New assays for botulinum toxin testing

Dr Dorothea Sesardic, from the National Institute for Biological Standards and Control, received an NC3Rs project grant to develop a cell-based assay for the detection of botulinum toxin. The assay has the potential to replace the use of around 70,000 mice each year in the UK alone. 

Research details

Principal Investigator: Dorothea Sesardic, Principal Scientist
Organisation: National Institute for Biological Standards and Control (a centre of Medicines and Healthcare Products Regulatory Agency)
Award: £337,708, in 2010, over 36 months (extension awarded)
Title: Development of cell-based assays as replacement assays for botulinum toxins and antitoxins

Read more about Dr Sesardic's research

Case study

Botulinum toxins are increasingly used as medical and cosmetic products

Botulinum toxin, one of the most acutely poisonous proteins known, is a neurotoxin produced by the bacterium Clostridium botulinum. There are eight serotypes which cause muscle paralysis by blocking the release of the neurotransmitter, acetylcholine.

In very small quantities, botulinum toxins can be used therapeutically to prevent muscle spasms and are increasingly available as licensed drugs for the treatment of a variety of disorders, such as dystonia (a neurological movement disorder), blepharospasm (involuntary eye muscle contractions) and hyperhidrosis (excessive sweating). ‘Botox’ injections are also increasingly used for cosmetic purposes to temporarily paralyse facial muscles to smooth the skin.

Batch testing involves thousands of mice

Before they can be used in humans, each batch of botulinum toxin is tested for safety, potency and stability, because the toxin is a biological product with inherent variability. These tests are required at a number of stages of the production process in order to meet regulatory requirements before marketing authorisation is granted. Typically, each batch is tested by both the manufacturer and the national competent authority.

Worldwide, the most commonly used test is the mouse lethal dose 50 (LD50) assay. This assay involves injecting groups of mice with dilutions of the botulinum toxin to determine the dose that will kill half of the animals at a defined time-point. The test causes severe suffering as the toxin induces paralysis of the respiratory muscles. In the UK, around 70,000 mice are used each year for botulinum toxin testing; globally the figure is estimated to be more than 600,000.

Current in vitro alternatives have limitations

The UK’s National Institute for Biological Standards and Control (NIBSC), which is a centre of the Medicines and Healthcare Products Regulatory Agency, has been part of a global effort to develop alternatives for in vitro botulinum safety and potency testing. Although good progress has been made many of the current tests are limited because they fail to mimic all of the mechanisms that contribute to the toxin’s action in vivo. In 2010, Dr Dorothea Sesardic, NIBSC, was awarded NC3Rs funding to develop improved cell-based assays for botulinum toxin serotype A, which is licensed for use in therapies.

Improved functional assays for botulinum toxin testing

Mouse embryonic stem cells (E14Tg2a), human neuroblastoma cells (SH-SY5Y) and commercially sourced neurons derived from human induced pluripotent stem cells have been differentiated into mature neuronal cells and fully characterised for their response to various concentrations of botulinum toxin using a range of functional assays such as electrical activity, synaptic vesicle trafficking and specific immunodetection of intracellular target protein. These assays offer the potential for the complete replacement of the mouse studies because their mode of action involves all of the key hallmarks of botulinum neurotoxicity.

Botulinum toxins contain enzymes that breakdown proteins essential for neurotransmitter release. The new assays include a highly sensitive measure of the cleavage of one of these, the presynaptic plasma membrane protein SNAP-25, which is selectively cleaved by botulinum neurotoxin (BoNTs) serotypes A, C and E. Dr Sesardic has demonstrated that incubation of the E14Tg2a-derived neurons with extremely low levels of purified BoNT/A in the presence of high concentrations of stabilising proteins and other excipients that are found in therapeutic products, results in dose-dependent breakdown of SNAP-25. The assay is specific to BoNT/A and the next step is to establish if the assay can be adapted for toxin neutralisation studies.

Dissemination, collaborations and further funding

There have been three publications arising from this grant. Collaborations have been established for sharing botulinum toxin sub-serotypes, cells and expertise, with the MRC Centre for Developmental Neurobiology, MRC Laboratory of Molecular Biology, Wellcome Trust Centre for Stem Cell Research, and members of an EU-funded consortium (called AntiBotABE) aimed at developing neutralising antibodies against botulinum toxins A, B and E.

The success of the initial NC3Rs-funded work has led to internal funding from NIBSC for a pilot project to initiate development of a cell-based assay for tetanus toxin. In addition, Dr Sesardic is leading a team to apply the 3Rs to the control of biological medicines, funded as part of a £5 million grant-in-aid to the NIBSC Regulatory Research Unit from the UK Department of Health.

This case study was published in a review of our research portfolio in November 2013.