A cell culture-based in vitro method for determining the activity of botulinum toxin

Background:

Botulinum toxin is a bacterial exotoxin. It is produced by the anaerobic, spore-forming bacterium Clostridium botulinum and is one of the most potent bacterial toxins in existence. The lethal dose in humans is approximately 1 ng/kg body weight. Botulinum toxin is a neurotoxin that inhibits the release of neurotransmitters from nerve endings, which stimulate muscle contraction. This leads to flaccid paralysis of the affected muscles. Death occurs as a result of paralysis of the respiratory muscles. Botulinum toxin is a protein consisting of two subunits. The large subunit binds to nerve cells and ensures that the small subunit can penetrate the nerve cells. Within the nerve cells, the small subunit destroys proteins that are necessary for the release of neurotransmitters from the nerve cells. There are different serotypes of botulinum toxin, which differ both in terms of the structures via which the toxin binds to nerve cells and in terms of the proteins that are cleaved by the small subunit.
The bacteria thrive in food that has not been properly sterilised and is stored in an airtight container, such as improperly prepared tinned food, the consumption of which leads to botulism, a condition that is often fatal. However, when administered locally in low doses, botulinum toxin can be used as a medicine to treat conditions caused by a sustained contraction (spasm) of a muscle or group of muscles, such as blepharospasm or spastic torticollis. However, botulinum toxin finds its widest therapeutic application in cosmetic medicine, where it is used to suppress the formation of skin wrinkles by selectively paralysing the skin muscles that cause them. To this end, it is injected in low concentrations into the relevant areas of skin. Botulinum toxin for therapeutic use is derived from Clostridium cultures. During the purification steps required for this, part of the protein is inactivated. Consequently, the finished preparation consists of varying proportions of functional and inactive protein. Due to its high toxicity, the activity of each preparation must be precisely determined. In doing so, the activity of both the large and small subunits must be measured. This is currently achieved most reliably by measuring the biological effect in whole animals. To do this, the concentration of the substance is determined at which half of the treated mice die from respiratory paralysis. For this test, approximately 300,000 mice worldwide die each year a painful death by suffocation (http://altweb.jhsph.edu/sebin/c/g/altex_2_10_bitz.pdf; accessed 14 June 2012).

Project:
The project established a cell-based method for measuring the activity of botulinum toxin, which can largely replace animal testing. It differs from all previously established alternative methods in that a reporter, which is released from nerve cells simultaneously with the neurotransmitter, is used to directly detect the endpoint of botulinum toxin action: the inhibition of neurotransmitter release from the nerve cell. This allows the activity of both the large and small subunits to be determined in a single step, and it should theoretically be possible to measure the toxin’s activity independently of the serotype. The principle (Fig. 1) is based on the fact that, within the cell, proteins can be directed to a specific location by certain amino acid sequences (address sequences). Thus, using suitable address sequences, proteins can also be directed into the storage vesicles from which the neurotransmitters of the nerve cell are released. These proteins are then released together with the neurotransmitters, or their release is inhibited when the release of the neurotransmitters is inhibited. If an enzyme that is not normally produced by the cells is selected as the protein, the release can be determined by measuring the enzyme’s activity in the cell culture supernatant; the stimulus-dependent activity in the supernatant decreases when the release of the neurotransmitter is inhibited by botulinum toxin.

A transgenic cell line was established based on a human nerve tumour cell line (neuroblastoma cell line) that synthesises luciferase (a light-producing enzyme) and releases it, together with the carrier substances, from storage vesicles when the cell is stimulated with a stimulation buffer. This stimulus-dependent release can be almost completely inhibited by botulinum toxin (Pathe-Neuschäfer-Rube et al. ALTEX 2015; 32: 297–306. http://www.altex.ch/All-issues/Issue.50.html?iid=155&aid=5). For botulinum toxin of serotype A, it has been shown that it inhibits the stimulus-dependent release of the reporter enzyme from nerve cells in a dose-dependent manner (Fig. 2).

Both sensitivity and dose range are very close to the results obtained in the mouse lethality test. The method is therefore fundamentally suitable as a replacement procedure. Botulinum toxin of serotype C could be detected with similar sensitivity to that of serotype A, whereas botulinum toxin of serotype B could only be detected with significantly lower sensitivity (Fig. 3).

In line with the 3Rs, the method represents a possibility for the extensive replacement of animal testing in the measurement of botulinum toxin activity. Where it appears necessary to further confirm the level of activity for safety reasons through animal testing, the number of animals required for this purpose can be reduced, as the range of doses to be tested can be significantly narrowed down by the cell-based assay.