P505-15, a Highly Selective SYK Inhibitor, Shows Significant Activity In Primary CLL Cells and Is Synergistic with Fludarabine at Low Concentrations

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World. Despite significant progress, CLL remains incurable so novel therapies are needed. Recent studies have highlighted the importance of B-cell receptor (BCR) associated kinases in the pathogenesis of B cell malignancies and as potential therapeutic targets. Spleen tyrosine kinase (SYK) is of particular interest as its activation results in enhanced proliferation and survival of B-cells. Analysis of non-Hodgkins lymphoma (NHL) cell lines and primary chronic lymphocytic leukemia (CLL) samples have shown that SYK is persistently phosphorylated and that SYK inhibition results in abrogation of downstream kinase activity, leading to apoptosis. The kinase inhibitor FosD (R788), which has shown clinical activity in heavily pre-treated NHL and CLL patients, exhibits inhibitory activity against SYK (IC₅₀ = 41nM) but also inhibits a broad spectrum of other kinase targets. By contrast, P505-15 is a novel small-molecule SYK inhibitor (SYK IC₅₀ = 1nM) that is far more selective than previously described SYK inhibitory compounds with anti SYK activity that is at least 80-fold greater than its affinity for other kinases. We applied P505-15 to primary CLL cells to evaluate the effect of P505-15 on cell viability alone or in combination with fludarabine.

Fresh primary CLL cells from 42 CLL patients were purified using a Ficoll gradient. Purified cells were then added to wells (5 × 10⁴ per well) containing four serial dilutions of P505-15 (ranging from 10 nM to 10 μM) with or without different concentrations of fludarabine (also ranging from 10nM to 10 μM). Three days after adding primary CLL cells to each well, we performed a tetrazolium-based cell viability assay (MTS) to evaluate the effect of P505-15 on CLL cells. The viability data are normalized to untreated controls and used to calculate IC₅₀ values. Synergy (P505-15 plus fludarabine) was evaluated using the Calcusyn program.

We saw significantly decreased cell viability (IC₅₀ < 3 μM) in 15/42 (36%) of primary CLL samples. Twelve (29%) of these samples had IC₅₀ drug concentrations < 1 μM (median 393.6 nM). One of seven samples with the 17p deletion exhibited significant sensitivity (IC₅₀ = 37.5 nM). In the presence of P505-15, significant decreases in cell viability were also seen in samples from relapsed patients and in those with additional poor risk features such as ZAP70 and/or CD38 expression, chromosome 11q deletion, and/or an unmutated IgVh. When P505-15 was combined with fludarabine (n=15), synergy was observed in 13 (87%) of samples including at the lowest concentrations of P505-15 (10 nM) and fludarabine (10 nM)-See Figure 1.

Figure 1:

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Isobologram showing synergy for P505-15 plus fludarabine in a primary CLL sample (09-248). The linear lines (A, for IC50 and IC75) represent the dose combination that is expected to yield an additive drug effect while the inset lines (B, for IC50 and IC75) illustrate the synergistic effect for this drug combination).

Figure 1:

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Isobologram showing synergy for P505-15 plus fludarabine in a primary CLL sample (09-248). The linear lines (A, for IC50 and IC75) represent the dose combination that is expected to yield an additive drug effect while the inset lines (B, for IC50 and IC75) illustrate the synergistic effect for this drug combination).

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These data demonstrate single agent activity of the highly specific SYK inhibitor P505-15 in CLL. Combination therapy with fludarabine is synergistic at very low concentrations of both P505-15 and fludarabine. Thus, P505-15 appears to be an attractive compound for clinical development especially given its high selectivity for SYK. Our results validate the rationale of targeting SYK in CLL. An initial human dose finding study in normal healthy volunteers is ongoing.

Druker:Molecular MD: Equity Ownership. Pandey:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment.