The Ageing Skin – Part 4g – Botulinum Toxin


The skin is the most superficial part of the body. The signs of ageing are most visible in the skin. Although, ageing skin is not a threat to a person, it can have a detrimental effect on the psychology of a person. A look into the causes of skin ageing, the available treatments and preventive measures for this inevitable change is important to help both the already aged, as well as, the youth.

This is a 4 part article in which:

  1. Part 1 – Discusses the structure of skin and its different components
  2. Part 2 – Discusses cutaneous ageing and its causes
  3. Part 3 – Discusses the characteristics of ageing skin and the changes in skin appearance
  4. Part 4 – Discusses products and treatments for skin ageing
    a) Sunscreen Agents
    b) Moisturizers
    c) Antioxidants
    d) Make Up
    e) Dermal Fillers
    f) Chemical Peels
    g) Botulinum Toxin (current article)
    h) Estrogen and Hormonal Treatments
    i) Plastic Surgery

Botulinum Toxin (Botox) 1,2

Toxin botulinum or Botox, is a sterile, vacuum dried purified form of botulinum toxin type A indicated for the treatment of strabismus, blepharospasm, and other related condition. It acts by inhibition of acetylcholine release of the motor endplates. It temporarily denervates specific muscles responsible for certain facial rhytids including the glabellar furrow, horizontal forehead lines, horizontal neck lines, and crow’s feet.

The genus Clostridium has more than one hundred and twenty seven species, grouped by morphology and function. The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals known as botulism in higher concentrations. However, in extremely low concentration the toxin is helpful as it causes flaccid paralysis of the muscles and thus relaxing the wrinkles and expression lines.


Clostridium botulinum and its spores are commonly found in soil and the bacterium can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.


Botulinum toxin type A is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) type A is a LD50 in mice. On a molar basis, botulinum toxin type A is 1.8 billion times more lethal than diphtheria, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobrotoxin and 12 million times more lethal than cholera. One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 20 grams each. In other words, one unit of botulinum toxin is the amount of botulinum toxin that kills 50% of a group of female Swiss Webster mice.


Seven generally immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F, and G, each of which is distinguished by neutralization with type‐specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. Additionally, botulinum toxin type B has been determined to be non‐toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for botulinum toxin type A.

Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. Botulinum toxin A is a zinc endopeptidase which can specifically hydrolyze a peptide linkage of the intracellular, vesicle associated protein SNAP‐25. Botulinum type E also cleaves the 25 kiloDalton (kD) synaptosomal associated protein (SNAP‐25), but targets different amino acid sequences within this protein, as compared to botulinum toxin type A. Botulinum toxin types B, D, F and G act on vesicle‐associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP‐ 25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes.


The botulinum toxins apparently bind with high affinity to cholinergic motor neurons, are translocated into the neuron and block the presynaptic release of acetylcholine.

Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages.

In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain (H chain) and a cell surface receptor; the receptor is thought to be different for each serotype of botulinum toxin and for botulinum toxin. The carboxyl end segment of the H chain, Hc, appears to be important for targeting of the toxin to the cell surface.

In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor‐mediated endocytosis, and an endosome containing the toxin is formed.

The toxin then escapes the endosome into the cytoplasm of the cell. This last step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin then translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the H and L chain. The entire toxic activity of botulinum and botulinum toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter‐containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Botulinum neurotoxin, botulinum toxin B, D, F, and G cause degradation of synaptobrevin (also called vesicle‐associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.


The botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium (e.g. a medium composed of meat and milk products). Raw toxin can be harvested by precipitation with sulfuric acid and concentrated by ultramicrofiltration. Purification can be carried out by dissolving the acid precipitate in calcium chloride. The toxin can then be precipitated with cold ethanol. The precipitate can be dissolved in sodium phosphate buffer and centrifuged. Upon drying there can then be obtained approximately 900 kD crystalline botulinum toxin type A complex with a specific potency of 3×107 LD50 U/mg or greater. This known process can also be used, upon separation out of the non‐toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1.2×108 LD50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1.2×108 LD50U/mg or greater, and; purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1.2×107 LD50 U/mg or greater.


Commercially available botulinum toxin containing pharmaceutical compositions include Botox® (Botulinum toxin type A neurotoxin complex with human serum albumin and sodium chloride) available from Allergan, Inc., in 100 unit vials as a lyophilized powder to be reconstituted with 0.9% sodium chloride before use), Dysport® (Clostridium botulinum type A toxin haemagglutinin complex with human serum albumin and lactose in the formulation), available from Ipsen Limited, as a powder to be reconstituted with 0.9% sodium chloride before use), and MyoBloc™ (an injectable solution comprising botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride at about pH 5.6, available from Elan Corporation).


  1. Elderly skin and its rejuvenation: products and procedures for the aging skin, Marcia Ramos‐e‐Silva et. al., Journal of Cosmetic Dermatology, 6, 40–50
  2. Botulinum Toxin Production Method, United States Patent 7189541.

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