Pilot Study to Evaluate Safety & Biological Effects of Orally Administered Reparixin in Early Breast Cancer

CSCs were measured in tissue samples by the following techniques: ALDH-1 by ALDEFLUOR and by immunohistochemistry (IHC); CD44/CD24 by flow cytometry or examination of RNA transcripts by RT-PCR and by immunohistochemistry (IHC); epithelial mesenchymal markers (Snail, Twist, Notch) by immunohistochemistry (IHC).

Pathway markers (AKT, FAK, PTEN and CXCR1) were measured in tissue samples at the pre-study (Day -14 to 0) and Day 21 (or within 24 hours of last dose). For the evaluation of the stain extent, the following semi-quantitative score method (0 to 4) was used: 0, no positive cells; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%. Hence for this scale, the higher the values, the worse is the outcome. For the evaluation of the stain intensity, the following score method (0 to 3) was used: 0, negative; 1, weak; 2, moderate, 3; strong. Also for this scale, the higher the score, the worse the outcome.

Serine-threonine protein kinase (AKT, also known as protein kinase B, PKB) Focal adhesion kinase (FAK) Chemokine receptor-1 (CXCR1)

Markers of inflammation (IL-1β, IL-6, IL-8, TNF-α, GM-CSF, VEGF, and b-FGF) were measured in plasma from peripheral blood.

IL = interleukins TNF-α = tumor necrosis factor-alpha GM-CSF = macrophage colony stimulating factor VEGF = vascular endothelial growth factor b-FGF = basic fibroblast growth factor

CD31 staining by IHC. For the evaluation of the stain extent, the following semi-quantitative score method (0 to 4) was used: 0, no positive cells; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%. Hence for this scale, the higher the values, the worse is the outcome. For the evaluation of the stain intensity, the following score method (0 to 3) was used: 0, negative; 1, weak; 2, moderate, 3; strong. Also for this scale, the higher the score, the worse the outcome.

CD31 is a transmembrane glycoprotein, 130-140 kDa, also know as platelet-endothelium cell adhesion molecule (PECAM-1). CD31 is ligand for CD38 and plays a role in thrombosis and angiogenesis. CD31 is strongly expressed in endothelial cells and weakly expressed in megakaryocytes, platelets, occasional plasma cells, lymphocytes (espc. marginal zone B-cells, peripheral T-cells) and neutrophils.

Autophagy is a lysosomal degradation and recycling process implicated in cancer progression and therapy resistance. Labeling of p62 serves as a useful marker for the induction of autophagy, clearance of protein aggregates, and the inhibition of autophagy. Labeling of LC3B serves to track the binding of p62 and subsequent recruitment of autophagosomes.

For the evaluation of the extent, the following semi-quantitative score method (0 to 4) was used: 0, no positive cells; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%. Hence for this scale, the higher the values, the worse is the outcome. For the evaluation of the intensity, the following score method (0 to 3) was used: 0, negative; 1, weak; 2, moderate, 3; strong. Also for this scale, the higher the score, the worse the outcome.

Once absorbed, reparixin is highly protein bound. By comparing Cmax and AUC for unbound drug to that for total drug, only < 0.1% to 0.2% of reparixin is available as unbound (free) drug. The intent of the PK outcomes was not to compare the results between the two cohorts; for this reason results for the PK outcomes are reported for the whole group of the treated patients.

Cmax = Maximum plasma concentration obtained directly from the data without interpolation, expressed in concentration units

Once absorbed, reparixin is highly protein-bound. By comparing Cmax and AUC for unbound drug to that for total drug, only < 0.1% to 0.2% of reparixin is available as unbound (free) drug. The intent of the PK outcomes was not to compare the results between the two cohorts; for this reason results for the PK outcomes are reported for the whole group of the treated patients.

tmax = Time to reach the maximum plasma concentration obtained directly from the data without interpolation t1/2 = Terminal elimination half-life calculated as ln(2)/ lambda z; calculated only if the coefficient of determination R2 in lambda z estimation is at least 0.8.

The intent of the PK outcomes was not to compare the results between the two cohorts; for this reason results for the PK outcomes are reported for the whole group of the treated patients.

AUC0-8 = The area under the plasma concentration-time curve from time 0 to 8 hours post-dose; AUClast = The area under the concentration-time curve from time 0 to last quantifiable concentration AUCtau = The area under the plasma concentration-time curve for dosing interval (dosing interval [tau] = 8 hours); AUCinf = The total area under the plasma concentration-time curve from time zero to time infinity; AUC0-inf = AUClast + Clast/lambda zeta, where Clast is the last observed concentration ≥ lower limit of quantitation at time tlast.

All these parameters were calculated by the linear trapezoidal rule.

The intent of the PK outcomes was not to compare the results between the two cohorts; for this reason results for the PK outcomes are reported for the whole group of the treated patients.

CL/F = Apparent oral clearance - for DF1681Y only, calculated as dose/AUCinf.; calculated only when the coefficient of determination R2 in lambda zeta estimation is at least 0.8 and percent AUC extrapolation is less than or equal to 20%.

CLss/F = Steady state apparent oral clearance - for DF1681Y only calculated as dose/AUCtau.

The Leukocytes subsets analyzed are the following: Lymphocyte in WBC, Total T cell in lymphocytes, B cells in lymphocytes, T-helper cell in lymphocytes, CTL in lymphocytes, NKT cell in lymphocytes, ADCC NK subsets in lymphocytes, Regulatory NK subsets in lymphocytes, Exhausted NK subsets in lymphocytes, CD56-CD16+ NK subsets in lymphocytes, CD11b in PMNs - IL-8, CD18 in PMNs - IL-8, MFI of CD11b - IL-8, MFI of CD66b - IL-8, MFI of CD18 - IL-8, CD11b in PMNs - US, CD18 in PMNs - US, MFI of CD11b - US, MFI of CD66b - US, MFI of CD18 - US, Percent Monocytes expressing IL6 - IL-8,Percent Monocytes expressing IL1b - IL-8, Percent Monocytes expressing IL8 - IL-8, Percent Monocytes expressing TNFa - IL-8, Percent Neutrophils expressing IL6 - IL-8, Percent Neutrophils expressing IL1b - IL-8, Percent Neutrophils expressing IL8 - IL-8, Percent Neutrophils expressing TNFa - IL-8,Percent Monocytes expressing IL6 - US,Percent Monocytes expressing IL1b - US, etc.