Can Zebrafish Testing Help Bring Your Drug To Market?

Zebrafish contribution to drug development reaching clinical stages

By Miriam Martínez, Marketing Manager at ZeClinics.

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from the Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. This year she joined ZeClinics with a branding and marketing strategy focus.

In this article, we will summarize the latest developments in zebrafish studies in the U.S. and Europe, focusing on currently ongoing clinical trials resulting from zebrafish pipelines. We will include studies that go from bench to bedside and present examples of drug discovery through zebrafish studies, such as clemizole to treat Dravet syndrome and ProHema for leukemia. We will complete this information with our current contribution to the field.

Zebrafish as a New Alternative Methodology (NAM) for drug discovery

The scientific and technological breakthroughs of the last decades have streamlined the drug discovery pipelines. Nevertheless, the clinical failure rate of novel compounds remains high because the preclinical phase provides a lower-than-expected prediction of toxic liabilities and therapeutic effects in human patients. 

Supranational organizations and agencies, like the U.S. Environmental Protection Agency (EPA), have declared their strong aim to reduce and eventually eliminate animal testing during the next decade. In this landscape, new alternative methodologies (NAMs) are focusing on novel research efforts in drug discovery and regulatory chemical hazard determination.

Among NAMs, zebrafish is one of the most promising models since it ensures, at the same time, high-throughput capabilities and the advantages of whole-organism biology. These capabilities are granted because of their high genetic and physiologic homology with mammals. As such, when compared with mammals, the zebrafish model:

  1. Presents the possibility of testing hundreds of organisms in parallel.
  2. Allows generating multiple disease models through the use of CRISPR and other technologies.
  3. Provides substantial experimental advantages for rapidly analyzing a broad variety of phenotypes – optical transparency, quick physiological maturation, homologous organs and tissues, small size, easy and fast genetic manipulation, etc.
  4. Permits the implementation of 3R measures, given most assays are performed under five days post fertilization (dpf), a stage considered as non-animal testing by EU legislation and integrated inside in vitro batteries by regulatory agencies. 

On the other hand, when compared with in vitro models, the zebrafish model:

  1. Provides the possibility of testing chemicals under a full ADME context
  2. Displays complex biological processes challenging to mimic by in vitro models, such as embryonic development, cardiovascular physiology, angiogenesis, locomotor behavior, or metastasis, to name a few. 

This combination of biological translatability and experimental throughput allows a high predictivity for addressing target and chemical/drug activities. 

Clinical trials resulting from zebrafish pipelines

The relevance of zebrafish studies in the clinical development of new therapies has become increasingly strategic in recent years. Despite being a relatively new model for drug discovery, and considering the long time from those early stages to the clinical development of drugs, a high number of successful cases could be found in the literature.

To date, drug screenings in zebrafish have brought to clinical trials nearly twenty compounds. Those compounds, expected to increase in coming years,  are now in different clinical phases, endorsing further the predictive potential of zebrafish.

A recent review published by Patton E. et al in Nature Reviews, Drug Discovery demonstrates a quick advance in zebrafish-based discoveries in the clinic. This information, combined with internal research conducted by ZeClinics, has allowed the gathering of the data presented in Table 1. It reports a list of drugs in clinical phases, with direct involvement of zebrafish-based assays.

Table 1. Clinical Trials related to the Zebrafish Model

TRIAL WITH LINK TO PAGECONDITION OR DISEASEINTERVENTION/
TREATMENT
PHASEROLE OF ZEBRAFISH
Xenotransplantation of Primary Cancer Samples in Zebrafish Embryos (xenoZ)Hepato-biliary-pancreatic cancers and gastrointestinal cancersChemotherapyN.A.Personalized medicine [1]
Xenotransplantation of Primary Leukemia Samples Into ZebrafishLeukemiaChemotherapyN.A.Personalized medicine [2]
Influence of Cortisone on QTc-intervalQTc-intervalCortisoneN.A.Modeling  disease;
Efficacy [3]
A Randomized Controlled Study of LiuWeiLuoBi Granule for the Treatment of Diabetic Peripheral NeuropathyDiabetic Peripheral Neuropathy;
Chinese Medicine;
Nerve Conduction
LiuWeiLuoBi GranuleEarly Phase ISafety [4]
Phase 2 Pharmacological Trial to Evaluate the Safety of Miglustat Administration in Subjects With Spastic Paraplegia 11 (TreatSPG11)Hereditary Spastic ParaparesisMiglustatPhase IIModeling  disease [5]
Gene Expression During Surgical Scar Remodeling by Fractional PhotothermolysisHypertrophic ScarsDevice: Fraxel Repair - Fractional Laser treatmentN.A.Efficacy [6]
Phase I/II, Open Label Study to Determine Safety of Trifluoperazine (TFP) in Adults With Red Blood Cell Transfusion-Dependent Diamond Blackfan AnemiaDiamond Blackfan Anemia; 
Pure Red Cell Aplasia
TrifluoperazinePhase I;
Phase II
Modeling  disease;
Efficacy [7]
Randomized Triple-blind Placebo Controlled Trial of Influence of Morphine or Ketamine or Saline Applied During In-hospital Cardiopulmonary Resuscitation on Early Survival and Neurological OutcomeCardiac ArrestMorphine; 
Ketamine
Phase IEfficacy [8]
Incidence of Cereblon in Intensive Care PatientsCritical Illness;
Sepsis
Immunomodulatory drugsN.A.Safety [9, 10]
Molecular Imaging of Primary Amyloid CardiomyopathyAmyloidosis, Primary;
Cardiomyopathy
Radiation: F-18 florbetapir/C-11 acetate PET, N-13 ammonia PETN.A.Safety [11, 12]
A Phase II Trial of All-trans Retinoic Acid (ATRA) in Advanced Adenoid Cystic CarcinomaAdenoid Cystic CarcinomaTretinoinPhase IIEfficacy [13]
Phase I Study of Olaparib and Temozolomide in Adult Patients With Recurrent/Metastatic Ewing's Sarcoma or Rhabdomyosarcoma Following Failure of Prior ChemotherapyEwing Sarcoma;
Rhabdomyosarcoma
Olaparib, Temozolomide,  IrinotecanPhase IIPersonalized medicine;
Efficacy [14]
A Phase 2 Controlled Trial of a Single ProHema®-CB Unit (Ex Vivo Modulated Human Cord Blood) As Part of a Double Umbilical Cord Blood Transplant Following Myeloablative or Reduced Intensity Conditioning For Patients Age 15-65 Years With Hematologic Malignancies.Hematologic MalignanciesProHema-CBPhase IIModeling disease [15]
A 20-Week Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial of EPX-100 (Clemizole Hydrochloride) as Adjunctive Therapy in Patients With Dravet SyndromeDravet SyndromeEPX-100 (Clemizole HCl)Phase IIModeling  disease;
Efficacy [16]
Leflunomide in Combination With Vemurafenib in Patients With V600 Mutant Metastatic MelanomaMelanomaVemurafenib, LeflunomidePhase IEfficacy [17]
Phase 1b Randomized, Double-Blind, Placebo-Controlled, Multicenter Study to Evaluate the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Repeated Doses of DB-020 in Patients Receiving CisplatinOtotoxicityDB-020Phase IEfficacy [18]
KER-047 for the treatment of anemia resulting from iron imbalance and for the treatment of fibrodysplasia ossificans progressivaFibrodysplasia ossificans progressiva; iron deficiency anemiaKER-047Phase IModeling  disease;
Efficacy [19]
ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitorLymphatic diseaseMEK inhibitorCompassionate use;
Clinical case study
Modeling  disease;
Efficacy [20]
Phase II Clinical Trial of MEK Inhibitor Trametinib in the Treatment of Complicated Extracranial Arterial Venous Malformation (VM)Arteriovenous malformationMAPK pathway inhibitorsPhase IIModeling  disease;
Efficacy [21]
Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2T cell acute lymphoblastic leukemiaPP2A activatorPreclinicalEfficacy [22]

The contribution of zebrafish studies in those clinically-relevant pipelines spans several levels of knowledge around drug development, which could be summarized in four types of contributions:

  1. Safety: evaluating toxic effects of molecules in zebrafish;
  2. Disease modeling: the creation of zebrafish lines with either genetic and/or other factors, reproducing the human disease and phenotypes;
  3. Efficacy: discovering compounds rescuing pathological phenotypes in zebrafish disease models;
  4. Personalized medicine: using zebrafish as the host organism for modeling individual patient conditions or mutations.

Importantly, ZeClinics has built an extensive portfolio of validated assays, already commercialized, covering these four main applications. Therefore, ZeClinics is well-positioned for contributing to each and every step of the value chain of the drug development process.

ZeClinics involvement in zebrafish drug development reaching clinical stages

ZeClinics has contributed massively in the last years to the validation, approval, and consolidation of the zebrafish model in the drug discovery and preclinical world. During its nine years of history and seven full years of commercializing services, the company has published 19 scientific articles that demonstrate the model's predictive value in several therapeutic areas and applications: toxicology, genetics, ophthalmology, cardiovascular, oncology, and CNS.

From the beginning, ZeClinics has participated in more than 276 studies from 112 customers, of which 14 are clinical-stage companies, and 15 have already products in the market. The clients range from academic centers to biotech, pharma, agrochemical, and other types of clients (cosmetics and industrial ones). Currently, the company operates in 24 countries and has constant conversations with the most important drug discovery centers, both public and private.

A perfect case study is our strong collaboration with Dompe Farmaceutici. We have recently co-published an article in which we demonstrated the retinal regenerative role of recombinant human NGF (rhNGF) in the zebrafish model of retinal degeneration. NGF is an FDA and EMA-approved drug for neurotrophic keratitis,  a rare eye disease that can lead to loss of sight. Dompe decided to test the compound in an alternative model to better understand the mechanism of action of the drug. Importantly, this initial collaboration has led to multiple unrelated projects.

In addition, ZeClinics has performed several repurposing projects for clients, interested in discovering new indications for their clinical or marketed drugs. We have also tested several drugs and biologics, currently in the preclinical stage, for several clients. Finally, ZeClinics is or has been involved in multiple target discovery projects, validating the association of genetic targets with specific diseases. These types of projects, based on our huge expertise in genetic manipulation and our commercial license for the CRISPR/Cas9 technology, have been performed for several companies and across multiple indications.

However, the direct impact of the work performed by ZeClinics in the discovery of additional targets and molecules toward their approval to enter clinical stages is a challenging aspect to evaluate. This is due to different reasons:

  1. The company generally receives blinded compounds from the sponsors.
  2. The clients tend to share little information about the nature of the therapies to protect their IP better.
  3. Given the commercial company growth, most of the projects have been completed during the last four years; thus, it is still too early for many of these tested targets and drugs to reach clinical phases.

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drug discoveryNAMspredictabilityR&DTranslatabilityvalidityZebrafish