Efficacy In Tumor Xenograft Models Research Articles

Primary Preclinical and Clinical Evaluation of 68Ga-DOTA-TMVP1 as a Novel VEGFR-3 PET Imaging Radiotracer in Gynecological Cancer.

Tumor periphery and lymph nodes of tumor-induced lymphangiogenesis often abundantly express VEGFR-3. In our previous study, we identified a 5-amino acid peptide named TMVP1, which binds specifically to VEGFR-3. The objective of this study was to develop a novel 68Ga-labeled TMVP1 for VEGFR-3 PET imaging and to investigate its safety, biodistribution, and tumor-localizing efficacy in xenograft tumor models and a small cohort of patients with recurrent ovarian and cervical cancer. The DOTA-conjugated TMVP1 peptide was labeled with radionuclide 68Ga. SPR and saturation binding assays were used for the receptor-binding studies. Gynecologic xenograft tumors were employed for small-animal PET imaging and biodistribution of 68Ga-DOTA-TMVP1 in vivo. In the clinical study, 5 healthy volunteers and 8 patients with gynecologic cancer underwent whole-body PET/CT after being injected with 68Ga-DOTA-TMVP1. DOTA-TMVP1 was successfully labeled with 68Ga. LECs showed higher binding capacity with 68Ga-DOTA-TMVP1 than LEC(shVEGFR-3) and human umbilical vein endothelial cells. In mice with subcutaneous C33-A and SKOV-3 xenografts, the tracer was rapidly eliminated through the kidney to the bladder, and the small-animal PET/CT helped to clearly visualize the tumors. In patients with recurrent ovarian cancer and cervical cancer, tracer accumulation well above the background level was demonstrated in most identified sites of disease; especially with recurrent endodermal sinus tumors, the diagnostic value of 68Ga-DOTA-TMVP1 was comparable with that of 18F-FDG PET/CT. 68Ga-DOTA-TMVP1 is a potential PET tracer for imaging VEGFR-3 with favorable pharmacokinetics.

Multi-responsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation

Herein, we developed a multifunctional nanoplatform based on the nanoassembly of gold nanoparticles (GNP) conjugated with lonidamine (LND) and aptamer AS1411 (AS-LAGN) as an effective cancer treatment. Conjugating AS1411 aptamer on the surface of the nanoparticle significantly improved particle accumulation in cancer cells via specific affinity toward the nucleolin receptors. In vitro study clearly revealed that laser irradiation-based hyperthermia effect enhanced the chemotherapeutic effects of LND. Combinational treatment modalities revealed significant apoptosis with higher cell killing effect due to increased ROS production and inhibition of cell migration. GNP's ability to convert the excited state photon energy into thermal heat enabled synergistic photothermal/chemotherapy with improved therapeutic efficacy in animal models. Moreover, immunohistochemistry staining assays confirmed the ability of AS-LAGN to induce cellular apoptosis/necrosis and ablation in tumor tissues, without causing evident damages to the surrounding healthy tissues. Altogether, this AS-LAGN nanoplatform could be a promising strategy for mitochondria-based cancer treatment. Statement of significanceWe have designed a facile biodegradable multifunctional nanocarrier system to target the mitochondria, the major “power house” of the cancer cells. We have constructed a multifunctional nanoassembly of protein coronated gold nanoparticles (GNP) conjugated with lonidamine (LND) and aptamer AS1411 (AS-LAGN) as an effective combination of phototherapy with chemotherapy for cancer treatment. The LND was conjugated with albumin which was in turn conjugated to GNP via redox-liable disulfide linkage to generate oxidative stress and ROS to kill cancer cells. GNP's ability to convert the excited state photon energy into thermal heat enabled synergistic photothermal/chemotherapy with improved therapeutic efficacy in animal models. Consistently, AS-LAGN showed enhanced antitumor efficacy in xenograft tumor model with remarkable tumor regression property.

Liposomal paclitaxel induces fewer hematopoietic and cardiovascular complications than bioequivalent doses of Taxol.

Paclitaxel (PTX) exhibits potent antineoplastic activity against various human malignancies; however, clinical application must overcome the inherent hydrophobicity of this molecule. The commercialized Taxol formulation utilizes Cremophor EL (CrEL)/ethanol as a solvent to stabilize and dispense PTX in an aqueous solution. However, adverse CrEL-induced hypersensitivity reactions have been reported in ~30% of recipients, and 40% of patients receiving premedication may also experience this adverse effect. Therefore, the development of a CrEL-free delivery system is crucial, in order to fully exploit the therapeutic efficacy of PTX. In the present study, a novel liposomal PTX (lipo-PTX) formulation was optimized with regards to encapsulation rate and long-term stability, arriving at a molar constituent ratio of soybean phosp hatidylcholine:cholesterol:N-(carbonyl-methoxy-poly-ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt:PTX at 95:2:1:2. Comparable doses of lipo-PTX and Taxol were bioequivalent in terms of therapeutic efficacy in xenograft tumor models. However, the systemic side effects, including hematopoietic toxicity, acute hypersensitivity reactions and cardiac irregularities, were significantly reduced in lipo-PTX-treated mice compared with those infused with reference formulations of PTX. In conclusion, the present study reported that lipo-PTX exhibited a higher therapeutic index than clinical PTX formulations.

Facile construction of bioreducible crosslinked polypeptide micelles for enhanced cancer combination therapy.

We have synthesized a biodegradable polypeptide block copolymer to construct a facile pH and redox-responsive polymeric micelle asan advanced therapeutic system for cancer therapy. We have designed a corona-crosslinked triblock copolymer (poly (ethylene glycol)-b-poly(aspartic acid)-b-poly(tyrosine) (PEG-b-pAsp-b-pTyr)) micelles co-loaded with docetaxel and lonidamine (cl-M/DL). The corona of triblock polymer was crosslinked to maintain its structural integrity in the physiological environment. The mitochondrial targeting LND is expected to generate ROS, oxidative stress and thereby synergize the chemotherapeutic efficacy of DTX in killing cancer cells. Consistently, cl-M/DL exhibited excellent antitumor efficacy in xenograft tumor model with remarkable tumor regression. Overall, we demonstrated the construction of bioreducible nanosystem for the effective synergistic delivery of DTX/LND in tumor tissues towards cancer treatment.

Abstract 62: CD19 - a potential target for Amanitin-based ADCs

Abstract Background ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of new ATACs targeting CD19 (also known as B4, CVID3) are presented. CD19, a class I transmembrane glycoprotein with no significant homology to any known protein, is expressed in B cells and B-cell malignancies like in B-cell acute lymphocytic leukemia (B-ALL) and B cell chronic lymphocytic leukemia (B-CLL). Therefore it is an ideal target for Amanitin based ADCs. Material and methods Cell lines: Raji (Burkitt Lymphoma), Nalm-6 (B-ALL) and HL-60 (CD19-negative cell line). Antibody: anti-CD19 Thiomab (licensed from the German Cancer Research Center; Heidelberg, Germany). Synthesis of CD19-Thiomab-ATACs: Maleimide amanitin compounds were conjugated site specifically to engineered cysteine residues of the anti-CD19 Thiomab. Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST-1 assay (Roche). Animal models: Mouse xenograft tumor models were performed. Tolerability was assessed in mice and non-human primates (NHP). Results CD19-Thiomab-ATACs showed in vitro cytotoxicity on CD19+ cell lines in picomolar range, whereas no cytotoxic activity on CD19- cells was observed. In mouse xenograft models, CD19 showed dose-dependent tumor regression and complete remission after single dose i.v. of 2mg/kg and 4mg/kg. In a disseminating Raji xenograft model, median survival was increased from 6 days (vehicle control) to 105 days (CD19-Thiomab-30.2115 treated group at 6mg/kg, single dose). On day 154, 4 animals were still alive and in good overall condition. Safety profiling in Cynomolgus monkey revealed a good tolerability after sequentially applied doses of 0.3, 1.0, 3.0 and 5.0 mg/kg. Hematology and clinical chemistry data show minor alterations: transient increase in liver enzymes (ALT and AST) in combination with a transient increase in LDH. The half-life of the ADC in serum is 7-11 days; the free toxin is detectable at serum levels close to the lower limit of quantification only (LLOQ = 1.2 nM). Conclusions In the current study, in vitro and in vivo data of Amanitin-ADCs targeting CD19 are presented. CD19 is expressed on cells of the B cell lineage, ranging from the pre-B cells until the terminal differentiation to plasma cells. It is expressed in most acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL) and B cell lymphomas (Kemeng et al. (2012); Exp Hematol Oncol 1: 36). The preclinical data show high cytotoxicity in picomolar range, very good efficacy in tumor xenograft models and tolerability in an exploratory tolerability study in NHP. The positive findings of these initial experiments encourage Heidelberg Pharma to further proceed with anti-CD19-Amanitin-based ADCs towards clinics. Citation Format: Torsten Hechler, Aniko Palfi, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. CD19 - a potential target for Amanitin-based ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2017-62

Prodrug AST-003 Improves the Therapeutic Index of the Multi-Targeted Tyrosine Kinase Inhibitor Sunitinib

Patients have responded well to the multi-targeted tyrosine kinase inhibitor (TKI) Sunitinib in the clinic. But the severe toxic side effects associated with Sunitinib limit its therapeutic index. To improve the therapeutic index of Sunitinib, a prodrug strategy was employed to modify Sunitinib. The inactive prodrug AST-003 can be converted to Sunitinib in vitro and in vivo. Compared with Sunitinib, AST-003 has unique biochemical, cellular and pharmacokinetic properties with improved tolerability in mice and yield higher efficacy in tumor xenograft models. This prodrug strategy may constitute a novel paradigm to improve the therapeutic index of Sunitinib and other TKI or anti-angiogenesis drugs in general.

Abstract CT139: Phase I study of GDC-0425, a checkpoint kinase 1 inhibitor, in combination with gemcitabine in patients with refractory solid tumors

Abstract Background: Checkpoint kinase 1 (Chk1) acts at S and G2/M checkpoints to allow time for high-fidelity DNA replication and repair before cell cycle progression. Chk1 inhibition converts a transient genotoxic insult from chemotherapy into a cytotoxic event by overriding cell cycle arrest, allowing cells mitosis with DNA damage. GDC-0425 is an oral, selective Chk1 inhibitor. GDC-0425 enhances gemcitabine (gem) efficacy in tumor xenograft models. Greater chemopotentiation is observed in cancer cell lines lacking p53 activity. Methods: A phase I dose-escalation trial (3+3+3 design) included patients (pts) with refractory solid tumors and ECOG performance status (PS) ≤ 1. Pts received a single dose of GDC-0425 on day -7 for PK evaluation followed by 21-day cycles of gem on Days 1 and 8 and GDC-0425 on Days 2 and 9 at gem (mg/m2) + GDC-0425 (mg) dose levels of 750 + 60, 1000 + 60, and 1000 + 80. p53 was evaluated in archival tumor tissue by gene sequencing, immunohistochemistry, and gene expression signature. Safety, pharmacokinetics (PK), pharmacodynamics, and tumor response were investigated. Results: Of 40 pts treated, 55% were female, median age was 56 years (range 33-82), and 68% had ECOG PS 0. Most common tumor types were breast (n = 10), non-small cell lung (n = 5), and cancer of unknown primary (CUP, n = 4). Maximum concentrations of GDC-0425 were achieved within 4 hours of dosing and its half-life was approximately 16 hours. Target exposures associated with checkpoint abrogation and anti-tumor activity in preclinical models were exceeded at 60 mg. No PK interaction was observed with GDC-0425 and gem. Dose escalation was halted at GDC-0425 80 mg with gem 1000 mg/m2 as 3 of 6 pts experienced Grade 4 thrombocytopenia as a dose-limiting toxicity (DLT); 1 pt also had Grade 3 neutropenia that delayed Cycle 2 (DLT). Blood counts recovered with treatment interruption and supportive care. The maximum tolerated dose of GDC-0425 was 60 mg with gem 1000 mg/m2. The most frequent adverse events (AEs) (all grades) related to GDC-0425 and/or gem were nausea (48%); anemia, neutropenia, vomiting (45% each); fatigue (43%); pyrexia (40%); and thrombocytopenia (35%). Serious AEs related to GDC-0425 and/or gem occurred in 8 pts: neutropenia and thrombocytopenia (n = 2 each); leukopenia, ALT/AST/GGT increased, pyrexia, rash, dyspnea, gastric ulcer, and gastroenteritis (n = 1 each). Median number of administered cycles was 3.5 (range 1-14). There were 3 partial responses in pts with triple-negative breast cancer (TNBC, TP53 mutated), melanoma, and CUP (n = 1 each). Conclusions: It is safe and feasible to administer GDC-0425 with a standard dose of gem. At the doses assessed, bone marrow suppression is common but manageable and exposures exceed those predicted by preclinical models to inhibit Chk1. Clinical activity was observed, including 1 patient with TP53 mutated TNBC. Citation Format: Jeffrey R. Infante, Antoine Hollebecque, Sophie Postel-Vinay, Todd Bauer, Beth Blackwood, Marie Evangelista, Sami Mahrus, Frank Peale, Xuyang Lu, Srikumar Sahasranaman, Rui Zhu, Yuan Chen, Xiao Ding, Elaine Murray, Jennifer Schutzman, Jennifer Lauchle, Jean-Charles Soria, Patricia LoRusso. Phase I study of GDC-0425, a checkpoint kinase 1 inhibitor, in combination with gemcitabine in patients with refractory solid tumors. [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 CT139. doi:10.1158/1538-7445.AM2015-CT139

Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy

The tumor microenvironment plays an important role in tumor growth and progression. Here we demonstrate that vaccinia virus-mediated, constitutively expressed intratumoral antibodies against vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) significantly improved tumor regression and oncolytic virotherapy through suppression of angiogenesis, cell proliferation, and stromagenesis in virus-colonized tumors. In contrast to the tumor growth inhibition by the three tumor growth-inhibiting antibodies individually, when two of the three antibodies were expressed simultaneously by single vaccinia virus strains tumor regression was further enhanced. These findings strongly indicate that interference with the two tumor growth-stimulating mechanisms did in fact result in enhanced therapeutic efficacy in tumor xenograft models and may lead to an effective therapy in patients with cancer.

Abstract 4587: Combined administration of Voluven® 10% and anti-cancer drugs increases anti-tumor efficacy

Abstract Background: Standard-of-care anti-cancer drugs exhibit anti-tumor activity but typically display dose-limiting toxicities in patients. The aim of the current studies was to investigate, whether a combined approach consisting of a pre-treatment with the hydroxyethyl starch solution Voluven® 10% and a subsequent treatment with suboptimal doses of selected anti-cancer drugs improves the anti-cancer efficacy in human tumor xenograft models in mice. Materials and methods: Female NMRI nu/nu mice bearing subcutaneous xenografts of the human lung squamous cell carcinoma LXFE 397 were treated i.v. with 20 ml/kg Voluven® 10% followed by a p.o. treatment with 100 mg/kg capecitabine 1 hour later on days 0, 4, 7, 11, and 14. In a separate study, female NMRI nu/nu mice bearing subcutaneous xenografts of the human head-and-neck carcinoma HNXF 1842 were treated i.v. with 20 ml/kg Voluven® 10% followed by an i.v. treatment with 10 mg/kg paclitaxel 1 hour later on days 0, 7 and 14. In both studies, dosing started on the day of randomization (day 0) when animals carried tumors of appropriate size (50-250 mm3). Saline (20 ml/kg) served as negative control and the anti-cancer drug alone as reference. From the day of first dosing, the tumor growth and body weight were monitored over a time period of 14 days in the lung carcinoma study and 18 days in the head-and-neck carcinoma study. Results: In mice bearing human lung squamous cell carcinoma xenografts, the median relative tumor volume increased approximately 19-fold in the saline group during the 14 days observation time, whereas the reference drug capecitabine moderately inhibited the tumor growth. Combining capecitabine treatment with a Voluven® 10% pre-treatment caused a much stronger inhibitory effect on tumor growth as evidenced by an 8.5-fold median relative tumor volume increase in contrast to a 15.2-fold increase in the reference group (capecitabine). In mice bearing human head-and-neck carcinoma xenografts, the median relative tumor volume of the saline group increased 12.1-fold during the observation period of 18 days. The treatment with paclitaxel caused a tumor growth reduction resulting in a 7.1-fold increase of the median relative tumor volume. This effect was further enhanced by the pre-treatment with Voluven® 10% which resulted in a 4-fold median relative tumor volume increase over 18 days. The combination of Voluven® 10% with capecitabine or paclitaxel did not increase toxicity as demonstrated by the body weight development. Conclusion: We showed for the first time that the pre-treatment with Voluven® 10% 1 hour prior to the administration of the anti-cancer drugs capecitabine or paclitaxel is useful to improve their efficacy in tumor xenograft models in mice. The enhancement of anti-cancer drug effectiveness might allow reducing the dosage of cytotoxic anti-cancer drugs and therefore minimizing unwanted side effects. Citation Format: Silke Baasner, Corinna Lupp, Stefanie Honndorf, Johannes Hermle, Martin Westphal. Combined administration of Voluven® 10% and anti-cancer drugs increases anti-tumor efficacy. [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 4587. doi:10.1158/1538-7445.AM2014-4587

Abstract 1618: New peptide-linked anilino-maytansinoid antibody-drug conjugates (ADCs) for the treatment of cancer

Abstract A number of ADCs consisting of a maytansinoid cytotoxic agent linked to an antibody via a cleavable disulfide linker or a non-cleavable thioether linker are in the clinic. In preclinical models, antibody-maytansinoid conjugates (AMCs) utilizing a cleavable disulfide linker have been shown to be able to kill both the targeted antigen-positive (Ag+) cancer cells and also adjacent Ag- “bystander” cancer cells because of intra-cellular generation of maytansinoid catabolites that can travel out of the Ag+ cells, into adjacent Ag- cells, and kill these cells when they divide. Such bystander killing of Ag- cancer cells can be advantageous for some tumor types, such as those with heterogeneous target expression on the cancer cells. We thus developed a new dipeptide linker designed to promote bystander killing more efficiently than current disulfide linkers. It is used to attach an aniline-bearing maytansinoid to the targeting antibody. To determine the effect of amino acid stereochemistry on the therapeutic index, dipeptide-anilino AMCs (dpa-AMCs) were prepared with the four possible L- and D-alanine configurations using an anti-EGFR antibody. These were compared to a dpa-AMC containing the L-Val-L-Cit dipeptide, which is a commonly utilized dipeptide for ADC linkage. Dipeptide stereochemistry had a significant effect on the in vitro potency and bystander killing activity of the AMCs. Based on the results, the three most potent designs - those bearing the L-Ala-L-Ala, D-Ala-L-Ala, and L-Val-L-Cit peptides - were further investigated. In tolerability experiments in mice, with AMC dosing based on linked maytansinoid, the MTDs of the D-Ala-L-Ala dpa-AMCs and a disulfide linked control AMC were both approximately 1.4 mg/kg, while the L-Val-L-Cit and L-Ala-L-Ala dpa-AMCs were not as well tolerated. The AMC with the disulfide linker and the three most potent dpa-AMCs had similar in vivo efficacy in tumor xenograft models in mice where the antigen was expressed homogeneously. The D-Ala-L-Ala dpa-AMC however was selected as the lead based on its favorable MTD and because its anti-CanAg conjugate had 2-3 fold greater efficacy in vivo than a disulfide linked anti-CanAg AMC in a HT-29 tumor model, where the antigen is expressed heterogeneously. Cellular processing studies revealed that the anti-CanAg D-Ala-L-Ala dpa-AMC produced a unique, highly potent anilino-maytansinoid catabolite that can diffuse into, and kill, bystander cells. In conclusion, we have developed a promising new type of maytansinoid ADC, one that provides a high degree of bystander killing, improved activity in a heterogeneous tumor model in vivo, and a different mechanism of cytotoxic catabolite release than current AMC designs. Citation Format: Wayne C. Widdison, Joe Ponte, Jennifer Coccia, Yulius Setiady, Ling Dong, Anja Skaletskaya, Nathan Fishkin, Yelena Kovtun, Rui Wu, Rajeeva Singh, Luke Harris, Greg Jones, Leanne Lanieri, Erin Maloney, Charlene Audette, Andre Dandeneau, Ravi Chari, Juliet Costoplus, Karen Veale, Sharon Wilhelm. New peptide-linked anilino-maytansinoid antibody-drug conjugates (ADCs) for the treatment of cancer. [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 1618. doi:10.1158/1538-7445.AM2014-1618

Modeling Sustained Treatment Effects in Tumor Xenograft Experiments

In cancer drug development, demonstrated efficacy in tumor xenograft models is an important step toward bringing a promising compound to human use. A key outcome variable is tumor volume measured over a period of time, while mice are treated with certain treatment regimens. A constrained parametric model has been proposed to account for special features, such as intrinsic tumor growth, or tumor volume truncations due to tumor size being either too large or too small to detect. However, since the drug concentration in the blood of a mouse or its tissues may be stabilized at a certain level and maintained during a period of time, the treatment may have sustained effects. This article extends the constrained parametric model to account for the sustained drug effects. The ECM algorithm for incomplete data is applied to estimating the dose-response relationship in the proposed model. The model selection based on likelihood functions is given and a simulation study is conducted to investigate the performance of the proposed estimator. A real xenograft study on the antitumor agent temozolomide combined with irinotecan against the rhabdomyosarcoma is analyzed using the proposed methods.

An Antibody That Locks HER3 in the Inactive Conformation Inhibits Tumor Growth Driven by HER2 or Neuregulin

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Clinical pharmacokinetics (PK) and translational PK-pharmacodynamic (PD) modeling and simulation to predict antitumor response of various dosing schedules to guide the selection of a recommended phase II dose (RP2D) and schedule for the investigational agent MLN0128.

2567 Background: MLN0128 (INK128) is an investigational oral, potent, and highly selective inhibitor of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) currently in clinical investigation. In the phase1 study INK128-001, MLN0128 was administered once daily (QD), once weekly (QW), QDx3D/week, and QDx5D/week, with respective MTDs of 6, 40, 16, and 10 mg. To guide selection of dose/schedule for further investigation, PD modulation in skin (pS6, p4EBP1, pNDRG1, pPRAS40) was put into context of clinical PK in INK128-001. A preclinical translational dynamic-PK efficacy model was used to describe the relationship and determine PK drivers of efficacy in tumor xenograft models. This model was implemented using human PK parameters to predict tumor volume-time curves, which was utilized to help determine the optimal MLN0128 dose/schedule. Methods: Phoenix NLME v1.1 was used for compartmental modeling of clinical and preclinical PK data, and modeling the preclinical PK-efficacy relationship of MLN0128. PD activity in skin was measured by immunohistochemistry, reported as H scores. Tumor growth curves were simulated using NONMEM v7.2; predicted tumor growth curves were plotted in S-Plus v8.1. Results: Clinical skin PD data suggests exposure dependent inhibition of pS6, and p4EBP1. A two compartment PK model adequately described the PK characteristics of MLN0128 [mean (%CV) ka: ~5.305 h-1 (114), k12: ~0.490 h-1(85), k21: ~0.67 h-1(69), V/F: ~180 L (44), Tlag: 0.317 h (73)]. Simulation of human tumor volume-time curves suggest efficacy is dependent on schedule and that MLN0128 administered in more frequent schedules (QD, QDx5D) provides stronger antitumor effect vs less frequent schedules (QW, QDx3D). Conclusions: The results indicate that per unit MLN0128 plasma exposure, QD and QDx5D may be optimal in comparison with QDx3D and QW dosing. However, these results will also need to be put into context with the overall safety profile and respective MTDs and RP2Ds for each schedule with their resultant achievable total cycle dose by schedule. Clinical trial information: NCT01058707.

Abstract 2465: A new polymer conjugated taxane shows improved efficacy in tumor xenograft models.

Abstract Introduction: Microtubule inhibitors are a mainstay of cancer therapy despite limitations in efficacy and tolerability. Such deficiencies are typical of cytotoxic drugs and a variety of strategies have been investigated to improve them, including the use of polymer conjugates. For example, etirinotecan pegol (EP, formerly NKTR-102) is a topoisomerase I inhibitor-polyethylene glycol (PEG) conjugate engineered by attaching irinotecan molecules to PEG using a biodegradable linker. EP has been studied in multiple clinical oncology studies and showed significantly higher response rates and decreased bone marrow toxicity in Phase 2 compared to historical data for irinotecan. EP is currently in a Phase 3 trial (BEACON; NCT01492101) for metastatic breast cancer. We here report a novel PEG-taxane conjugate, PEG-TX1, which exhibits improved tumor growth inhibition compared to a recently approved taxane, cabazitaxel (CBZ). We have explored the basis for the improved preclinical activity, and show increased intratumoral exposure of the released active taxane (TX1) following administration of PEG-TX1, compared with that achieved with CBZ. Methods: Tumor growth inhibition was examined using subcutaneous NCI-H460 tumor xenografts in nu/nu athymic mice. Test articles were administered intravenously via tail vein, q7dx3, at the maximum tolerated dose with a study endpoint of 2000 mm3. In PK/PD experiments to determine tissue and plasma concentrations of CBZ, TX1, and PEG-TX1, a single dose of test article was administered intravenously, and samples were collected at various time points to 14 d. Drug concentrations in tissue and plasma were quantified using LC/MS. Results: In the NCI-H460 xenograft model, PEG-TX1 (administered q7dx3) delayed tumor growth by 336%, producing 90% partial and 10% complete tumor regressions. In comparison, CBZ delayed tumor growth by 213% and produced only 40% partial tumor regressions and 0% complete regressions. Following a single administration of PEG-TX1 vs. CBZ, significantly greater tumor growth inhibition was observed for PEG-TX1. This improved activity correlated well with increased tumor accumulation of PEG-TX1 and TX1 than was achieved for CBZ. Throughout the study, the concentration of TX1 was at least 10-fold higher in tumor tissue than in plasma or all other tissues examined. CBZ did not show such a sustained increase in tumor exposure relative to other tissues. Conclusions: We demonstrate that PEG-TX1, a PEG-taxane prodrug utilizing Nektar's proprietary polymer conjugation technology, achieved superior tumor growth suppression in a murine xenograft model. Tumor growth inhibition is consistent with the enhanced concentration of taxane detected in tumors following administration of PEG-TX1. These data further demonstrate the utility of polymer conjugation to modify the biodistribution of drug conjugates and thereby enhance the therapeutic properties of an active pharmacophore. Citation Format: Dennis G. Fry, Christine Brew, Steve Lee, Ute Hoch, Wen Zhang, Antoni Kozlowski, Fadil Dahhani, Nagabhusan Tangudu, Stephen D. Harrison, Jennifer Riggs-Sauthier. A new polymer conjugated taxane shows improved efficacy in tumor xenograft models. [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 2465. doi:10.1158/1538-7445.AM2013-2465

Structure-Based Design of Novel Class II c-Met Inhibitors: 2. SAR and Kinase Selectivity Profiles of the Pyrazolone Series

As part of our effort toward developing an effective therapeutic agent for c-Met-dependent tumors, a pyrazolone-based class II c-Met inhibitor, N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (1), was identified. Knowledge of the binding mode of this molecule in both c-Met and VEGFR-2 proteins led to a novel strategy for designing more selective analogues of 1. Along with detailed SAR information, we demonstrate that the low kinase selectivity associated with class II c-Met inhibitors can be improved significantly. This work resulted in the discovery of potent c-Met inhibitors with improved selectivity profiles over VEGFR-2 and IGF-1R that could serve as useful tools to probe the relationship between kinase selectivity and in vivo efficacy in tumor xenograft models. Compound 59e (AMG 458) was ultimately advanced into preclinical safety studies.

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer.

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

Discovery of an Orally Efficacious Imidazo[5,1-f][1,2,4]triazine Dual Inhibitor of IGF-1R and IR.

This report describes the investigation of a series of 5,7-disubstituted imidazo[5,1-f][1,2,4]triazine inhibitors of insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (IR). Structure-activity relationship exploration and optimization leading to the identification, characterization, and pharmacological activity of compound 9b, a potent, selective, well-tolerated, and orally bioavailable dual inhibitor of IGF-1R and IR with in vivo efficacy in tumor xenograft models, is discussed.

The Akt/PKB Family of Protein Kinases: A Review of Small Molecule Inhibitors and Progress Towards Target Validation: A 2009 Update

This article describes recent advances in the development and biological evaluation of small molecule inhibitors for the serine/threonine kinase Akt (PKB) as a reprise of our 2005 review with new data from the 2006-2009 time period. Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors. Recent studies indicate that unregulated activation of the PI3K/Akt pathway is a prominent feature of many human cancers and Akt is over-expressed or activated in all major cancers. Akt is considered an attractive target for cancer therapy and inhibition of Akt alone or in combination with standard cancer chemotherapeutics has been postulated to reduce the apoptotic threshold and preferentially kill cancer cells. Recently, several series of small molecule, ATP-competitive inhibitors have been reported with a range of Akt potencies and selectivities. Phosphatidylinositol (PI) analogs have been reported to inhibit Akt, but these inhibitors may also have specificity problems with respect to other pleckstrin homology (PH) domain containing proteins and may have poor bioavailability. In addition, novel allosteric inhibitors have been reported which are PH domain dependent, exhibit selectivity for the individual Akt isozymes and inhibit the activity and the activation of Akt. Compounds within these classes Akt inhibitors have sufficient potency and specificity which have culminated in recent reports of efficacy in tumor xenograft models. Moreover, Merck just disclosed positive Phase I data with an oral allosteric Akt inhibitor (MK-2206).

ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.