Theranostic evaluations of bioreductively-activated prodrugs for the management of hypoxic solid tumors

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Theranostic evaluations of bioreductively-activated prodrugs for the management of hypoxic solid tumors

Summary: The project pursues the translational explorations of glucose-tirapazamine (TPZ)-based conjugates to evaluate their potential in future clinical management of solid hypoxic tumors in cancer diseases.TPZ is a highly potent hypoxia-selective clinical drug that was used in several clinical trials, but was withdrawn from the clinic due to its severe neurotoxic manifestations and poor patient population selection. Our approach transforms TPZ into ‘next generation’ mutimodal (diagnosis, radiosensitization, chemotherapy, molecular radiotherapy) theranostics (therapy+diagnostic), which would turn ‘hypoxia from problem to advantage’.

Background data:

Rationale for the project:

Solid tumors frequently exhibit rapid growth and aberrant vasculature which leads to oxygen depletion (hypoxia) and poor drug and nutrient’s supply.1,2 Hypoxia alters cellular metabolism, which can trigger transcriptional responses, inducing alterations in gene expression, mostly mediated by hypoxia-inducible factors 1 & 2 (HIF).3,4 At low oxygen levels, HIFs trigger the activation of genes involved in glycolysis, cell survival, replication, angiogenesis, tissue invasion and metastatis.5-7 As a consequence, the malignant potential of hypoxic tumors is altered which makes them resistant to conventional radio- and chemotherapy (due to impaired drug delivery) interventions.10 Therefore tumor hypoxia presents significant human health challenges and contributes to poor overall survival of cancer patients. TPZ, is a hypoxia targeting bioreductive anticancer drug, but was withdrawn from clinic due to its severe toxicity. What happens in TPZ is, it gets metabolized and lysed before reaching the targeted cells. Hence, conjugates of glucose and an analogue of TPZ, that is less toxic is being developed in our lab, in order to control these tumors. Glucose moiety will facilitate their transport (through upregulated glucose transporters) in hypoxic cancer cells where TPZ will bioreductively activate in hypoxic atmosphere selectively and bind to cytoplasmic macromolecules therein to impart four-fold theranostic features. Healthy cells are oxygenated therefore the drug will not be retained therein, and minimal toxicity will be experienced by them. Overall, the goal of this study, is to examine the multimodal theranostic potential of TPZ-glucose conjugates and the corresponding radiohalogenated pharmaceuticals for the management of hypoxic tumors.

Role of HIF1-α in tumor hypoxia:

The phenomenon of tumor hypoxia has been studied since several decades and was first investigated and discussed by Gray et al 11,1953. Since then, enormous research has been conducted in order to control the hypoxic effects in solid tumors thereby to improve the efficacy of treatment given to cancer patients. The estimated level of oxygenation in normal tissues is said to be 40mmHg, whereas the level reduces to 10mmHg in hypoxic condition, and decelerating to 1% at anoxia (at extreme hypoxia)12. Reduction in oxygen levels are mainly due to poor vascularization in tumor tissues which renders it difficult to satisfy the high demand of oxygen created due to rapid and aggressive proliferation of cells 13. This condition, ultimately leads to a reduction in nutrient supply to tumor cells 14. The tumor cells in order to survive in a low intra tumoral pH, in the tumor microenvironment, undergo a lot of adaptive and cellular changes regulated by HIF-1 3,4. HIF1, a transcription factor, identified in 1991, is a heterodimeric protein that binds to hypoxia-responsive element (HRE) in the erythropoietin gene. 15,16 Under normal O2 conditions, Von-Hippel Lindau (VHL) tumor suppressor gene, binds to the α-sub unit of HIF-1, initiating ubiquitination, and thus HIF-1 undergoes proteolytic degradation 17. But in hypoxic conditions, HIF-1α gets stabilized and translocates from cytoplasm to nucleus and heterodimerizes with its binding partner, HIF-1β, thus evading the VHL-regulated proteasomal degradation 16. Hence, the stable transcritionally active heterodimerized complex, (HIF-1+HIF-2) induces gene expression by binding to HRE 18(Fig 1) 21.These effects finally results in the activation of increased expression of facilitative glucose transporters, adaptation to low pH in hypoxic tumor environment and increased angiogenesis 20. In a earlier study, increased mRNA expression of HIF-1α was observed in hypoxic cells when compared with normoxic cells at two different time points (4 and 24hr) analyzed using RT-PCR. These results correlated with the results obtained by western blot analysis (Fig 2). Hence, these data confirms the role of HIF-1α in hypoxic condition 19.

Response of GLUT1 at hypoxic conditions:

The crucial step in the production of ATP in cells, is oxidative phosphorylation, where the hydrogen atoms and electrons are transferred to oxygen. But naturally in hypoxic atmosphere, due to low O2, cells choose an alternative pathway such as anaerobic glycolysis to release energy, and this pathway is regulated by HIF-1. Basically hypoxia induces a shift from aerobic to anaerobic glycolysis, which leads to increase in expression of genes encoding key glycolytic enzymes and glucose transporters (GLUT1) 8,9. Thus, glucose transport regulated by hypoxia, is said to be triphasic. Initially, GLUT-1 gets activated on the pre-existing plasma membrane due to decreased oxidative phosphorylation and ATP production, and if hypoxia still exists, GLUT-1 molecules are translocated from intracellular vesicles to plasma membrane, and in chronic hypoxic cells, de novo synthesis of GLUT-1 is mediated by HIF-1 (Fig 3) 10. This regulation pattern, strongly renders GLUT-1 as a means of targeting hypoxic tissue in tumours. High expression of membranous Glut1 was observed in head and neck carcinomas after infusion of pimonidazole, an exogenous hypoxic cell marker (Fig 4).22 Hence, GLUT1 has been regarded as a diagnostic hallmark of tumor hypoxia.

Use of analogues and conjugates of GLUT-1 to target hypoxic cells:

Studies have been carried out on the use of analogue of glucose namely, 2-deoxy-D-glucose (2-DG), that would inhibit glycolysis 23. Due to the enhanced uptake of glucose analogues in cancer cells, high toxicity was observed in hypoxic cells. But, since these studies, show non- specific inhibition of glycolysis in tumors, more research was conducted on the basis of GLUT-1 expression. Preclinical studies were carried out on 2-halogen substituted analogues of glucose, 2-deoxy (2-DG), 2- chloro (2-CG) and 2- bromo (2-BG) which specifically target HIF- regulated enzyme hexokinase rendered better tumor response 24(Fig 5). Offlate, a better alternative strategy, to use GLUT1 molecules as a tumor marker has been developed. Innovative structural improvements in the glucose conjugates by linking it with a cytotoxic species have been developed, so that the conjugate gets selectively transported into tumor cells by GLUT-1. In a earlier study, 2- glu-SNAP, glucose molecule conjugated with a nitric- oxide donor, (S-nitroso-N-acetyl-penicillamine) SNAP on a glioblastoma cell line showing increased activity of tumor cytotoxicty when compared to unconjugated SNAP molecule 25. Another study shows the use of combined treatment of Glufosfamide, a conjugate of glucose and isophoramide mustard, a metabolite of ifosfamide (which exhibits DNA- alkylating properties) and gemcitabine (anti-cancer agent) in phase-I clinical trials in pancreatic cancer 26. Hence, this idea, marks the foundation of our study to use GLUT1 conjugates in addition with a hypoxia selective bioreductive pro-drug-TPZ (Fig 6).

Recent advancements in Tirapazamine, a hypoxia-dependent bioreductive agent:

Oxygen acts as a radiosensitizer by enhancing the efficiency of ionizing radiation dose during radiotherapy, by formation of DNA- damaging free radicals in cancer cells. Thus hypoxic tumors are resistant to radio and chemotherapy. Although, several hypoxia- targeting oxygen mimetic drugs have been developed to make the hypoxic tumors sensitive to therapies, still several studies are being carried out to identify a better bioreductive agent, that possess high hypoxic differential. In our study, Tirapazamine, a hypoxic cytotoxin, that gets bioreductively activated at hypoxic atmosphere is being used. TPZ, a heterocyclic di-N-oxide (belongs to benzotriazine family) undergoes enzymatic metabolism at hypoxic atmosphere from a dioxide to a mono-N-oxide metabolite, which exhibits oxygen mimetic (radiosensitizing) properties.28.29 The activation of TPZ is catalysed by cytochrome P450 enzymes which reduces the prodrug to cytotoxic radical by exhibiting DNA-strand breaking properties 25. Phase-II clinical trials conducted on TPZ, in combination with cisplatin, etoposide and radiotherapy, in small cell-lung cancer showed increased toxicity and reduced survival benefit 30. In phase-III clinical trial of head and neck cancer type, effect of TPZ, cisplatin and radiotherapy were compared with effect of cisplatin conjugated with fluorouracil, and radiation. Positron emission tomography (PET) was used to examine the hypoxic tumors before and after treatment and results suggest, that patients with hypoxic tumors who had received TPZ, found no survival benefit 31. Thus, TPZ should be structurally altered to induce low toxicity to render them as feasible targets for therapeutic response. In our lab, an analogues of TPZ (ie., less toxic) are being synthesized in order to explore the potencity of this prodrug. Recent study conducted by Johnson et al, 2014, suggests that addition of nitrogen mustards, cytotoxic chemotherapeutic agents to TPZ, might activate a DNA-alkylating species selectively in hypoxic tissues, which in order will induce DNA damage to hypoxic tumor cells 11 (Fig 7).

Theranostic approach of Tirapazamine:

Several convincing results have demonstrated the use of PET (18F [FDG] as radiotracer), in diagnosing hypoxic tumors after treatment with TPZ. Many researchers have also examined the role of TPZ as a chemotherapeutic agent. When combined with radiotherapy, TPZ exhibits increased anti-tumor effect. A combined study of x-ray radiotherapy (XRT) and TPZ was found to be ineffective, due to the deactivation of TPZ before reaching the target. Hence, in this study, the potencity of glucose- TPZ based conjugates will be explored further (Fig 8).

Hypothesis: We hypothesize that our prodrugs, when containing a diagnostic radionuclide (e.g., F-18, I-124), will allow diagnosis of hypoxic tumors by PET and, when labelled with a therapeutic radioiodine (e.g., I-131), will impart hypoxia-targeted in situ molecular radiotherapy (MRT). The drug can also be used as a radiosensitizer and possible chemotherapy agent. The aim is to provide better theranostic options for cancer patients diagnosed with therapy-resistant solid tumors.

Specific Aims:

1.Determine the pharmacodynamics and kinetics of the GLUT-1 expression in selected cancer cell lines followed by investigating the cytotoxicity of TPZ in vitro.

2.Determine the effect of key proteins involved in the synthesized glucose-TPZ conjugates.

3.Investigate the cytotoxic effect and theranostic features of glucose-TPZ conjugates in hypoxic tumor- bearing animal models.

Basically, validation of a series of theranostic studies will determine the anti-cancer potential of the conjugates, thereby providing a basis for future clinical trials with curative response.

Experimental approach:

Aim 1. Determine the pharmacodynamics and kinetics of the GLUT-1 expression in selected cancer cell lines followed by investigating cytotoxicity of TPZ in vitro.

Initially, to determine the dynamics and kinetics of GLUT1 expression, two cell lines of head and neck cancer cell types- oral squamous carcinoma cells (SCC) and human hypopharyngeal squamous carcinoma cells (FaDu) are considered. The cells will be incubated at different O2 conditions, namely normoxic – 20% O2 and hypoxic – 5%, 2%, 1% and 0.5% O2 for 24 hours. Confluence and acidity will be taken into account so that they don’t exhibit a negative impact on the experimental processes. Protein lysates and RNA will be collected from the incubated cells. Western blotting will be carried out to determine the levels of GLUT-1 and HIF-1 relative to β-actin. Real time RT-PCR will be used to determine the levels of expression of GLUT-1 and LOX when compared to housekeeping gene RPLPO. After determination of an O2 condition that best upregulates the GLUT-1 expression, a time-course will be run by incubating cells in selected O2 condition (near anoxia) at different time points such as 0 min, 6hrs, 12 hrs, 24 hrs and 48 hrs. Western blotting and Real time RT-PCR will be carried out from the extracted protein cell lystaes and RNA as described above. In order to study the cytotoxicity of the drug (TPZ), cells will be incubated at hypoxic/anoxic condition at time established in kinetics, and will be treated with different concentrations of drug (TPZ). Output will be studied by the live/dead assay by examining the colony forming ability of cells.

Aim 2. Determine the effect of key proteins involved in the synthesized glucose-TPZ conjugates.

Radiation induced cell death usually results from DNA damage.17 Inhibition of DNA damage repair proteins will be studied to investigate the importance of key proteins known to influence DNA responses to radiation and hypoxia after treatment with drug. Key proteins such as p53, a tumor suppressor is known to play a major role in regulation of DNA repair in irradiated cells. Effect of p53 inhibitor, pfithirin on normal and hypoxic cells before and after the treatment of prodrugs will be studied.18 Effect and expression of HIF1-α will also be investigated under normoxic and hypoxic conditions. Extraction of proteins followed by western blot analysis will be performed. Cells will be exposed to increasing doses of radiosensitizer drugs followed by X- ray irradiation (as described by Barranco et al 19) with and without prodrug treatment. In order to study the DNA repair caused by radiation, γ H2A-X assay (as described by Luca et al 20) will be carried out with and without treatment of pfithirin. Cell sensitivity to irradiation will be determined using clonogenic cell survival assay. Immunohistochemical staining of GLUT-1, HIF1-α and p-53 protein expression will be performed in prodrugs after infusion of pimonidazole, tumor hypoxic marker.

Aim 3. Investigate the cytotoxic effect and theranostic features of glucose-TPZ conjugates in hypoxic tumor- bearing animal models.

Cytotoxicity and theranostic features (diagnosis, radiosensitization, chemotherapy, molecular radiotherapy) will be examined individually on the glucose-TPZ based conjugates in-vivo. Murine model of FaDu hypoxic tumor xenografts will be established in nude mice, as described by Chowdhury et al15 and different doses of prodrugs will be injected into the tail-vein of xenografts. Histopathological studies of the hypoxic tumor tissues will be studied to examine the cytotoxicity of the administered pro drugs and the highest dose with no visible side-effect will be selected. Radioactive- isotopes (F-18, I-124, I-127, I-131) will be produced through specific nuclear reactions in a cyclotron at MICF (Medical Isotope and Cyclotron Facility, South campus). Then, these isotopes will be labelled to the synthesized Glucose-TPZ based conjugates. Initially, well known- diagnostic radionuclide, 18F labelled with the synthesized prodrugs, will be injected into the xenografts. Diagnosis of radiotherapy- resistant hypoxic tumors will be carried out by PET due to the high uptake of glucose metabolism in hypoxic cancer areas. Radionuclide 124I, has been regarded as a good radiotracer, since they possess long half-life (4.2 days), high clearance of non-specific radioactivity, high positron emission energy and its labelling chemistry have been studied in detail.16 So, our prodrugs will be labelled with 124I, to investigate whether they can exhibit better tumor imaging characteristics in PET when compared to 18F. Radiation dosimetry estimates of our pro-drugs will be carried out by our group, to determine their in situ MRT potential in hypoxic tumors. Our prodrugs, labelled with therapeutic radioiodine (131I) will be administered into hypoxic tumor xenografts and PET studies will be carried on to examine their potency to impart hypoxia-targeted in situ MRT. A radiotherapeutic agent 177Lu DOTA-octreotate, will also be labelled with the synthesized conjugates to examine whether it can facilitate in situ MRT potential. To explore the ability of our prodrugs as a radiosensitizer for XRT, time of the uptake of prodrugs (maximal dose) will be established by PET. Studies suggest that 19F (non-radioactive isotope) and 127I is a radiosensitizer of hypoxic cells to external beam XRT. Hence different doses of x-ray will be given to tumor xenografts containing different doses of 19F labelled prodrugs. Maximal accumulation of radiosensitizer will be determined using PET imaging. Comparative studies will be carried on by labelling the conjugates with 127I, in order to determine the best radiosensitizer.

Novelty: No hypoxia targeting drug has been investigated in a multimodal theranostic approach for the management of hypoxic tumors. Hence, our research is highly innovative.

Collaborative Structure: Our multidisciplinary team (basic, translational and clinical sciences) further strengthens the research progression, and provides excellence in all aspects to successfully achieve current objectives.

Potential Outcome and Value: Our study contributes to the diagnosis and treatment of hypoxic solid tumors leading to improved patient care, reduced morbidity and mortality, substantial health care cost savings, and thereby consequently improving the quality of life of cancer patients.