charcoal-stripped serum to remove potential androgens from the serum. Under these conditions, cell growth is dependent on exogenous androgens (eg, R1881). The androgen receptor antagonist Compound A inhibits the growth of LNCaP cells in a concentration-dependent manner in the presence of synthetic androgen (0.1 nMR1881). Similarly, Compound B also inhibited the growth of LNCaP cells under these conditions. When LNCaP cells were treated concomitantly with both compounds, there was an additive antiproliferative effect ( figure 1 ).
[0292] Cell Proliferation Assay
[0293] The androgen-dependent prostate cancer cell line LNCaP was cultured at a density of 1,000 cells / well in 96-well tissue culture plates for 24 hours in RPMI1640 medium supplemented with 10% ...
Embodiment 2
[0295] Effects of Compound A and Compound B on Cell Signaling in Prostate Cancer Cells
[0296] LNCaP cells were treated with compound B (3 or 10 uM) alone or in the presence of compound A (3 uM), both in the presence of 0.1 nM of synthetic androgen (R1881).
[0297] Compound B inhibits AKT phosphorylation, suggesting inhibition of PI3 kinase activity in cells. The AKT inhibitor used as a control in this experiment (it was either alone or in combination with compound A, also labeled ENZA) demonstrated a reduction in phosphorylation of downstream signaling as evidenced by a reduction in phospho-PRAS40 and phospho-S6 . Compound B treatment alone and in the presence of Compound A demonstrated inhibition of phospho-S6, a marker of downstream pathway regulation.
[0298] The combination of the two compounds resulted in a greater decrease in phospho-S6 levels compared to Compound B alone. Decrease in S6 phosphorylation has been associated with greater anti-proliferative effects...
Embodiment 3
[0302] Effects of Compound A and Compound B on Caspase 3 / 7 Induction in Prostate Cancer Cells
[0303] Caspase 3 / 7 activity, a marker of apoptosis, was measured using a luminescent caspase 3 / 7 assay in LNCaP cells treated with Compound A (5 uM), Compound B (5 uM), or both ). Caspase 3 / 7 activity was normalized and plotted as a percentage of untreated control samples. Data from 2 independent experiments (N=1, N=2) are shown and represent mean ± standard deviation from duplicate treatments.
[0304] In LNCaP cells, there was a slight induction of caspase 3 / 7 activity after 5 days of treatment with compound B or compound A, while treatment with the combination had a further increase (1.4-2.0 fold in growth medium, 2.5-4 times in culture medium)( image 3 ).
[0305] Luminescent caspase 3 / 7 assay
[0306]Tumor cells were inoculated in 100 μL of growth medium (medium containing 10% FBS) or CSS medium (medium containing 10% activated carbon-absorbed fetal calf serum) in 96-...
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