European Journal of Medicinal Chemistry 74 (2014) 302e313

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

b-Glucuronidase-responsive prodrugs for selective cancer chemotherapy: An update Isabelle Tranoy-Opalinski a, Thibaut Legigan a, Romain Barat a, Jonathan Clarhaut a, b, Mikaël Thomas a, Brigitte Renoux a, Sébastien Papot a, * a

Université de Poitiers, UMR-CNRS 7285, Institut de Chimie des Milieux et des Matériaux de Poitiers (IC2MP), Groupe “Systèmes Moléculaires Programmés”, 4 rue Michel Brunet, 86022 Poitiers, France INSERM CIC 0802, CHU de Poitiers, 2 rue de la Milétrie, 86021 Poitiers, France

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a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2013 Received in revised form 22 December 2013 Accepted 23 December 2013 Available online 11 January 2014

The design of novel antitumor agents allowing the destruction of malignant cells while sparing healthy tissues is one of the major challenges in medicinal chemistry. In this context, the use of non-toxic prodrugs programmed to be selectively activated by beta-glucuronidase present at high concentration in the microenvironment of most solid tumors has attracted considerable attention. This review summarizes the major progresses that have been realized in this field over the past ten years. This includes the new prodrugs that have been designed to target a wide variety of anticancer drugs, the prodrugs employed in the course of a combined therapy, the dendritic glucuronide prodrugs and the concept of bglucuronidase-responsive albumin binding prodrugs. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Cancer chemotherapy Tumor targeting Glucuronide prodrugs Self-immolative linkers

1. Introduction In recent years, the design of novel antitumor agents allowing the destruction of malignant cells while sparing healthy tissues has become one of the major challenges in medicinal chemistry. Within this framework, numerous research efforts focused on the development of self-responsive chemical systems programmed to deliver potent cytotoxics selectively at the tumor site [1,2]. Such systems are usually complex molecular assemblies that build into their structure (1) a targeting unit enabling the recognition of a tumor-associated specificity and (2) either an enzymatic or a chemical trigger that can be activated exclusively in cancerous tissues to induce the release of the drug in a stringently controlled fashion. The vast majority of the drug delivery systems that have been developed until now were designed to target cancer cell surface specificities (e.g. a membrane receptor or an antigen) [3]. In this approach, the molecular assembly includes either a monoclonal antibody or low-molecular-weight ligand that displays a high affinity for the corresponding tumor-associated cell surface marker. When cancer cell surface is detected by the targeting unit, the

* Corresponding author. Tel.: þ33 549 453 862. E-mail address: [email protected] (S. Papot). 0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.12.045

whole system is internalized via receptor-mediated endocytosis [4]. Once inside the cell, activation of the trigger leads to the release of the active drug selectively in the intracellular medium. Several drug delivery systems of this type are currently being assessed clinically for diverse applications in oncology [5,6]. Furthermore, the antibodyedrug conjugate Adcetris (formally Brentuximab vedotin) reached the market in 2011 for the treatment of Hodgkin lymphoma [7,8]. However, the “Achilles’heel” of drug delivery systems designed to target cell surface specificities relies on the heterogeneity of cancerous tissues. Indeed, all the cells of a tumor mass are not identical exhibiting different concentrations of a given cell surface marker. Thus, only cancer cells that express the selected tumorassociated marker at a sufficient level are directly affected by this class of targeting systems. In this context, the use of enzymeresponsive prodrugs that can be selectively activated by the corresponding enzyme naturally overexpressed in the tumor microenvironment [9] offers a valuable alternative to this targeting approach. In this case, the anticancer agent is released in the extracellular medium and can further penetrate passively inside various types of surrounding malignant cells whatever their membrane characteristics. As early as 1947, Fishman and Anlyan have reported the presence of elevated b-glucuronidase activity in tumors as compared to normal tissues [10,11]. This observation was confirmed thereafter

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Fig. 1. General action mode of prodrugs containing a self-immolative spacer.

by several research groups who detected high concentrations of this enzyme in a wide range of malignancies including breast, lung, ovarian and gastrointestinal tract carcinomas as well as melanomas [12e14]. In tumors, b-glucuronidase is secreted extracellularly in necrotic areas by inflammatory cells (monocytes/granulocytes) while in healthy tissues its activity is confined in lysosomes [14]. In 1988, Tietze [15] was the first who proposed to target this enzymatic specificity of the tumor microenvironment by means of bglucuronidase-responsive prodrugs in the course of a prodrug monotherapy (PMT [13]). Since then, numerous prodrugs have been investigated with the aim to deliver potent anticancer drugs selectively in the vicinity of malignant cells. Not surprisingly, since high level of b-glucuronidase can be found in most solid tumors, this strategy was applied for the targeting of various classes of cytotoxics such as anthracyclines, taxanes, camptothecin derivatives, nitrogen mustards, histone deacetylase inhibitors, hedgehog inhibitors, auristatins and duocarmycins. With a few exceptions, these prodrugs include a selfimmolative linker [16] between the carbohydrate trigger and the drug. The glucuronide moiety is thereby sufficiently far away from the antitumor agent to allow an easy recognition of the enzymatic substrate by b-glucuronidase. The release of the drug proceeds then via a two steps process including (1) the enzymatic hydrolysis of the glycosidic bound and (2) the spontaneous decomposition of the linker leading to the expulsion of the active compound (Fig. 1). The linker plays a major role in the success of this targeting strategy. Besides facilitating the enzymatic reaction, the linker must decompose rapidly after b-glucuronidase-catalyzed activation of the trigger. This allows avoiding the diffusion of the linker-drug intermediate outside of the tumor site that could result in nonspecific delivery of the cytotoxic compound in all the body. Furthermore, a suitable design of the linker can improve

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significantly prodrug properties such as toxicity, pharmacokinetics, organ distribution or bioavailability. As a general statement, b-glucuronidase-responsive prodrugs are far less toxic than the parent drugs. The reduced toxicity is mainly due to the hydrophilicity imparted by the glucuronide moiety that prevents passive cellular uptake and further intracellular prodrug activation by lysosomal b-glucuronidase within nonmalignant cells. As a result, glucuronide prodrugs can be administered at relatively high doses without inducing side effects with respect to the corresponding anticancer agents. Thus, by combining a low toxicity and a selective activation in the tumor microenvironment, glucuronide prodrugs allow the increased drug deposition in the tumor while reducing drug concentration in normal tissues. To date, several glucuronide prodrugs have been evaluated in vivo leading to superior efficacy compared to standard chemotherapy. These results have already been summarized in excellent reviews up to 2003 [17,18]. In this paper, we present an update of the major progresses that have been realized in this field over the past ten years. This includes the new prodrugs that have been designed to target a wide variety of anticancer drugs, the prodrugs employed in the course of a combined therapy, the dendritic glucuronide prodrugs and the concept of b-glucuronidase-responsive albumin binding prodrugs.

2. Glucuronide prodrugs of paclitaxel Paclitaxel (TaxolÒ) [19] is a member of the taxanes family currently used in clinic to treat a variety of malignancies including ovarian, breast and non-small-cell lung cancer. At therapeutic doses this drug induces a number of undesirable side effects such as myelosuppression and sensory neuropathy. Moreover, as paclitaxel exhibits poor water solubility, it has to be co-injected with a detergent, Cremophor EL, which causes hypersensitivity reactions. To circumvent these problems, Schmidt and co-workers have studied the glucuronide prodrugs 1e3 where the 20 -OH of paclitaxel is linked to different self-immolative linkers through either a carbamate or a carbonate (Fig. 2) [20,21]. Elongated linkers [22] were chosen in this study to limit steric hindrance of the glycosidic linkage by the bulky drug. Indeed, previous works have shown inefficient enzymatic activation of glucuronide prodrugs of paclitaxel containing shorter linkers [23]. In the presence of b-

Fig. 2. Glucuronide prodrugs of paclitaxel 1e3.

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Fig. 3. Glucuronide prodrugs of paclitaxel 4 and 5.

glururonidase, prodrugs 1e3 led to the release of the free drug via the mechanism illustrated in Fig. 2. However, by comparing the kinetics of transformation of these prodrugs into the active compound, it was observed that 2 and 3 bearing a carbonate function were cleaved faster than the carbamate analog 1. Since the kinetics of drug release is a key parameter for the efficiency of this targeting strategy, glucuronides 2 and 3 were selected as the more promising targeting systems. The cytotoxicity of both prodrugs were compared to that of paclitaxel on HT-29 (colon cancer) cell line. As expected, 2 and 3 were 180 and 70 fold less toxic than the parent drug confirming that derivatization of paclitaxel in the form of glucuronide prodrugs markedly reduced its toxicity (IC50: 2 ¼ 28.9 nM, 3 ¼ 11.3 nM, paclitaxel ¼ 0.16 nM). On the other hand, when incubated with b-glucuronidase the antiproliferative activities of both prodrugs were identical to that recorded for the free drug, as the consequence of its efficient release in the culture medium. Stability of glucuronides 2 and 3 were then measured in a 10% fetal calf serum in a phosphate buffer solution at 37  C. While prodrug 3 (X ¼ NH2) was stable over 24 h, the analog 2 bearing a nitro group (X ¼ NO2) on the linker was decomposed rapidly under these conditions. In the light of these results, the authors concluded that the glucuronide prodrug 3 was the best candidate for further in vivo evaluations. As N-phenyl b-O-glucuronyl carbamates [24] are known to be excellent substrates for b-glururonidase [25], the same group synthesized the prodrugs 4 and 5 programmed to release paclitaxel through the chemical cascades depicted in Fig. 3 [26]. Unfortunately, glucuronide 5 was unstable under physiological conditions leading to spontaneous expulsion of the free drug. In contrast, prodrug 4 proved to be stable in phosphate buffer containing 10% fetal calf serum with less than 5% of decomposition over 24 h. Such a difference in stability was attributed to the presence of the benzylic carbonate that promoted the premature 1,6-elimination and subsequent drug release directly from prodrug 5 in the

absence of b-glururonidase. These results clearly ruled out the potential use of 5 in the course of targeted therapy. Interestingly, prodrug 4 exhibited an IC50 value of 26.3 nM that was 164-fold less than that measured for paclitaxel on HT-29 colon cancer cell line. However, enzymatic hydrolysis assays showed incomplete transformation of the glucuronide into the active drug due to partial inhibition of b-glururonidase by the azaquinone methide 6 (Fig. 3). While prodrug 3 presents all the favorable criterions to be used in PMT, the efficacy of this targeting device in animal models has not been published yet. Despite numerous efforts, none glucuronide prodrug of paclitaxel has been evaluated in vivo so far. 3. Glucuronide prodrugs of histone deacetylase inhibitors Recently, the inhibition of histone deacetylases [27] has emerged as a new strategy in cancer chemotherapy [28]. Over the past 15 years, an increasing number of structurally diverse histone deacetylases inhibitors (HDACi) have been studied as potential therapeutic agents [29e32] and more than ten different HDACi were evaluated clinically [33]. Among them, the suberoylanilide hydroxamic acid or SAHA (ZolinzaÔ), reached the market for the treatment of cutaneous T-cell lymphoma in October 2006 [34,35]. However, the use of SAHA is often associated with severe side effects such as anorexia, anemia and thrombocytopenia [36]. Thus, in order to improve the therapeutic index of this promising class of anticancer agents, our group designed the first glucuronide prodrug of SAHA 7 (Fig. 4) [37,38]. In this case, the hydroxamic acid function of the drug was directly linked to the carbohydrate trigger without the need of a linker. We have anticipated that prodrug 7 would be less toxic than SAHA since crystallographic studies demonstrated that this hydroxamate-based HDACi acted by binding the zinc ion in the active site of the enzyme in a bidentate fashion, via its CO and OH groups [39e41]. To verify this hypothesis, the antiproliferative activity of 7 was evaluated on non-small cell

Fig. 4. Glucuronide prodrug of SAHA.

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lung cancer H661 and compared to that of SAHA. In these experiments, prodrug 7 did not present any detectable cytotoxicity at the highest tested dose of 25 mM whereas the free drug exhibited an IC50 value of 1 mM. However, when incubated with a large excess of b-glucuronidase, 7 did not lead to the release of SAHA. This result was surprising since the glucuronide of TrocadeÔ, a bulkier hydroxamate-containing drug, was reported as a good substrate for b-glucuronidase [42]. Nevertheless, this result clearly showed that the glucuronide of SAHA 7 is inappropriate for b-glucuronidasemediated PMT. We decided then to pursue our investigations in this area with CI-994, a potent member of the benzamide class of HDACi. This compound has demonstrated significant antitumor activity against a broad spectrum of tumor models [43e45]. Furthermore, CI-994 was assessed clinically in combination with other standard anticancer drugs [46e48]. Unfortunately, the use of this HDACi in cancer chemotherapy is hampered by its unselective toxicity as well as its poor aqueous solubility. Thus, with the aim to solve these problems, we designed the two b-glucuronidase-responsive prodrugs of CI-994 8 and 9 [49] (Fig. 5). The prodrug 9 included the well-known nitrobenzylphenoxy carbamate linker [16] whereas in the case of prodrug 8, the drug was directly attached to the glucuronide moiety through a carbamate functional group. These two compounds appeared highly stable either in phosphate buffer (pH 7) or in cell culture medium with no detectable decomposition over a period of 72 h under these conditions. In the course of enzymatic hydrolysis assays CI-994 was released slightly faster from prodrug 8 than prodrug 9. However, kinetic profiles were very similar indicating that the linker decomposition step of 9 was not rate-determining. As expected, these prodrugs were at least 12.5 fold more soluble in aqueous media than the parent drug therefore allowing their i.v. administration (solubility in water: CI-994 ¼ 0.08 mg/mL, 8 and 9 > 1 mg/ mL). In vitro biological evaluation demonstrated that the carbamoyl derivatisation of the amino group of CI-994 reduced significantly its toxicity. Indeed, while CI-994 exhibited an IC50 value of 20 mM on H661 cancer cells, prodrugs 8 and 9 did not affected viability of the cells even when tested at doses as high as 300 mM. In contrast, addition of b-glucuronidase with 8 and 9 induced a dramatic antiproliferative effect (IC50 for 8 and 9 ¼ 20 mM), restored HDAC inhibition and E-Cadherin expression showing the efficient enzymecatalyzed release of the active drug in the culture medium. All these results suggested that both glucuronide prodrugs 8 and 9 possess the necessary prerequisites for further in vivo experiments. However, since the cytotoxicity of CI-994 remains relatively low, the use of these prodrugs in a tumor targeting strategy should be envisioned exclusively in combination with a highly toxic drug.

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4. Glucuronide prodrugs of cyclopamine Aberrant activation of the Hedgehog (Hh) signaling pathway [50] has been observed in a wide range of malignancies such as breast [51], prostate [52], gastric [53], lung [54] and brain tumors [55]. As a result, many efforts have been devoted to the discovery of small-molecule Hh inhibitors for cancer chemotherapy [56]. Cyclopamine, a natural alkaloid from Veratrum californicum (the hellebore or corn-lily), was the first Hh inhibitor to be identified [57]. This compound already demonstrated antitumor activity in the course of preclinical and clinical evaluations [58]. Although this Hh inhibitor is a promising chemotherapeutic agent, cyclopamine could induce serious damages in normal tissues since somatic stem cells are also Hh-dependent. Moreover, the use of cyclopamine is limited by its very low aqueous solubility [59]. Thus, the selective deposition of cyclopamine in the tumor microenvironment by the means of hydrophilic glucuronide prodrugs seems to be an attractive alternative to the systemic administration of this Hh inhibitor. In this context, our group has undertaken the study of the two prodrugs 10 [60] and 11 [61] depicted in Fig. 6. When the glucuronide 10 was placed in the presence of bglucuronidase, the glycosidic bond was rapidly cleaved to generate the corresponding phenol 12. This intermediate underwent a 1,6elimination followed by a spontaneous decarboxylation leading to the full release of cyclopamine within 28 h. The slow liberation of the active compound was due to the precipitation of the lipophilic phenol 12 as soon as it was produced by the enzymatic hydrolysis. Consequently, the kinetics of drug release was limited by the gradual solubilization of 12 in aqueous medium. Since in this targeting strategy it is well admitted that the expulsion of the anticancer agent has to occur quickly after the enzymatic activation step to avoid the diffusion of the linker-drug intermediate outside of the tumor site, we decided to continue our investigations with the prodrug 11. In contrast with its analog 10, this compound comprises a hydrophilic glycosylated poly(ethylene glycol) side chain [62] introduced at the benzylic position of the selfimmolative linker via “click chemistry” [63,64]. With this design, the kinetics of drug release was markedly improved as compared to that recorded from prodrug 10. Indeed, starting from glucuronide 11, enzymatic hydrolysis yielded the corresponding phenol 12 which was readily water soluble thanks to the presence of the hydrophilic side chain. Thus, cyclopamine was totally liberated in less than 2 h after the addition of b-glucuronidase in the medium. Prodrug 11 was tested for its anti-proliferative activity on U87 glioblastoma cells. When incubated alone in the culture medium, 11 did not affect the viability of cells whereas the free cyclopamine was highly toxic with an IC50 value of 16.5 mM. On the other hand, in

Fig. 5. Glucuronide prodrugs of CI-994.

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Fig. 6. Glucuronide prodrugs of cyclopamine.

the presence of the activating enzyme, the cytotoxicity of the Hh inhibitor was restored as the result of its quick release through the mechanism illustrated in Fig. 6. The antitumor efficacy of the glucuronide prodrug 11 is currently evaluated in mice for the treatment of glioblastoma. 5. Glucuronide prodrugs of camptothecin derivatives Camptothecin (CPT) is an alkaloid isolated from Camptotheca acuminate [65] that displays potent antitumor activity by inhibition of topoisomerase I [66]. While CPT showed promising results in preliminary clinical trials, its use was limited by extremely poor water solubility. This drawback led to the development of more water-soluble derivatives among which Topotecan [67] and Irinotecan [68] have been approved for clinical use. However, these two CPT analogs induce serious side effects such as diarrhea and immunosuppression. In 1999, Roffler and Chern have proposed the study of the glucuronide prodrug of 9-aminocamptothecin 13 to improve both the safety and aqueous solubility of CPT derivatives [69] (Fig. 7). This prodrug was 20e80-fold less toxic than 9-aminocamptothecin when evaluated alone on a wide range of human tumor cell lines. Furthermore, simultaneous addition of 13 and b-glucuronidase in the culture mediums resulted in an antiproliferative activity comparable to that of the free drug showing that the prodrug was converted to 9-aminocamptothecin. These promising preliminary results motivated the authors to pursue the study of prodrug 13 in vivo [70]. When tested against LS174T human colon cancer xenografts in nude mice, a single i.v. injection of 50 mg kg1 glucuronide 13 resulted in a good antitumor activity with acceptable levels of toxicity. In comparison, 9aminocamptothecin administrated at 5 mg kg1 induced a similar antitumor effect but was highly toxic, killing half of the mice

included in this study. Prodrug 13 was even more effective against CL1-5 human lung adenocarcinoma leading to the cure of 70e85% of mice with established tumors. In this model, either 9aminocamptothecin or topotecan produced significantly lower antitumor activities. The superior efficacy of prodrug 13 towards CL1-5 compared with LS174T tumors was elucidated in 2009 by Roffler and Cha who demonstrated that inflammatory cell (neutrophils and macrophages) infiltration was insufficient in the colon tumors to secrete adequate level of b-glucuronidase in the microenvironment [71]. In this remarkable study, the authors also showed that the antiangiogenic monoclonal antibody DC101 enhanced the accumulation of neutrophils and macrophages leading to an increased concentration of b-glucuronidase at the tumor site. Most importantly, combined therapy with DC101 and a single dose of prodrug 13 (50 mg.kg1) resulted in long-term tumor suppression and mice survival. Overall, this study provided evidence that antiangiogenic agents can synergize the antitumor activity of b-glucuronidaseresponsive prodrugs which represents an advance of prime importance in the field. Chern and co-workers have studied the derivatization of 10hydroxycamptothecin in the form of the prodrug 14 [71] (Fig. 7). In topoisomerase I inhibition assays, this CPT derivative was found to be more potent than topotecan [72]. Moreover, 10-hydroxy camptothecin was effective against multidrug-resistant cancer cell lines, whereas topotecan and 9-aminocamptothecin were relatively inefficient [73]. In the design of prodrug 14, the drug was connected to the self-immolative linker via an ether linkage which was unprecedented in this area. This compound was 80 times more water-soluble than 10-hydroxycamptothecin and stable in human plasma. Enzymatic kinetics assays revealed that b-glucuronidase exhibited 520 higher catalytic efficiency for prodrug 14 than for the analog 13. Furthermore, 14 was about 15-fold less toxic than the

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Fig. 7. Glucuronide prodrugs of 9-aminocamptothecin and 10-hydroxycamptothecin.

parent drug against four human tumor cell lines when incubated alone, but induced a similar cytotoxic effect than that of 10hydroxycamptothecin in the presence of the activating enzyme. These data suggested that glucuronide 14 may be useful for selective cancer chemotherapy. However, the antitumor efficacy of this targeting device has not been published yet. 6. Glucuronide prodrug of duocarmycin derivatives The duocarmycins are a family of antineoplastic agents with low picomolar potency against a wide range of cancer cell lines [74,75]. The cytotoxic activity is believed to derive from their ability to bind and alkylate DNA in AT-rich region of the minor groove [76], although other modes of action have been suggested recently [77e 80]. However, the duocarmycins display side effects in vivo including hepatotoxicity and myelosuppression which represent a major limitation for their clinical use [81,82]. Thus, Tietze and coworkers have proposed to study the glucuronide prodrug of a seco-analog of duocarmycin 15 with the aim to develop more selective therapeutic agents [83e85] (Fig. 8). As phenyl b-glucuronides are known to be excellent substrates for b-glucuronidase, the

presence of a linker was not necessary and the seco-drug was directly attached to the carbohydrate trigger. In this approach, enzymatic cleavage of the glycosidic bond releases first the secodrug which is rapidly transformed in situ into the corresponding duocarmycin derivative that is responsible for the biological activity [86]. In vitro cytotoxicity of prodrug 15 was evaluated on several human cancer cell lines with IC50 values ranging from 10 to 82 nM indicating that 15 was thousand-fold less toxic than the drug [85]. In contrast, addition of b-glucuronidase in the culture mediums produced a dramatic increase of toxicity leading to IC50 values comparable to that of the free drug (from 12 to 46 pM). The antitumor activity of prodrug 15 was then tested in mice for the treatment of a CL1-5 human lung cancer xenograft and compared to the efficacy of carboplatin, a commonly used antineoplastic agent. In these experiments, glucuronide 15, administered at 2.5 mg kg1, induced better tumor suppression with less toxicity than carboplatin injected at 50 mg kg1. However, although significant, the antitumor activity remained modest probably due to insufficient level of b-glucuronidase in the tumor microenvironment. Therefore, the authors hypothesized that increasing the extracellular concentration of this enzyme should further enhance

Fig. 8. Glucuronide prodrug of duocarmycin derivative.

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the therapeutic effect of prodrug 15. For this purpose, prior to the administration of 15, the mice received intratumoral injections of an adenoviral vector expressing membrane-anchored b-glucuronidase [87,88]. In this experiment, complete tumor remission was observed in seven of nine mice treated with this combined protocol. As expected, pretreatment of cancer tissues by the adenoviral vector strongly potentiated the antitumor activity of prodrug 15. Other approaches aimed to increase the concentration of bglucuronidase in the tumor microenvironment have been developed [89e93]. These strategies could represent valuable alternatives to PMT in order to enhance the therapeutic index of glucuronide prodrugs, especially for the treatment of tumors expressing insufficient levels of b-glucuronidase as well as metastatic tumors. 7. Glucuronide prodrug of monomethylauristatin E Monomethylauristatin E (MMAE) is a potent inhibitor of tubulin polymerization with subnanomolar cytotoxic activity in vitro. Because of its high toxicity, MMAE has only limited efficacy in vivo at a dose that is not lethal to animals. However, this drug already shown a remarkable efficiency in human when targeted to cancer cells in the form of Brentuximab Vedotin [7,8]. This alternative also proved its validity in vivo with other enzyme-responsive drug carriers of MMAE where the therapeutic index of this drug was greatly enhanced when used in a targeted strategy [94e97]. In this context, our group has undertaken the study of the glucuronide prodrug 16 designed for the selective delivery of MMAE in the tumor microenvironment [98] (Fig. 9). In the presence of b-glucuronidase, this prodrug was rapidly cleaved leading to the full release of the drug in less than 40 min. This was accompanied by the formation of the 4-hydroxy-3nitrobenzyl alcohol, confirming that the disassembly of 16 proceeded through the self-immolative mechanism illustrated in Fig. 9. This prodrug was 40e110 fold less toxic than MMAE on several types of cancer cells including A549 (human lung adenocarcinoma), KB (human squamous carcinoma) and MDA-MB-231 (human breast adenocarcinoma) cells. On the other hand, when the targeting system 16 was incubated with b-glucuronidase, the antiproliferative activity of the drug was restored with IC50 values ranging from 0.16 to 0.52 nM. The cytotoxicity of glucuronide 16 was also tested on primary cultures of patients who underwent surgical resection for primary non-small cell lung cancer. While 16 did not affect the viability of primary cells when incubated alone,

addition of b-glucuronidase with the prodrug in the medium produced a strong cytotoxic effect at a dose as low as 1 nM, killing 75% of the cells. With these promising results in hands, we pursued the evaluation of prodrug 16 in mice for the treatment of a subcutaneous murine Lewis Lung Carcinoma (LLC) xenograft. The animals received three i.v. injections of the glucuronide 16 at 0.5 mg kg1 (days 7, 11 and 14 after tumor implantation). Prodrug 16 was well tolerated without any sign of overt toxicity at the tested dose. On day 20, mice were euthanized and tumor weights were measured. In these experiments, the tumor weights were 1.5-fold lower in mice treated with 16 compared to that recorded in the control group (untreated animals). These results clearly showed the therapeutic benefit brought by this targeting system. However, this study remains preliminary and other in vivo assays need to be carried out to determine the full potential of this glucuronide prodrug. 8. Glucuronide prodrugs of anthracyclines Anthracyclines, like daunorubicin, doxorubicin and epirubicin are potent antitumor agents that have been widely used in clinic for the treatment of numerous malignancies such as leukemias, lymphomas, soft-tissues sarcomas, breast, uterine and ovarian cancers [99]. Anthracyclines are however notorious for causing cumulative and irreversible cardiotoxicity, which considerably limits their usefulness [100,101]. Thus, b-glucuronidase-responsive prodrugs of anthracyclines have received considerable attention over the past three decades to decrease the toxicity of this class of anticancer drugs towards healthy tissues [13,14,90,102e116]. Among them, DNR-GA3 [104,105], DOX-GA3 [108] and HMR 1826 [13,14,114,117] have been intensively investigated for their therapeutic activity in the course of a PMT (Fig. 10). These glucuronide prodrugs showed superior antitumor efficacy associated with reduced toxicity compared to standard chemotherapy. Nevertheless, the administration of very high doses was usually required to achieve a significant therapeutic effect, therefore representing a major problem for their use in clinic. Indeed, the therapeutic efficiency of these prodrugs was limited by two major drawbacks: (1) The reduced turnover of b-glucuronidase in the tumor microenvironment. The optimal pH for activity of this enzyme is approximately 4 whereas that of the tumor extracellular medium is 6e7. Above a certain threshold dose

Fig. 9. Glucuronide prodrug of monomethylauristatin E.

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Fig. 10. Glucuronide prodrugs of doxorubicin and daunorubicin.

of prodrug, b-glucuronidase is saturated and consequently the amount of drug liberated in targeted tissues is not sufficient to induce total and lasting tumor shrinkage. (2) The reduced plasmatic half-life of b-glucuronidase-responsive prodrugs. Like most of the glucuronides [118], these prodrugs are rapidly eliminated by the kidneys, lowering their action along the time. Recently, new approaches have been proposed to overcome these weaknesses with the goal to enhance the therapeutic index of

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glucuronide prodrugs of anthracyclines. Within this framework, our group developed the first dimeric glucuronide prodrug of doxorubicin 17 programmed to release two molecules of drug after a single enzymatic activation step [119] (Fig. 11). This study relied on the concept of self-immolative dendrimers introduced in 2003 simultaneously by Shabat [120], de Groot [121] and McGrath [122]. The targeting system 17 comprised a glucuronide trigger, a linker and the two drug molecules articulated around a chemical amplifier. With this design, enzymatic hydrolysis of the glycosidic bond resulted in the formation of the phenol intermediate 18 which induced the release of the aniline 19 through an 1,6 elimination. The amplifier unit then conducted to the expulsion of the two doxorubicins via 1,4 and 1,6 elimination processes as illustrated in Fig. 11. The dimeric prodrug 17 was tested for its antiproliferative activity against H661 lung cancer cell line and compared to that of HMR 1826 in the presence of the same quantity of b-glucuronidase. In these experiments, the cytotoxicity of glucuronide 17 was approximately 2-fold higher than that obtained for its monomeric analog confirming the enzyme-catalyzed double drug release in the cell culture medium (IC50: 17 ¼ 110 nM; HMR 1826 ¼ 280 nM; doxorubicin ¼ 250 nM). In the same field of investigation, we also developed the heterodimeric glucuronide prodrug 20 designed to target two different anticancer agents: the well-known histone deacetylase inhibitor MS-275 and the potent doxorubicin [123] (Fig. 12). When the heterodimer 20 was placed in the presence of b-glucuronidase, doxorubicin was released within 5 h, whereas MS-275 was

Fig. 11. Dimeric glucuronide prodrug.

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liberated significantly slower (within 18 h). In contrast with the observations reported in a previous study [124], this result demonstrated that the 1,4 elimination occurred faster than the 1,6 elimination reaction from the aniline 21 (Fig. 12). Prodrug 20 was then evaluated for its antiproliferative activity against H290 lung mesothelioma cell line. While the heterodimer alone did not exhibit any toxicity, addition of the activating enzyme in the culture medium produced a strong antiproliferative effect with an IC50 value of 48 nM. As expected, glucuronide 20 was about 8-fold more toxic than HMR 1826 when incubated with the same quantity of b-glucuronidase. However, the cytotoxicity of prodrug 20 was surprisingly 5-fold higher than that recorded for the combination of doxorubicin and MS-275 (IC50 ¼ 221 nM), suggesting that this antiproliferative activity cannot be solely attributed to the enzyme-catalyzed release of the two drugs. Similar results were also observed on H661 and H157 lung cancer cell lines. Further experiments conducted to explain this phenomenon clearly demonstrated that the amplifier unit was involved in the cytotoxicity of 20 via its transformation into the azaquinone methide 22 (Fig. 12). In the light of these results, it appeared that glucuronide prodrug 20 could lead to selective and potent cancer tritherapy by releasing three different active species at the tumor site. The studies of prodrugs 17 and 20 revealed that targeting systems allowing the release of several drugs after a single enzymatic triggering event could permit to overcome the poor turnover of bglucuronidase in the tumor microenvironment. The potential of this novel approach has however to be confirmed in vivo. As rapid renal excretion of glucuronide prodrugs represents a major limitation for their therapeutic efficacy, Boven and coworkers proposed the study of the methyl ester analog of DOXGA3, DOX-mGA3 [125] (Fig. 13). The authors expected that this more lipophilic derivative would result in improved circulation half-life. Furthermore, they hypothesized that DOX-mGA3 would be slowly converted to DOX-GA3 by esterase activity in blood, prior to the b-glucuronidase-mediated activation of this latter in the tumor.

Fig. 13. Methyl ester glucuronide prodrug of doxorubicin.

Intravenous administration of DOX-mGA3 in mice led to prolonged circulation associated with a 2.7-fold higher doxorubicin concentration in tumor tissues as compared to administration of DOX-GA3. Nonetheless, treatment with DOX-mGA3 was hampered by its poor aqueous solubility precluding effective doses to be administered. Indeed, the highest dose that could be injected to the animals was 20 mg kg1 whereas a 250 mg kg1 dose of DOX-GA3 was needed to produce a significant tumor growth inhibition [108]. More recently, our research team developed the concept of bglucuronidase-responsive albumin-binding prodrugs [126], as an extension of the works described earlier by Kratz and co-workers. The Kratz’s group studied highly innovative albumin-binding prodrugs [127] that accumulated selectively in tumors as the consequence of the EPR effect [128]. The originality of this targeting strategy relies on the formation of a macromolecular drug carrier in the blood stream resulting from the selective coupling of a maleimide-containing prodrug with the cysteine 34 position of circulating albumin after intravenous administration [129]. Several

Fig. 12. Heterodimeric glucuronide prodrug.

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prodrugs of this type demonstrated remarkable antitumor activity in vivo [130e134] and one among them, INNO-206, is currently being assessed clinically [135,136]. In addition to their discriminating accumulation in malignant tissues, these prodrugs exhibit a favorable pharmacokinetic profile due to the prolonged half-life of albumin. Thus, we decided to combine this approach with the targeting of b-glucuronidase in the tumor microenvironment to enhance the therapeutic efficacy of glucuronide prodrugs. In this context, we designed the b-glucuronidase-responsive albumin-binding prodrug of doxorubicin 23 (Fig. 14). With this design, we expected that the in situ binding of 23 to the thiol at the cysteine 34 position of plasmatic albumin through Michael addition will produce the macromolecular drug carrier 24. Once in the targeted tissues, the b-glucuronidase-catalyzed cleavage of the glycoside bond should then trigger the release of doxorubicin via the self-immolative mechanism illustrated in Fig. 14. The in vivo efficacy of prodrug 23 was evaluated in a subcutaneous murine Lewis lung carcinoma xenograft and compared to that of HMR 1826 and doxorubicin. The animals received two i.v. injections of 43.6 mmol kg1 of 23 or HMR 1826 at days 4 and 11 after tumor implantation. Doxorubicin was tested at its maximal tolerate dose (2  13.7 mmol kg1) following the same therapeutic protocol. Prodrug 23 produced a good antitumor response (T/C ¼ 24%) which was similar to that obtained with doxorubicin (T/C ¼ 31%). In contrast, HMR 1826 was poorly active at the tested dose (T/C ¼ 69%). Furthermore, the free drug induced a body weight loss of 21% due to nephrotoxicity whereas glucuronide 23 was well tolerated without any sign of overt toxicity. These results

Fig. 14. b-Glucuronidase-responsive albumin-binding prodrug of doxorubicin.

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demonstrated unambiguously the benefit brought by b-glucuronidase-responsive albumin-binding prodrugs compared to either standard chemotherapy or the use of glucuronide prodrugs which cannot bind covalently to circulating albumin. Further studies in this field are expected to confirm the potential of this approach. 9. Conclusion Since Tietze has proposed to target extracellular b-glucuronidase in the tumor microenvironment, a wide number of structurally diverse glucuronide prodrugs have been designed with the aim to enhance the selectivity of cancer chemotherapy. Conducted in the nineties, the first evaluations of this strategy led to encouraging outcomes in several animal models. These studies also highlighted the problems which have to be solved in order to improve this therapeutic approach, such as the insufficient concentration of bglucuronidase in some tumors, the poor turnover of this enzyme in the tumor microenvironment and the rapid excretion of glucuronide prodrugs by kidneys. Over the last decade, the concept of b-glucuronidase-responsive prodrugs has been extended to new classes of anticancer drugs including histone deacetylase inhibitors, hedgehog inhibitors, duocarmycin and auristatin derivatives. The prodrugs of duocarmycin and auristatin showed significant therapeutic efficacy when administrated at low doses. These results point out the benefit brought by the targeting of highly potent cytotoxic agents with respect to b-glucuronidase saturation in the tumor area. Homo and heterodimeric glucuronide prodrugs have also emerged as a promising alternative to overcome the relatively low enzymatic activity of b-glucuronidase in some tumors. Furthermore, by releasing different anticancer agents after a single enzymatic activation step, heterodimeric prodrugs could open the door towards a selective polychemotherapy of solid tumors. Numerous efforts have been devoted to increase the level of bglucuronidase at the tumor site. Within this framework, the demonstration that antiangiogenic agents can synergize the antitumor activity of b-glucuronidase-responsive prodrugs by enhancing the concentration of the activating enzyme selectively in the tumor microenvironment represents a major advance. This is probably the simplest strategy that has been reported in this area so far. Thus, it seems that investigations in this direction deserve to be pursued further. The concept of b-glucuronidase-responsive albumin-binding prodrugs appeared as a promising approach to limit the rapid renal clearance observed with previous glucuronide prodrugs. The first and unique example of such drug delivery system led to very encouraging results in vivo. Other prodrugs of this type have to be studied to validate the potential of this targeting strategy. In summary, there is no doubt that several significant advances have been realized in the field of glucuronide prodrugs over these past years. In a near future, the association of several approaches presented in this review could be a valuable area of investigation to increase the potential of this therapeutic strategy. For instance, one can imagine that the use of a dimeric b-glucuronidase-responsive albumin-binding, programmed to target two highly potent drugs such as duocarmycin or auristatin derivatives, in combination with an approach designed to enhance the concentration of b-glucuronidase in the tumor microenvironment should lead to an improved antitumor effect. Thus, further progresses are expected shortly to confirm the validity of this therapeutic approach in human. If successful, the use of b-glucuronidase-responsive prodrugs would give rise to more selective chemotherapies of solid tumors.

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