HOW DOES IT WORK? Chemical denervation, Dermal

fillers

Constantinos Laskarides DMD, DDS, PharmD, FICD ORAL & MAXILLOFACIAL SURGERY

Assoc i a te Professor , TUFTS UNIVERSITY

At tend i ng Surgeon, TUFTS MEDICAL CENTER

D i p l omate , Amer i can Board of Ora l & Max i l l o fac ia l Surgery

Fe l l ow, Amer i can Assoc i a t i on of Ora l & Max i l l o fac ia l Surgeons

Fe l l ow, Amer i can Co l l ege of Ora l & Max i l l o fac ia l Surgeons

Clostridium botulinum

• Anaerobic, Gram+ spore forming bacterium, commonly found in soil, that under certain conditions can germinate and create a toxin.

• First recognized and isolated in 1895 by Emile van Ermengem from home cured ham implicated in a botulism outbreak. Named Bacillus botulinus (botulus = sausage in Latin)

• Symptoms: double vision, blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness. Progress to paralysis of the respiratory muscles, arms, legs, and trunk.

Botulinum Toxin A

• Exotoxin. Total of 7 subtypes of botulinum toxin: A, B, C1, D, E, F, G

• Types A, B, and E are most commonly associated with botulism in human

• Only A & B available for clinical use. Type A for cosmetic use.

• Type B has been studied for cosmetic use, it generally

elicits more pain upon injection. Faster onset but shorter

duration of action than type A

• Definition: one unit of toxin is the calculated median intraperitoneal LD50 for a group of female Swiss Webster mice, weighting 18-20 g.

• Activity: 20 Units/nanogram

• The estimated LD50 for humans is 40 U/kg or 2,500-3,000 U (25-30 vials) for a 70 kg man.

• The potency Units of BOTOX are specific to the preparation and assay method utilized. They are not interchangeable with other preparations of botulinum toxin products

• Duration of effect: 3-4 months

Botulinum Toxin A

History of the Cosmetic Use of Botulinum A Exotoxin

• 1973

– Dr. Alan Scott (Ophthamologist) treated for the first time strabismus. Smith Kettlewell Research

Institute in San Francisco.

• 1979

– First preparation of Botulinum toxin-A. Limited FDA-approval to use of BTX-A for strabismus.

• 1985

– FDA-approval for blepharospasm.

• 1987

– Dr. Jean Carruthers. One patient with blepharospasm pointed out the smooth, relaxed

appearance she had after treatment of the glabellar area

• 1989

– Oculinum Inc. (Dr. Scott’s company) acquired by Allergan, Inc. Product named Botox. FDA-

approval for hemifacial spasm.

• 1992 – Carruthers JDA, Carruthers JA. Treatment of glabellar frown lines with C. botulinum-A exotoxin.

J Dermatol Surg Oncol. 1992;18(1):17–21

• 2003 – FDA-approval for treatment of glabellar rhytides.

Mechanism of action

• BTA is a dichain molecule with a

heavy and light chain

• The heavy chain is responsible for

selective binding of the neurotoxin

to cholinergic nerve terminals, and

the light chain acts intracellular to

prevent acetylcholine release

• BTA blocks neuromuscular

transmission by binding to acceptor

sites on motor or sympathetic nerve

terminals, entering the nerve

terminals, and inhibiting the release

of acetylcholine.

The action of botulinum toxin on the neuro-muscular junction. A. S. V. Burgen et al.

J Physiol. 1949 August 1; 109(1-2): 10–24

Hathaway. In: Hauschild AHW, ed. Clostridium Botulinum: Ecology and Control in Foods New York, NY: Marcel Dekker, Inc.;1993;54:pp 3-20.

Inoue et al. Infect Immun 1996;64:1589-1594. Lacy et al. Nat Struct Biol 1998;5:898-902

Non-toxic,

non-hemagglutinin (NTNH)

Hemagglutinin (HA)

Motor end plate

BTA blocks neuromuscular transmission by binding to acceptor

sites on motor or sympathetic nerve terminals, entering the

nerve terminals, and inhibiting the release of acetylcholine.

BTA cleaves synaptosomal membrane-associated protein 25 kDa SNAP-

25, a protein integral to the successful docking and release of

acetylcholine from vesicles situated within nerve endings.

Muscle atrophies - axonal sprouting may occur - extrajunctional

acetylcholine receptors may develop - reinnervation of the muscle

BOTOX® BOTOX®

Cosmetic/

Vistabel®/

Vistabex®

Dysport®/

Reloxin®

Dysport®

Cosmetic

Myobloc®/

NeuroBloc

®

NT-201/

XEOMIN®

PurTox®

Company Allergan Inc.

85% of

market

Allergan Inc. Ipsen Inc./

Medicis Inc.

Ipsen Inc./

Medicis Inc.

Solstice

Neuroscience

s Inc.

Merz

Pharmaceutic

al

Mentor

Corporation

Type Type A-Hall

strain

Type A-Hall

strain

Type A Type A Type B Type A-Hall

strain

Type A-Hall

strain

Approvals In over 75

countries

worldwide,

including US

and Canada

In over 16

countries,

including US,

Canada, Italy,

France

In over 65

countries;

approved in

US

Germany,

other

European

countries

Some

European

countries,

US, Canada

Germany,

other

European

countries,

Mexico,

Argentina,

US

None (clinical

trial phase)

BOTOX® BOTOX®

Cosmetic/

Vistabel®/

Vistabex®

Dysport®/

Reloxin®

Dysport®

Cosmetic

Myobloc®/

NeuroBloc

®

NT-201/

XEOMIN®

PurTox®

Active

Substance

(molecular

weight)

Botulinum

toxin type A

complex

(900kD)

Botulinum

toxin type A

complex

(900kD)

Botulinum

toxin type A

complex

(900kD)*

Botulinum

toxin type A

complex

(900kD)*

Botulinum

toxin type B

complex

(700kD)

Botulinum

toxin type A,

free from

complexing

proteins

(150kD)

Botulinum

toxin type A,

free from

complexing

proteins

(150kD)

Strength of

Action

(BTX-

A:Product)

1:1 1:1 1:2 - 1:4

(approximate)

1:2 - 1:4

(approximate)

1:50 - 1:100 1:1 1:1.5?

* The formulation contains complexes of variable size between

500kD-900kD

BOTOX® BOTOX®

Cosmetic/

Vistabel®/

Vistabex®

Dysport®/

Reloxin®

Dysport®

Cosmetic

Myobloc®/

NeuroBloc

®

NT-201/

XEOMIN®

PurTox®

Indications Blepharospas

m; cervical

dystonia;

glabellar

lines;

hyperhidrosis

Glabellar

lines Crow’s

feet

Blepharospas

m; cervical

dystonia

Glabellar

lines

Cervical

dystonia

Blepharospas

m; cervical

dystonia;

glabellar lines

in Argentina

Phase 3 for

glabellar

lines; Phase

1 for

spasmodic

torticollis/cerv

ical dystonia

Mode of

Action

SNAP-25

SNAP-25 SNAP-25 SNAP-25 VAMP SNAP-25 SNAP-25

Pharmaceuti

cal

Form

Powder

dissolved in

solution for

injection

Powder

dissolved in

solution for

injection

Powder

dissolved in

solution for

injection

Powder

dissolved in

solution for

injection

Solution Powder

dissolved in

solution for

injection

?

• SNAP-25 (synaptosomal associated protein with the molecular mass of 25kD) = An intracellular protein that is essential for synaptic vesicle transmission; it has been identified as the molecular target of BTX-A.

• VAMP (synaptobrevin) = A protein involved in synaptic vesicle movement that has been identified as the molecular target of BTX-B.

• ? = Data unavailable or unconfirmed

BOTOX® BOTOX®

Cosmetic/

Vistabel®/

Vistabex®

Dysport®/

Reloxin®

Dysport®

Cosmetic

Myobloc®/

NeuroBloc

®

NT-201/

XEOMIN®

PurTox®

Units/vial 100 100, 50 500 300 or 500 2,500; 5,000;

10,000

100 ?

Volume 10mL

maximum

1.25mL or

2.5mL

recommend

2.5mL

recommend

5mL

maximum

0.5mL; 1mL;

2mL

8mL

maximum

?

Re-

constitution

0.9% NaCl

solution

0.9% NaCl

solution

0.9% NaCl

solution

0.9% NaCl

solution

Prepared

solution,

dilutable

0.9% NaCl

solution

0.9% NaCl

solution

Storage 2-8°C or < -5°C

2-8°C or < -5°C

2-8°C 2-8°C 2-8°C do not freeze

Up to 25°C ?

Other BTX Products Under Global Development

• CBTX-A (Lanzhou Institute of Biological Products)

People’s Republic of China; marketed in Brazil as Prosigne®; bovine gelatin protein is added; potential to induce allergic reactions, or possibly bovine spongiform encephalopathy

• CNBTX-A (Nanfeng Medical Science and Technology Development Co., Ltd.)

neither licensed nor approved in any country, Shijiazhuang Hebei China

• Neuronox® (Medy-Tox Inc.)

South Korea; worldwide licensing of its cosmetic use has been acquired by Q-Med Inc. (Sweden)

• Topical • RT001 (Revance Therapeutics, CA) BTA Topical Gel. In Phase 3.

Has developed a proprietary platform that enables transcutaneous flux of large medicinal payloads

• Linurase (Prollenium Medical Technologies, Canada) topical BTA serum

New Generic Names FDA acknowledge that toxins are produced by different biological manufacturing

processes, obtained by different isolation and purification techniques, and

derived from different Clostridium batches

active neurotoxin is 150 kDa. formulations differ because of

presence or absence of complexing proteins.

Dilution and Storage of Botulinum Toxin

• Each vial of Botox is freeze-dried, has approx. 100 U (5 ng protein) of

toxin, 0.5 mg of human albumin, and 0.9 mg of sodium chloride (or 50

Units, 0.25 mg, and 0.45 mg) in a sterile, vacuum-dried form without a

preservative.

• Diluent: normal saline with no preservatives

• Technique: thoroughly evaporate the alcohol applied to the cap,

introduce the saline into the vial by vacuum and not by squirting, avoid

bubbles/foaming by not agitating the solution during dilution and filling

of the syringe, and do not shake the vial.

Dilution:

• 5cc of NS / vial, 1cc syringe with 30-gauge needle (2units

/ 0.1cc)

• This amount is more that recommended but permits even

distribution. Greater dilution decrease potency of Botox,

greater chance of toxin migration.

B O T O X (100u)

DILUENT

(NS)

CONCENTRATION U per

0.1ml

1ml 100 U/ml 10

2ml 50 U/ml 5

2.5ml 40 U/ml 4

4ml 25 U/ml 2.5

5ml 20 U/ml 2

8ml 12.5 U/ml 1.25

D Y S P O R T (300u)

DILUENT

(NS)

CONCENTRATION U per

0.1ml

1ml 300 U/ml 30

2ml 150 U/ml 15

2.5ml 120 U/ml 12

4ml 75 U/ml 7.5

5ml 60 U/ml 6

8ml 37.5 U/ml 3.75

Dilution and Storage of Botulinum Toxin

Dilution and Storage of Botulinum Toxin

• Un-reconstituted BTA can be stored up to 36 months (indefinitely) in a

conventional freezer

• Storage: once reconstituted, should be stored at 2-8 ºC (refrigeration).

The length of viability of reconstituted product is subject to debate.

Most clinicians (46%) store it for up to 1 week, 23% for 24 hours, 15%

for 2 weeks, 8% for 10 days, and 8% for 4 days. (Allergan: 24h)

Allergan: BOTOX Cosmetic vials are for single-use only. Discard any remaining solution.

• Up to 6 weeks at 4 ºC without loosing efficacy

Hexsel et al. Dermatol Surg. 2009;35(6):993

Tray for neurotoxin treatment

• Neurotoxin vial

• Normal Saline (0.9% NaCl), single use, injectable,10cc vial

• Alcohol pads

• 2 x 2 gauzes

• 5cc syringe

• 1cc syringes

• 30G or 31G needles

• Disposable ice-packs

• EMLA or Lidocaine 4% cream (optional)

• Skin marker (optional)

• Gloves, mask

• Autologous fat transfer was first described by Neuber in 1893

• Medical-grade liquid silicone was used successfully in thousands of

patients in the United States and elsewhere since the 1960s for off-label

cosmetic purposes

• 1981: FDA approval of bovine collagen

• Medically useful hyaluronic acid was first isolated and purified in 1962. In

the 1980s, cross-linking was perfected to support the stability and

viscoelastic properties.

• Hylaform (Inamed Corporation), the first hyaluronic acid skin filler; this

product was released in Europe 1996.

Dermal Fillers Historical overview

• Resorbable materials (collagen, hyaluronic acid,

hydroxyethyl methacrylate, dextran, and polylactic

acid) enzymatically metabolized or phagocytized

slowly, with minimal histologic reaction.

• Permanent and semipermanent fillers (last longer

than 1 to 2 years) such as silicone, Teflon and

PMMA. Some particles may have an irregular surface that cannot be phagocytosed and may eventually form foreign body

granulomas.

Particles

Fillers

Hyaluronic acid

• In humans, HA serves as the ground substance of

dermis, fascia, and most fluid mediums because of its

viscoelastic properties. A 70kg man has 15gr dry HA.

• Natural HA has a half-life in tissue of only 1 to 2 days

before undergoing aqueous dilution and enzyme

degradation.

• There is no antigenic specificity for species or

tissues; and thus, it has a low potential for allergic or

immunogenic reaction

• Cross-linking native HA forms larger, more stable

molecules with longer residue time in tissues.

• Consists of polyanionic di-saccharide units of glucuronic acid and

N-acetyl-glucosamine connected by alternating β 1–3 and β 1–4 bonds

• Cross-linking agents: butanediol-diglycidyl-ether (BDDE)

divinyl sulfone

bis-epoxides

Trace amounts may be found in the final product

• Biphasic gels:

Selectively sized particles of cross-linked HA suspended within non-cross-linked HA used

as a carrier

• Monophasic gels:

Varied degree of high-molecular weight HA and low-molecular weight HA.

Varied degree of cross-linking

Adjusted hardness, size, hydration

Hyaluronic acid

Hyaluronic acid

Elastic modulus (G’) of HA fillers

• Animal derived (Hylaform)

• Non-animal derived (streptococcus species biofermentation) (Restylane,

Juvederm)

• Total HA concentration in filler: ~24mg/ml

• Percentage of uncross-linked: HA ~10%

• Formulation: smooth cohesive viscoelastic gel

• Needle: 27 or 30G

• Highly hydrophilic. Swelling until equilibrium for bound water is reached.

Depends on concentration, cross-link density, gel hydration

Hyaluronic acid

• No single parameter defines the use of a HA filler

• Results are also depended on implantation technique and the response of

the biological host

Hyaluronic acid

Genzyme

Biosurgery

Captique Hyaluronic

Acid

FDA approved

Anika

Therapeutics

Hydrelle – Elevess

Hyaluronic

Acid with

Lidocaine

FDA approved

Fibrocell

Science

Laviv azficel-T autologous fibroblast cellular product FDA approved

Aesthetic

Factors

Selphyl

Platelet Rich

Plasma

Matrix

“Vampire facelift”

CE mark

Tray for dermal filler (HA) treatment

• Dermal filler package

• Alcohol pads

• 2 x 2 gauzes

• Disposable ice-packs

• EMLA or Lidocaine 4% cream (optional)

• Skin marker (optional)

• Dental syringe for local anesthesia, disposable needles

• Local anesthetic ampules (lidocaine, etc)

• Topical anesthesia gel swabs for mucosa (optional)

• Gloves, mask

Non-animal Stabilized Hyaluronic Acid (NASHA)

Calcium Hydroxylapatite Facial Filler (Radiesse)

• Ca-HA microspheres (25-45 μm) suspended in a gel

carrier.

• Gel is dissipated in vivo and replaced with collagen,

whereas Ca-HA remains at the site.

• Average longevity: 18 – 24 mo.

• FDA approved (12-06)

Calcium Hydroxylapatite Facial Filler (Radiesse)

Radiographic appearance

• Non-animal, non-bacterial, not chemically cross-linked

absorbable polymers

• Protein-free, reduced immunogenic potential

• Ultra-smooth, non-particulate gel

• Better rheologic properties in higher concentrations

• Currently available to practitioners in the European

Union

Carboxymethylcellulose (CMC) and polyethylene oxide (PEO) (Laresse)

• Approved for the treatment of of HIV-associated facial lipoatrophy

• Sculptra Aesthetic approved by FDA for nasolabial folds and other facial wrinkles .

• Elicit the stimulation of fibroblasts, which in turn produce collagen, adding volume to the skin gradually.

• 3 injection sessions over a few months.

• Results can last up to 2 years.

• Reconstituted at room temperature with 3 to 5mL of sterile water for injection, 2 to 72 hours before administration

Poly-L-lactic acid (PLLA) (Sculptra)

• PMMA microspheres (20% by volume), 30–50 μm in diameter, suspended in 3.5% bovine collagen solution (80% by volume) and 0.3% lidocaine.

• collagen carrier is absorbed within 1 month after injection and completely replaced by the patient’s own connective tissue within 3 months. “PERMANENT” FILLER

• Bovine collagen allergy testing is required, 4 months prior.

Polymethylmethacrylate (PMMA) (ArteFill)

http://rds.yahoo.com/_ylt=A0WTefZXCddL4yYAj9KjzbkF/SIG=13aiie004/EXP=1272470231/**http://www.realself.com/files/imagecache/full/Artefill-Syringe-01-wcopyright-26358.jpg

Growth of dermal fillers

Growth of dermal fillers

Growth of dermal fillers

Growth of dermal fillers

Growth of dermal fillers