GnRH-gemcitabine conjugates for the treatment of androgen-independent prostate cancer : pharmacokinetic enhancements combined with targeted drug delivery

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone- Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG, showed a significant advantage in tumor growth inhibition when compared to gemcitabine.A.G.Leventis foundation and the General Secretariat for Research & Technology of the Greek Ministry of Education (LS7- 1682/17156/6.12.10).MRC and National Research Foundation of South Africa, and the Universities of Pretoria and Cape Townhttp://pubs.acs.org/bc2015-02-28hb201

Βιοδιαθεσιμότητα και φαρμακοκινητική αξιολόγηση συζευγμένων αναλόγων της ορμόνης GnRH

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this thesis was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in castration resistant prostate cancer models. In vitro cytotoxicity, cellular uptake and metabolite formation of the conjugates were examined in prostate cancer cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely GSG, displaying high antiproliferative activity in prostate cancer cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG, showed a significant advantage in tumor growth inhibition when compared to gemcitabine.Η χρήση της γεμσιταβίνης, ενός αντικαρκινικού φαρμάκου με καθιερωμένη αποτελεσματικότητα εναντίον διαφόρων συμπαγών όγκων, χαρακτηρίζεται από συγκεκριμένα μειονεκτήματα που σχετίζονται με την ταχεία μεταβολική αδρανοποίησή της. Ο σκοπός της διδακτορικής διατριβής ήταν η ανάπτυξη μιας καινοτόμου στρατηγικής για την παραγωγή ενός μεταβολικά σταθερού ανάλογου γεμσιταβίνης που θα μπορούσε επίσης να αποδοθεί σε καρκινικά κύτταρα του προστάτη με βάση την έκφραση του υποδοχέα της γοναδοεκλυτίνης (GnRH-R) στην κυτταρική επιφάνεια. Η αξιολόγηση συζευγμένων μορίων, που αποτελούνται από γεμσιταβίνη συνδεδεμένη με έναν αγωνιστή της γοναδοεκλυτίνης (GnRH) παρουσιάζεται στην παρούσα διατριβή, με αποτελέσματα σε μοντέλα μη ορμονοεξαρτώμενου καρκίνου του προστάτη. Η ιn vitro κυτταροτοξικότητα, η κυτταρική πρόσληψη και ο σχηματισμός μεταβολιτών των συζευγμένων μορίων εξετάστηκαν σε κυτταρικές σειρές καρκίνου του προστάτη. Επιλεγμένα μόρια σύζευξης ήταν αποτελεσματικά στις in vitro δοκιμασίες με ένα από αυτά, συγκεκριμένα το GSG, να εμφανίζει υψηλή αντιπολλαπλασιαστική δραστικότητα σε κυτταρικές σειρές καρκίνου του προστάτη, μαζί με σημαντικά μεταβολικά και φαρμακοκινητικά πλεονεκτήματα σε σύγκριση με τη γεμσιταβίνη. Τέλος, χορήγηση του GSG σε μύες με ξενομοσχεύματα καρκίνου του προστάτη τα οποία εκφράζουν τον GnRH-R, έδειξαν ένα σημαντικό πλεονέκτημα στην αναστολή της αύξησης του όγκου σε σύγκριση με τη γεμσιταβίνη

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy

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 article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC–MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to 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 <i>in vitro</i> colony forming assay together with <i>in vivo</i> whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. 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. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic 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

Peptide-Drug conjugate gnrh-sunitinib targets angiogenesis selectively at the site of action to inhibit tumor growth

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [D-Lys6]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphor-ylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.</p

GnRH-Gemcitabine Conjugates for the Treatment of Androgen-Independent Prostate Cancer: Pharmacokinetic Enhancements Combined with Targeted Drug Delivery

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. <i>In vitro</i> cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the <i>in vitro</i> assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine