Exploiting tumor hypoxia for cancer treatment

Hypoxia, i.e. low blood oxygen levels, is a common feature of solid tumours. Hypoxia causes tumour cells to become even more malignant. It is also associated with a poor prognosis and problems occurring during cancer treatment. This dissertation evaluates two potential cancer treatments targeting tumour hypoxia in a preclinical setting. We show that new substances targeting CA-IX, a protein occurring in hypoxic tumour regions, have promising anti-cancer effects. We also studied a new potential drug called CP-506. This substance is activated specifically in hypoxic regions. We demonstrate that CP-506 is a promising new substance with characteristics favourable for future clinical use. Our results confirm that treatment targeting tumour hypoxia is an interesting and promising approach to cancer

Prognostic Significance of Carbonic Anhydrase IX Expression in Cancer Patients: A Meta-Analysis

Hypoxia is a characteristic of many solid tumors and an adverse prognostic factor for treatment outcome. Hypoxia increases the expression of carbonic anhydrase IX, an enzyme that is predominantly found on tumor cells and is involved in maintaining the cellular pH balance. Many clinical studies investigated the prognostic value of carbonic anhydrase IX expression, but most have been inconclusive, partly due to small numbers of patients included. The present meta-analysis was therefore performed utilizing the results of all clinical studies to determine the prognostic value of carbonic anhydrase IX expression in solid tumors. Renal cell carcinoma was excluded from this meta-analysis due to an alternative mechanism of upregulation. 958 papers were identified from a literature search performed in Pubmed and Embase. These papers were independently evaluated by two reviewers and 147 studies were included in the analysis. The meta-analysis revealed strong significant associations between CAIX expression and all endpoints: overall survival (HR=1.76, 95%CI 1.58 – 1.98), disease-free survival (HR=1.87, 95%CI 1.62 – 2.16), locoregional control (HR=1.54, 95%CI 1.22 – 1.93), disease-specific survival (HR=1.78, 95%CI 1.41 – 2.25), metastasis-free survival (HR=1.82, 95%CI 1.33 – 2.50), and progression-free survival (HR=1.58, 95%CI 1.27 – 1.96). Subgroup analyses revealed similar associations in the majority of tumor sites and types. In conclusion, these results show that patients having tumors with high carbonic anhydrase IX expression have higher risk of locoregional failure, disease progression, and higher risk to develop metastases, independent of tumor type or site. The results of this meta-analysis further support the development of a clinical test to determine patient prognosis based on CAIX expression and may have important implications for the development of new treatment strategies

New approach of delivering cytotoxic drugs towards CAIX expressing cells : A concept of dual-target drugs

Abstract Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies

The sulfamate small molecule CAIX inhibitor 1 S4 modulates 2 doxorubicin efficacy

Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 -CAIX high and HT29 -CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 µM, p = 0.0003). Similar results were observed for HT29-CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 µM, p<0.0001) and in the HT29 -CAIX low cells (IC50 is 0.09 µM, p<0.0001). In vivo doxorubicin treatment was only effective in the MDA-MB-231 xenografts, but the efficacy of doxorubicin was decreased when combined with S4. In conclusion, the efficacy of doxorubicin treatment can be increased when combined with the selective sulfamate CAIX inhibitor S4 in vitro in certain cell lines. Nevertheless, in xenografts S4 did not enhance doxorubicin efficacy in the FaDu and HT29 tumor models and decreased doxorubicin efficacy in the MDA-MB-231 tumor model. These results stress the importance of better understanding the role of CAIX inhibitors in intratumoral pH regulation before combining them with standard treatment modalities, such as doxorubicin

Hypoxia-activated prodrugs and (lack of) clinical progress: The need for hypoxia-based biomarker patient selection in phase III clinical trials

Hypoxia-activated prodrugs (HAPs) are designed to specifically target the hypoxic cells of tumors, which are an important cause of treatment resistance to conventional therapies. Despite promising preclinical and clinical phase I and II results, the most important of which are described in this review, the implementation of hypoxia-activated prodrugs in the clinic has, so far, not been successful. The lack of stratification of patients based on tumor hypoxia status, which can vary widely, is sufficient to account for the failure of phase III trials. To fully exploit the potential of hypoxia-activated prodrugs, hypoxia stratification of patients is needed. Here, we propose a biomarker-stratified enriched Phase III study design in which only biomarker-positive (i.e. hypoxia-positive) patients are randomized between standard treatment and the combination of standard treatment with a hypoxia-activated prodrug. This implies the necessity of a Phase II study in which the biomarker or a combination of biomarkers will be evaluated. The total number of patients needed for both clinical studies will be far lower than in currently used randomize-all designs. In addition, we elaborate on the improvements in HAP design that are feasible to increase the treatment success rates. Keywords: Hypoxia-activated prodrugs, Phase III clinical trial, Biomarker, Hypoxi

Novel fluorinated carbonic anhydrase IX inhibitors reduce hypoxia-induced acidification and clonogenic survival of cancer cells

Human carbonic anhydrase (CA) IX has emerged as a promising anticancer target and a diagnostic biomarker for solid hypoxic tumors. Novel fluorinated CA IX inhibitors exhibited up to 50 pM affinity towards the recombinant human CA IX, selectivity over other CAs, and direct binding to Zn(II) in the active site of CA IX inducing novel conformational changes as determined by X-ray crystallography. Mass spectrometric gas-analysis confirmed the CA IX-based mechanism of the inhibitors in a CRISPR/Cas9-mediated CA IX knockout in HeLa cells. Hypoxia-induced extracellular acidification was significantly reduced in HeLa, H460, MDA-MB-231, and A549 cells exposed to the compounds, with the IC50 values up to 1.29 nM. A decreased clonogenic survival was observed when hypoxic H460 3D spheroids were incubated with our lead compound. These novel compounds are therefore promising agents for CA IXspecific therapy

New ways to image and target tumour hypoxia and its molecular responses

International audienceTumour hypoxia and its molecular responses have been shown to be associated with poor prognosis. Detection of hypoxia, preferably in a non-invasive manner, could therefore predict treatment outcome and serve as a tool to individualize treatment. This review gives an overview of recent literature on hypoxia imaging markers currently used in clinical trials. Furthermore, recent progress made in targeting hypoxia (hypoxia-activated prodrugs) or hypoxia response (carbonic anhydrase IX inhibitors) is summarized. Last, window-of-opportunity trials implementing non-invasive imaging are proposed as an important tool to prove anti-tumour efficacy of experimental drugs early during drug development

Synthesis and in Vivo Biological Evaluation of Ga-68-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography

Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [68Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing 68Ga for preclinical CA IX imaging are scarce, and this is one of the first effective 68Ga compounds reported for PET imaging of CA IX.Griffith Sciences, School of Natural SciencesNo Full Tex