Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer

Abstract

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. This study evaluated the efficacy and safety profile of combining T with gemcitabine (G) and nab-paclitaxel (nP) in human pancreatic ductal adenocarcinoma (PDAC) xenograft models in mice. Antitumor activity of the G + nP + T triplet was assessed and compared with T-alone or the G + nP doublet in the Hs766t, MIA PaCa-2, PANC-1, and BxPC-3 PDAC xenograft models. Efficacy was assessed by tumor growth kinetic analysis. Body weight, blood cell counts, blood chemistry, and the von Frey neuropathy assay were analyzed to evaluate safety profiles. Pharmacodynamic changes after the treatment were determined by immunohistochemistry of cell proliferation, DNA damage, apoptosis, hypoxia, and tumor stroma density. The G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP doublet. Compared with vehicle (V), G + nP induced body weight loss, reduced neutrophil and lymphocyte counts, increased the levels of liver function parameters, and induced neurotoxicity. However, when T was added to G + nP, there was no statistically increased impairment compared to G + nP. The triplet significantly increased DNA damage, apoptosis, and tumor necrosis. Furthermore, the triplet further inhibited cell proliferation and reduced stroma density and intratumoral hypoxia. The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to the G + nP doublet in this study. This study provides a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. A Phase I clinical trial of the triplet combination is currently underway (NCT02047500).

Keywords: BW, body weight; Br-IPM, a brominated analog of isophosphoramide mustard; CAF, cancer-associated fibroblast; CAIX, carbonic anhydrase IX; CR, complete response; EMT, epithelial to mesenchymal transition; G, gemcitabine; HF, hypoxic fraction; ILS, increased life span; MT, median time to reach the size of 1000 mm3; MTD, maximum tolerated dose; NF, necrotic fraction; PDAC, pancreatic ductal adenocarcinoma; T, TH-302; TGD1000, tumor growth delay compared to Vehicle reaching the size of 1000 mm3; TGI, tumor growth inhibition; TH-302; V, vehicle; gemcitabine; hypoxia; hypoxia-activated prodrug; nP, nab-paclitaxel; nab-paclitaxel; pancreatic cancer; pharmacodynamics, biomarker; smooth muscle actin; xenograft; α-SMA, α.

Figures

Figure 1.

Antitumor efficacy of TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) in 4 PDAC xenograft models: Hs766t (A and B), MIA PaCa-2 (C and D), PANC-1 (E and F) and BxPC-3 (G and H), n = 10 for each group. T was given at 50 mg/kg, ip, G was given at 60 mg/kg ip and nP was given at 30 mg/kg, iv; all drugs were dosed at a Q3Dx5 regimen. A, C, E, and G, tumor growth was monitored and quantified twice a week. Data represent Mean ± SEM. B, D, F and H, Kaplan-Meier analysis plot using tumor size of 1000 mm3 as end-point. V, vehicle; Arrow, dosing time.

Figure 2.

Body weight change of animals treated with TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) in tumor-bearing immunocomprised mice and immunocompetent mice. T was given at 50 mg/kg, ip, G was given at 60 mg/kg ip and nP was given at 30 mg/kg, iv; all drugs were dosed at a Q3Dx5 regimen. (A-D), in Hs766t, MIA PaCa-2, PANC-1 and BxPC-3 tumor-bearing nu/nu mice, respectively, n = 10 per group; (E), in CD-1 female mice, n = 6 per group; and (F), in CD-1 male mice, n = 10 per group. Data represent Mean ± SEM. Arrow, dosing time.

Figure 3.

Effect of TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) on hematologic change and blood chemistry changes PANC-1 tumor bearing nude mice and CD1 mice. The means and standard errors from the 6 mice per group are presented. (A) blood samples were collected 24 hrs after the last treatment from non-tumor bearing CD-1 mice. (B) blood samples were collected 24 hrs after the last treatment from PANC-1 bearing nude mice. C, blood samples were collected 30 days after the last treatment from non-tumor bearing CD-1 mice. (A-C), y axis label is cell number (x103/μl). (D), plasma samples were collected 24 hrs after the last treatment from non-tumor bearing CD-1 mice. T was given at 50 mg/kg, ip, G was given at 60 mg/kg ip and nP was given at 30 mg/kg, iv; all drugs were dosed at a Q3Dx5 regimen. *, P < 0.05 as compared to Vehicle (V).

Figure 4.

Effect of TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) on mechanical hyperalgesia, analyzed by von Frey Assay. T was given at 50 mg/kg, ip, G was given at 60 mg/kg ip and nP was given at 30 mg/kg, iv; all drugs were dosed at a Q3Dx5 regimen. Data represent Mean ± SEM of 10 male CD-1 mice each group. Arrow, dosing time.

Figure 5.

Effect of TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) on tumor cells. PANC-1 tumor bearing animals received T, 50 mg/kg, ip; G, 60 mg/kg ip; and nP 30 mg/kg, iv, at a Q3Dx5 regimen. Tumors were collected 24 hrs after the last treatment. (A), representative images of Masson's Trichrome histology staining, TUNEL staining, Caspase-3, γH2AX, and Ki67 immunostaining in vehicle (V), T alone, G + nP doublet and G + nP + T triplet groups. Morphometric analysis of (B), necrotic fraction in the whole tumor by Masson's Trichrome; (C), number of TUNEL-positive apoptotic cells; (D), percentage of Caspase-3 positive apoptotic cells; (E), number of γH2AX positive DNA damage cells; and (F) number of Ki67 positive proliferative cells. *, P < 0.05 as compared to Vehicle (V). a, P < 0.05 as compared to G + nP; b, P < 0.05 as compared to T alone.

Figure 6

(See previous page). Effect of TH-302 (T) in combination with gemcitabine (G) and nab-paclitaxel (nP) on tumor microenvironment. PANC-1 tumor bearing animals received T, 50 mg/kg, ip; G, 60 mg/kg ip; and nP 30 mg/kg, iv, at a Q3Dx5 regimen. Tumors were collected 24 hrs after the last treatment. (A), representative images of Picrosirius red histology staining, α-smooth muscle actin (α-SMA) and pimonidazole immunostaining in vehicle (V), T alone, G + nP doublet and G + nP + T triplet groups. Morphometric analysis of (B) percentage of extracellular collagen by Picrosirius red staining; (C) percentage of α-SMA positivity inside the tumor; and (D) hypoxic fraction in the whole tumor by pimonidazole immunostaining. *, p < 0.05 as compared to Vehicle (V). a, P < 0.05 as compared to G + nP; b, P < 0.05 as compared to T alone. (E), representative images of pimonidazole and Caspase-3 immunostaining on consecutive sections. H, hypoxic compartment; O, oxic compartment; N, necrotic region; arrow, indicating the positive cells.