A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity

Abstract

AST-3424/OBI-3424 (denoted by 3424) is a novel prodrug bis-alkylating agent activated by AKR1C3. AKR1C3 is overexpressed in many types of cancer, particularly in liver, non-small cell lung, gastric, renal and CRPC cancer. Currently 3424 is being studied in phase 1/2 clinical trials for the treatment of solid and hematologic cancers, and it represents potentially a novel, selective anti-cancer agent for multiple indications. In this study, AKR1C3-dependent activation of 3424 was investigated in vitro using recombinant human AKR1C3. AKR1C3-dependent cytotoxicity of 3424 was determined in a wide range of human cancer cell lines with different AKR1C3 expression levels. In addition, anti-tumor activity of 3424 was also investigated in a broad panel of CDX and PDX models. AKR1C3-dependent activation of prodrug 3424 was evident by monitoring the decrease of 3424 and generation of the active form, 2660. Kinetic analysis indicated that AKR1C3 exhibited higher catalytic efficiency towards 3424 compared to the physiological substrates. There was a strong correlation between 3424 cytotoxic potency and AKR1C3 expression. The racemic mixture induced DNA cross-linking in a concentration dependent manner. Tumor growth inhibition of 3424 was shown to be better than or comparable to the standard of care chemotherapy at clinically achievable doses as a single agent in various CDX models with high expression of AKR1C3, including liver HepG2, lung H460, castration-resistant prostate VCaP, gastric SNU-16, and kidney A498 cancer cell lines. The excellent anti-tumor efficacy of 3424 was further demonstrated in PDX models which have high level of AKR1C3 expression, but not in a model with low level of AKR1C3 expression. In the combination therapy, we showed that 3424 could enhance the efficacy of the standard care of chemotherapy in the CDX models. The results described here highlight that 3424 exhibits AKR1C3-dependent cytotoxicity in vitro and anti-tumor activity in vivo in a wide range of human cancer types, which support further development of 3424 as an anti-cancer agent for treating different types of cancers and the use of AKR1C3 as a biomarker to profile cancer patients and further guide patient selection for therapy with 3424.

Keywords: AKR1C3; alkylating agent; cancers and anti-cancer agent; prodrug.

Figure 1

Structure and 3424 activation. A. Chemical structure of 3424 (S-enantiomer), 3423 (R-enantiomer) and 2870 (racemic mixture of 3424 and 3423 at 1:1 ratio). B. Schema of 3424 reductive activation pathway.

Figure 2

AKR1C3-dependent 3424 activation. A. 3424 reduction. B. 2660 production.

Figure 3

AKR1C3-dependent in vitro cytotoxicity of 3424. A. Correlation between AKR1C3 protein expression and 3424 IC₅₀ in liver cancer cells (left); Correlation between AKR1C3 RNA expression and 3424 IC₅₀ in liver cancer cells (middle); Correlation between AKR1C3 RNA expression and 3424 IC₅₀ in NSCLC cancer cells (right). B. AKR1C3-specific inhibitor, 3021, efficiently inhibited cytotoxicity of 3424 (left), 3423 (middle) and racemic mixture 2870 (right). The data are the representative of three independent experiments. C. Compound 2870 induced concentration-dependent DNA cross-linking. The data are the representative images of cell nuclei stained with SYBR Green after electrophoresis of two independent experiments. Original magnification: 10 ×, scale bar: 100 μm.

Figure 4

In vivo anti-tumor activity of 3424 in HepG2 and H460 CDX models. A. Fluorescence area of 3424 and sorafenib in GFP-HepG2 orthotopic CDX model. B. GFP-HepG2 tumor fluorescent images at the end of the experiment by using FluorVivo Model-100 fluorescence imager. Scale bar: 10 mm. C. Fluorescence area of 3424 and Taxol in GFP-H460 subcutaneous CDX model. Animals were monitored daily and tumor growth was quantified twice a week. Data are expressed as Mean ± SEM of 10 or 8 animals per group in HepG2 and H460, respectively.

Figure 5

In vivo anti-tumor efficacy of 3424 as monotherapy or in combination with standard of care therapy in VCap, SNU-16 and A498 CDX models. A. Tumor growth of 3424 and abiraterone plus prednisolone as a monotherapy or in combination in VCaP. B. Measurement of serum prostate specific antigen (PSA) after treatment at the times indicated in VCap. C. Tumor growth of 3424 and 5-flurouracie as a monotherapy or in combination in SNU-16. D. Tumor growth of 3424 and sunitinib as monotherapy or in combination in A498. E. Tumor growth of 3424 and gemcitabine as a monotherapy or in combination in A498. Data are expressed as Mean ± SEM of 8 animals per group. AA, abiraterone acetate; P, prednisolone; 5-FU, 5-flurouracie; S, sunitinib; G, Gemcitabine.

Figure 6

In vivo anti-tumor activity of 3424 against a panel of PDXs. A. Tumor growth of 3424 in pancreatic cancer PA1280. B. Tumor growth of 3424 in gastric cancer GA6201. C. Tumor growth of 3424 in lung cancer LU2505 with higher AKR1C3 expression. D. Tumor growth of 3424 in lung cancer LU2057 with low AKR1C3 expression. Animals were monitored daily and tumor growth was quantified twice a week. Data are expressed as Mean ± SEM of 5-6 animals per group.