Entinostat

Athymic nu/nu mice (Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China) were maintained in accordance with the IACUC procedures and guidelines. Five × 10⁶ A549-Fluc or H460-Fluc cells were suspended in 100 μL of PBS, mixed with Matrigel (BD Biosciences) and injected subcutaneously into the flanks of 5-week-old female mice. Tumor formation was assessed by palpation and measured with fine calipers three times a week. Tumor volume was calculated by the formula: Volume = (Length × Width²)/2, which was statistically analyzed as we described previously [18 (link), 52 (link)]. When tumors reach ~150 mm³, mice were randomly assigned into four groups (n = 4 and n = 3 for A549-Fluc and H460-Fluc cells, respectively): 1) control mice received intraperitoneal (i.p.) injection of 100 μl of PBS; 2) mice received i.p. injection of entinostat (25 mg/kg for A549-Fluc or 12.5 mg/kg for H460-Fluc) in 100 μl PBS twice a week; 3) mice received i.p. injection of paclitaxel (7.5 mg/kg) in 100 μl PBS twice a week; 4) mice received i.p. injection of entinostat and paclitaxel in 100 μl PBS twice a week. At the end of study, mice were euthanized according to approved protocol. Tumors were excised and subjected to immunohistochemistry analyses and qRT-PCR measurement of miRNA expression.

The amount of RG7388 and Entinostat within NPs
was assessed using
high performance liquid chromatography (HPLC) and absorbance spectrometry,
respectively. The NP pellet was lysed using a mixture of 1:1 acetonitrile
and dimethyl sulfoxide (DMSO) to release any entrapped drug. RG7388
entrapment was detected by HPLC using a C18 reverse phase column (Phenomenex,
150 × 4.6 mm, 5 μM). The flow rate was set to be constant
at 1 mL/min at 25 °C. 30 μL of 1 mg/mL of sample was injected
per run, and the absorbance was detected at 273 nm and compared to
a series of standards prepared by spiking known amounts of free RG7388
into blank NPs (BNPs) in 1:1 acetonitrile:DMSO. Entinostat entrapment
was quantified by measurement of absorbance at 330 nm using a plate
reader (Biotek) and again compared to a series of standards prepared
by spiking known amounts of free Entinostat into BNPs in 1:1 acetonitrile:DMSO
(Supplementary Figure 1). Drug loading
was calculated using the formula below.

C57BL/6 mice
(8–12 weeks old) were treated with dual-loaded NPs via intravenous
injection (2 mg of polymer per animal in PBS), equivalent doses of
free RG7388 and Entinostat mixed with BNPs via intraperitoneal injection
(2 mg of polymer, Entinostat, and RG7388 per animal in 2% DMSO, 10%
kolliphor, 30% PEG 400, and 58% saline), or corresponding vehicle
controls on day 0 of the study. This dosing was repeated on day 5
of the study. Prior to commencing treatment, and also at 48 h after
each dose, blood samples were collected via tail vein puncture using
EDTA-coated capillary tubes (Greiner Bio-One). A complete blood count
analysis (CBC) with white blood cells differential was then carried
out in Belfast City Hospital using a XE-2100 automated hematology
analyzer (Sysmex Corporation, Kobe, Japan) equipped with Sysmex Work
Area Manager software. The body weight was monitored routinely to
guarantee the animal welfare.

The first phase of this trial was designed to determine the maximum tolerated dose combination (MTDC), defined by an acceptable toxicity profile of the combination, from among the dose combinations in Table 1.
This study was implemented at two large academic medical centers, the University of Virginia Health System and University of Rochester Medical Center. The study was an open-label pilot study. For the first lead-in phase, patients with metastatic disease were treated with capecitabine and entinostat at varied dosing combinations. Notably the existing dose for entinostat monotherapy at the time of study conception was 5 mg po weekly. The starting dose for entinostat used in this combination study was 3 mg po weekly based on preclinical and phase 1 data [20 (link), 21 (link)]. The starting dose for capecitabine for combination in this study was 800 mg/m² twice daily for 14 days followed by 7 days off (which falls on the low end of the range of clinically utilized and validated doses for capecitabine in combination). This starting dose was chosen as a safe dose with reasonable expectation of potential for clinical activity. The maximum target doses were entinostat 5 mg po weekly and capecitabine 1000 mg/m² po bid 14 days on, 7 days off. The maximum target dose for capecitabine was selected based on prevailing clinical practice at the time of study design and in anticipation of possible overlap of toxicities.
Dose escalation was conducted using the Bayesian partial order continual reassessment method (POCRM) for drug combinations [22 (link)]. Dose-limiting toxicity (DLT) was determined by adverse events occurring during the first cycle of treatment. The MTDC per protocol was specified to be the drug dose combination with a rate of DLT nearest to the target rate of 25%. Patients were monitored for toxicity using the standard NCI CTCAE version 4.03 tool. Individual patients experiencing DLT were required to interrupt therapy and reduce dose. Each occurrence of a DLT was then used to recalculate probabilities of further toxicity at all dose levels and to guide assignment of subsequent patients to treatment levels. The statistical model was set to allocate subsequent participants to the dose combination with an estimated future DLT rate closest to 25%.
Per published designs for a two-drug combinatorial study in cancer, a 90% confidence interval would be calculated around the DLT probability for each two-drug combination level studied. The final recommended dose combination to move forward to the second phase would then be the best dose level with a DLT rate under 25%. Estimates were made using the continual reassessment method (CRM) models [23 (link)]. The second phase of the study was designed to continue until 30 eligible participants with high-risk residual disease had been treated with protocol treatment at the recommended MTDC. The maximum target sample size was based upon acquiring sufficient information to assess the goal of determining the MTDC in participants with high-risk residual disease, obtaining an estimate of treatment tolerance and preliminary assessment of disease-free survival. The enrollment goal of 30 breast cancer subjects was calculated/powered to test for tolerance. The null rate of treatment tolerability was 60% and was to be compared to an alternative rate of 80% with a one-sided type I error rate of 0.094 and power of 0.871 with a binomial test. The choices of the null and alternative rates were based upon results reported in the CREATE-X trial [14 (link)] which reported 75% of participants (95% CI [69, 80%]) treated with 8 cycles of capecitabine did not discontinue treatment. For this study, data supporting a tolerance rate of 60% (below the lower limit of the confidence bound) would be considered unacceptable. At study conclusion, frequency, proportion, and severity of adverse events and DLTs by treatment combination were tabulated.
The study was an investigator-initiated trial funded by the UVA Cancer Center and philanthropic funds. The trial was overseen by the institutional review board, the protocol review board, and the data safety board at the UVA Cancer Center. The study was compliant with ICH-GCP guidelines. All consented patients have been included in this report.