Constantin Tamvakopoulos, PhD, Pharmacology - Pharmacotechnology, BRFAA, Athens, Greece

Transcription

1 Strategies for the treatment of castrationresistant prostate cancer: Reduced metabolic inactivation combined with targeted drug delivery of gemcitabine based pro-drugs Constantin Tamvakopoulos, PhD, Pharmacology - Pharmacotechnology, BRFAA, Athens, Greece

2 Prostate Cancer Epidemiology Prostate Cancer Incidence Mortality 1 in 7 men will develop prostate cancer Most common malignancy in Western World Risk factors: - Age - Genetic predisposition - Diet Leading New Cancer Cases and Deaths 2014 Estimates Estimated New Cases (American Cancer Society) Estimated Deaths Male Female Male Female Prostate Breast Lung and bronchus Lung and bronchus 233,000 (27%) #1 232,670 (29%) 89,930 (28%) 72,330 (26%) Lung and bronchus Lung and bronchus Prostate Breast #2 116,000 (14%) 108,210 (13%) 29,480 (10%) 40,000 (15%) Survival rates at early stages reach 100% Severity of the disease increases significantly in later stages

3 Prostate Cancer (CaP) Progression (Iwata T. et al, 2010, Plos One, 5 (2)) Gleason Score Grading (Epstein JI., 2010, J Urol,183(2)) 1. Small, uniform glands 2. More space between glands 3. Distinct infiltration of cells from glands 4. Irregular masses of neoplastic cells 5. Lack of glands, sheets of cells Well Differentiated Moderately differentiated Poorly Differentiated, Anaplastic

4 Prostate Cancer (CaP) Molecular Biology Prostate s functional and structural integrity depends on androgen receptor (AR) activation by testosterone binding Activation of AR results in the production of andromedins Andromedins secretion is regulated by a paracrine signaling pathway produced by stromal cells act in epithelial cells Confirmation of CaP histologic diagnosis : loss of the basal cell layer In CaP, paracrine AR signaling is replaced by an autocrine signaling pathway AR stimulates direct production of andromedins Androgen suppression results in prostate cancer cells senescence and eventual cell death Androgen Deprivation Therapy (ADT) is the main therapy for prostate cancer

5 Castration Resistant Prostate Cancer (CRPC) ADT results in prostate cancer remission in approx. 90% of patients In advanced CaP patients cancer progresses to castration resistant prostate cancer (CRPC) In CRPC ADT can not be used as a treating option as AR might be activated by alternative pathways CRPC = unmet medical need (Tsao et al, 2010, Curr. Opin. Urol. 22)

6 Prostate cancer therapies

7 GnRH and its Implication to Prostate Cancer Synthesized in the hypothalamus Released in synchronized pulses from approximately 1000 neurons in the hypophyseal portal system every minutes Characteristic of the hypothalamic-pituitary-gonadal (HPG) axis system Acts on the pituitary Main Regulator of Reproductive Hormonal Pathway Therapeutic target of pathological conditions (e.g. prostate cancer, hormone related disorders) (Mason M., 2009, Future Prescriber 10 (1)) ADT is achieved with the use of GnRH analogues

8 GnRH in Prostate Cancer Pharmacology Androgen Deprivation Therapy (ADT): Main Prostate Cancer Therapy since 1941 Chemical Castration based on GnRH peptide analogues (Agonists-Antagonists) GnRH Peptide Agonists + High affinity binding GnRH Receptor in the pituitary + Desensitization of the receptor Decrease of LH, FSH + Low Immunogenic burden - Initial Flare Effect (Stimulate GnRH Receptor) ( GnRH Peptide Antagonists + Bind to GnRH Receptor without activation (No flare effect) - More Immunogenic - Less Potent (Millar et al., (2004), 25(2): )

9 GnRH Extrapituitary Implication in Cancer GnRH Analogues have been shown to directly inhibit tumor cells: Growth Invasion Metastasis Angiogenesis Opportunity to treat cancer by targeting the GnRH Receptor beyond the pituitary

10 Extrapituitary Localization of GnRH and GnRH-R GnRH and GnRH-R are expressed in extrapituitary tissues of the male and the female reproductive systems such as endometrium, ovaries, breast and prostate Significant expression of GnRH and GnRH-R has been confirmed in various cancer types such as prostate cancer and in breast cancer Prostate cancer tissues exhibit high binding affinity sites for GnRH analogues Prostate cancer tissue has the highest levels of GnRH-R mrna compared to normal prostate or other peripheral tissues Opportunity to treat cancer by targeting the GnRH Receptor beyond the pituitary Targeted therapies that would benefit from GnRH-R overexpression in prostate cancer could lead to therapies with enhanced efficacy and reduced peripheral toxicities

11 Tumor cell targeted therapy using GnRH conjugates GnRH Receptor is a GPCR It can internalize into the cell together with its ligand upon activation Conjugation Delivery of active drug selectively in cancer cells

12 Targeted therapy using GnRH conjugates Chemotherapeutic peptide conjugates GnRH conjugate = GnRH analogue + linker + small molecule anticancer drug Anticancer drug Linker ANTICANCER Millar, R.P. et al (2004) Endocrine Reviews, 25: 235 GnRH peptide analogue

13 Gemcitabine Potent antimetabolite anticancer agent used for the treatment of several solid tumors such as colon, lung, pancreatic cancer Transferred into the cell by nucleoside transporters, undergoes phosphorylation and blocks DNA synthesis Main limitation: Rapid metabolic inactivation through deamination and formation of dfdu Improving gemcitabine poor pharmacokinetics would improve its therapeutic potential

14 Gemcitabine Induced Resistance Another important gemcitabine therapy drawback is that after initial tumor regression, tumors develop different forms of drug resistance Nucleoside transporter deficiency Higher levels of CDA Reducing gemcitabine inactivation and suggesting an alternative entrance route for gemcitabine could be beneficial

15 Gemcitabine in Prostate Cancer Use of gemcitabine in prostate cancer has not been proved beneficial In vitro studies demonstrated that gemcitabine has exceptional antiproliferative effects in androgenindependent prostate cancer cells and in xenograft models In the clinic, a modest clinical benefit was observed when tested as monotherapy or combination therapy, mainly due to its peripheral toxicities

16 Research Aims Design and evaluation of gemcitabine prodrugs (GnRH-gemcitabine conjugates) aiming to: 1)Reduce gemcitabine s metabolic inactivation gemcitabine prodrugs can be designed specifically to affect its interaction with cytidine deaminase 2) lead gemcitabine specifically to the tumor site through conjugation to a peptide with a strong affinity for a cell surface receptor overexpressed in the tumor cell. 3) Provide gemcitabine an alternative entrance route through conjugation with a peptide that could enter the cell using an alternative route (e.g. a GPCR)

17 LC-MS/MS Method Development Biological sample (plasma, kidneys) HPLC/nano UPLC DionexUltimate 3000 Triple quadrupole configuration Mass Spectrometer System AB Sciex 4000 QTRAP High Accuracy Quantification of small molecules and peptides in biological samples MW (m/z) based analysis Essential tool for Pharmacokinetics and Drug Metabolism

18 LC-MS/MS Characterization of GnRH-gemcitabine Conjugates Several GnRH-gemcitabine analogues have been synthesized and tested containing different linkers and different gemcitabine conjugation sites

19 % Cell Viability Development of In Vitro and In Vivo Tools for the Evaluation of GnRH-gemcitabine Conjugates Cell Lines Used DU145 PC3 WPE1-NB26 WPE1-NB26-3 Androgen Independent derived from brain metastasis Androgen Independent derived from bone metastasis Tumorigenic prostate cell line Clone of the above cell line, stably transfected to overexpress the GnRH-R Evaluation of GnRH-R Implication in Cell Proliferation Development of In Vivo Efficacy Models [D-Lys]-GnRH in WPE1-NB26-3 cells Concentration (nm)

20 Evaluation of GnRH-gemcitabine conjugates Several GnRH-gemcitabine analogues have been synthesized and tested so far including different linkers and different conjugation sites Evaluation in cell cultures IC 50 (nm) Gemcitabine 3G 3G 2 GSG GSG 2 DU ± ± ± ± ± 217 PC3 585 ± ± ± ± ± 125 GnRH-R binding assay Superimposed 2D-NMR Compound IC 50 Triptorelin nm GSG 1.96 nm

21 PK studies of GnRH-gemcitabine conjugates in mice GSG administration led to higher and more extended levels of gemcitabine GSG administration led to lower levels of dfdu - GSG High gemcitabine AND low dfdu levels

22 Biodistribution in Xenografted Mice GSG or gemcitabine (6.3 μmol/kg) were administered of in NOD-SCID bearing DU145 tumors Blood and tumors were collected (1h, 4h, 8h, 24h) Gemcitabine:dFdU AUC (0-24 h) Gemcitabine Administration GSG Administration Blood Tumor GSG has tumor bioavailability (184 ng/g or 115 nm at 1 h) even at a low dose (6.3 μmol/kg) dfdu formation in blood was less extensive after GSG administration dfdu formation in tumor was less extensive after GSG administration but not as pronounced as in blood

23 Measuring intracellular levels of gemcitabine, dfdu AUCs (h -1 ) Gemc = 2.80 GSG = 4.55 Incubated 10μΜ of Gemcitabine and Gemcitabine-GnRH conjugate in DU145 cells for 1,4,8 hr gemcitabine degrades rapidly forming dfdu GSG slowly forms gemcitabine incubation with gemcitabine leads to higher dfdu levels in comparison to dfdu formed from GSG suggesting a protective effect of GSG on gemcitabine s rapid metabolic inactivation

24 GSG area counts/is area counts Evaluation of GSG Cell Uptake through the GnRH-R DU145 cells were preincubated with [D-Lys6]-GnRH in order to assess whether GSG enters the cell through the GnRH-R 1.60E E E E-02 * 8.00E E E E E+00 GSG10μM 60' [D-Lys6]-GnRH 1μΜ 60' + GSG10μM 60' A 40% reduction in intra-cellular concentrations of GSG was measured in the pre-treated cells selective GSG entrance to the cell through the GnRH-R Confirmation of alternative entrance route for gemcitabine

25 Plasma testosterone (ng/ml) Assessment of the Agonistic Potential of GSG for the GnRH-R GSG vs [D-Lys6]-GnRH binding and agonistic potential for the GnRH-R Evaluate acute testosterone release in mice *** *** vehicle GSG 1 mg/kg [D-Lys6]-GnRH 1 mg/kg Conjugation of gemcitabine to [D-Lys6]-GnRH does not affect its agonistic potential GSG could also be used in androgen dependent prostate cancers as an androgen deprivation therapy

26 Efficacy of GSG in DU145 xenografted mice DU145 cells were subcutaneously injected in NOD-SCID mice Treatment started when tumors were ~250 mm 3 GSG efficacy was achieved with a significantly lower dose compared to gemcitabine (approx. 25 times)

27 Ongoing work Future Plans LC-MS/MS Characterization of Gemcitabine Active Metabolites Assess effect of GnRH-gemcitabine conjugates in gemcitabine activation a bioanalytical methodology for the identification and quantification of dfdcdp and dfdctp was developed gemcitabine dfdcdp dfdctp

28 Ongoing work Future Plans LC-MS/MS Monitoring of GnRH-R Activation Pathways GnRH-R activation in cancer cells results in an antitumor effect through inhibition of cyclic AMP (camp) Activation of the GnRH-R results in the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP3) LC-MS methodologies for the quantification of camp and IP3 in response to GnRH-R activation are currently being developed

29 Conclusions Developed bioanalytical methodologies for the identification and quantification of novel GnRHgemcitabine conjugates Evaluated the in vitro antiproliferative effect of the of the novel GnRH-gemcitabine conjugates Studied the stability of GnRH conjugates in vitro in order to be able to predict of the in vivo stability In vivo pharmacokinetics showed that GSG produces more gemcitabine and less dfdu Cell uptake studies showed GSG selective entrance through the GnRH-R Testosterone relase studies in mice confirmed GSG s binding and agonistic potential Monitoring of the active metabolites of gemcitabine and molecules related with GnRH-R activation is ongoing Cell uptake studies confirmed the PK findings about GSG reduced metabolic inactivation possibly due to interaction with CDA GSG efficacy in an in vivo xenograft model was achieved with a significantly lower dose compared to gemcitabine

30 Pharmacology Pharmacotechnology (Current/Past members) -Theodoros Karampelas MSc -Katerina Pyrillou MSc -Eva Kouvari MSc -Dr Orestis Argyros -Dr Olga Tsigkou -Dr Panagiwta Agrafiotou -Dr Alexandros Siskos -Dr Theodora Katsila -Dr Sophia Hatziieremia -Irene Baira MSc -Nikolaos Lemonakis Msc -Zacharias Sofianos Msc -Dr. Kostas Dimas -Dr Panayotis Pantazis University of Ioannina -Dr Demosthenes Fokas -Dr Andreas Tzakos -Dr Nisar Sayyad -Dr Theodoros Fotsis -Dr Savvas Christoforidis University of Crete -Dr Georgios Liapakis University of Patras -Dr Theodore Tselios -Dr Ioannis Matsoukas University of Pretoria -Prof Robert Millar University of Hull -Dr Kevin Morgan Funding Agencies -European Commission-FP7 -GGET, Leventis Foundation -MJ Fox Foundation Acknowledgements University of Athens -Prof Leandros Skaltsounis -Prof Emanouel Mikros -Prof Nikos Papadopoulos University of Thrace -Prof Georgios Kolios Demokritos Dr Georgios Kordas Dr Eleni Euthymiadou