Evaluation of the antitumor activity of NOV202, a novel microtubule targeting and vascular disrupting agent

Figures & data

Table 1 Pharmacokinetics of NOV202 in male C57BI/6 mice

Parameter	NOV202 IV	NOV202 PO
Dose (mg/kg)	9.5	28.6
Cmax (ng/mL)	1,775	406
Tmax (h)	0.08	0.25
AUC0–t (ng h/mL)	1,423	1,576
AUC0–inf (ng h/mL)	1,485	1,607
λz range (h)	2–10	6–24
t½z (h)	2.4	4.8
Vd (L/kg)	22	–
Vss, area (L)	16	–
CL (L/h/kg)	6.4	–
Bioavailability (%) based on AUC0–inf	100	36

Abbreviations: λ_z, terminal disposition rate constant/terminal rate constant; t½z, terminal disposition rate constant/terminal rate constant half-life; Vd, apparent volume of distribution; Vss, apparent volume of distribution at steady state; CL, apparent total body clearance of the drug from plasma; AUC, area under the curve; IV, intravenous; PO, oral.

Table 2 Caco-2 permeability of NOV202

Compound	Caco-2, Papp A-to-B (10^−6 cm/s)	Caco-2, Papp B-to-A (10^−6 cm/s)	Permeability classification	Efflux ratio	Efflux substrate
NOV202	15.8±0.2	16.1±2.2	High	1.0	No

Notes: Permeability coefficiency and efflux ratio of a bi-directional Caco-2 permeability assay. The data are from two biological replicates (mean ± SD).

Abbreviation: SD, standard deviation.

Table 3 The mean plasma and brain levels of NOV202

Route	Plasma (ng/mL)	Brain (ng/g)	Plasma:brain ratio
PO
2 h	329	589	1.8
6 h	61	NA	–
24 h	NA	NA	–
IV
2 h	246	1,667	6.8
6 h	58	NA	–
24 h	NA	NA	–

Notes: The data are from three biological replicates. NA, the concentration was below the limit of reliable quantification (<25 ng/g in brain).

Abbreviations: IV, intravenous; NA, not available; PO, oral.

Table 4 Antiproliferative activity (IC₅₀, nM) of NOV202 and reference compounds in various cancer cell lines

Cell line	Disease	NOV202	PTX	VinC	CYT997
BXPC-3	Pancreatic adenocarcinoma	8.0	4.0	2.0	37.1
CCRF-CEM	Acute lymphoblastic leukemia	10.0	NA	10.4	3.6
HCT116	Colorectal carcinoma	9.2	1.9	1.7	47.7
HT29	Colorectal adenocarcinoma	3.9	3.5	0.4	39.3
RPM I8226	Multiple myeloma	12.0	NA	17.9	18.3
U-937	Histiocytic lymphoma	8.0	NA	7.6	33.1

Abbreviations: NA, not available; PTX, paclitaxel; VinC, vincristine; CYT997, lexibulin.

Table 5 IC₅₀-values (nM) and ratios of NOV202 and reference compounds in drug sensitive and resistant ovarian carcinoma cell lines

Cell line	NOV202	PTX	VinC	CYT997
A2780 (drug sensitive)	2.40	0.26	0.22	54.60
A2780/Adr (drug resistant)	2.30	810	210	182.70
IC50 ratio (resistant vs sensitive cells)	0.96	3,115	955	3.35

Abbreviations: PTX, paclitaxel; VinC, vincristine; CYT997, lexibulin.

Figure 1 Analysis of NOV202 and reference compound effects on viability and mitotic fate of human HCT116, HCT8 and MCF10A cells in vitro.

Notes: The cell confluency measurements (A) indicate clear growth suppression by the 50 nM concentration of NOV202 and reference drugs. Analysis of mitotic duration (B) shows that all compounds induce a long-lasting mitotic arrest. The fate of drug-treated mitotic cells (C) varies in a cell line dependent manner. All drug treatments significantly increased frequency of cell death (D) at each analysis time point and elevated the mitotic indices (E), especially at the 24 h time point. ***Statistical significance of P≤0.001.
Abbreviations: DMSO, dimethyl sulfoxide; EpoB, epothilone-B; PTX, paclitaxel; VBL, vinblastine.

Figure 2 Analysis of NOV202 and reference compounds effects on cell death and mitotic indices in ovarian carcinoma cell lines.

Notes: Treatment of OvCar-8, OvCar-4 and CaOv-3 cells with 12.5 nM NOV202 elevated markedly the frequencies of mitotic cells (A) at 12 and 24 h time points and increased cell death (B) at 24, 48 and 72 h time points in comparison to DMSO.
Abbreviations: DMSO, dimethyl sulfoxide; PTX, paclitaxel.

Figure 3 Effect of NOV202 on tubulin polymerization in vitro.

Notes: Data from a representative tubulin polymerization assay indicating the suppressive potency of NOV202 at 1–10 µM concentrations. Vincristine and paclitaxel were used as positive controls at 3 µM concentration for tubulin polymerization inhibition and stabilization, respectively.
Abbreviation: DMSO, dimethyl sulfoxide.

Figure 4 Inhibitory effects of NOV202 on endothelial cell cord formation and disruption in vitro.

Notes: (A) Over the entire concentration range (4.6 nM–10 µM), NOV202 suppressed completely the VEGF-induced cord formation determined as relative cord length (left hand diagram) and branching (right hand diagram). Combretastatin A4, a known inhibitor of angiogenesis, was equally effective at concentrations above 6.0 nM. (B) Representative micrographs for assay media control, VEGF (20 ng/mL), NOV202 (1.1 µM) and Combretastatin A4 (0.1 µM)-treated cells. The scale bar equals 1,000 µm. (C) Disruption of established cords by NOV202 and Combretastatin A4 determined as relative cord length (left hand diagrams) and branch points (right hand diagrams). The drugs were added at 96 h time point. (D) Concentration response curves for NOV202 and Combretastatin A4 caused disruption of VEGF stimulated established cords. The table shows pIC₅₀, Hill coefficient (slope) and maximal cord disruption (%) values that were determined for each replicate with respect to the maximal control VEGF (20 ng/mL) response. For the controls see also Figure S2.
Abbreviations: AUC, area under curve; VEGF, vascular endothelial growth factor; pIC₅₀, −log10 (IC₅₀).

Figure 5 Effects of NOV202 on developing blood vessels of chick embryo.

Notes: (A) A 50 nM NOV202 suppressed significantly the growth of the blood vessels at 24 and 48 h time points in comparison to controls. The antivascular impact of NOV202 was comparable to Combretastatin A4. (B) Representative micrographs showing the developing blood vessels for DMSO, NOV202 and Combretastatin A4-treated eggs. The white half-circles are the paper filters housing the experimental compounds. (C) Breakage of existing chick embryo blood vessels by NOV202 and Combretastatin A4. The scale bar equals 2,000 µm. *, **, and *** denote statistical significances of P≤0.05, P≤0.01 and P≤0.001, respectively.
Abbreviations: DMSO, dimethyl sulfoxide; SD, standard deviation.

Figure 6 In vivo efficacy of NOV202 on A2780 xenograft model and impact on body weight of NMRI nu/nu female nude mice.

Notes: (A) NOV202 induced a clear reduction in the volume of A2780 tumors in comparison to controls. (B) The NOV202 therapy was well tolerated according to the body weight measurements.
Abbreviations: IV, intravenous; PTX, paclitaxel; NMRI, Naval Medical Research Institute; PO, oral administration.

Figure S1 Cell population growth suppression by NOV202 and PTX in OC cell lines in vitro.

Notes: Representative images of OvCar-8, OvCar-4 and CaOv-3 cells cultured in the presence of 12.5 nM NOV202 or 12.5 nM PTX. The scale bar equals 1,000 µm.
Abbreviations: DMSO, dimethyl sulfoxide; OC, ovarian cancer; PTX, paclitaxel.

Figure S2 Neoangiogenic and established cord formation control data.

Notes: Time-courses of (A) neoangiogenic cord length and (B) branch point values (n=4–8 per assay point). Samples were first added at t=0 h (4 h post coculture creation). Suramin (100 µM), a known VEGF inhibitor, markedly attenuated the formation of cords. Time-courses of established cord length (C) and branch point (D) values (n=4–8 per assay point). Samples were first added at t=96 h. Addition of suramin (100 µM) resulted in complete regression of established cords.
Abbreviations: DMSO, dimethyl sulfoxide; VEGF, vascular endothelial growth factor.

Figure S3 High concentrations of NOV202 and Combretastatin A4 inhibit VEGF-induced cord formation without impairing cell viability.

Notes: Representative phase contrast and fluorescence micrographs showing the impact of the two compounds on endothelial cell cord structures and survival of cocultured cells. The images were taken 72 h posttreatment. The scale bar equals 1,000 µm.
Abbreviations: GFP, green fluorescent protein; VEGF, vascular endothelial growth factor.