Clinical Trial Phases and Regulatory Approval: The Final Steps of Drug Development

Developing a new drug takes approximately 12-15 years with an average cost of 2.8$ billion. It begins with basic research that leads to identifying a promising molecule. After that,  in vitro experiments and animal studies are conducted to understand its mechanism of action, toxicity, and pharmacokinetics. These preclinical results are critical to determining whether the compound is safe enough to be tested in humans. Once preclinical data support a reasonable safety profile, the compound enters the last step in the drug development process, the clinical phase. 

Understanding the Four Phases of Clinical Trials

Clinical trial phases in drug development are structured as follows: 

• Phase 1 in clinical trials represents the first time a new drug is tested in humans. Typically involving 20 to 100 healthy volunteers, the primary aim is to assess safety, determine safe dosage ranges, and identify potential side effects. These studies are generally non-therapeutic and focused on pharmacokinetics and pharmacodynamics.
• Phase 2 involves a larger group of participants (100-300), usually a few hundred patients who have the condition the drug is designed to treat. The goal here is to evaluate the drug’s effectiveness while continuing to monitor its safety. Phase 2 trials often compare the new drug to a placebo or standard therapy and help refine dosing regimens.
• Phase 3 involves hundreds or even thousands of participants (300-3000) in multiple centers and is designed to confirm efficacy, monitor adverse reactions, and collect data that will support the New Drug Application (NDA) for regulatory approval. At this stage, the investigational drug is often compared directly to current gold-standard treatments to demonstrate added value.
• Phase 4 conducted after regulatory approval, is also known as post-marketing surveillance. These studies track long-term effects and rare adverse events, helping regulators and developers refine their understanding of the drug's safety profile in the general population.

Figure 1. Clinical trials phases flow chart 

Key Regulatory Requirements Before and After Clinical Trial Approval

Before any clinical trial can begin in the United States, the sponsor must file an Investigational New Drug (IND) application with the Food and Drug Administration (FDA). This includes preclinical data, study protocols, and manufacturing information. The FDA reviews the application within 30 days, during which time the trial cannot start unless explicitly authorized.

​​In the European Union, all clinical trials that are ongoing after 30 January 2025 must comply with the Clinical Trials Regulation (CTR) and should have been submitted to the Clinical Trials Information System (CTIS). Under the Clinical Trials Regulation (CTR EU No 536/2014), which came into force in 2022, the European Medicines Agency (EMA) has introduced this centralized portal for submitting and assessing trial applications across EU member states. This harmonized framework aims to simplify trial approval and improve transparency while maintaining high safety and scientific standards.

Once clinical trials begin, both the FDA and EMA require continuous reporting on safety events and study progress. The EMA also assesses pediatric investigation plans (PIPs) and provides scientific advice that can be crucial for a drug’s development path in Europe.

Zebrafish in Enhancing Drug Safety Predictions and Clinical Success

In preclinical drug discovery, regulatory agencies like the FDA and EMA typically require testing in at least two animal species before a compound can proceed to human clinical trials. The standard practice involves a rodent (commonly rats or mice) and a non-rodent species (often dogs, rabbits, or non-human primates). 

Nevertheless, ICH guideline M3(R2) clearly states that “This guidance should reduce the use of animals in accordance with the 3Rs principles. [...] Although not discussed in this guidance, consideration should be given to use of new in vitro alternative methods for safety evaluation.”

Zebrafish has emerged as particularly valuable in early-phase drug safety and efficacy screening. According to the EU Directive 2010/63, zebrafish larvae are classified as in vitro models during the first 5 days post-fertilization. By this time, they already have fully developed organ systems, and systemic in vivo data can be obtained without the ethical and regulatory constraints of vertebrate models, supporting the principle of Replacement.

Their genetic similarity to humans, rapid development, and transparent embryos make them ideal for observing organ-specific toxicity, developmental impacts, and neurobehavioral outcomes in real-time.  Their predictive validity for cardiotoxicity, hepatotoxicity, and teratogenicity has contributed to more informed decisions before clinical trials, potentially reducing the failure rates in later human phases.

Figure 2. Examples of validated compounds using zebrafish as a model that made it to clinical trials. 

By integrating zebrafish models into the preclinical workflow, we can refine compound selection and dosing strategies, reducing the number of mammals used in later, regulated stages and improving the chances that a candidate will succeed through clinical trials and reach approval.

Regulatory Approval Process: From Phase 3 to FDA Authorization

Once clinical study phase 3 data confirm a drug’s benefit-risk profile, sponsors in the US can submit a New Drug Application (NDA) to the FDA. This comprehensive dossier contains preclinical and clinical data, a summary of formulation development and manufacturing processes, and proposed labeling information to be included in the drug’s packaging. It is reviewed by multidisciplinary experts and may be subject to Advisory Committee evaluation.

In Europe, sponsors submit a Marketing Authorization Application (MAA) to the EMA. Unlike the FDA, the EMA does not directly grant marketing authorizations. Instead, it evaluates the application and issues a scientific opinion to the European Commission, which makes the final decision. The centralized procedure allows for drug approval across all EU and EEA countries simultaneously. This is mandatory for advanced therapies, orphan drugs, and treatments for certain serious conditions, and optional for others.

The EMA’s Committee for Medicinal Products for Human Use (CHMP) plays a key role in this process, reviewing the application’s quality, safety, and efficacy data. The agency also evaluates risk management plans and can request post-marketing obligations or additional monitoring depending on the therapy’s profile.

What Happens After Approval? Post-Marketing Surveillance and Phase 4 Studies

With regulatory approval secured, a drug enters the market, but real-world usage often reveals new insights. Clinical phase 4 definition includes studies and post-marketing surveillance programs that aim to detect rare or long-term adverse effects not captured in controlled trials. In fact, 4% of drugs are withdrawn for safety reasons, and 20% acquire new black box warnings post-marketing.  These studies can also assess effectiveness in broader patient populations, drug interactions in polypharmacy scenarios, and patterns of off-label use. 

Clinical phase 4 can also include observational studies, registries, and pragmatic trials designed to explore outcomes in routine clinical practice. These efforts not only safeguard public health but also provide pharmaceutical companies with critical data to inform lifecycle management and future innovation.

Navigating the final steps of drug development requires meticulous planning, rigorous data, and continuous oversight. From clinical study phase 1 to post-marketing studies, every stage is governed by robust regulatory frameworks designed to ensure that only safe and effective therapies reach patients. Innovative tools like the zebrafish model are further enhancing the predictive power of preclinical studies, improving the chances that a candidate will succeed through clinical trials and reach approval, and helping to close the gap between lab and clinic. 

References

European Medicines Agency. Clinical Trials Information System (CTIS) [Internet]. Amsterdam: EMA; [cited 2025 Jul 4]. Available from: https://www.ema.europa.eu/en/human-regulatory-overview/research-development/clinical-trials-human-medicines/clinical-trials-information-system

European Medicines Agency. ICH M3(R2): Non‑clinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals [Internet]. Amsterdam: EMA; 2013 [cited 2025 Jul 4]. Available from: https://www.ema.europa.eu/en/ich-m3-r2-non-clinical-safety-studies-conduct-human-clinical-trials-pharmaceuticals-scientific-guideline

European Parliament and Council. Directive 2010/63/EU of 22 September 2010 on the protection of animals used for scientific purposes [Internet]. Official Journal of the European Union. 2010 Sep 22 [cited 2025 Jul 4];L276:33–79. Available from: https://eur-lex.europa.eu/eli/dir/2010/63/oj/eng

European Parliament and Council. Regulation (EU) 2014/536 of 16 April 2014 on clinical trials on medicinal products for human use [Internet]. Official Journal of the European Union. 2014 Apr 27 [cited 2025 Jul 4];L158:1–76. Available from: https://eur-lex.europa.eu/eli/reg/2014/536/oj/eng

Umscheid CA, Margolis DJ, Grossman CE. Key concepts of clinical trials: a narrative review. (2011). Postgrad Med. 123(5):194-204. doi: 10.3810/pgm.2011.

University of Cincinnati College of Medicine. Clinical trial phases 1, 2, 3 defined [Internet]. Cincinnati (OH): University of Cincinnati; [cited 2025 Jul 4]. Available from: https://med.uc.edu/depart/psychiatry/research/clinical-research/crm/trial-phases-1-2-3-defined

U.S. Food and Drug Administration. Investigational New Drug (IND) Application [Internet]. Silver Spring (MD): U.S. Food and Drug Administration; [cited 2025 Jul 4]. Available from: https://www.fda.gov/drugs/types-applications/investigational-new-drug-ind-application

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.