Hypoxia-Targeting Carbonic Anhydrase IX Inhibitors by a New Series of Nitroimidazole-Sulfonamides/Sulfamides/Sulfamates

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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

Targeting carbonic anhydrase IX by nitroimidazole based sulfamides enhances the therapeutic effect of tumor irradiation: A new concept of dual targeting drugs

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Cibler CAIX en utilisant de petites molécules : conception, synthèse et efficacité biologique

Hypoxia is a salient feature in many solid tumors and arises due to an inadequate and immature vascular supply resulting in a decreased delivery of oxygen and nutrients. These hypoxic regions show resistance towards conventional treatment modalities such as radio- and chemotherapy and are associated with poor survival. Under hypoxic conditions HIF-1α enhances the expression of many target genes, one of them being carbonic anhydrase IX (CAIX).CAIX is a transmembrane enzyme, which is involved in reversible hydration of carbon dioxide to bicarbonate and a proton.The aim of this thesis was to target CAIX using various dual target drugs combined with radiosensitizers, cytotoxic drugs and bio-reducible drugs.In this thesis several CAIX targeting approaches have been investigated.Since nitroimidazoles are good hypoxic cell sensitizers,we have designed several dual target compounds existing out of a combination of a nitroimidazole and a carbonic anhydrase IX inhibitory moiety (Chapter 3). It has been shown that extracellular acidity limits the uptake of weak basic chemotherapeutic drugs, such as doxorubicin, and thereby decreases its efficacy. We hypothesized that combining these nitroimidazole moieties with a sulfonamide/sulfamide/sulfamate to target CAIX results in a decrease in extracellular acidosis and sensitizes hypoxic tumors to chemo- and radiotherapy. Previously, our group has shown that the sulfamide-based derivative 7 enhanced the therapeutic efficacy of irradiation in a CAIX dependent manner with a sensitization enhancement ratio of 1.50,which is higher than several clinically tested radiosensitizers such as misonidazole and nimorazole. Chemosensitization efficacy was observed upon combination of 7 with doxorubicin in HT29 tumor-bearing mice (Chapter 3).A similar dual target approach may be exploitable to deliver cytotoxic drugs towards CAIX expressing cells, resulting in a specific tumor targeted delivery and consequently reduced normal tissue toxicity. Chapter 4 describes the design and synthesis of new series of dual target compounds combining several anti-cancer drugs, Only one compound, i.e. an ATRi derivative, showed a higher efficacy in combination with radiation in CAIX overexpressing cells as compared to cells lacking CAIX expression.Chapter 5 describes the design and synthesis of various bio-reducible nitroimidazole derivatives, nitrogen mustard alkylating agents and N-oxide derivatives combined with a carbonic anhydrase IX inhibiting moiety. All these compounds showed weak to moderate inhibition profiles towards several tested CA isoforms. We have observed that different substitutions and linkers within the same family of compounds influence the binding capacity to CAIX. For example derivatives 17 and 20 belong to same family, but have a different linker and substitution of the aromatic ring, leading to a different binding capacity towards CAIX.To expand our study on bio-reducible drugs, Chapter 6 describes the design and synthesis of 2-, 5-nitroimdazole and nitrogen mustards combined with carbonic anhydrase IX inhibitors by a carbamate linker. The 2-nitroimidazole derivative 1b revealed cytotoxicity in HT29 and HCT116 cell lines and might be explain by the higher reduction potential of 2-nitroimidazoles compared to 5-nitroimidazoles, since our results did show no cytotoxicity of the 5-nitroimidazole derivatives. Our future studies aim to optimize the radiosensitizing efficacy of 2b and 2c and further explore the cytotoxic properties of 1b.In conclusion, this thesis showed that the dual target nitroimidazole combined with CAIXi increases the efficacy of standard treatment modalities such as chemo and radiotherapy. Targeting CAIX with combination of cytotoxic drugs continues to be an interesting approach to target hypoxic tumors in future. Bio-reducible drugs with higher reduction potentials would serve as potential cytotoxic agents to hypoxic tumors thereby decreasing the normal tissue toxicity.L'hypoxie est une caractéristique saillante de nombreuses tumeurs solides et provient d'un apport vasculaire inadéquat et immature, entraînant une diminution l'apport d'oxygène et nutriments. Ces régions hypoxiques montrent une résistance aux modalités de traitement classiques telles que la radio et la chimiothérapie et sont associées à une survie insuffisante. Dans des conditions hypoxiques,le HIF-1α améliore l'expression de nombreux gènes cibles,l'un d'eux étant l'anhydrase carbonique IX (CAIX). La CAIX est une enzyme transmembranaire,impliquée dans l'hydratation réversible du dioxyde de carbone au bicarbonate et au proton. L'objectif de cette thèse était de cibler CAIX en utilisant divers médicaments à double cible combinés avec des radiosensibilisateurs, des médicaments cytotoxiques etdes médicaments bio-réductibles. Dans cette thèse,plusieurs approches de ciblage CAIX ont été étudiées.Étant donné que les nitroimidazoles sont de bons sensibilisants cellulaires hypoxiques, nous avons conçu plusieurs composés à double cible existant par combinaison d'un nitroimidazole et d'une fraction inhibitrice d'anhydrase carbonique IX (chapitre 3). Auparavant, notre groupe a montré que le dérivé à base de sulfamide 7 a amélioré l'efficacité thérapeutique de l'irradiation de manière dépendante la CAIX avec un rapport d'amélioration de la sensibilisation (SER) de 1,50, ce qui est plus élevé que plusieurs radiosensibilisateurs testés cliniquement tels que le misonidazole et le nimorazole. L'efficacité de la chimiosensibilisation a été observée lors de la combinaison de 7 avec doxorubicine chez des souris porteurs de tumeur HT29 (chapitre 3).Une approche à double cible similaire peut être exploitable pour délivrer des médicaments cytotoxiques vers des cellules exprimant CAIX, ce qui entraîne une administration ciblée spécifique de la tumeur et par conséquent réduit la toxicité normale des tissus. Le chapitre 4 décrit la conception et la synthèse de nouvelles séries de composés à double cible combinant plusieurs médicaments anti-cancéreux. Un seul composé,c'est-à-dire un dérivé ATRi, a montré une efficacité supérieure en association avec un rayonnement dans des cellules surexprimant CAIX par rapport aux cellules dépourvues d'expression CAIX.Le chapitre 5 décrit la conception et la synthèse de divers dérivés de nitroimidazole bio-réductibles,des agents alkylants de moutarde d'azote et des dérivés de N-oxyde combinés avec une fraction inhibitrice de l'anhydrase carbonique IX. Tous ces composés présentent des profils d'inhibition faibles à modérés vers plusieurs isoformes CA testées. Nous avons observé que différentes substitutions et liens dans la même famille de composés influencent la capacité de liaison à CAIX.Pour élargir notre étude sur les médicaments bio-réductibles, le chapitre 6 décrit la conception et la synthèse de 2, 5-nitroimdazole et moutarde d'azote combinées avec des inhibiteurs de l'anhydrase carbonique IX par un agent de liaison de carbamate. Le dérivé 2-nitroimidazole 1b a révélé une cytotoxicité dans les lignées cellulaires HT29 et HCT116 et pourrait être expliqué par le potentiel de réduction des 2-nitroimidazoles par rapport aux 5-nitroimidazoles, car nos résultats n'ont montré aucune cytotoxicité des dérivés du 5-nitroimidazole. Nos futures études visent à optimiser l'efficacité radiosensibilisante de 2b et 2c explorent davantage les propriétés cytotoxiques de 1b.En conclusion, cette thèse a montré que le double nitroimidazole cible combiné à la CAIXi augmente l'efficacité des modalités de traitement standard telles que la chimiothérapie et la radiothérapie. Le ciblage de la CAIX avec la combinaison de médicaments cytotoxiques continue d'être une approche intéressante pour les tumeurs hypoxiques cibles à l'avenir. Les médicaments bio-réductibles avec des potentiels de réduction plus élevés serviraient d'agents cytotoxiques potentiels aux tumeurs hypoxiques, ce qui réduirait la toxicité tissulaire normale

Hypoxia-Activated Prodrug Derivatives of Carbonic Anhydrase Inhibitors in Benzenesulfonamide Series: Synthesis and Biological Evaluation

International audienceHypoxia, a common feature of solid tumours’ microenvironment, is associated with an aggressive phenotype and is known to cause resistance to anticancer chemo- and radiotherapies. Tumour-associated carbonic anhydrases isoform IX (hCA IX), which is upregulated under hypoxia in many malignancies participating to the microenvironment acidosis, represents a valuable target for drug strategy against advanced solid tumours. To overcome cancer cell resistance and improve the efficacy of therapeutics, the use of bio-reducible prodrugs also known as Hypoxia-activated prodrugs (HAPs), represents an interesting strategy to be applied to target hCA IX isozyme through the design of selective carbonic anhydrase IX inhibitors (CAIs). Here, we report the design, synthesis and biological evaluations including CA inhibition assays, toxicity assays on zebrafish and viability assays on human cell lines (HT29 and HCT116) of new HAP-CAIs, harboring different bio-reducible moieties in nitroaromatic series and a benzenesulfonamide warhead to target hCA IX. The CA inhibition assays of this compound series showed a slight selectivity against hCA IX versus the cytosolic off-target hCA II and hCA I isozymes. Toxicity and viability assays have highlighted that the compound bearing the 2-nitroimidazole moiety possesses the lowest toxicity (LC50 of 1400 µM) and shows interesting results on viability assays

Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes

Carbonic anhydrase (CA) IX is a hypoxia inducible enzyme that is highly expressed in solid tumours. Therefore, it has been considered as an anticancer target using specific chemical inhibitors. The nitroimidazoles DTP338 and DTP348 have been shown to inhibit CA IX in nanomolar range in vitro and reduce extracellular acidification in hypoxia, and impair tumour growth. We screened these compounds for toxicity using zebrafish embryos and measured their in vivo effects on human CA IX in Xenopus oocytes. In the toxicity screening, the LD50 for both compounds was 3.5 mM. Neither compound showed apparent toxicity below 300 µM concentration. Above this concentration, both compounds altered the movement of zebrafish larvae. The IC50 was 0.14 ± 0.02 µM for DTP338 and 19.26 ± 1.97 µM for DTP348, suggesting that these compounds efficiently inhibit CA IX in vivo. Our results suggest that these compounds can be developed as drugs for cancer 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