Science Pool

Rapidly growing tumor cells need a lot of oxygen and nutrients to proliferate, which requires access to the bloodstream. Therefore, tumors induce the formation of new blood vessels through a variety of molecular mechanisms. In normal tissue, there is a delicate balance of pro- and anti-angiogenic factors. In cancers, a process known as the "angiogenic switch" is initiated. As a result, pro-angiogenic signaling becomes dominant, allowing tumors to induce anarchic blood vessel formation. This switch is a critical step in the rapid growth of malignant cells, accompanied by the formation of new blood vessels.

One of the most important initiators of angiogenesis is the family of pro-angiogenic vascular endothelial growth factors (VEGF) and their receptors (VEGFR), which play an important role in both physiological and cancer angiogenesis. All members of the VEGF family stimulate cellular responses by binding to specific tyrosine kinase receptors, the VEGFRs, on the cell surface, causing them to dimerize and become activated. While VEGF-A regulates angiogenesis and vascular permeability by activating VEGFR-1 and VEGFR-2, VEGF-B seems to play a role in the maintenance of newly formed blood vessels under pathological conditions, while VEGF-C and VEGF-D and their corresponding receptor VEGFR-3 regulate lymphangiogenesis, i.e., the formation of lymphatic vessels.

This critical involvement of VEGFRs and their associated signaling pathways in the orchestration of (lymph)angiogenesis makes VEGFRs attractive targets for the treatment of tumors and the prevention of metastasis. However, existing therapies targeting VEGFRs are not very specific and inhibit a broad spectrum of receptor tyrosine kinases including the entire VEGFR family, causing many side effects such as hypertension, proteinuria, hand-foot syndrome, anorexia, and fatigue. While they show good response rates and short-term efficacy, their impact on overall survival is limited, in part because side effects limit the effective dose. This also results in only partial or transient inhibition of VEGFR-3, allowing lymphangiogenesis to serve as a tumor escape mechanism.

Evotec and Kazia therapeutics has therefore started to develop more selective VEGFR-3 receptor tyrosine kinase inhibitors. Its lead candidate, EVT801, an orally available VEGFR-3 inhibitor, is not only highly selective for VEGFR-3, but also the only inhibitor known to inhibit both VEGFR-3 homodimers and VEGFR-3:VEGFR-2 heterodimers. The compound shows low nanomolar inhibitory activity and high selectivity over kinases, various receptors, and ion channels. In November 2022, Evotec scientists reported in Cancer Research Communications that EVT801 showed potent antitumor activity in various in vitro and in vivo models. Moreover, the compound's in vivo efficacy was at least as good as that of the marketed pankinase inhibitors sorafenib and pazopanib. However, unlike sorafenib, EVT801 did not increase blood pressure in monkeys during regulatory toxicology studies or in a rat model of hypertension at doses up to 500 mg/kg, an order of magnitude higher than the pharmacological doses.

The investigators also observed that EVT801 reduced tumor (lymph)angiogenesis, apparently affecting small tumor vessels more significant than larger ones. 

The efficacy of EVT801 is expected to depend on the level of VEGFR-3 expression. Interestingly, expression level of VEGFR-3 did not appear to be affected by sorafenib treatment, suggesting that EVT801 could be used in patients previously treated with any VEGFR tyrosine kinase inhibitor. However, these findings need to be confirmed in clinical trials to determine the minimum threshold of VEGFR-3 expression for effective clinical application of EVT801 and for future patient stratification. 

The proposed mechanism of action of EVT801 involves three sequential anti-cancer mechanisms, all of which contribute to the inhibition of tumor growth and metastasis:

• It prevents tumor growth by impairing both tumor angiogenesis and (lymph)angiogenesis, thereby stabilizing the tumor vasculature, reducing metastasis, and reducing hypoxia in the tumor microenvironment.
• It enhances anti-cancer immunity as reflected by a decrease in immunosuppressive cytokines and cells in the circulation and tumor environment.
• It promotes T-cell infiltration into the tumor, ultimately supporting an enhanced and long-lasting anti-tumor immune response.

Taken together, these studies demonstrate that EVT801 is a novel anti(lymph)angiogenic agent that selectively targets VEGFR-3, modulates the tumor microenvironment to induce tumor vasculogenesis (i.e., fewer and overall larger vessels), and enhances immunotherapy.

Based on these promising results, EVT801 was selected to enter clinical trials. It is currently being evaluated as a single agent in a Phase I trial (NCT05114668) sponsored by Kazia and managed by Evotec Clinical Operations. The first stage of this Phase I study is designed as an open‑label, dose escalation trial to assess the safety, tolerability, and pharmacokinetics of EVT801 in up to 48 patients with advanced solid tumors. Details of the study were presented at the AACR Annual Meeting 2023 (Orlando, FL) (Abstract #1015). This dose escalation part will be followed by a biomarker and pharmacodynamics expansion cohort (second stage), including patients with high VEGFR-3 expressing cancers. These study sections may then be followed by a second dose escalation study, in combination with cancer immunotherapies.

The plan is to establish a patient stratification based on VEGFR-3 expression assessed by immunohistochemical imaging and immunofluorescence on tumor tissues before and after treatment. To enable this patient stratification analysis in a clinical setting, Evotec has established and validated a highly specific protocol for VEGFR-3 immunohistochemistry labeling and scoring strategy that is readily transferable to clinical sites.

To refine the VEGFR3 immunohistochemistry signature and to improve patient characterization, Evotec has developed a VEGFR3 mRNA gene signature consisting of 23 genes highly correlated with VEGFR-3 expression. The gene signature will be analyzed using the Fluidigm platform on matched FFPE patient samples. The relationship of key markers at protein and mRNA level will be investigated to potentially establish biomarkers for patient stratification and selection. 

We expect that the ambitious biomarker strategy will help to better understand the effects of EVT801 in humans and may also help to select the most responsive patients and provide early indications of clinical efficacy as a monotherapy (e.g. clear renal cell carcinoma, soft tissue sarcoma and ovarian cancer) or in combination with standard of care (e.g.immune checkpoint therapies). Evotec and Kazia recently presented a scientific poster on this topic.

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Hepatic transporter inhibition, mitochondrial dysfunction, and reactive metabolite formation are some of the most common mechanisms associated with intrinsic DILI. The cytochrome P450 (CYP450) superfamily of enzymes play an important role in phase 1 metabolism within the liver. For certain chemical entities, reactive metabolites may form with increased toxicity compared to the parent. These reactive metabolites may result in hepatotoxicity through the formation of reactive oxygen species, DNA damage, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. In our research the pan-specific CYP450 inhibitor, 1-aminobenzotriazole (1-ABT), was used in combination with high-content imaging to evaluate the effects of potential reactive metabolites on cell health parameters in hepatocytes. The endpoint assessed included nuclear features, glutathione (GSH) content, mitochondrial dysfunction, and reactive oxygen species (ROS) formation, as well as cellular ATP content. A calculated fold-shift in cell health features between the plus and minus 1-ABT dosing conditions was used to determine reactive metabolite formation. A panel of known DILI reference compounds associated with the formation of reactive metabolites were assessed through this HCI bioactivation assay within metabolically competent HepaRG cells, primary human hepatocytes (PHH) and primary mouse hepatocyte (PMH).

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Peripheral neuropathy can be induced by many chemotherapeutics. Symptoms include numbness, tingling or abnormal sensations which can impact on the long-term quality of a patient’s life. Animal models used to evaluate these side effects may be difficult to interpret and are labor-intensive. In this study, we further optimized a previously developed DRG assay by comparing the neurite outgrowth responses of a group of chemotherapeutics from different classes at 24 h and 72 as an in vitro cell-based model for peripheral neuropathy. Cytotoxicity was assessed alongside neurite outgrowth. Taxanes (paclitaxel, docetaxel), microtubule interfering agents (vincristine, vinblastine, colchicine, nocodazole) and epothilones (ixabepilone) were assessed.

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The generation of anti-drug antibodies (ADA) towards a therapeutic agent can have severe implications with respect to drug safety and efficacy. Therefore, identification of this risk during the initial phase of development is imperative to improve both patient outcomes and downstream attrition rates. As preclinical species often fail to mimic the complexity of the human immune system, the immunogenic potency of therapeutic agents and their potential to elicit a human-specific proinflammatory response can be grossly underestimated during in vivo safety assessment. In our research, a high-throughput in vitro approach to immunogenicity screening was developed using two distinct models: i) peripheral blood mononuclear cells (PBMC) and ii) monocyte-derived dendritic cells (MoDC) isolated from the blood of healthy human donors. The effect of serum-free culture conditions was also evaluated. The immunogenicity of monoclonal antibodies, small molecules associated with delayed-onset hypersensitivity reactions and oligonucleotides were assessed in each model through lymphocyte proliferation and cytokine release.

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The periodic activation and inactivation of various cardiac ion channels enables the regular and synchronous contraction of the heart. Therefore, any disruption of the cardiac ion channel signaling network can result in a change in membrane potential and in the shape of cardiac action potential (AP). As a result, the cardiac excitation-contraction coupling can be modified which may lead to arrhythmias and potential patient death. Local Extracellular Action Potential (LEAP) is a technique which enables non-invasive, label-free monitoring of cardiac action potential in a high-throughput real-time format. In our research, we used the Axion LEAP MEA assay to analyse the effect of various selective and non-selective ion channel inhibitors on cardiac AP by quantification of action potential morphology, repolarization irregularities, and arrhythmic risk factors such as triangulation.

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Drug-induced cardiotoxicity may result from a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or from a structural change, resulting in damage to the cardiac tissue. In our research, the effects of 42 reference compounds in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were investigated in a combined risk assessment strategy. Functional cardiotoxicity was evaluated through kinetic monitoring of calcium transients (CaT), while structural morphology changes and gross cytotoxicity were assessed using high-content imaging (HCI) and cellular ATP measurements. In addition, whole genome high-throughput RNA-sequencing (ScreenSeq) was performed in matched-sister plates. Data were analysed to determine differentially expressed genes (DEGs) and any associated perturbed pathways.

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Our Drug Transporter Sciences team at Cyprotex are delighted to announce publication of a review article in Expert Opinion on Drug Metabolism and Toxicology entitled ‘Studying the Right Transporter at the Right Time: An In Vitro Strategy for Assessing Drug-Drug Interaction Risk during Drug Discovery and Development’.

As our understanding of the role of drug transporters in clinical drug-drug interactions (DDIs) has developed, the list of transporters requiring in vitro study by regulators has grown to accommodate assessment of risk for new drugs.  Currently, ten transporters require routine study prior to regulatory NDA submission.  Getting the timing wrong for these investigations could result in in vitro data being generated either 1) too early in the drug discovery/development timeline and potentially becoming surplus to requirements if the investigational drug fails for reasons of poor pharmacokinetics (and efficacy) or toxicity, or 2) too late to influence finalisation of the clinical development plan resulting in perhaps unnecessary comedication exclusions that impact patient recruitment and thus delay clinical trials.  In either case, there will be a cost and resource penalty, with the overall impact being considerably cheaper for the former compared with the latter.  To minimize these development risks, project teams should study the right transporters at the right time for their investigational drug and the authors (Dr’s Robert Elsby, Hayley Atkinson, Philip Butler and Rob Riley) have tried to address this in their review article by proposing in vitro strategies that could be employed to either mitigate/remove transporter DDI risk during development through frontloading certain studies, or to manage (contextualize) DDI risk to patients in the clinical setting.

In the article, an overview of clinically relevant drug transporters and observed DDIs is provided, alongside presentation of key considerations/recommendations for in vitro study design when evaluating drugs as inhibitors or substrates of transporters.  Guidance on identifying critical victim comedications and their clinically relevant disposition pathways, and using mechanistic static equations for quantitative prediction of DDI (demonstrating a 97% predictive accuracy for 28 statin DDIs) is also compiled.  To truly alleviate or manage clinical risk, the industry would benefit from moving away from current regulatory qualitative basic static equation approaches to quantitative mechanistic DDI prediction, thereby contextualising risk to ascertain whether a transporter DDI is simply pharmacokinetic or clinically significant requiring intervention.  Furthermore, such a mechanistic approach can be used towards either mitigating perpetrator DDI risk early during candidate selection, or managing clinical risk and aiding patient recruitment by informing labels and potentially providing an alternative to conducting costly clinical interaction studies with co-medications in the future. 

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Date: 10th -13th September 2023

Location: The Westin Boston Seaport District Boston, Massachusetts

Booth Location: 206

Attending: Stephen Madden, Chris Strock, Erin Koester, Ralf Geiben Lynn, Sravani Adusumalli, Li Li & Anu Marahatta

Presentation: : CYP46A1 Inhibition and Activation: An In Vitro High Throughput Screening Assay To Assess Possible Drug Interactions Using FDA Approved Chemical Library - Presented by Sravani Adusumalli & High Throughput and Fully Automated Sample Preparation for Quantitative Bioanalysis Using LC/MS/MS - Presented by Anu Marahatta

Learn more about 25th North American ISSX Meeting

Date: 06 July2023

Location: Alderley Park Conference Centre, Alderley Park, Congleton Road, Alderley Edge, Macclesfield SK10 4TG

Attending: Tim Potter, Mark Wenlock, Simon Thomas, Hannah Crowson, Stephen Madden & Phil Butler

Presentation: 1-3 The prediction of human pharmacokinetics in early-stage drug discovery - Simon Thomas

Learn more about SMR-DMPK