Preclinical Evaluation of the Novel, Orally Bioavailable Selective Inhibitor of Nuclear Export (SINE) KPT-335 in Spontaneous Canine Cancer: Results of a Phase I Study

The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability.Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35-256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35-354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities.This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer

IC₅₀ (± S.D.) of SINE for human and canine lymphoma cells.

<p>IC₅₀, 50% inhibitory concentration; DLBCL, diffuse large B-cell lymphoma;</p><p>NP, not performed.</p

Subject demographics.

<p>Subject demographics.</p

Hepatic and hematologic toxicities.

<p>Hepatic and hematologic toxicities.</p

Trends in quality of life in dogs treated with KPT-335.

<p>An overall score was created based on answers to questions on the quality of life questionnaire. The scores from each question were summed resulting in an overall quality of life score which could range from 23 to 115. These are represented graphically in the figure above where scores for each patient are graphed over time (each line represents a patient). Trends in quality of life during the study were examined using linear mixed models. The overall quality of life did not change significantly in dogs treated in either the (A) dose escalation study (p = 0.64) or (B) dose expansion study (p = 0.47).</p

Biologic activity of SINE compounds against canine tumor cell lines.

<p>Canine tumor cell lines C2 (mast cell), OSA16 (osteosarcoma) and 323610-3 were cultured in 96 well plates in triplicate with serial dilutions of KPT-214 for 92 hours after which the plates were collected, media removed, and the plates were frozen at −80°C. Analysis for effects on cell proliferation was then performed using the CyQUANT assay according to the manufacturer’s specifications. Experiments were repeated three times; the IC₅₀ for each cell line is shown.</p

Pharmacokinetics of KPT-335 in healthy dogs.

<p>Pharmacokinetics of KPT-335 in healthy dogs.</p

Constitutional and gastrointestinal toxicities.

<p>Constitutional and gastrointestinal toxicities.</p

Biologic activity of SINE compounds against canine NHL cells.

<p>(A) Jurkat cells and primary canine DLBCL cells (sample #1–5, #5 was independently tested twice) were cultured in 96-well plates for 72 hours with log serial dilutions of KPT-185 and the cell viability was analyzed using the MTS assay. Each experiment was performed in duplicate wells and experiments were repeated three times (B) Human and canine DLBCL cells were cultured in a 96-well plate for 72 hours with 3-fold serial dilutions (0–1000 nM) of KPT-335 and analyzed using the MTS assay. Each experiment was performed in duplicate wells and experiments were repeated three times. (C) CLBL1 cells and primary canine DLBCL cells (sample #1) were treated with 100 nM KPT-335 for 24 hours and analyzed for apoptosis using flow cytometry. Experiments were performed three times independently and the average results are shown. (D) Expression of XPO1 in human and canine DLBCL cell lines. Protein lysates prepared from OCI-Ly3, OCI-Ly10, and CLBL1 were separated by SDS-PAGE and subjected to immunoblotting for XPO1; β-actin served as the control.</p