In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

Abstract The inability to target distant metastases limits the treatment of late-stage cancers. At least in part, this is due to the lack of optimal targeted therapies and treatment resistance to the remaining standard of care consisting of chemo-/radio- therapy. Here, we show that cancer cells that are irreparably damaged can withstand cell death through a process we define as “scarring”. In scarred cells, p53 is phosphorylated at Ser46 and Ser392 and localizes into progressive multifocal leukemia (PML) nuclear bodies. We have identified scarring in over a quarter of metastatic breast, colon, stomach and ovarian cancer. Thus, pivoting scarred p53 towards cell death in scarred cancer could establish a novel broad-range therapeutic opportunity against metastatic cancer. Targeting p53 with small molecules has proven challenging. We developed cell-penetrating peptides that selectively bind scarred p53. One of our lead compounds, CL04177 dissociates p53 from its PML scaffold leading to its a nuclear exclusion and initiation of the transcription-independent mitochondrial apoptosis. To examine the role of scarring as a therapeutic target, we focused on triple-negative breast cancer (TNBC). TNBC cells are marked by the expression of p53 and PML compared to other breast cancer subtypes. In cytotoxicity experiments, CL04177 specifically targeted TNBC over other breast cancer subtypes. Further investigation into how this compound can target metastatic cells revealed that scarred features are upregulated in metastasis-initiating cells of organoids when induced to invade in contact with collagen and that CL04177 can induce apoptosis specifically in these invading strands. In vivo, CL04177 showed favorable pharmacokinetics and -dynamics and a clear safety profile. Importantly, when dosed at 10-fold below its Maximally Tolerated Dose, CL04177 strongly reduced primary tumor mass and metastatic dissemination to the liver of TNBC cell line-derived orthotopic xenografts. This data indicates a new concept of scarred p53 in cancer progression and metastasis. Moreover, peptides that release scarring markers from nuclear structures can eliminate aggressive cancers in vitro and, most importantly, in vivo, e.g., in p53-mutated, metastatic TNBC. This ability introduces a conceptually new therapeutic option, especially for types of cancer for which there is a high unmet medical need. Citation Format: Diana Putavet, Marjolein P. Baar, Johannes H. Lehmann, Antoine Khalil, Tao Shi, Thijs Koorman, Tobias B. Dansen, Patrick WB Derksen, Michael Teifel, Tobias Madl, Nicholas Sarlis. Peptide-based pivoting of p53 to target metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2095.

In recent years, a novel treatment method for cancer emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the L-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata . Previously isolated from the ink, the L-amino acids oxidase was described to oxidate the essential amino acid L-lysine and L-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of amino acid oxidase Aplysia Punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG which lead to deprivation of arginine was well tolerated.

Background/Aims: Diagnosis of growth hormone deficiency (GHD) in children requires the use of provocative growth hormone (GH) stimulation tests, which can have limited reliability and are potentially contraindicated in some patients. This is the first paediatric study to test the safety, tolerability, and pharmacokinetics (PK)/pharmacodynamics (PD) of macimorelin, an oral GH secretagogue, approved for diagnosis of adult GHD. Methods: In this open-label, group comparison, single-dose escalation trial (EudraCT 2018-001988-23), sequential cohorts of patients (C1–C3) received ascending single doses of macimorelin: 0.25 (C1), 0.5 (C2), and 1.0 (C3) mg/kg. Primary endpoints were safety and tolerability, and secondary endpoints were PK/PD. Results: Twenty-four patients aged between 2 and <18 with suspected GHD participated in the study. No macimorelin-related adverse events were reported, and macimorelin was well tolerated. Plasma macimorelin concentrations increased with dose: mean areas under the curve were 6.69 (C1), 18.02 (C2), and 30.92 (C3) h × ng/mL; mean maximum concentrations were 3.46 (C1), 8.13 (C2), and 12.87 (C3) ng/mL. GH concentration increased following macimorelin administration: mean times of maximum measured concentration were 52.5 (C1), 37.5 (C2), and 37.5 (C3) min. Conclusion: All 3 doses of macimorelin had excellent safety and tolerability with PK/PD profiles in expected ranges. These results support the use of 1.0 mg/mL macimorelin in a Phase 3 test validation trial in children.

Growth hormone deficiency (GHD) in children is a rare, aetiologically diverse condition that results in growth failure and short stature. Inadequate response to two different growth hormone stimulation tests (GHST) is required for the diagnosis of GHD. Macimorelin acetate, a potent, orally administered growth hormone (GH) secretagogue, is approved by the FDA and EMA for the diagnosis of adult GHD. Study AEZS-130-P01 is the first of two studies to investigate macimorelin acetate as a diagnostic test in children with suspected GHD. This was an open-label, group comparison, dose escalation trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of single-dose 0.25, 0.5 and 1 mg/kg oral macimorelin acetate in paediatric subjects with suspected GHD. The macimorelin GHST was administered between two standard GHST, conducted as per local clinical practice, with a recovery period of 7-28 days between tests. Blood samples were collected pre-dose (±15 min) and 15, 30, 45, 60, 90, 120 and 360 minutes after macimorelin acetate intake. Overall, 24 paediatric subjects (8 per cohort [C1, C2, C3]) were included in the pharmacokinetic/pharmacodynamic (PK/PD) analysis. Five males and 3 females were observed in C1 and C2, 7 males and 1 female in C3. In all three cohorts, at least 3 subjects represented Tanner stages I or II. All 24 subjects (100%) were white, with a median age of 9.8, 9.0 and 10.5 years (range 4-15 years) and a median body-mass index of 16.1 kg/m2 (12.4-21.4 kg/m2) at screening. Overall, 88 adverse events were reported, many related to the standard GHST; none were considered related to the macimorelin test. Maximum plasma concentrations for macimorelin were mainly observed between 30-45 min. The mean Cmax values were 3.46, 8.13 and 12.87 ng/ml for C1, C2, and C3, respectively. The AUCs increased with dose; the mean AUC0-6 values were 6.69, 18.02 and 30.92 h*ng/mL. The mean elimination half-lives were 1.22, 1.61 and 1.71 h, respectively. PK and PD profiles for all three cohorts were comparable, with peak GH levels mainly observed within 30-60 min following macimorelin intake. Macimorelin acetate was safe and well tolerated in all dosing cohorts. A dose-dependent increase in macimorelin Cmax and AUC in children and adolescents correlated well with data from adult subjects. A robust dose-proportional GH response was also achieved. PD results showed that GH response was comparable in all dose groups, with a slight shift to earlier tmax at higher macimorelin doses.

Zoptarelin doxorubicin is a fusion molecule of the chemotherapeutic doxorubicin and a luteinizing hormone-releasing hormone receptor (LHRHR) agonist, designed for drug targeting to LHRHR positive tumors. The aim of this study was to establish a physiologically based pharmacokinetic (PBPK) parent-metabolite model of zoptarelin doxorubicin and to apply it for drug–drug interaction (DDI) potential analysis.

Urea and aniline derivatives were active at low micromomolar IC50 values against a panel of seven cancer-related protein kinases.

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hormone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored. We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring. Herein we report the design and the structure–activity relationships study of these new ghrelin receptor ligands.

Perifosine treatment exhibits a complex molecular response including the inhibition of Akt or the induction of apoptosis via clustering of death receptors in lipid rafts. However, the molecular response can vary between different tumor entities and the contribution of each target pathway to the activity of Perifosine might be distinct depending on the tumor entity or the agent combined with Perifosine. In this review we discuss the current view on the mechanism of action of perifosine in cancer and the contribution of the molecular targets of Perifosine to its activity.

Abstract The MAPK pathway represents a prime target for therapeutic intervention in cancer. Recent approvals of B-Raf and Mek inhibitors lead to exciting antitumor activities and survival benefits. However the observation of resistance to such Raf and Mek inhibitors has been documented in both clinical and preclinical trials. These phenomena stimulated further research into the regulation of the MAPK pathway and led to the suggestion to target the downstream kinase Erk, as well as to combine its targeting with Raf and Mek inhibitors. Here we report that Erk inhibitors result in efficient growth inhibition of tumor cells that are resistant to Raf and Mek inhibitors. AEZS-134 - a highly potent and selective ATP competitive Erk inhibitor - overcomes the Raf inhibitor induced paradoxical cell activation and the aquired resistance to Mek inhibitors in tumor cells. We demonstrate potent antiproliferative activity of AEZS-134 in B-Raf wildtype, B-RafV600E mutant, Ras wildtype and K-Ras mutant tumor cell lines in comparison to common Raf inhibitors. Furthermore we show that AEZS-134 is efficacious in Mek inhibitor resistant Hct116 and MDA MB231 cells which have been well characterized in terms of Mek F129L allosteric binding pocket mutation, varying degrees of K-Ras amplification, cellular proliferation assays and MAPK pathway phosphorylation studies. Our data provide a rational for new therapeutic opportunities by using Erk inhibitors in oncology. In order to maximize therapeutic benefit for patients and considering that Erk is directly downstream of B-Raf and Mek, targeting Erk may be more effective than targeting Raf or Mek in a variety of MAPK resistance settings. AEZS-134 and related compounds provide potent and selective tools to help address this question. Development of the Erk inhibitors by Æterna Zentaris is integral part of our inhouse kinase research program comprising the investigation of different compounds for single Erk inhibition, single PI3K inhibition and dual kinase inhibitions of Erk and PI3K. All compounds are exclusively synthesized by our medicinal chemistry department. Citation Format: Irene Seipelt, Peter Schmidt, Helene Maerzhaeuser, Matthias Gerlach, Kai Jung, Tilmann Schuster, Michael Teifel. Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2014-3711

A novel series of (7-aryl-1,5-naphthyridin-2-yl)ureas was discovered as dual ERK2 and Aurora B kinases inhibitors. Several analogues were active at micromolar and submicromolar range against ERK2 and Aurora B, associated with very promising antiproliferative activity toward various cancer cell lines. Synthesis, structure activity relationship and docking study are reported. In vitro ADME properties and safety data are also discussed.

As part of our research projects to identify new chemical entities of biological interest, we developed a synthetic approach and the biological evaluation of (7-aryl-1,5-naphthyridin-4-yl)ureas as a novel class of Aurora kinase inhibitors for the treatment of malignant diseases based on pathological cell proliferation. 1,5-Naphthyridine derivatives showed excellent inhibitory activities toward Aurora kinases A and B, and the most active compound, 1-cyclopropyl-3-[7-(1-methyl-1H-pyrazol-4-yl)-1,5-naphthyridin-4-yl]urea (49), displayed IC50 values of 13 and 107nM against Aurora kinases A and B, respectively. In addition, the selectivity toward a panel of seven cancer-related protein kinases was highlighted. In vitro ADME properties were also determined in order to rationalize the difficulties in correlating antiproliferative activity with Aurora kinase inhibition. Finally, the good safety profile of these compounds imparts promising potential for their further development as anticancer agents.

Abstract Background For drug-targeting aimed at the treatment of LHRH receptor overexpressing cancers the LHRH receptor agonistic peptide D-Lys6-LHRH has been conjugated to the novel highly cytotoxic natural compound Disorazol Z. As shown previously by early proof of concept in an ovary cancer xenograft model, differentially linked conjugates possess a high potential regarding the treatment of LHRH-R positive tumors [1]. Here we present further characterization of these conjugates with respect to PK/PD parameter and provide evidence that LHRH receptor targeting significantly contributes to their mechanism of action. Materials and Methods LHRH-R competitive binding, calcium release and cytotoxic activity were measured by Tag-Lite technology (Cisbio), and Fluo-4 (Invitrogen) or Resazurin-based detection, respectively. PK parameter were assessed by standard procedures followed by LC-MS/MS analysis. For the xenograft studies, tumor fragments were transplanted subcutaneously in female nude mice and treatment was started at a tumor size of approx. 100 mm3. Results Disorazol Z - D-Lys6-LHRH conjugates showed varying cytotoxic activity from single digit nanomolar to higher submicromolar EC50 values but comparable LHRH receptor binding and activation in the low nanomolar EC50 range. Comparison in ovarian and triple negative breast cancer xenograft models revealed potent inhibition of tumor growth for the conjugates, whereas equimolar dosing of Disorazol Z failed to reach statistical significance. PK analysis showed substantial plasma levels for the conjugates with only minor release of Disorazol Z, pointing to stabilization by conjugation and demonstrating reasonable half-life of the intact conjugates as prerequisite for tumor targeting. In the same tumor models, competition by previous administration of D-Lys6-LHRH provides evidence for LHRH receptor targeting as mechanism of action. Increased sensitivity of LHRH receptor overexpressing cells towards conjugate cytotoxicity, i.e. leading to about 30 fold decreased EC50 values for the conjugate AEZS-125, further supports the LHRH-R dependency of conjugate efficacy. Conclusions The presented LHRH receptor-dependent efficacies of Disorazol Z - D-Lys6-LHRH conjugates in vitro and in mouse xenograft models support the principle of tumor targeting by the LHRH receptor as already employed by the drug candidate AEZS-108, which is currently in phase II clinical studies. Preclinical development of Disorazol Z conjugates will be started in the first half of 2013. Citation Format: Babette Aicher, Tilmann Schuster, Lars Blumenstein, Antje Schubert, Carsten Gründker, Joerg B. Engel, Olaf Ortmann, Rolf Mueller, Eckhard Guenther, Matthias Gerlach, Michael Teifel. LHRH receptor targeting as mechanism of anti-tumor activity for cytotoxic conjugates of Disorazol Z with the LHRH receptor agonistic peptide D-Lys6-LHRH. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2013-5476

Überexpression von ERK wurde in 34% der trripelnegativen Mammakarzinomen beschrieben und ist mit Anthtrazyklinresistenz assoziiert. (1). AEZS-131 ist ein oral wirksamer, hochselektiver Inhbitor von ERK ½. Der in in vivo Modellen des Kolonkarzinoms Antitumoraktivität gezeigt hat. Die vorliegende Studie untersucht den Wirkmechanismus und die Effektivität in in vitro Modellen des tripelnegativen Mammakarzinoms.