Clostridium botulinum

4 – 9 μm long0.9-1.2 μm large

Spores

Anaerobe which produces a strong

NEUROTOXIN

which causes

BOTULISM

In humans, Botulism is a rare disease but it is severe and potentially lethal(incidence of 1 to 5 cases per 10 millions, but it is believed that many cases of botulism go unreported in

Asia and Africa)

Botulism: paralysis of skeletal andautonomic cholinergic nerve terminals.

Rossetto et al., Nature Rev. Microbiol. 2014

MAIN BOTULISM SYMPTOMS in humans:- Paralysis of respiratory muscles - Diplopia, ptosis - Dysphagia - paralysis of facial muscles which progressively descends to the

trunk, visceral muscles, arms and legs- Reduced salivation and lacrimation, - nausea, vomiting, and abdominal pain.

The botulism patient is fully conscious but cannot operate cholinergic dependent peripheral body functions !

If properly treated (intensive care unit with mechanical ventilation) patients survive botulism, but their complete recovery may require several months.

Botulism is reversible

Reports of 5-6 millions salmons or of 2-300.000 ducks that diedin a single botulism outbreak

i.p. MLD50: 0.01 - 5 ng/Kgmeasured in the animal house

Few thousands molecules in a mouse !

≈ femtograms/Kg !!!!

Lethal dose for humans is not known, but it is estimated from primate studies to be for a 70-kg human:

intravenously or intramuscularly 0.09 – 0.15 µg

Inhalational 0.70 – 0.90 µg

orally 70 µg

Selection on the basis of criteria such as…

- prior use as a biological weapon

- use might cause mass casualties

- potential economic impact

- U.S. enemies had looked into

using the agent as a weapon

Botulinum neurotoxins are classified as Dangerous Select agents

U.S. Federal Experts Security Advisory Panel

recently revisited Select agent list

Clinical isolation

Bacterial cultivation

Purification of the toxin

from sterile supernatant

Serological, biochemical, and

toxicological characterization

Genome determination

Botulinum toxin sequence

determined

Botulism outbreak

Many toxinvariants

7 Serotypes and mosaics:A,B,C,D,E,F,G,DC,CD,FA

Brunt et al., FEBS Lett. 2018

The complex of BoNT/A with accessory proteins ( NTNHA e HA70, HA17, HA33) rapidly dissociates at the neutral pH of tissues freeing BoNT/A which can bind to cholinergic terminals.

MERZ

ALLERGAN

DYSPORT

Meditox,

Lanzhou,

Daewong

The Five Steps of BoNT intoxication of Nerve Terminals

Pirazzini et al., Pharmacol. Rev. 2017

The discovery of the cellular and molecular mechanism of action of the botulinum neurotoxins was important for several reasons:

1.It provided the molecular basis of the pathogenesis of botulism.

2.It proved that the three SNARE proteins: VAMP, SNAP-25 and Syntaxin are the core of the nanomachine which mediates neurotransmitter release at the synapse, and of exocytosis in general.

3.It provided the conclusive prove of the correctness of the “Quantal hypothesis of neurotransmitter release”.

4.It provided the basis to identify drugs that prevent or block botulism after the toxin is inside neuron, when it

canNOT be neutralized by Antibodies.

Pirazzini et al., Cell Rep. 2014

Mouse DAS assay

Electrophysiologycal recording at the neuromuscular junction

Ebselen and PX-12 could be effective in treatinginfant botulism and to prevent the other forms of botulism irrespectively of the type of BONT

BoNT intoxication is fully reversible

The time course of the recovery of the function of acholinergic nerve terminal poisoned by BoNT depends on:

1. The animal species

1. the BoNT serotype and the DOSE

3. the type of terminal (skeletal “rapid”, autonomic “slow”)

0 10 20 30 40 50 60

0

10

20

30

40

50

60

70

80

90

100

110

Wei

ght %

of t

he C

TR

Days

B: BoNT/A

D: BoNT/C

F: BoNT/E

Duration of the muscle paralysis induced

by BoNT/A, /C and /E in mice and humans

Eleopra et al. 1997

EE

A A A AAA

C C C C C C

E

Morbiato et al. 2007

BoNT is a formidable drug to treat all human syndromesor conditions generated by hyperfunction of cholinergicperipheral nerve terminals because of:

1) Its neurospecificity and potency

2) Its reversibility and the long duration of action (in humans: 3-4 months in skeletal nerves and > 1 year in autonomic nerve terminals).

3) Limited diffusion from the site of injection

4) Very low immunogenicity at the extremely low dosesused in human therapy

Pirazzini et al., Pharmacol. Rev. 2017

2. Limited diffusion from the site of injection

Black bars, BoNT/A; light gray bars, BoNT/B; dark gray bars, BoNT/C; white bars, BoNT/F; striped bars, placebo.

There is little if any spreading into adjacent muscles

Eleopra et al. 2004

Carli et al., Muscle & Nerve 2009

Tibialis Soleus Gastrocnemius Quadriceps

BOTOX

Dysport

Xeomin

Carli et al., Muscle & Nerve 2009

Tissue Diffusion of BoNT/A1 from the site of inoculation

BoNT/A1 diffusion around the site of injection depends

on the injected volume.Small Volumes ≈ lower diffusionLarge Volumes ≈ larger diffusion

The extent of diffusion of BoNT/A1 depends on:

1. dose of BoNT/A1: higher dosage larger diffusion.

2. site of injection (large muscles, small or flat muscles) and

3. mode on injection (im, subcutaneous).

4. position and number of cholinergic terminals that are

ready to rapidly bind BoNT/A1 before it enters in the fluid

circulation with consequent diffusion, followed by dilution

and loss of capacity of binding.

After a subcutaneous injection, albumin is estimated to enter the venous circulation after 4 seconds, whilst the contrary takes about 25 second.

BoNT/A1 is believed to diffuse laterally.Part of it binds to cholinergic nerve terminals of underlying muscles paralysising them, but a sizeable part is rapidly diluted into the lymphatic and venous circulations.

This is at the basis of the multi-sites treatment in order to achieve a better targetting.

3) Very low immunogenicity at the extremely low doses used in human therapy

In cosmetics even lower doses are used and, therefore, the possibility of the immunization is even lower.

Protein content of BoNT/A preparations

0

5

10

15

20

25

30

1995 2005

Year of development

To

tal

pro

tein

co

nte

nt

(ng

/via

l b

otu

lin

um

to

xin

typ

e A

) Botox®1

Dysport®2

XEOMIN®3

Blepharospasm

Orbicularis oculi

orbital portion

Orbicularis oculi

pretarsal portion

Campos, 1992

Minor test to quantify sweating After treatment with BoNT/A1

Hyperhydrosis is defined as generalized or localized overproduction by eccrine sweat glands

BoNT/A1 treatment of axillary hyperhydrosis

Before treatment

After treatment

Esthetics

(D. De Grandis, 1992)

Serendipitously, it was observed that BoNT/A1, when used therapeutically for blefarospasm, smoothed facial lines and this led to study the toxin effect on glabellar lines, which are perceived as a sign of aging and of negative emotions (Carruthers and Carruthers, 1992).

This led to the ever increasing use of BoNT/A1 in aesthetic medicine, which has become the major use of BoNT/A1 nowadays.

Serendipitously, it was observed that individuals treated forfacial wrinkles or ridges and that were at the same time suffering from migrane experienced a remission of symptomsfor 3-4 months, in 30-40% cases.

An extensive meta-analysis concluded that BoNT/A1 may reduce the mean number of headache days per month in chronic migraine and chronic daily headache, in comparison with placebo. BoNT/A1 was not beneficial in the treatment of episodic migraine or tension-type headache. (Jackson et al, 2012; Simpson et al., 2016)

Accordingly, chronic migraine is the only pain disorder with FDA approved BoNT/A1 use, notwithstanding the fact that the mechanism of action is still not completely elucidated.(Chen, 2012)

Possible mechanisms of transport of BoNT/A1 to the spinal cord

Two sources of reduced motoneuron output after BoNT/A:

• Peripheral neuroparalysis

• Reduced excitability via central effect on cholinergic afferents

Rossetto et al., Nature Rev. Microbiol. 2014

The minimal lethal dose for a 70 Kg person is estimated to be around 0.1 μg.This is indeed a very high toxicity. However, perhaps surprisingly, the number of cases of jatrogenic botulism islimited to few dozens of cases that is to be compared withthe millions and millions of patients treated over the yearssince the begin of the 80s.

BoNT/A1 is a very very safe pharmaceutical drug !

A systematic review and meta-analysis of randomized clinical trials did not find reports of serious side effects due to BoNT treatment .

Contraindications to BoNT therapy are limited to a hypersensitivity to the toxin or an infection at the site of injection. An increased monitoring is recommended in patients treated simultaneously with aminoglycosides, anticholinergics, or other neuromuscular blocking agents.

BoNT/A1 is a very very safe pharmaceutical drug when

administered by authorized medical personell, using licensed

toxin preparations.

In contrast, the handling of BoNTs by

unskilled/nonauthorized persons or the use of

counterfeit or unapproved agents that have permeated the

market worldwide, or their combination, may place patients

at risk for potentially devastating consequences.

These considerations are valid

for pharmaceutical as well as cosmetic treatments

Botulinum neurotoxin preparation for human injection are dosed in Units.A Unit is the median lethal dose 50 in mice and it correspondsto 4.5 – 12 pg depending on the commercial preparations.

In human therapy doses in the 10 – 800 Uare used depending on the pathological condition treated.

This correponds to:50 – 9600 pg to be compared with the 100,000 pg of theestimated minimal lethal dose.

In cosmetics.......we are on the lower side, but we are on thesafe side only if BoNT/A1 is administered by authorized medical personell, using licensed toxin preparations.

We gratefully acknowledge the financial support of:

Dipartimento Sanità

Marco Pirazzini Ornella Rossetto

Paola Caccin

Giorgia D‘Este

Elisa Duregotti

Aram Megighian

Federico Fabris

Samuele Negro

Michela Rigoni

Morena Simonato

Marco Stazi

Giulia Zanetti-

Neurotoxins, Neurodegeneration and Regeneration Lab