Bioreductive Prodrugs Research Articles

Perfluorocarbon regulates the intratumoural environment to enhance hypoxia-based agent efficacy

Hypoxia-based agents (HBAs), such as anaerobic bacteria and bioreductive prodrugs, require both a permeable and hypoxic intratumoural environment to be fully effective. To solve this problem, herein, we report that perfluorocarbon nanoparticles (PNPs) can be used to create a long-lasting, penetrable and hypoxic tumour microenvironment for ensuring both the delivery and activation of subsequently administered HBAs. In addition to the increased permeability and enhanced hypoxia caused by the PNPs, the PNPs can be retained to further achieve the long-term inhibition of intratumoural O2 reperfusion while enhancing HBA accumulation for over 24 h. Therefore, perfluorocarbon materials may have great potential for reigniting clinical research on hypoxia-based drugs.

Hybrid Nanomedicine Fabricated from Photosensitizer-Terminated Metal-Organic Framework Nanoparticles for Photodynamic Therapy and Hypoxia-Activated Cascade Chemotherapy.

During photodynamic therapy (PDT), severe hypoxia often occurs as an undesirable limitation of PDT owing to the O2 -consuming photodynamic process, compromising the effectiveness of PDT. To overcome this problem, several strategies aiming to improve tumor oxygenation are developed. Unlike these traditional approaches, an opposite method combining hypoxia-activated prodrug and PDT may provide a promising strategy for cancer synergistic therapy. In light of this, azido-/photosensitizer-terminated UiO-66 nanoscale metal-organic frameworks (UiO-66-H/N3 NMOFs) which serve as nanocarriers for the bioreductive prodrug banoxantrone (AQ4N) are engineered. Owing to the effective shielding of the nanoparticles, the stability of AQ4N is well preserved, highlighting the vital function of the nanocarriers. By virtue of strain-promoted azide-alkyne cycloaddition, the nanocarriers are further decorated with a dense PEG layer to enhance their dispersion in the physiological environment and improve their therapeutic performance. Both in vitro and in vivo studies reveal that the O2 -depleting PDT process indeed aggravates intracellular/tumor hypoxia that activates the cytotoxicity of AQ4N through a cascade process, consequently achieving PDT-induced and hypoxia-activated synergistic therapy. Benefiting from the localized therapeutic effect of PDT and hypoxia-activated cytotoxicity of AQ4N, this hybrid nanomedicine exhibits enhanced therapeutic efficacy with negligible systemic toxicity, making it a promising candidate for cancer therapy.

Antitumor activity of the bioreductive prodrug 3-(2-nitrophenyl) propionic acid-paclitaxel nanoparticles (NPPA-PTX NPs) on MDA-MB-231 cells: in vitro and in vivo.

Background3-(2-Nitrophenyl) propionic acid-paclitaxel (NPPA-PTX) is a paclitaxel (PTX) bioreductive prodrug synthesized by our lab. We hypothesize that NPPA-PTX can self-assemble to form nanoparticles (NPs).Materials and methodsIn the present research, the theoretical partition coefficient (XlogP) and Hansen solubility parameters of NPPA-PTX were calculated. NPPA-PTX nanoparticles prepared by NPPA-PTX and DSPE-PEG (NPPA-PTX:DSPE-PEG =1:0.1, w/w) (NPPA-PTX@PEG NPs) were prepared and characterized. The cellular uptake, in vitro antitumor activity, in vivo targeting effect, tumor distribution, in vivo antitumor activity, and safety of NPPA-PTX@PEG NPs were investigated.ResultsOur results indicate that NPPA-PTX can self-assemble to form NPPA-PTX@PEG NPs. Both the cellular uptake and safety of NPPA-PTX@PEG NPs were higher than those of Taxol. NPPA-PTX@PEG NPs could target tumor tissues by a passive targeting effect. In tumor tissues, NPPA-PTX@PEG NPs could completely transform into active PTX. The in vivo antitumor activity of NPPA-PTX@PEG NPs was confirmed in MDA-MB-231 tumor-bearing nude mice.ConclusionThe bioreductive prodrug NPPA-PTX could self-assemble to form NPs. The safety and antitumor activity of NPPA-PTX@PEG were confirmed in our in vitro and in vivo experiments. The NPPA-PTX@PEG NPs developed in this study could offer a new way of preparing bioreductive prodrug, self-assembled NPs suitable for antitumor therapy.

Hypoxia-Activated, Small-Molecule-Induced Gene Expression.

Hypoxia, a condition of reduced oxygen, occurs in a wide variety of biological contexts, including solid tumors and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated, small-molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-β-d-galactopyranoside. As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production of any given protein of choice.

4-Hydroxy-3-nitro-5-ureido-benzenesulfonamides Selectively Target the Tumor-Associated Carbonic Anhydrase Isoforms IX and XII Showing Hypoxia-Enhanced Antiproliferative Profiles.

Human carbonic anhydrases (CA, EC, 4.2.1.1) IX and XII are overexpressed in cancer cells as adaptive response to hypoxia and acidic conditions characteristic of many tumors. In addition, hypoxia facilitates the activity of specific oxido-reductases that may be exploited to selectively activate bioreductive prodrugs. Here, new selective CA IX/XII inhibitors, as analogues of the antitumor phase II drug SLC-0111 are described, namely ureido-substituted benzenesulfonamides appended with a nitro-aromatic moiety to yield an antiproliferative action increased by hypoxia. These compounds were screened for the inhibition of the ubiquitous hCA I/II and the target hCA IX/XII. Six X-ray crystallographies with CA II and IX/mimic allowed for the rationalization of the compounds inhibitory activity. The effects of some such compounds on the viability of HT-29, MDA-MB-231, and PC-3 human cancer cell lines in both normoxic and hypoxic conditions were examined, providing the initiation toward the development of hypoxia-activated antitumor CAIs.

Design, synthesis and biological evaluation of a novel platinum(II) complex possessing bioreductive groups for cancer therapy

Cisplatin is one of the most successful antitumor agents, yet also restricted by its poor cellular uptake and low selectivity. Since 3-(2-nitrophenyl) propionic acid (NPPA) has been reported as a bioreductive prodrug moiety, herein we combined NPPA with cisplatin (compound 1) to improve its lipophilicity and targetability and then to improve the antitumor outcomes. In addition, compound 2 possessing 3-phenyl propionic acid (PPA) was also synthesized as a comparison to test the influence of the NPPA to the cytotoxicity, since PPA was not a bioreductive moiety. Bioevaluations showed that 1 displayed more potent antitumor potency than cisplatin and 2, suggesting Pt(II) complexes possessing NPPA groups may be a good strategy for future platinum drug discovery.

Antagonism in effectiveness of evofosfamide and doxorubicin through intermolecular electron transfer

Hypoxic cells pose a problem in anticancer chemotherapy, in which often drugs require oxygen as an electron acceptor to bring about the death of actively cycling cells. Bioreductive anticancer drugs, which are selectively activated in the hypoxic regions of tumours through enzymatic one-electron reduction, are being developed for combination with chemotherapy-, radiotherapy- and immunotherapy-containing regimens to kill treatment-resistant hypoxic cells. The most clinically-advanced bioreductive drug, evofosfamide (TH-302), which acts by releasing a DNA-crosslinking mustard, failed to extend overall survival in combination with doxorubicin, a topoisomerase II inhibitor, for advanced soft tissue sarcoma in a pivotal clinical trial. However, the reasons for the lack of additive efficacy with this combination are unknown. Here, we show that the radical anion of evofosfamide undergoes electron transfer to doxorubicin in kinetic competition to fragmentation of the radical anion, thus suppressing the release the cytotoxic mustard. This electron transfer process may account, at least in part, for the lack of overall survival improvement in the recent clinical trial. This study underlines the need to consider both redox and electron transfer chemistry when combining bioreductive prodrugs with other redox-active drugs in cancer treatment.

Prodrug-embedded angiogenic vessel-targeting nanoparticle: A positive feedback amplifier in hypoxia-induced chemo-photo therapy.

Photodynamic therapy (PDT) induced hypoxia can significantly upregulate the expression of vascular endothelial growth factor (VEGF) at the tumor-stromal interface, resulting in a promoted angiogenesis. Thus, an angiogenesis vessel-targeting nanoparticle (AVT-NP) consisting of photosensitizer, angiogenic vessel-targeting peptide, and bioreductive prodrug is developed for a chemo-photo synergistic cancer therapy, with which anti-cancer effect is achieved first by PDT and immediately followed with hypoxia-activated cytotoxic free radicals. With targeting capability, the AVT-NPs can effectively accumulate at the tumor site due to the promoted angiogenesis in response to PDT-induced hypoxia. The more nanoparticles delivered to the tumor tissue, the higher efficacy of PDT can be achieved, resulting in a more severe hypoxia and increased angiogenesis. Therefore, the prodrug embedded AVT-NP functions as a positive feedback amplifier in the combinational chemo-photo treatment and indeed achieves an enhanced anti-tumor effect in both invitro and invivo studies.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies.

Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co(III) to paramagnetic Co(II). Cobalt pro-drugs built upon the tris(2-pyridylmethyl)amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic 1H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co(II)TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies.

Anaerobe‐Inspired Anticancer Nanovesicles

AbstractAnaerobic bacteria, such as Clostridium and Salmonella, can selectively invade and colonize in tumor hypoxic regions (THRs) and deliver therapeutic products to destroy cancer cells. Herein, we present an anaerobe nanovesicle mimic that can not only be activated in THRs but also induce hypoxia in tumors by themselves. Moreover, inspired by the oxygen metabolism of anaerobes, we construct a light‐induced hypoxia‐responsive modality to promote dissociation of vehicles and activation of bioreductive prodrugs simultaneously. In vitro and in vivo experiments indicate that this anaerobe‐inspired nanovesicle can efficiently induce apoptotic cell death and significantly inhibit tumor growth. Our work provides a new strategy for engineering stimuli‐responsive drug delivery systems in a bioinspired and synergistic fashion.

Anaerobe-Inspired Anticancer Nanovesicles.

Anaerobic bacteria, such as Clostridium and Salmonella, can selectively invade and colonize in tumor hypoxic regions (THRs) and deliver therapeutic products to destroy cancer cells. Herein, we present an anaerobe nanovesicle mimic that can not only be activated in THRs but also induce hypoxia in tumors by themselves. Moreover, inspired by the oxygen metabolism of anaerobes, we construct a light-induced hypoxia-responsive modality to promote dissociation of vehicles and activation of bioreductive prodrugs simultaneously. In vitro and in vivo experiments indicate that this anaerobe-inspired nanovesicle can efficiently induce apoptotic cell death and significantly inhibit tumor growth. Our work provides a new strategy for engineering stimuli-responsive drug delivery systems in a bioinspired and synergistic fashion.

Abstract 732: The impact of aerobic exercise on the tumor microenvironment

Abstract Intratumoral hypoxia (pO2 < 10mmHg) is associated with aggressive tumor progression, radiation resistance, suppression of the immune anti-tumor response and poor patient outcome. It is often found in solid tumors, and can constitute a major obstacle to anticancer therapy. The primary cause of abnormal oxygen tensions in tumors is the aberrant tumor vasculature, characterized by tortuous, leaky and immature vessels. A number of intervention strategies designed to overcome tumor hypoxia and improve patient outcome have been investigated including high oxygen content breathing and the use of bioreductive prodrugs, but these have had only moderate success in the clinic. The goal of the present investigations was to determine whether aerobic exercise could be applied to improve tumor perfusion, oxygenation and the immune anti-tumor response in breast cancer and fibrosarcoma models. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of mild and moderate intensity treadmill running were studied in mice bearing syngeneic murine mammary carcinomas (4T1 and EMT6) or fibrosarcomas (KHT). The exercise intensities were determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and exercise commenced when tumors reached a size of ∼500 mm3 (single) or ∼200 mm3 (repeated bouts). Once size was attained, mice were exposed to either mild (12 m/min) or moderate (18 m/min) intensity exercise. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of the exercise period tumors were analyzed by histology to assess for physiological changes. Specifically, blood was collected and tumors were harvested, sectioned and evaluated by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion and while the hypoxia marker (EF5) was used to determine the level of tumor hypoxia. Both markers were quantified using a Chalkley counter and ImageJ NIH software. In addition, the evaluation of tumor infiltrating immune cells after exercise compared to rest is under active investigation by histological analysis. Furthermore, plasma samples from exercised mice are being tested for immune related cytokines, chemokines and growth factors and compared to those from sedentary controls. Results to date indicate no difference in tumor growth rate between sedentary and exercising mice. Mild daily bouts of aerobic exercise do not affect the total number of tumor blood vessels but do increase the number of blood vessels that are actively perfused. These findings suggest that exercise may have potential utility in overcoming the aberrant microenvironmental conditions associated in solid tumors with therapeutic resistance. Citation Format: Jennifer M. Wiggins, Jennifer Lee, Lori Rice, Sharon Lepler, Christine Pampo, Dietmar Siemann. The impact of aerobic exercise on the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 732.

The anti-tumor efficacy of 3-(2-Nitrophenyl) propionic acid-paclitaxel (NPPA-PTX): a novel paclitaxel bioreductive prodrug.

Hypoxia is an important microenvironmental pressure present in the majority of solid tumors and, so, tumor hypoxia might be considered an attractive target for tumor therapy. One strategy for targeting hypoxia is to develop bioreductive prodrugs. In the present research, we synthesized a bioreductive paclitaxel prodrug, 3-(2-Nitrophenyl) propionic acid-paclitaxel (NPPA-PTX). The stability of NPPA-PTX in PBS and rat plasma was investigated. The anti-tumor activity of NPPA-PTX was also evaluated in vitro and in vivo. The results of our stability study indicated that NPPA-PTX was stable in PBS and rat plasma as well as in the blood circulation. The in vitro and in vivo anti-tumor activity of NPPA-PTX was confirmed in both KB cells and MDA-MB-231 cells. Our results also indicated that NPPA-PTX could completely convert to active PTX in tumor tissues and produced the anti-tumor activity in both KB and MDA-MB-231 tumor-bearing nude mice. We suggest that the dissociated PTX which converted from NPPA-PTX in tumor tissues played a key role in producing anti-tumor activity. Considering all our results, we suggest that NPPA-PTX is a novel bioreductive PTX prodrug which could undergo further evaluation.

Hypoxia-activated prodrugs: paths forward in the era of personalised medicine.

Tumour hypoxia has been pursued as a cancer drug target for over 30 years, most notably using bioreductive (hypoxia-activated) prodrugs that target antineoplastic agents to low-oxygen tumour compartments. Despite compelling evidence linking hypoxia with treatment resistance and adverse prognosis, a number of such prodrugs have recently failed to demonstrate efficacy in pivotal clinical trials; an outcome that demands reflection on the discovery and development of these compounds. In this review, we discuss a clear disconnect between the pathobiology of tumour hypoxia, the pharmacology of hypoxia-activated prodrugs and the manner in which they have been taken into clinical development. Hypoxia-activated prodrugs have been evaluated in the manner of broad-spectrum cytotoxic agents, yet a growing body of evidence suggests that their activity is likely to be dependent on the coincidence of tumour hypoxia, expression of specific prodrug-activating reductases and intrinsic sensitivity of malignant clones to the cytotoxic effector. Hypoxia itself is highly variable between and within individual tumours and is not treatment-limiting in all cancer subtypes. Defining predictive biomarkers for hypoxia-activated prodrugs and overcoming the technical challenges of assaying them in clinical settings will be essential to deploying these agents in the era of personalised cancer medicine.

Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs.

Regions of insufficient oxygen supply-hypoxia-occur in diverse contexts across biology in both healthy and diseased organisms. The difference in the chemical environment between a hypoxic biological system and one with normal oxygen levels provides an opportunity for targeting compound delivery to hypoxic regions by using bioreductive prodrugs. Here we detail a protocol for the efficient synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which is a key intermediate that can be converted into a range of 1-methyl-2-nitro-1H-imidazole-based precursors of bioreductive prodrugs. We outline methods for attaching the bioreductive group to a range of functionalities, and we discuss the strategy for positioning of the group on the biologically active parent compound. We have used two parent checkpoint kinase 1 (Chk1) inhibitors to exemplify the protocol. The PROCEDURE also describes a suite of reduction assays, of increasing biological relevance, to validate the bioreductive prodrug. These assays are applied to an exemplar compound, CH-01, which is a bioreductive Chk1 inhibitor. This protocol has broad applications to the development of hypoxia-targeted compounds.

Abstract 3198: The impact of aerobic exercise on oxygenation and vascularity in breast cancer models

Abstract Background: Intratumoral hypoxia (pO2 <10mmHg) is associated with aggressive tumor progression and poor patient outcome. It is often found in solid tumors, and can constitute a major obstacle to anticancer therapy. The primary cause of abnormal oxygen tensions in tumors is the aberrant tumor vasculature, characterized by tortuous, leaky and immature vessels. A number of intervention strategies designed to overcome tumor hypoxia have been investigated including high oxygen content breathing and the use of bioreductive prodrugs, but these have had only moderate success in the clinic. Purpose: The goal of the present investigations was to determine whether aerobic exercise could be applied as a means to improve tumor physiology and decrease tumor hypoxia in breast cancer models. Methods: The effects of single and repeated bouts of moderate intensity exercise (treadmill running) were studied in mice bearing syngeneic murine mammary carcinoma models (EMT6 and 4T1). The exercise intensities were determined by measuring the anaerobic threshold, which is reflected by the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and once the tumors reached a size of 250 mm3, the mice were exposed to either a single bout of exercise or daily exercise bouts performed for 5 consecutive days. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. Following cessation of each exercise regime tumors were harvested, sectioned and evaluated by immunofluorescence. Vessel density (MECA-32), blood perfusion (Hoechst-33342) and hypoxia (EF5) were quantified and significance was established at p<0.05. Results: Preliminary data indicate that even a single exercise bout may begin to demonstrate improvements in blood vessel perfusion and tumor oxygenation. Importantly, after 5 daily bouts of exercise, there was a significant increase in the number of patent vessels and improvement in oxygenation in the tumors of exercising mice compared to those of sedentary controls. Conclusion: The results indicate that moderate aerobic exercise can improve the oxygenation status of solid tumors. These findings suggest that such a non-toxic intervention may have potential utility in overcoming hypoxia-associated treatment resistance in cancer patients and improve patient outcome. Citation Format: Jennifer M. Wiggins, Jennifer A. Lee, Lori Rice, Dietmar Siemann. The impact of aerobic exercise on oxygenation and vascularity in breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2015-3198

Hypoxia Induced by Upconversion-Based Photodynamic Therapy: Towards Highly Effective Synergistic Bioreductive Therapy in Tumors.

Local hypoxia in tumors is an undesirable consequence of photodynamic therapy (PDT), which will lead to greatly reduced effectiveness of this therapy. Bioreductive pro-drugs that can be activated at low-oxygen conditions will be highly cytotoxic under hypoxia in tumors. Based on this principle, double silica-shelled upconversion nanoparticles (UCNPs) nanostructure capable of co-delivering photosensitizer (PS) molecules and a bioreductive pro-drug (tirapazamine, TPZ) were designed (TPZ-UC/PS), with which a synergetic tumor therapeutic effect has been achieved first by UC-based (UC-) PDT under normal oxygen environment, immediately followed by the induced cytotoxicity of activated TPZ when oxygen is depleted by UC-PDT. Treatment with TPZ-UC/PS plus NIR laser resulted in a remarkably suppressed tumor growth as compared to UC-PDT alone, implying that the delivered TPZ has a profound effect on treatment outcomes for the much-enhanced cytotoxicity of TPZ under PDT-induced hypoxia.

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma

PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.

Biological evaluation of the activity of some benzimidazole-4,7-dione derivatives.

The study presented here is a follow up of the authors’ interest in the approach to selective and cytotoxic bioreductive anticancer prodrugs. The current work is devoted to explore both the biological activity of some previously obtained compounds and the search for an explanation of their target(s) in hypoxic pathways. In this work the biological activity of some benzimidazole-4,7-diones was evaluated. These compounds were examined as potential bioreductive agents specific for the hypoxic environment found in tumor cells. The main aim was concerned with establishing their cytotoxic properties by using proliferation, apoptosis and DNA destruction tests on selected tumor cells. Their cytotoxic effects on two tumor cell lines (human lung adenocarcinoma A549 cells line and human malignant melanoma WM115) was compared by means of a WST-1 test. Next, the mode of cytotoxicity behind the selected tumor cells’ death was determined by the caspase 3/7 test. The last point referred to the DNA destruction of A549 and WM115 cells and the in situ DNA Assay Kit test was applied. The cytotoxic tests confirmed their activity against the tumor cells and target hypoxia (compounds 2b, 2a, 2d). The screening test of the caspase-dependent apoptosis proved that the exposure of the tested tumor cells in hypoxia to these benzimidazole-4,7-diones promoted the apoptotic cell death. Additionally, the DNA damage test established that benzimidazole-4,7-diones can be potential hypoxia-selective agents for tumor cells, especially compound 2b. All results classify the tested benzimidazole-4,7-diones as promising, lead molecules and provide a rationale for further molecular studies to explain their usefulness as potential inhibitors of the hypoxia-inducible factor 1 (HIF1).