A gemcitabine based peptide conjugate with improved metabolic

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A gemcitabine based peptide conjugate with improved metabolic properties and dual mode of efficacy Theodoros Karampelas, Eleni Skavatsou, Orestis Argyros, Demosthenes Fokas, and Constantin Tamvakopoulos Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00961 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Molecular Pharmaceutics

A gemcitabine based peptide conjugate with improved metabolic properties and dual mode of efficacy Theodoros Karampelas†, Eleni Skavatsou†, Orestis Argyros†, Demosthenes Fokas‡, Constantin Tamvakopoulos*,† †

Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou Street, 11527, Athens, Greece. ‡

Laboratory of Medicinal Chemistry, Department of Materials Science and Engineering, University of Ioannina, Greece.

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*Address correspondence to this author at: Address: Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou Street, 11527, Athens, Greece. E-mail: [email protected]. Phone: +30-210-6597475. Fax: +30-210-6597545.

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Keywords: gemcitabine, prostate cancer, GnRH, targeted therapies

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Abstract

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Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide, and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current manuscript provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic.

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LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake and release of gemcitabine and quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG.

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Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine’s bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity.

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In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single non-toxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution and safety, while also agonistically ablating testosterone levels.

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Introduction

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Gemcitabine [2′,2′-difluorodeoxycytidine (dFdC)] is a potent antimetabolite anticancer agent that is used for the treatment of various malignancies such as pancreatic, breast and lung cancer.1 Despite its clinical benefit, gemcitabine is also characterized by limitations that result in either lack of efficacy and/or severe side effects, with hematotoxicity being the most important among them.2 Gemcitabine is typically administered in 4-week cycles, intravenously (IV), in 30 min infusions weekly for three weeks with one week of rest at the end of each cycle.3 The pharmacology of gemcitabine is of particular interest with respect to its metabolic profile, with a relatively short (42-94 min) half-life that has a direct negative impact on its efficacy.3 Upon administration, approximately 90% of gemcitabine is transformed to its inactive metabolite, 2’deoxy-2’,2’ difluorouridine (dFdU).4,5 The enzyme responsible for this conversion is cytidine deaminase (CDA), an enzyme that is highly abundant in the liver as well as in the plasma.6,7 Intracellular uptake of gemcitabine is achieved by different types of nucleoside transporters.8 Once inside the cell, gemcitabine is either inactivated by CDA or is subjected to consecutive phosphorylations that generate its active metabolites, namely 2′,2′-difluorodeoxycytidine diphosphate (dFdCDP) and 2′,2′-difluorodeoxycytidine triphosphate (dFdCTP). dFdCTP is the most active metabolite as it has a direct effect on DNA synthesis by being incorporated in the DNA strand, a process that results in inhibition of proliferation and subsequent cell death by apoptosis. dFdCDP inhibits ribonucleotide reductase (RR), an enzyme that is responsible for the conversion of cytidine diphosphate (CDP) to deoxycytidine diphosphate (dCDP), a process that is necessary for DNA synthesis.3,5

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Although gemcitabine is being routinely used for the treatment of several solid tumors, its use has not been as effective against all cancer types. More specifically in prostate cancer (CaP), although several preclinical studies with gemcitabine had promising outcomes,9 clinical data have shown that administration of gemcitabine in CaP patients has a therapeutic benefit similar to that of docetaxel, the standard of care for CaP but with an unfavorable toxicity profile mainly due to hematotoxicity.6 CaP is rather manageable in earlier stages of the disease typically by blocking the testosterone secretion pathway that is achieved with the use of analogues of the gonadotropin releasing hormone (GnRH) (agonists or antagonists).10 Nevertheless, in many cases CaP advances to a condition known as castration resistant prostate cancer (CRPC).11 Although recent advances12,13 allowed us to have an improved understanding of the molecular mechanisms of CRPC and novel drugs are regularly assessed in the clinic, CRPC is still considered an unmet medical need. We have previously shown that gemcitabine acting as the pharmacophore of a targeted peptide-drug conjugate is efficacious in GnRH-R expressing preclinical models of CRPC, but also as a prodrug of gemcitabine with pharmacokinetic advantages when compared to unconjugated gemcitabine.14 The rational design this type of conjugates14,15 utilized various linkers for linking an anticancer agent to a peptide ligand, agonistic analogue of the GnRH, with binding affinity to the GnRH receptor (GnRH-R) that is found to be overexpressed in CaP.16–18

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The lead compound that emerged from these studies (GSG), showed a metabolic advantage regarding the bioavailability of gemcitabine systemically (in blood of mice administered with GSG or gemcitabine) but also locally (in cancer cells) (Figure 1). More importantly, GSG appears to provide gemcitabine protection from its rapid inactivation. Since the transformation of gemcitabine to its inactive metabolite (dFdU) is a key limiting step of gemcitabine’s efficacy that



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leads to the necessity of high dose administration, our approach appears to provide an alternative to gemcitabine that requires further elucidation.

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Figure 1: GSG is a peptide-drug conjugate that targets specifically CaP cells that overexpress the GnRH-R in the cell surface but also acts as a gemcitabine prodrug that prevents its rapid inactivation from cytidine deaminase (CDA).

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In the current study, we provide in depth mechanistic insights about the previously identified metabolic and efficacy advantages of GSG over gemcitabine. To this end, we designed assays that would provide us with information about key processes related to GSG pharmacology including formation and kinetics of active over inactive metabolites of gemcitabine, stability and formation of metabolites in clinically relevant systems as well as uptake and intracellular release of gemcitabine from GSG. In addition, since the ligand molecule used for this study is an analogue of GnRH, we confirmed its GnRH-R binding capacity but also GSG’s potential to be used as an anticancer agent that can also act systemically, by targeting the GnRH-R in the pituitary gland (hormonal therapy). Finally, we evaluated GSG in an in vitro hematoxicity assay to predict clinical hematotoxicity in order to assess whether conjugation of gemcitabine to the peptide ligand results in reduced toxicity and correlated these findings in vivo with blood measurements from animals dosed daily with GSG.


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Results Metabolism of GSG in human liver microsomes: GSG is metabolized to a more stable metabolite, GSG-(1-9). In an effort to assess the metabolism of GSG in a clinically relevant framework, we performed studies in human liver microsomes and in plasma. Incubation of GSG with human liver microsomes resulted in the production of one major metabolite that appears to correspond to the nonapeptide molecule resulting from the breakdown of the amide bond between Pro-9 and Gly-10 of GSG (Figure 2A). This metabolite was named GSG-(1-9), according to GnRH-(1-9). The proposed metabolite structure was confirmed by the MS/MS fragmentation patterns of its two ionized forms (doubly and triply charged ions, shown in Figures 2B, C, and D). In a multiple time point stability study, incubation of GSG in human liver microsomes showed that GSG-(1-9), produced as a metabolic product of GSG after incubation in human microsomes presented improved stability compared to GSG (Figure 2E). Although GSG(1-9) is not expected to have activity as a GnRH-R agonist19, due to the fact that terminal Gly-10 is essential for GnRH-R activation, it could act as a gemcitabine prodrug, contributing to the prolongation of gemcitabine’s systemic exposure, as observed in previous pharmacokinetic studies in mice.14 Furthermore, the described microsomal incubation assay showed that GSG incubation resulted into lower levels of dFdU compared to equimolar gemcitabine (Figure 2G), confirming our previous preclinical pharmacokinetic data in mice and cell uptake studies and showing that GSG appears to offer a protection for gemcitabine’s rapid deactivation.14 In particular, at 4h, where important GSG degradation has already occurred (81.5%) forming gemcitabine, the gemcitabine over dFdU ratio is significantly higher for GSG compared to gemcitabine (16.4 ± 2.9 vs. 0.2 ± 0.1). Importantly, plasma stability studies revealed that GSG was significantly more stable in human plasma compared to mouse plasma (Supplementary Figure S.I.1), supporting the clinical prospect of GSG and demonstrating that the metabolic advantage observed in mice could be extended and ideally improved in humans.



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Figure 2: GSG-(1-9) MS characterization and stability after incubation in human liver microsomes. A. GSG proposed metabolite formation in microsomes. GSG can be metabolized either to GSG(1-9) or to gemcitabine that is further metabolized to its inactive metabolite dFdU. GSG-(1-9) can be also metabolized to gemcitabine. B. LC-MS Analysis of GSG-(1-9) after incubation of GSG with human liver microsomes. The spectrometric footprint of the doublet of ions (m/z 772.3, 515.5) suggests a molecule with multiple charged states (singly, doubly, triply charged) C. MS/MS analysis of the doubly charged ionized forms of GSG-(1-9) (m/z 772.3). The fragment ions detected correlate well with the suggested fragments in Figure 2A. D. MS/MS analysis of the triply charged ionized forms of GSG-(1-9) (m/z 515.5). The fragment ions from the triply charged ion suggest that GSG’s metabolic degradation occurs in the Gly-10 end since the m/z 264.3 fragment corresponds to gemcitabine (MW=263.2) and the m/z 249.6, 221.2 ions have been previously described to result from the pGlu-1 end of GnRH analogues.20 E. GSG degradation and GSG-(1-9) formation following incubation of GSG in human liver microsomes. Values are expressed as MS analyte/IS area counts. F. Gemcitabine degradation or formation after incubation of gemcitabine or GSG in human liver microsomes respectively. Values are expressed as % of control. Control (100 %) represents the value for gemcitabine (1 µM) at t= 0 h. G. dFdU formation after incubation of gemcitabine or GSG in human liver microsomes. Values expressed in LC-MS/MS analyte/IS area counts. Experiments were performed in triplicates (+/SD). ∗: P

A gemcitabine based peptide conjugate with improved metabolic

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