Theodoros Karampelas

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.

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-Lys(6)]-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 phosphorylation 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.

Nurr1:RXRα heterodimer activation as monotherapy for Parkinson’s disease

Significance In Parkinson’s disease (PD), dopamine (DA)-producing neurons gradually degenerate, leading to DA deficiency and to the main symptoms of PD. Current medications do not impede neurodegeneration, but relieve symptoms by replenishing DA; however, their chronic use causes serious side effects. We targeted a protein required for the development and function of DA neurons by designing a chemical compound that, by activating this protein, increases DA and improves symptoms without current treatment side effects while simultaneously preventing neuron loss in PD mice. Our findings point to a monotherapy that can both impede PD progression and concurrently improve symptoms of PD. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.

Versatile quarto stimuli nanostructure based on Trojan Horse approach for cancer therapy: Synthesis, characterization, in vitro and in vivo studies

Nanostructured delivery and diagnostic systems that induces specific targeting properties by exploiting the local physicochemical tumour characteristics will be evaluated is the present work. It is well known that cancer cells have specific physicochemical characteristics, which can be taken into consideration for the design of a broad spectrum of drug delivery systems (DDS). Some of those characteristics including the different temperature environment their susceptibility when temperature ranges between 40 and 43°C where cell apoptosis is induced, the intra- and extra-cellular pH which varies from 6.0 to 6.8, for cancer cells, and 6.5 to 7.4 for normal cells respectively, (lysosomes acidic pH ranges 4-5). Additional significant factors are the overexpressed receptors on the tumour surface. Loading and release studies were carried out by using the anthracycline drug Doxorubicin and their cytotoxicity was evaluated by using the MTT assay in healthy and diseased cell lines. The highlight of this work is the in vitro and in vivo studies which were performed in order to evaluate different nanostructures as for their biodistribution, pharmacokinetic and toxicity per se.

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 in vitro colony forming assay together with in vivo 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 gemcitabines 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.

PO-444 Evaluation of novel nucleoside analogues for lung cancer treatment: an approach based on metronomic chemotherapy

Introduction Lung cancer presents a global pandemic responsible for an estimated 20% of cancer cases with Non-Small Cell Lung Cancer (NSCLC) being the most prevalent form. Chemotherapy is the preferred treatment modality for NSCLC, focusing on the disruption of the abnormal proliferation of cancer cells. Nevertheless, this approach causes patients to experience unpleasant side effects and more importantly, results in cancer recurrence. Gemcitabine (Gemc) is a nucleoside analogue used against NSCLC, which inhibits cell cycle and prevents tumour growth. Although gemc is approved for the treatment of various cancer types, its efficacy is still limited due to its lack of efficiency, which is caused by rapid metabolic inactivation. Metronomic chemotherapy (MTR), relying on the daily oral administration of a drug, at low doses, is a multi-targeted therapy, as it inhibits tumour angiogenesis, modulates immunity pathways and effects tumour initiating cells reducing the toxicity of traditional maximal tolerated dose chemotherapy (MTD). Our goal is to provide a new angle in the MTR approach, by administering an oral prodrug of gemc, Oral Gem, to improve gemc’s therapeutic properties, but also cover patients‘ quality of life. Material and methods The A549 lung cancer cell line was used to establish an in vitro model that simulated the MTD versus the MTR conditions. Cells were cultured either in presence of a high concentration of gemc or in medium in which lower concentrations were added daily in order to study alterations in the expression of various angiogenic factors. Additionally, an in vivo xenografted animal model was set up to study the effects of MTR chemotherapy on tumour’s expansion, toxicity of the drug and angiogenesis. Results and discussions Daily addition of gemc in A549 cells led to a decreased expression of VEGFA, a well-established angiogenic factor, compared to the high dose incubation. In NOD/SCID xenografted mice, the MTR administration of Oral Gem led to a decreased expression of VEGFA and CD31, a marker found on endothelial cells, suggesting a suppressed angiogenic profile. Finally, MTR administration of Oral Gem led to an increase in the expression levels of Thrombospondin-1, an anti-angiogenic factor, compared to MTD chemotherapy. Conclusion MTR administration of Oral Gem limits the formed vessels around the tumour combining restriction of angiogenesis and vessel normalisation. In contrast, MTD chemotherapy seems to enhance the angiogenic potential around the tumour site, serving tumour’s establishment and expansion.

Targeting of the breast cancer microenvironment with a potent and linkable oxindole based antiangiogenic small molecule

The clinical efficacy of antiangiogenic small molecules (e.g., sunitinib) in breast carcinoma has largely failed with substantial off-target toxicity. We rationally designed and evaluated preclinically a novel sunitinib analogue, SAP, with favourable pharmacological properties and the ability to be readily conjugated to a targeting peptide or antibody for active tumour targeting. SAP was evaluated in silico and in vitro in order to verify target engagement (e.g., VEGFR2). Pharmacokinetic and biodistribution parameters were determined in mice using LC-MS/MS. SAP efficacy was tested in two breast cancer xenograft and two syngeneic animal models and pharmacodynamic evaluation was accomplished using phosphokinase assays and immunohistochemistry. Cardiac and blood toxicity of SAP were also monitored. SAP retained the antiangiogenic and cytotoxic properties of the parental molecule with an increased blood exposure and tumor accumulation compared to sunitinib. SAP proved efficacious in all animal models. Tumors from SAP treated animals had significantly decreased Ki-67 and CD31 markers and reduced levels of phosphorylated AKT, ERK and S6 compared to vehicle treated animals. In mice dosed with SAP there was negligible hematotoxicity, while cardiac function measurements showed a reduction in the percentage left ventricular fractional shortening compared to vehicle treated animals. In conclusion, SAP is a novel rationally designed conjugatable small antiangiogenic molecule, efficacious in preclinical models of breast cancer.

Abstract 2618: Gemcitabine based peptide conjugates: Overcoming the pitfalls of conventional therapies by targeted approaches

Although gemcitabine has been proved to be a valuable ally against several types of cancer, it is characterized by some weaknesses that limit its anticancer potential. These limitations are mainly associated with gemcitabine rapid inactivation as well as the different types of resistance that are related with its use. We have previously shown that conjugation of gemcitabine to a ligand peptide (molecule GSG) enhanced its metabolic stability systemically and locally (within the cancer cell), and lead to improved efficacy in a castration resistant prostate cancer (CRPC) animal model. In order to refine our previous findings, a bioanalytical methodology based on mass spectrometry that allows the simultaneous monitoring of gemcitabine as well as its active (dFdCDP, dFdCTP) and inactive (dFdU) metabolites was developed in the context of this study. The balance of gemcitabine and its active/inactive metabolites was evaluated in in vitro and in vivo systems after treatment with gemcitabine or selected gemcitabine based peptide conjugates (GSG). Furthermore, since GSG9s structure contains a potent agonist peptide of the GnRH-R we investigated whether it could have a central effect through the pituitary by performing testosterone measurements in animals dosed with GSG. Interestingly, GSG appears to offer metabolic advantages in comparison to gemcitabine, an important attribute if we had to overcome gemcitabine acquired resistance. In addition, GSG is a potent GnRH-R agonist based on our in vivo biomarker model, suggesting that testosterone ablation might also represent a key attribute of the mechanism of action for our conjugate of interest. These findings along with the targeted delivery nature of this approach could ameliorate the therapeutic status of gemcitabine and expand the use of gemcitabine based conjugates in other types of cancers. Citation Format: Constantin Tamvakopoulos, Theodoros Karampelas, Orestis Argyros. Gemcitabine based peptide conjugates: Overcoming the pitfalls of conventional therapies by targeted approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2015-2618