Botulinum Toxins
Botulinum Toxins
Cosmetic and Clinical Applications
Edited by
Joel L. Cohen, MD (FAAD, FACMS)
Director
AboutSkin Dermatology and DermSurgery
Greenwood Village and Lone Tree
CO, USA
David M. Ozog, MD (FAAD, FACMS)
Chair, Department of Dermatology
C.S. Livingood Chair in Dermatology
Director of Cosmetic Dermatology
Division of Mohs and Dermatological Surgery
Henry Ford Hospital
Detroit, MI, USA
This edition first published 2017
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Library of Congress Cataloging-in-Publication Data
Names: Cohen, Joel L. (Dermatologist), editor. | Ozog, David M., editor.
Title: Botulinum toxins : cosmetic and clinical applications / edited by Joel L. Cohen, David M. Ozog.
Description: Chichester, UK ; Hoboken, NJ : John Wiley & Sons Inc., 2017. | Includes
bibliographical references and index.
Identifiers: LCCN 2017004474 (print) | LCCN 2017005656 (ebook) | ISBN
9781444338256 (cloth) | ISBN 9781118661864 (Adobe PDF) | ISBN 9781118661857 (ePub)
Subjects: | MESH: Botulinum Toxins, Type A–therapeutic use | Cosmetic Techniques
Classification: LCC RL120.B66 (print) | LCC RL120.B66 (ebook) | NLM QV 140 | DDC 615.7/78–dc23
LC record available at
Cover image: (Left) © SEBASTIAN KAULITZKI/Gettyimages; (Right) © iconogenic/Gettyimages
Cover design: Wiley
Set in 10/12pt WarnockPro by Aptara Inc., New Delhi, India
10 9 8 7 6 5 4 3 2 1
v
Contents
List of Contributors
vii
About the Companion Website
xiii
Video Table of Contents
xv
History of Botulinum Toxin for Medical and Aesthetic Use
1
Alastair Carruthers, FRCPC and Jean Carruthers, MD (FRCS(C), FRCOphth)
Anatomy and Aesthetic Principles
13
Timothy M. Greco, MD (FACS), Ch´erie M. Ditre, MD, and David M. Ozog, MD (FAAD, FACMS)
Botulinum Toxin: From Molecule to Medicine
37
Conor J. Gallagher, PhD and Alan Ackerman, PhD
Myobloc
53
Neil S. Sadick, MD (FACP, FAAD, FAACS, FACPh) and Suveena Manhas-Bhutani, MD
Abobotulinumtoxin: Development and Aesthetic Usage
65
Gary D. Monheit, MD
IncobotulinumtoxinA (Xeomin®/Bocouture®)
79
Ulrich K¨uhne, MD (DALM) and Matthias Imhof, MD (DALM)
Future Injectable Toxins
97
Michael H. Gold, MD
Reconstitution, Dilution, Diffusion, and Migration of Botulinum Toxin
109
Murad Alam, MD (MSCI, MBA), Hayes B. Gladstone, MD, and David M. Ozog, MD (FAAD, FACMS)
Patient Selection
121
Ryan M. Greene, MD (PhD, FACS), John P. Arkins, BS, and Steven H. Dayan, MD (FACS)
Treatment of the Glabella
133
Neal D. Varughese, MD (MBA) and David J. Goldberg, MD (JD)
Treatment of the Forehead
147
Joel L. Cohen, MD (FAAD, FACMS) and Ramin Fathi, MD
vi
Contents
Treatment of the Periocular Area – Crow’s Feet, Brow, and Bunny Lines
165
Girish S. Munavalli, MD (MHS, FACMS), Anthony V. Benedetto, DO (FACP, FCPP),
Brian S. Biesman, MD (FACS), and Carolee M. Cutler Peck, MD
Contouring of the Lower Face and of the Lower Leg and Calf
177
Mee young Park, MD (PhD), Dennis A. Porto, MD, and Ki Young Ahn, MD (PhD)
Treatment of the Perioral Area
191
Shawn Allen, MD (FAAD, FACMS), Roberta Sengelmann, MD, and Rachel Simmons, MD (FAAD)
Neck Rejuvenation
199
Koenraad De Boulle, MD, Lakhdar Belhaouari, MD, and Julia D. Kreger, MD
Correction of Facial Asymmetry
213
Scott Rickert, MD (FACS), Lesley F. Childs, MD, and Andrew Blitzer, MD (DDS, FACS)
Complications and Diffusion
221
Matteo C. LoPiccolo, MD, Farhaad R. Riyaz, MD, and David M. Ozog, MD (FAAD, FACMS)
Combination Therapy of Botulinum Toxin with other Nonsurgical Procedures
233
Amy Forman Taub, MD and Lauren Fine, MD (FAAD)
Peri-Procedure Botulinum Toxin for Skin Cancer Patients and Scars
253
Timothy Corcoran Flynn, MD, Molly C. Powers, MD, and David M. Ozog, MD (FAAD, FACMS)
Achieving a Natural Look
263
D´oris Hexsel, MD, Camile L. Hexsel, MD (FAAD, FACMS), and Carolina Siega, BSc
Special Considerations in Darker Skin
275
Cher´e Lucas Anthony, MD and Marta I. Rendon, MD (FAAD, FACP)
Axillary Hyperhidrosis
285
Ada Regina Trindade de Almeida, MD, Joel L. Cohen, MD (FAAD, FACMS),
and Chinobu Chisaki, MD
Primary Focal Palm, Sole, Craniofacial, and Compensatory Hyperhidrosis
299
Dee Anna Glaser, MD and Adam R. Mattox, DO (MS)
Topical Botulinum Toxin
317
Richard G. Glogau, MD and Eileen Axibal, MD
Exciting New Uses of Botulinum Toxin Type A: Dermatology/Dermatologic
Surgery and Beyond
329
Donna Bilu Martin, MD (FAAD) and Stephen Mandy, MD (FAAD)
Modulating Affect and Mood with Botulinum Toxin Injections: Psychosocial
Implications of Neuromodulators
345
James L. Griffith, MD (MSci), Kevin C. Smith, MD (FRCPC (DERM)), and Murad Alam,
MD (MSCI, MBA)
OnabotulinumtoxinA (Botox®) in Dermatology
357
Jason J. Emer, MD, Eileen Axibal, MD, Ellen S. Marmur, MD (FAAD), and Heidi Waldorf, MD
Index
369
vii
List of Contributors
Alan Ackerman, PhD
Master Medical Scientific Liaison, Retired
Ackerman LLC
Greeley, USA
Ki Young Ahn, MD (PhD)
Director
Dr. Ahn’s Aesthetic & Plastic Surgical Clinic
Daegu, South Korea
Murad Alam, MD (MSCI, MBA)
Professor and Vice-Chair
Department of Dermatology
Section of Cutaneous and Aesthetic Surgery
Departments of Dermatology
Otolaryngology, and Surgery
Northwestern University
Chicago, USA
Shawn Allen, MD (FAAD, FACMS)
Director and Founder
Dermatology Specialists
Boulder, USA
Assistant Clinical Professor
University of Colorado
Department of Dermatology
Boulder, USA
Ada Regina Trindade de Almeida, MD
Medical Assistant
Dermatology Clinic
Hospital do Servidor P´ublico Municipal de
S˜ao Paulo
S˜ao Paulo, Brazil
Cher´e Lucas Anthony, MD
Medical Director
Rendon Center for Dermatology and
Aesthetic Medicine
Boca Raton, USA
Voluntary Faculty
Dermatology and Cutaneous Surgery
University of Miami, Miller School of
Medicine
Miami, USA
John P. Arkins, BS
DeNova Research
Chicago, USA
Eileen Axibal, MD
Department of Dermatology
University of Colorado
Aurora, USA
Lakhdar Belhaouari, MD
Director
Centre de Chirurgie Esth´etique et Medecine
Esth´etique Jules Guesde
Toulouse, France
Anthony V. Benedetto, DO (FACP, FCPP)
Clinical Professor of Dermatology
Perelman School of Medicine
University of Pennsylvania
PA, USA
Medical Director
Dermatologic SurgiCenter
Philadelphia and Drexel Hill
PA, USA
viii
List of Contributors
Brian S. Biesman, MD (FACS)
Assistant Clinical Professor Ophthalmology
Dermatology, Otolaryngology
Vanderbilt University Medical Center
Nashville, TN, USA
Donna Bilu Martin, MD (FAAD)
Dermatologist, Premier Dermatology
Aventura, USA
Volunteer Professor of Dermatology and
Cutaneous Surgery
Miller School of Medicine University of
Miami
Miami, USA
Andrew Blitzer, MD (DDS, FACS)
Director
NY Center for Voice and Swallowing
Disorders
Senior Attending Physician
St. Luke’s/Roosevelt Hospital
Professor of Clinical Otolaryngology
Columbia University College of Physicians
and Surgeons
New York, USA
Alastair Carruthers, FRCPC
Clinical Professor
Department of Dermatology and Skin
Science
University of British Columbia
Vancouver, Canada
Jean Carruthers, MD (FRCS(C), FRCOphth)
Clinical Professor
Department of Ophthalmology and Visual
Sciences
University of British Columbia
Vancouver, Canada
Lesley F. Childs, MD
Attending Physician
Assistant Professor of Laryngology,
Neurolaryngology, and Professional Voice
UT Southwestern, Dallas, TX
Chinobu Chisaki, MD
Medical Assistant
Dermatology Clinic
Hospital do Servidor P´ublico Municipal de
S˜ao Paulo
S˜ao Paulo, Brazil
Joel L. Cohen, MD (FAAD, FACMS)
Director
AboutSkin Dermatology and DermSurgery
Greenwood Village and Lone Tree
Colorado, USA
Associate Clinical Professor
University of Colorado Department of
Dermatology
Denver, USA
Assistant Clinical Professor
University of California Irvine Department
of Dermatology
Irvine, USA
Carolee M. Cutler Peck, MD
Ophthalmic and Plastic and Reconstructive
Surgeon
SouthEast Eye Specialists
Knoxville, USA
Steven H. Dayan, MD (FACS)
Clinical Assistant Professor of
Otolaryngology
Chicago Centre for Facial Plastic Surgery
University of Illinois Chicago
Chicago, USA
Koenraad De Boulle, MD
Aalst Dermatology Clinic
Aalst, Belgium
Ch´erie M. Ditre, MD
Associate Professor
Department of Dermatology
University of Pennsylvania School of
Medicine
Philadelphia, USA
List of Contributors
ix
Jason J. Emer, MD
Cosmetic Dermatology and Body
Contouring
Private Practice
Beverly Hills, CA
Ramin Fathi, MD
Resident Physician
Department of Dermatology
University of Colorado
Aurora, USA
Lauren Fine, MD (FAAD)
Associate Dermatologist & Cosmetic Fellow
Advanced Dermatology, LLC
Chicago, USA
Timothy Corcoran Flynn, MD
Clinical Professor of Dermatology
University of North Carolina at Chapel Hill
Chapel Hill, USA
Medical Director
Cary Skin Center
Cary, USA
Conor J. Gallagher, PhD
Executive Director Medical Affairs
Facial Aesthetics
Allergan plc
Irvine, USA
Hayes B. Gladstone, MD
Gladstone Clinic
San Ramon, USA
Dee Anna Glaser, MD
Professor and Interim Chairman
Department of Dermatology
Saint Louis University School of Medicine
Saint Louis, USA
Richard G. Glogau, MD
Clinical Professor of Dermatology
University of California San Francisco
USA
Michael H. Gold, MD
Medical Director
Gold Skin Care Center
Nashville, USA
David J. Goldberg, MD (JD)
Clinical Professor of Dermatology
Department of Dermatology
Icahn School of Medicine at Mount Sinai
New York, USA
Skin Laser and Surgery Specialists of New
York and New Jersey
New York, USA
Timothy M. Greco, MD (FACS)
Clinical Assistant Professor
Department of Otolaryngology-Head and
Neck Surgery
Division of Facial Plastic Surgery
University of Pennsylvania School of
Medicine
Philadelphia, USA
Ryan M. Greene, MD (PhD, FACS)
Director
Plastic Surgery & Laser Center
Fort Lauderdale, USA
James L. Griffith, MD (MSci)
Dermatology Resident
Department of Dermatology
Henry Ford Hospital
Detroit, USA
Camile L. Hexsel, MD (FAAD, FACMS)
Dermatologist and Dermatologic Surgeon
Madison Medical Affiliates
Mohs Surgery
Glendale and Waukesha
USA
D´oris Hexsel, MD
Dermatologist and Dermatologic Surgeon
Brazilian Center for Studies in Dermatology
Porto Alegre, Brazil
x
List of Contributors
Matthias Imhof, MD (DALM)
Board Certified Dermatologist and
Allergologist
Aesthetische Dermatologie im Medico Palais
Bad Soden, Germany
Julia D. Kreger, MD
University of Colorado Dermatology
Colorado, USA
Ulrich K¨uhne, MD (DALM)
Board Certified Dermatologist and
Allergologist
Aesthetische Dermatologie im Medico
Palais
Bad Soden, Germany
Matteo C. LoPiccolo, MD
Henry Ford Health System
Department of Dermatology
Detroit, USA
Stephen Mandy, MD (FAAD)
Volunteer Professor of Dermatology and
Cutaneous Surgery
Miller School of Medicine University of
Miami
Miami, USA
Premier Dermatology
South Beach Dermatology
Miami Beach, USA
Suveena Manhas-Bhutani, MD
Sadick Dermatology and Research
New York, USA
Ellen S. Marmur, MD (FAAD)
Director, Marmur Medical
Mount Sinai School of Medicine
Department of Dermatology
New York, USA
Adam R. Mattox, DO (MS)
Micrographic Surgery & Dermatologic
Oncology Fellow
Department of Dermatology
Saint Louis University School of Medicine
Saint Louis, USA
Gary D. Monheit, MD
Total Skin and Beauty Dermatology Center
PC Private Practice
Associate Clinical Professor
Department of Dermatology
Department of Ophthalmology
University of Alabama at Birmingham
Birmingham, USA
Girish S. Munavalli, MD (MHS, FACMS)
Medical Director, Dermatology, Laser, and
Vein Specialists of the Carolinas, PLLC
Charlotte, USA
David M. Ozog, MD (FAAD, FACMS)
Chair, Department of Dermatology
C.S. Livingood Chair in Dermatology
Director of Cosmetic Dermatology
Division of Mohs and Dermatological
Surgery
Henry Ford Hospital
Detroit, MI, USA
Mee young Park, MD (PhD)
Department of Neurology
Yeungnam University
College of Medicine
Daegu, South Korea
Dennis A. Porto, MD
Department of Dermatology
Henry Ford Hospital
Detroit, USA
Molly C. Powers, MD
Dermatology Senior Resident
Department of Dermatology
Henry Ford Hospital
Detroit, USA
Marta I. Rendon, MD (FAAD, FACP)
Medical Director, Rendon Center for
Dermatology and Aesthetic Medicine
Boca Raton, USA
Voluntary Associate Clinical Professor
University of Miami
Dermatology Department
Miami, USA
List of Contributors
xi
Scott Rickert, MD (FACS)
Attending Physician
Assistant Professor of Otolaryngology,
Pediatrics, and Plastic Surgery
NYU Langone Medical Center
New York, USA
Farhaad R. Riyaz, MD
Henry Ford Health System
Department of Dermatology
Detroit, USA
Neil S. Sadick, MD (FACP, FAAD, FAACS, FACPh)
Clinical Professor
Weill Cornell Medical College
Cornell University
New York, USA
Roberta Sengelmann, MD
President and Owner
Santa Barbara Skin Institute
Associate Clinical Professor
UCI Dermatology
Santa Barbara, USA
Carolina Siega, BSc
Biologist
Brazilian Center for Studies in Dermatology
Porto Alegre, Brazil
Rachel Simmons, MD (FAAD)
Dermatologist
Dermatology Specialists
Boulder, USA
Kevin C. Smith, MD (FRCPC (DERM))
Private Practice Dermatologist
Niagara Falls
Ontario, Canada
Amy Forman Taub, MD
Director
Founder
Advanced Dermatology, LLC
Assistant Professor
Northwestern University Medical School
Chicago, USA
Assistant Clinical Professor
Northwestern University
Lincolnshire, USA
Neal D. Varughese, MD (MBA)
Skin Laser and Surgery Specialists of New
York and New Jersey
New York, USA
Heidi Waldorf, MD
Mount Sinai School of Medicine
Department of Dermatology
New York, USA
xiii
About the Companion Website
Don’t forget to visit the companion website for this book:
This site hosts valuable video materials to enhance your learning:
Dr Cohen and Dr Ozog present several patient cases, focusing on patient evaluation, prepa-
ration for toxins, and specific injection techniques. Each patient is appraised carefully and
optimal injection techniques are discussed, along with methods for avoiding adverse effects,
and ways to minimize injection points and related bruising. One week follow up videos will
highlight optimization of results.
xv
Video Table of Contents
1. Introduction from Dr Joel L. Cohen and
Dr David M. Ozog
2. Discussion on reconstitution techniques
from Dr Joel L. Cohen and Dr David M.
Ozog
Patient Cases
Patient 1, 66-year old female, Dr Joel L.
Cohen, Dr David M. Ozog
a)
Evaluation
b)
Glabellar Complex
c)
Lateral Canthal Area
d)
One Week Follow Up
Patient 2, 43-year old female, Dr Joel L.
Cohen, Dr David M. Ozog
a)
Evaluation
b)
Forehead
c)
Glabellar Complex
d)
Lateral Canthal Area
e)
One Week Follow Up
Patient 3, 69-year old female, Dr Joel L.
Cohen, Dr David M. Ozog
a)
Evaluation
b)
Glabellar Complex
c)
Lateral Canthal and Infraorbital Area
d)
One Week Follow Up
Patient 4, 48-year old female, Dr Joel L.
Cohen, Dr David M. Ozog
a)
Evaluation
b)
Glabellar Complex
c)
Lateral Canthal Area
d)
One Week Follow Up
Patient 5, 72-year old female, Dr Joel L.
Cohen, Dr David M. Ozog
a)
Evaluation
b)
Glabellar Complex
c)
Lateral Canthal Area
d)
One Week Follow Up
Patient 6, Treatment of Platysmal Bands in
female, Dr Koen De Boulle (narrated by
Dr Dennis A. Porto)
Patient 7, Dr David M. Ozog
a)
Depressor Septi Nasi
b)
Mentalis
and
Depressor
Anguli
Oris
c)
Platysmal Bands
d)
One Week Follow Up for Platysmal
Bands
Patient 8, Treatment of Lower Face and Neck
in 63-year old female patient, Dr Gary
D. Monheit (narrated by Dr Dennis A.
Porto)
History of Botulinum Toxin for Medical and Aesthetic Use
Alastair Carruthers, FRCPC1 and Jean Carruthers, MD (FRCS(C), FRCOphth)2
1Clinical Professor, Department of Dermatology and Skin Science, University of British Columbia, Vancouver, Canada
2Clinical Professor, Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, Canada
Sausage Poisoning
In the late 1700s in Europe, outbreaks of a
deadly illness from contaminated foods swept
across the continent, fueled in part by the
poverty from the Napoleonic War (1795–
1815) that led to unsanitary food produc-
tion [1]. The primary source of food-borne
illness of the time: smoked blood sausages.
One of the biggest outbreaks occurred in
1793 in Wildebrad, Southern Germany; by
1811, the Department of Internal Affairs of
the Kingdom of W¨urtemberg named “prus-
sic acid” as the culprit in sausage poison-
ing [2]. Intrigued, the district medical offi-
cer and poet, Dr. Justinus “W¨urst” Kerner
(1786–1862), began what would become a
lifelong quest to uncover the mysteries of
the poison. He would later be considered
the godfather of botulinum toxin (BoNT)
research for his early, intensive work. In 1817
and 1820, Kerner identified and described
the first accurate descriptions of botulism (a
term coined in 1871 from the Latin botu-
lus, meaning “sausage”) [2, 3]. In 1822, he
compared contaminated sausage ingredients
and concluded that the toxin must occur
in fat, leading him to call the suspicious
substance “sausage poison,” “fat poison,” or
“fatty acid,” and published the first complete
monograph of the “fatty toxin” from blood
sausages [2].
In his monograph, Kerner described the
symptoms of botulism – including vom-
iting, intestinal spasms, mydriasis, ptosis,
dysphagia, and respiratory failure – and
recommended methods for the treatment
and prevention of food poisoning. Through
animal
and
self-experimentation,
Kerner
observed that the toxin developed under
anaerobic conditions and was lethal in small
doses. Since the effects of this blood poi-
son were similar to atropine, scopolamine,
nicotine, and snake venom, Kerner surmised
that sausage poison was likely biological
in nature – remarkable in that microscopic
pathogens had not yet been discovered at that
time – and interrupted signal transmissions
within the peripheral and autonomic ner-
vous system. Indeed, some would call Kerner
prophetic: he suggested that small amounts
of this sausage poison might be used to lower
sympathetic nervous system activity asso-
ciated with movement disorders (i.e., treat
St. Vitus’ dance or Sydenham’s chorea, a dis-
order characterized by jerky, uncontrollable
movements, either of the face or of the arms
and legs) and hypersecretion of bodily fluid,
as well as to treat ulcers, delusions, rabies,
plague, tuberculosis, and yellow fever [4].
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:
Botulinum Toxins
Identification of C. botulinum
Microbiologist Professor Emile Pierre van
Ermengem
(1851–1922)
trained
under
Robert
Koch,
who
discovered
anthrax,
tuberculosis, and cholera and was the first
researcher to prove that microorganisms
could cause disease in animals [5]. In 1897,
Van Ermengem identified the bacterium
Clostridium
botulinum
(originally
called
Bacillus botulinus) as the causative agent of
botulism after examining postmortem tissue
of patients in Belgium who had contracted
gastroenteritis and died from eating raw,
salted pork [6]. Over the next twenty years,
different strains of the bacterium that pro-
duced serologically distinct types of toxins
were
recognized;
these
were
eventually
classified alphabetically into seven serotypes
(A, B, C1, D, E, F and G) [7]. In 1928, Dr.
Herman Sommer (University of California,
San Francisco) isolated the most potent
serotype – BoNT type A (BoNTA) – in
purified form as a stable acid precipitate,
paving the way for future studies [8].
Biological Weapon of Warfare
During the First World War, Germany
unsuccessfully attempted to produce chem-
ical and biological weapons. As World War
II approached, the American government
learned that multiple countries were engaged
in bio-warfare programs. In response, and
on orders from President Franklin Roosevelt,
the US National Academy of Sciences and
Fred Ira Baldwin, chairman of the bacte-
riology department of the University of
Wisconsin,
gathered
bacteriologists
and
physicians
in
a
laboratory
named
Fort
Detrick (Maryland). The purpose of Fort
Detrick: the investigation of dangerous infec-
tious bacteria and toxins to use as offensive
and defensive biological weapons [1].
In 1946, Carl Lamanna and James Duff
developed concentration and crystallization
techniques for the toxin that were subse-
quently used by Dr. Edward J. Schantz, a
young US army officer stationed at Fort Det-
rick to produce the first BoNTA lot for human
use (the basis of the later clinical product) [9,
10]. The US Office of Strategic Services (OSS)
developed a plan using Chinese prostitutes to
assassinate high-ranking Japanese officials via
gelatin capsules containing the newly puri-
fied BoNTA. The government abandoned the
plan when test donkeys that received the cap-
sules survived [1]. Ironically, though BoNT
today is considered one of the deadliest poi-
sons in the world – 1 g has the potential to
kill 1 million people – the toxin is not an ideal
biological weapon, since large amounts must
be ingested and mortality rates vary).
In 1972, President Richard Nixon signed
the Biological and Toxic Weapons Conven-
tion, effectively putting an end to all inves-
tigations on biological agents for use in
war. Schantz took his research to the Uni-
versity of Wisconsin, where he produced a
large amount of BoNTA (batch 79–11) that
remained in clinical use until December of
1997 [11].
Human Experimentation
Clinical use of the toxin began in the late
1960s and early 1970s, when Dr. Alan Scott
(Smith-Kettlewell Eye Research Foundation,
San Francisco; Figure 1.1) began experiment-
ing with BoNTA, supplied by Dr. Schantz,
and other chemical agents in monkeys, with
the hope that one of the compounds could
be used for the nonsurgical treatment of stra-
bismus in humans [12, 13]. Scott published
his first primate studies proving that BoNTA
could weaken extraocular muscles in 1973,
and postulated that the toxin could be used
for a wide variety of musculoskeletal disor-
ders and spasticity, even before conducting
any human studies [13, 14]. In 1978, Scott
received Food and Drug Association (FDA)
approval to begin testing small amounts
of the toxin (then named Oculinum) in
human volunteers; his landmark paper, pub-
lished in 1980 [15], showed that intramuscu-
lar injections of BoNTA could correct gaze
1 History of Botulinum Toxin for Medical and Aesthetic Use
Figure .Dr Alan Scott, the original user of
botulinum toxin A initially in monkeys and then in
humans, seen in 2010.
misalignment in humans. In 1989, one year
after manufacturer Allergan Inc. (Irvine,
CA) acquired the rights to distribute Scott’s
Oculinum in the United States, BoNTA
was approved for the nonsurgical correc-
tion of strabismus, blepharospasm, hemifa-
cial spasm, and Meige’s syndrome in adults,
and clinical use expanded to include the treat-
ment of cervical dystonia and spasmodic tor-
ticollis [13, 16, 17]. Shortly thereafter, Aller-
gan bought Scott’s company and renamed the
toxin. Botox® was born.
The Cosmetic Connection
In the mid-1980s, Dr. Jean Carruthers, an
ophthalmologist
in
Vancouver,
Canada,
noticed that her patients injected with
BoNTA for blepharospasm experienced a
reduction in glabellar rhytides, and discussed
the findings with both Scott and her der-
matologist spouse, Dr. Alastair Carruthers,
who was attempting to soften the forehead
wrinkles of his patients using soft-tissue
augmenting agents available at that time.
Intrigued by the possibilities, the Carruthers
used
the
toxin
experimentally
in
their
receptionist’s
forehead
and
subsequently
published the first report of BoNTA for the
treatment of glabellar frown lines in 1992 [18]
(Figure 1.2). Other reports soon followed
[19, 20], including the first double-blind,
placebo-controlled study for the treatment of
hyperkinetic facial lines [21].
Properties, Mechanism of Action,
and Clinical Effect
Clostridium
botulinum
is a rod-shaped,
gram-positive anaerobic bacterium. Of the
seven serotypes, A, B, and E are commonly
involved in human botulism [22]. BoNT is
a high-molecular-weight protein of 150,000
daltons with nonconvalent proteins pro-
tecting it from digestive enzymes, making
it a lethal cause of food poisoning [1]. The
symptoms of botulism include disturbances
in vision, speech, and swallowing, with
asphyxia and death sometimes occurring
18–36 hours after ingestion (mortality rate:
10–65%) [22].
Researchers gained an understanding of
mechanism of action in the late 1940s, when
they discovered that BoNT blocks neuro-
transmitter release at the neuromuscular
junction [23]. The follow-up discovery in
the mid-1950s that BoNT blocks the release
of acetylcholine from motor nerve endings
when injected into hyperactive muscles led
to a renewed interest in the neurotoxin as a
potential therapeutic agent [3].
Although all seven serotypes block neuro-
muscular motor transmission by binding to
receptor sites on motor nerve terminals and
inhibiting the release of acetylcholine, pro-
ducing temporary chemodenervation of the
muscles, each differs with regard to cellular
mechanism of action and clinical profile [24,
25]. The commercially available subtypes –
type A (BoNTA) and type B (BoNTB) –
are
both
150 kDa
dichain
polypeptides
Botulinum Toxins
(a)
(b)
(c)
(d)
Figure .The Carruthers’first patient treated in the glabella area for cosmetic reasons alone. Seen (a) before
frowning; (b) after frowning; (c) before at rest; (d) after at rest.
comprising heavy and light chains linked by
disulfide bonds. The light chain of BoNTA
cleaves to a 25 kDa synaptosomal associated
protein (SNAP-25), a protein integral to the
successful docking and release of acetyl-
choline from vesicles situated within nerve
endings, while the light chain of BoNTB
cleaves
to
vesicle-associated
membrane
protein (VAMP or synaptobrevin). This
difference may be responsible for some of
the differences witnessed in the clinical
effect of the subtypes [12]. When injected
intramuscularly at therapeutic doses, BoNT
produces temporary chemical denervation of
the muscle, resulting in a localized reduction
of muscle activity. The process of cellular
recovery after injection of BoNT is only par-
tially understood. Initial recovery of muscle
contraction is accompanied by collateral
sprouting of active terminal buds near the
parent terminal. However, research indicates
that these new sprouts are only transitory;
neurotransmission is eventually restored at
the original nerve ending, accompanied by
the elimination of the dispensable sprouts
[26], suggesting that treatment with BoNT
does not permanently alter the neuromuscu-
lar junction. Recommended doses of injected
neurotoxin do not result in systemic clinical
effects in patients without other neuromus-
cular dysfunction. Studies of human and
animal tissue show that in the first 2 weeks
postinjection with BoNTA, the target muscle
begins to atrophy, with changes in individual
muscle fibers [27]. The paralytic effect of
the toxin is dose-related, with initial effects
occurring within 2–3 days and peaking
approximately 1–2 weeks after treatment
[28]. Atrophy continues for approximately
4 weeks before stabilizing; clinical recovery of
function occurs 3–6 months posttreatment
[29]. There is an area of denervation associ-
ated with each point of injection due to toxin
spread of about 1–1.5 cm (diameter, 2–3 cm).
Repeated injections can extend the clinical
effect for up to 12 months [29]; it is possible
1 History of Botulinum Toxin for Medical and Aesthetic Use
that over the course of treatment, individuals
alter their habitual use of muscles that cause
expression
lines.
Long-term
remodeling
of the dermis and epidermis that helps to
sustain the cosmetic effects also occurs in
most individuals, because the tissue is no
longer subjected to the same forces of muscle
contraction.
A Multitude of Formulations
Until recently, one product – at least for
cosmetic purposes – dominated the mar-
ket:
onabotulinumtoxinA
(Botox®/Botox
Cosmetic®/Vistabel®/Vistabex®;
Allergan,
Inc., Irvine, CA). Now, however, a host of
other agents have joined the original for-
mulation to fight the signs of aging. Of the
formulations of BoNTA available or in devel-
opment, the original, onabotulinumtoxinA,
is the most recognized and discussed in peer-
reviewed literature. Botox Cosmetic, which
was approved by the US FDA in 2002 for the
treatment of glabellar rhytides [30], has gone
on to receive approval for 20 indications
in more than 75 countries [31]. Now three
formulations of botulinumtoxin type A are
approved for cosmetic use in North America.
The original onabotulinumtoxin A has been
joined by abobotulinumtoxinA (Dysport®)
and IncobotulinumtoxinA (Xeomin®). Ini-
tially approved in over 65 countries for
therapeutic
indications
(Dysport®;
Ipsen
Ltd., United Kingdom/Medicis, Scottsdale,
AZ; and Azzalure® in 15 European countries;
Galderma,
France),
abobotulinumtoxinA
received FDA approval for cosmetic applica-
tions in North America in 2009 (Dysport®;
Ipsen Ltd). Although produced from the
same serotype, abobotulinumtoxinA differs
from onabotulinumtoxinA in purification
procedures,
dosing,
injection
schedules,
and clinical effect [32]. Units of abobo-
tulinumtoxinA are less powerful than those
of
onabotulinumtoxinA;
most
cosmetic
injectors use a multiple of two to three
times the number of units. Overall, abobo-
tulinumtoxinA is safe and well tolerated
[33, 34]. A third BoNTA (Xeomin®/NT-201;
Merz Pharmaceuticals, Frankfurt, Germany)
is approved for therapeutic indications in
Germany and other European countries,
the United States, Canada, Mexico, and
Argentina, and has been approved for the
treatment of glabellar rhytides in Argentina
and the United States. Clinically, Xeomin and
onabotulinumtoxinA appear to behave in a
similar fashion, with equal levels of potency,
safety, and duration of effect [35–40]. Xeomin
is free of complexing proteins, which some
believe may result in purer formulations
with greater efficacy and a reduced risk of
sensitization and antibody formation [37].
One formulation of BoNT type B (BoNTB)
is also available in North America. Rimabo-
tulinumtoxinB
(Myobloc®/NeuroBloc®;
Solstice Neurosciences Inc./Eisai Co., Ltd.)
was FDA-approved in 2000 for the treat-
ment of cervical dystonia but has been used
off-label to treat facial wrinkles with some
success [41–44]. BoNTB works faster than
but does not last as long as BoNTA [45],
although duration has been shown to be
dose dependent [46]. BoNTB tends to diffuse
more widely than BoNTA and injections can
be more painful and may lead to additional
side effects [45]; however, a close examina-
tion of several doses found all to be safe and
effective for cosmetic use [46].
Cosmetic Applications
Hyperkinetic lines result from the repeated
contraction of muscles perpendicular to the
wrinkles. Weakening or relaxing these mus-
cles with BoNTA can smooth these lines,
including horizontal lines on the forehead
(from frontalis contraction), vertical lines in
the glabellar region between the eyebrows
(caused by the corrugator muscles), hori-
zontal creases across the bridge of the nose
(from procerus contraction), “crow’s feet” and
lateral lines along the lower eyelid (caused
by contraction of the lateral orbicularis
oculi), and perioral lines (from contraction of
the orbicularis oris). Deep grooves or folds
Botulinum Toxins
(a)
(b)
Figure .Treatment with 25 units to each masseter muscle: (a) before and (b) after.
elsewhere that are exacerbated by muscle
activity are also amenable to treatment.
Patients 30–50 years of age may be most
responsive to BoNTA, because their wrin-
kles are more likely to be caused by muscle
activity than by the loss of skin elasticity that
occurs during aging. Clinicians now use the
neurotoxin to treat a variety of hyperkinetic
facial lines in the upper face, including crow’s
feet, horizontal forehead lines, and glabellar
rhytides, as well as folds and lines in the lower
face, neck, and chest with a high level of effi-
cacy and patient satisfaction [12,47–52].
Facial Sculpting
Facial rejuvenation with BoNT has expanded
to involve a more artistic shaping and sculpt-
ing of the face. Now, in addition to targeting
simple dynamic rhytides, careful injection of
the toxin can be used to lift and shape the
brow [53], widen the eyes [12, 54], correct
facial asymmetry due to nerve palsies [55],
dystonias [17, 20], surgery [56], or trauma
[57], and to reduce muscle thickness of the
jaw in patients with masseteric hypertrophy
(Figure 1.3) [58–61].
Adjunctive Therapy
BoNT is used increasingly in combination
with other facial rejuvenation procedures,
such as soft-tissue augmentation [28, 62–66]
and laser or light-based therapies [12,28,67–
71], particularly for the treatment of deeper,
more static rhytides and folds. BoNT is also
used during surgery to prolong or enhance
the aesthetic results and as an aid in wound
healing and minimizing scars (Figure 1.4) [12,
73–77].
(a)
(b)
Figure .Scar forehead (a) shortly after injury and (b) 3 months after BTX. Source: Carruthers 1992. Reproduced
with permission of Lippincott Williams & Wilkins.
1 History of Botulinum Toxin for Medical and Aesthetic Use
Therapeutic Applications
Intramuscular injections of BoNTA have
become the treatment of choice for a num-
ber of disorders characterized by muscular
hyperactivity, such as strabismus [15], ble-
pharospasm and hemifacial spasm [17], cer-
vical dystonia [78], focal dystonia (writer’s
cramp) [79], and spasticity due to stroke [80,
81], and cerebral palsy [82]. In addition, the
ability of BoNT to block acetylcholine release
from autonomic nerve endings innervating
glandular tissue or smooth muscle has led to
investigation of its use for other indications,
including Frey’s syndrome [83] and hyper-
hidrosis [84–89], as well as various gastroin-
testinal, genitourinary, and sphincter disor-
ders [90], dyshidrotic hand eczema [91, 92],
and allergic rhinitis [93, 94]. Flushing of the
face and chest can be successfully treated
with BoNT due to its ability to regulate blood
vessel constriction [95, 96]. Clinicians con-
tinue to investigate the use of BoNT for the
treatment of chronic pain disorders, includ-
ing chronic lumbar [97], temporomandibular
dysfunction [98], myofascial [99], and neu-
ropathic pain [100], although the toxin’s effi-
cacy in the treatment of headache disorders
is under debate [101]. More recent research
includes applications of BoNT to relieve the
pain of arthritis [102,103].
Future Directions
It is interesting to note that what once began
as a potential – rather daring – treatment
for a single disorder has translated into a
worldwide phenomenon. And one cannot
help but wonder what Justinus Kerner would
think of his “sausage poison” now that so
consumed his time and became his life’s
research. BoNT has become the treatment
of choice for smoothing hyperkinetic lines
and shaping the face, alone or in combination
with other rejuvenating procedures. Thera-
peutic applications include a variety of move-
ment, pain, autonomic nervous system, and
gastrointestinal and genitourinary disorders,
among others. Current recruitment for clin-
ical trials includes everything from arthritis
and clubfoot to acne and depression, with
new products emerging or on the horizon.
Indeed, BoNT seems to have invaded nearly
every aspect of clinical medicine, at least in
some way, and there is no doubt that the
range of indications will only continue to
expand.
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Anatomy and Aesthetic Principles
Timothy M. Greco, MD (FACS),1 Ch´erie M. Ditre, MD,2 and David M. Ozog, MD (FAAD, FACMS)3
1Clinical Assistant Professor, Department of Otolaryngology-Head and Neck Surgery, Division of Facial Plastic Surgery,
University of Pennsylvania School of Medicine, Philadelphia, USA
2Associate Professor, Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, USA
3Chair, Department of Dermatology; C.S. Livingood Chair in Dermatology; Director of Cosmetic Dermatology, Henry Ford Hospital, Detroit, MI, USA
Anatomy of Youthful versus
Aging Skin
Apparent age is readily judged by several fac-
tors including the presence and numbers of
facial wrinkles, dyschromia, and skin laxity
among other anatomic aging skin changes.
To understand treatment with neuromodula-
tors, it is important for the reader to familiar-
ize themselves with basic facial skin anatomy
as well the anatomy of aging skin and its
underlying structures. The neurotoxin injec-
tor can then precisely target the intended
underlying muscles being treated with an
understanding of the expected improvement
from the toxin as well as the potential need
for adjuvant therapies.
Youthful skin is characteristically smooth
in texture, having a dewy luster, even tone,
high elasticity, and pleasant balanced con-
tours due to appropriate tissue volume.
Rhytides are absent or minimal at rest. Youth-
ful skin is by definition, normal or unaltered
in anatomical configuration and histology.
Aging skin, in contrast, exhibits surface
irregularities such as textural roughness, sal-
lowness, pigment alterations, and inelasticity
as well as the appearance of wrinkles/rhytids.
In addition, subsurface changes, namely loss
of volume due to dermal atrophy, fat atrophy
and redistribution, and biometric volume loss
due to deeper compartmental changes such
as bony and cartilaginous resorption, ensue.
Aging skin is viewed as an alteration of the
skin’s normal anatomy.
Wrinkles themselves are one modification
of the aging process, but nevertheless are
typically the most characteristic hallmark.
Despite this, there are few studies dedicated
to defining wrinkles both clinically and
histologically. These factors have muddied
the anatomic and histologic definition of
wrinkles.
Kligman attempted to define wrinkles by
histologic examination of 58 patients with
a variety of wrinkle types from the cheeks,
crow’s feet, temporal frown lines, upper verti-
cal lip lines and other body areas of crinkling
skin such as the abdomen and the back of the
neck. He concluded that wrinkles are not a
histological entity, as the microanatomic fea-
tures did not distinguish them from their sur-
rounding skin, but rather a configurational
change due to mechanical stresses on the
skin. He noted that these changes occurred
more prominently in actinically damaged
regions due to deterioration of the elas-
tic fibers. Kligman proposed that the facial
frown lines occurred primarily through mus-
cular contraction and that the facial mus-
cles are inserted into the overlying skin. Their
muscular contraction throws the skin into
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:
Botulinum Toxins
folds because while the muscles can contract,
the skin does not. He stated that in youth,
dynamic expression lines disappear imme-
diately when the muscles relax since elastic
fibers are not yet altered; however, muscu-
lar contractions on a degraded dermal matrix
result in permanent wrinkles [1].
Bosset et al. analyzed the histological fea-
tures of the pre-auricular wrinkle compared
to retroauricular skin in 16 subjects undergo-
ing face lifts (ages 36–94 years). In doing so,
they defined four types of facial skin depres-
sions and classified them based on their
depths: (i) invaginations of the skin struc-
tures from 250 to 400 μm deep were folds
such as nasolabial and melolabial, (ii) perma-
nent wrinkles are invaginations of the skin
structures of 100 μm deep, (iii) reducible
wrinkles (frown lines, crow lines and preau-
ricular wrinkles) are seen in vivo but not
after histologic processing, and (iv) microre-
lief (nonspecific frown lines due to aging)
are shallow depressions (10–30 μm deep)
involving the horny and granular layers of
the epidermis [2]. Histological analysis of the
epidermis and dermis of the skin specifically
under
and
surrounding
permanent
and
reducible wrinkles actually demonstrated
normal skin morphology; however, deep per-
manent wrinkles showed a heavier accumula-
tion of basophilic fibers representing actinic
elastosis – which involves the entire depth of
the superficial dermis in contrast to reducible
wrinkles. This suggests that the development
of wrinkles could be furthered by actinic elas-
tosis and the disappearance of microfibrils
and collagen fibers at the dermal–epidermal
junction (DEJ). The authors concluded that
a diminished skin resistance at the DEJ and
upper superficial dermis due to sun damage
is a prerequisite for wrinkle formation.
However, Pierard and Lapiere [3] con-
cluded that the histological changes neces-
sary to produce a wrinkle began with the
changes in the hypodermal connective tissue
septae below the wrinkles and not actually
in the epidermis or dermis. Underneath each
wrinkle there were hypodermal septae that
were shorter and thicker than those outside
the grooves. They postulated that wrinkling
was the result of skin remodeling of the
hypodermal connective tissue by repetitive
mechanical
stimuli
induced
by
striated
muscle [3].
Facial Fat Compartments
Neuromodulators are frequently injected
with filling agents as part of a comprehen-
sive facial rejuvenation plan. Knowledge of
facial fat compartments is paramount for
expert injectors to properly restore volume
with filling agents. Knowledge of cutaneous
anatomy in general is necessary to visualize
areas in patients that need to be relaxed
(neuromodulators), volume restored (fillers),
and resurfaced (lasers/chemical peels). This
should be the goal of every injector to create
a natural, youthful, and rested appearance.
Understanding the following key principles
regarding fat compartments will help to
further that goal (Figure 2.1).
Forehead and Temporal Fat Compartments
Subcutaneous forehead fat is composed of
three anatomical units – central, middle and
lateral temporal/cheek fat.
Figure .Facial fat compartments. Source: Rohrich
2007 [14]. Reproduced with permission of Wolters
Kluwer Health, Inc.
2 Anatomy and Aesthetic Principles
The central fat is in the midline region of
the forehead and is bounded inferiorly at the
nasal dorsum and is bounded laterally by a
dense fascial plane that appears to be the
central temporal septum. This dense fibrous
band septum underlies the fascia of the
frontalis muscle and inserts into the dermis.
The middle forehead fat sits on either side
of the midline central fat, and is medial to the
superior temporal septum. The middle fore-
head fat compartment is bound inferiorly by
the orbicularis oculi retaining ligament of the
superior orbit.
The lateral temporal/cheek compartment
connects the lateral forehead fat to the lat-
eral cheek and cervical fat. The lateral tem-
poral cheek septum spans the forehead with
the neck [14].
The Orbital Fat Compartment
There
are
also
three
periorbital
fat
compartments-superior
orbital
fat,
infe-
rior orbital fat, and lateral orbital fat. Loss of
periorbital fat contributes to the appearance
of rhytids and lax skin, which are treated
with combinations of toxins, fillers, and
resurfacing.
The superior orbital fat is bounded by the
orbicularis oculi retaining ligament of the
superior orbit. The orbicularis retaining liga-
ment is circumferential and spans the supe-
rior and inferior orbits blending into the
medial and lateral canthi; however, the supe-
rior and inferior compartments are distinct
from each other.
The inferior orbital fat is a thin layer that
lies directly below the inferior lid tarsus.
It is bounded inferiorly by the orbicularis
oculi retaining ligament or malar septum,
and medially and laterally by the respective
canthi.
The lateral orbital fat is bounded supe-
riorly by the inferior temporal septum and
inferiorly by the superior cheek septum. The
zygomaticus major muscle adheres to this
compartment. To elevate medial cheek fat or
jowl fat, one has to traverse the zygomaticus
muscle. There is the potential, while chasing
inferior crow’s feet wrinkles with botulinum
toxin, of inadvertently injecting the supe-
rior aspect of the zygomaticus muscle, which
would then result in depression of the cheek
and oral commisure.
The Nasolabial Fat Compartment
Prominence of the nasolabial fold is seen
in patients with more severe facial volume
loss. The nasolabial fold is one discrete unit
with distinct anatomical boundaries. The
nasolabial fat compartment lies immediately
anterior to the medial compartment of malar
cheek fat and overlaps jowl fat. It is bounded
superiorly by the orbicularis oculi retaining
ligament of the inferior orbit and by the sub-
orbicularis fat laterally. The inferior border
of the zygomaticus major muscle is teth-
ered to the nasolabial fat compartment. The
medial cheek septum separates the nasolabial
fat from the medial cheek fat.
The Cheek Fat Compartment
The Malar fat compartment is divided into
three units – medial, middle, and lateral tem-
poral cheek fat.
The medial cheek fat is lateral to the
nasolabial fold. It is bounded superiorly by
the orbicularis oculi retaining ligament of the
inferior orbit, laterally by the middle cheek
septum and inferiorly by jowl fat. The medial
cheek septum then separates the nasolabial
fat from the medial cheek fat.
The middle cheek fat lies between the
medial and lateral temporal-cheek compart-
ments and is anterior and superficial to the
parotid gland. The zygomaticus major muscle
adheres to its superior portion. The medial
and middle cheek fat compartments abut
each other but are clearly separated by the
middle cheek septum. Their septal bound-
aries fuse forming a dense fascial network
known as the zygomatic ligament.
The most lateral component of the cheek
fat is the lateral temporal-cheek compart-
ment. The lateral temporal cheek fat is
directly superficial to the parotid gland con-
necting the temporal fat to the cervical
Botulinum Toxins
subcutaneous fat. Anterior to this compart-
ment, the lateral cheek septum appears as
a vertical septal barrier. It is the first zone
encountered surgically during a face lift pro-
cedure after a preauricular incision moving
medially.
The Jowl Fat Compartment
Jowl fat is the most inferior facial fat.
Nasolabial fat and medial cheek fat are
located superiorly to jowl fat. Its medial
boundary is the depressor anguli oris (DAO)
muscle (lip depressor) while the inferior
boundary is the membranous fusion of the
platysma. The fusion point of the DAO and
the platysma is the mandibular retaining lig-
ament. It is not known how this particular
compartment behaves during the aging pro-
cess, but it is probably the most important
with regards to midfacial aging.
Superficial Musculoaponeurotic
System
The
superficial
musculoaponeurotic
sys-
tem (SMAS) is a complex fibromuscular
network which serves a distinct role in the
face. Superiorly, it is comprised of the galea
aponeurotica in the region of the forehead.
Here it divides and envelops the frontalis
muscle. Laterally, the fascia becomes the
temporoparietal fascia as it approaches the
zygomatic arch. It is here in the temporopari-
etal fascia where the temporal branch of the
facial nerve resides. This branch is in close
relationship to the periosteum of the zygo-
matic arch. In the cheek, there is a distinct
SMAS layer which becomes contiguous with
the platysma in the neck. Embryologically the
SMAS develops from an upper division com-
prising the frontalis, orbicularis oculi, lip ele-
vators, and the orbicularis oris. There is also a
lower division made up of the actual platysma
muscle along with the depressor anguli oris
and risorius [16].
The SMAS separates the subcutaneous
fat into superficial and deep layers. The
superficial layer, as mentioned, is divided into
fat compartments. In between these com-
partments are complex fascial condensations
forming a network of retaining ligaments. It is
this network that helps to transmit the forces
of the fascial musculature to the skin sur-
face – allowing for a myriad of complex facial
expressions which characterize the human
face as well as being responsible for the for-
mation of hyperfunctional rhytids.
The Facial Nerve
Found below the SMAS in the deep fat layer
is the facial nerve. It is this nerve that inner-
vates the distribution of the hyoid arch mes-
enchyme (second branchial arch) – which is
the embryologic precursor of the facial mus-
culature. The facial nerve (seventh cranial
nerve) arises from the stylomastoid foramen
of the base of the skull. As it exits the foramen
it immediately gives off a posterior auricu-
laris branch, which supplies the occipitalis,
posterior and superior auricular muscles. It
also gives off a branch to the posterior belly
of the digastric muscle and the stylohyoid
muscle. Next, as the nerve passes as a main
trunk anteriorly, it then divides into two main
divisions in the body of the parotid gland.
The upper division is comprised of the tem-
porofacial segment, and the lower division
is comprised of the cervicofacial segment.
However, as these divisions proceed more
anteriorly, extensive variability of branching
patterns may develop with variable patterns
of innervation ensuing. The temporal branch
runs in the temporoparietal fascia after
crossing over the zygomatic arch. It becomes
the frontal branch as it enters the lateral
and deep aspect of the frontalis muscle. The
temporal branch supplies the anterior and
superior auricular muscles and the superior
portion of the orbicularis oculi as well as the
corrugators. The corrugator muscles are also
supplied by a branch of the zygomatic nerve.
The zygomatic nerve supplies the lower
portion of the orbicularis oculi, the corru-
gators, the muscles of the nasal aperture
2 Anatomy and Aesthetic Principles
including the nasalis and the upper lip eleva-
tors. The buccal branch innervates the upper
lip sphincteric mechanism with contribu-
tions also innervating the upper lip elevators.
The marginal mandibular nerve innervates
the musculature of the lower lip: the orbicu-
laris oris, the lower lip depressors, depressor
anguli oris (DAO), depressor labii inferioris
(DLI) and mentalis. The cervical branch
innervates the platysma. There is extensive
and significant anastomosis between the
buccal and zygomatic branches. However,
the temporal and marginal branches lack
such anastomotic connections. As such,
injury to the temporal or marginal nerves
during surgery may result in longer lasting
paresis. Neurotoxin may be injected into the
contralateral musculature to maintain sym-
metry until the paresis resolves which may
or may not occur. For example, if a temporal
nerve is unilaterally compromised limiting
ipsilateral brow elevation, restoration of sym-
metry can occur if the neurotoxin is injected
into the contralateral frontalis muscle until
the temporal nerve function returns on the
affected side. The same principle applies
to injury of the marginal mandibular nerve
and treating the contralateral DAO and DLI
muscles to create a more symmetric smile
until nerve function returns. If the nerve has
been injured and not transected, function
usually returns within 6 months to 1 year.
Vasculature of the Face
The vasculature of the face is essentially com-
prised of branches from the internal and
external carotid arteries. Examples include
the supratrochlear, supraorbital, infraorbital
and mental arteries – which accompany their
corresponding nerves and exit the corre-
sponding foramina of the central face.
The external carotid artery feeds primar-
ily the maxilla, mandible, occipital and tem-
poral regions of the face. The main branches
of the external carotid artery include the
facial, internal maxillary, occipital, superficial
temporal, posterior auricular and transverse
facial arteries. The internal maxillary artery
branches into the infraorbital, the inferior
alveolar, which eventually becomes the men-
tal artery, and the posterior superior alveo-
lar arteries. The facial artery branches into
the superior and inferior labial arteries and
eventually becomes the angular artery as it
approaches the lateral aspect of the nose.
The internal carotid artery gives off the
ophthalmic artery, which then supplies the
supratrochlear
and
supraorbital
vessels.
Attention should be given to the watershed
area that exists between the supratrocholear
arteries in the glabellar region. This area of
the glabella is prone to necrosis if injecting
large volumes of fillers that, in turn, generates
external tamponade of the microvasculature
in this watershed region. Direct cannuliza-
tion of vessels has also occurred with fillers in
this area, resulting in complete intravascular
occlusion [17].
The venous structures of the face tend
to follow the arterial pattern, although the
venous pattern may have more variability and
less predictability. The venous plexus of the
lateral orbital and anterior temporal regions
drain into the sentinel veins. Care must be
taken when injecting neuromodulator agents
in the lateral orbital area for crow’s feet treat-
ments to avoid bruising in this region. Mag-
nification and enhanced lighting to identify
these vessels as best as possible, as well as
injecting tangentially and superficially cre-
ating dermal wheals, is the optimal way of
injecting this region with botulinum toxin to
try to avoid bruising.
Muscles of Facial Expression
To have a successful understanding of the
injection technique of neurotoxins for facial
reshaping and rhytids, it is imperative to
have a working understanding of the mus-
cles of facial expression. Thorough knowl-
edge of muscular anatomy also minimizes
complications and allows us to precisely tai-
lor injections to the patient’s aesthetic desires.
Knowing the origin, insertion, caliber or size,
Botulinum Toxins
Frontalis
Corrugator supercilli
Procerus
Orbicularis oculi
Depressor labii inferioris
Depressor anguli oris
Mentalis
Platysma
Levator labii superioris
alaeque nasi
Levator labii
superioris
Zygomaticus minor
Zygomaticus major
Levator anguli oris
Brow Depressors
Lip Depressors
Brow Elevator
Lip Elevators
Figure .Protagonist/antagonist muscles of facial expression. Redrawn based on an original drawing by
Margaret Ditr´e.
course, functional and aesthetic action of
each muscle is paramount to successfully
treating the facial muscles with neurotoxins.
Most facial muscles originate on the facial
skeleton and insert on the dermis of the
skin with assistance from the superficial mus-
culoaponeurotic system – thus transmitting
their forces to the overlying skin and signifi-
cant facial structures, such as the brows and
oral commissures.
The following is an in-depth look at the
anatomy of the facial musculature with clin-
ical correlation. The face will be divided
into upper, middle, and lower thirds. The
neck primarily has the platysma muscle as
its major contributor of facial expression and
will be discussed last. Although the discus-
sion involves individual analyses, it is impor-
tant to remember that an intricate interplay
exists between the muscles of facial expres-
sion. Each facial expression involves multi-
ple muscles, creating antagonist/protagonist
activity to result in a unique expression
(Figure 2.2).
The Upper Face
Before discussing the specific muscles of the
upper one-third of the face, it is critical to
understand the ideal aesthetic position of the
overlying skin and soft tissue, particularly the
brow. Much media attention has focused on
incorrect brow placement after botulinum
toxin injection over the past several years.
The terms “Nicholson brow,” “Spock brow” or
“mephisto-look” are now part of the vernacu-
lar of those familiar with cosmetic toxin injec-
tion, and the astute clinician understands
2 Anatomy and Aesthetic Principles
A
B
C
A
B
C
D
D
E
Female Brow
Male Brow
E
Figure .Female and male brow position.
Redrawn based on an original drawing by
David M. Ozog.
the importance of minimizing this type of
appearance.
There are more similarities than differ-
ences between the male and female brow (Fig-
ure 2.3). The edge of the eyebrow lies in a
vertical plane passing through the alar base.
The lateral eyebrow ends at an oblique line
drawn from the alar base through the lat-
eral canthus. The medial and lateral ends of
the eyebrow lie at the same horizontal level
in both males and females. The apex of the
eyebrow lies vertically above the lateral lim-
bus of the eye at approximately the junction
of the medial two thirds and the lateral third
of the eyebrow. The main differences are that
the eyebrow arches above the supraorbital
rim in women and lies at the rim in men and
the male eyebrow tends to be heavier and less
arched.
The muscles of the upper one-third of the
face are critical with respect to the shape and
position of the brows and the presence of
hyperfunctional rhytids (Table 2.1). These
rhytids include the transverse rhytids of the
forehead, the vertical and horizontal glabel-
lar frown lines, as well as lateral canthal
crow’s-feet. The shape and size of the palpe-
bral aperture is intimately associated with
function of the orbicularis oculi muscle. The
shape and position of the brow exemplifies
the antagonist and protagonist relationship
that occurs in the musculature of the upper
one-third of the face. The frontalis muscle
is a paired muscle that forms the anterior
part of the occipitofrontalis (epicranius) mus-
cle. It is connected to the posterior occipi-
talis muscle via the galea aponeurotica, and
therefore does not have a bony origin as most
facial muscles do. It inserts into the skin of
the lower forehead in the region of the brows
and interdigitates with the corrugator, pro-
cerus and orbicularis oculi muscles. It is this
interdigitation with these muscles that allows
it to have a unique role in determining not
only the position of the brow but also the
shape of the brow. The frontalis muscle is the
only elevator of the brow complex, and there-
fore should be treated conservatively when
using neurotoxins. Avoiding injection of neu-
rotoxin to the lower one-third of the frontalis
in general is helpful in many patients to pre-
venting brow ptosis and maintaining some
brow movement. The frontalis muscle is also
responsible for the creation of the transverse
rhytids of the forehead. These rhytids may
become deeper in time as the frontalis mus-
cle becomes more active to compensate for
eyelid ptosis. This hyperfunctional compen-
satory mechanism is seen in older patients
as they try to compensate for ptosis that is
occurring from the dis-insertion of the leva-
tor aponeurosis from the superior aspect of
Botulinum Toxins
Table .Muscles of the brow complex.
Muscle
Origin
Insertion
Action
Cosmetic
consequence
Eyebrow depressors
Corrugator
supercilli
Medial inferior aspect
of frontal bone near
nasofrontal suture
and medial superior
orbital rim
Skin above medial
brow
Adducts and
depresses medial
brow
Vertical frown
lines of glabella
Procerus
Inferior aspect of
nasal bones and
upper lateral nasal
cartlidges
Dermis of glabella
and lower mid
forehead
Downward pull
on skin of medial
forehead
Transverse
rhytids between
brows
Orbicularis oculi
– Orbital portion
Medial palpebral
ligament, nasal
portion of frontal
bone and frontal
process of maxilla
Cheeks, forehead
and temples;
interdigitates with
surrounding
muscles (frontalis
and corrugator
supercilli)
Depresses medial
and lateral aspets
of brow; adducts
brow
Lateral canthal
rhytids (crow’s
feet)
– Palpebral
portion:
preseptal and
pretarsal
Preseptal: medial
palpebral ligament
Pretarsal: medial
palpebral ligament
Preseptal: lateral
palpebral raphe
Pretarsal: lateral
orbital tubercle
Medial
attachments allow
for pumping
mechanism for
tears
Rhytids of lower
eyelid
Eyebrow elevators
Frontalis
Galea aponeurotica
Skin of lower
forehead;
interdigitates with
corrugator,
procerus and
orbicularis oculi
Elevates brows
Transverse
rhytids of
forehead
the tarsal plate. Although the frontalis has
been classically described as a paired mus-
cle complex, it frequently is contiguous across
the forehead, thus explaining the continu-
ous rhytids that are seen in many patients
(Figure 2.4). When there is a dehiscence of
the muscle, a double arched transverse rhytid
pattern may be seen in those patients when
raising their eyebrows.
The lateral extent of the frontalis muscle
is seen at the temporal fusion line – which
is also the medial extent of the temporalis
muscle. This is usually the region where the
anterior hairline exists. This relationship is
important to identify because it explains why
lateral brow ptosis occurs with aging. This
will be discussed further in detail later.
The corrugator supercilli muscle originates
on the medial inferior aspect of the frontal
bone near the nasofrontal suture as well as the
medial superior orbital rim. It runs laterally
and superiorly to insert onto the skin above
the medial brow. Its insertion can be seen as
a dimple in the skin above the medial brow as
the patient frowns (Figure 2.5). This may be
seen medial, at or lateral to the mid-pupillary
line – depending on the length of the muscle.
There may, in fact, be multiple insertions in
this region. This corruguator supercilli mus-
cle runs deep to the procerus, frontalis, and
2 Anatomy and Aesthetic Principles
(a)
(b)
Figure .Transverse rhytids of the forehead (a) relaxed and (b) in animation showing continuous horizontal
rhytids across forehead consistent with contiguous frontalis muscle.
Figure .Insertion of corrugator supercilli onto
dermis of skin of medial brow as depicted by the
dimple.
orbicularis oculi medially and becomes more
superficial as it courses laterally [18]. The
supraorbital and supratrochlear neurovascu-
lar bundles run through the belly of the cor-
rugator muscle. This explains why migraine
headaches may be treated with an injection
of the corrugator muscle since the supra-
trochlear nerve can be considered one of
the triggers of migraine headaches [19]. The
corrugator muscle functions to adduct and
depress the medial brow thus creating verti-
cal frown lines of the glabella (Figure 2.6). The
corrugator is innervated by the temporal and
zyogomatic branches of the facial nerve.
The procerus muscle is a triangular shaped
muscle which originates from the inferior
aspect of the nasal bones and upper lateral
nasal cartilages and inserts onto the der-
mis of the skin of the glabella and lower
mid forehead. It pulls the skin of the medial
forehead downward thus creating transverse
Figure .Vertical frown lines of glabella created by
the action of corrugator supercilli.
rhytids seen between the brows (Figure 2.7).
Although having been described previously,
the existence of a distinct depressor super-
cilli muscle was not identified in the recent
anatomic dissections of Macdonald, et al.
[20].
Figure .Horizontal frown lines of glabella created
by the action of the procerus muscle.
Botulinum Toxins
Orbital
orbicularis oculi
Preseptal
orbicularis oculi
Pretarsal
orbicularis oculi
Figure .Divisions of the orbicularis oculi muscle into orbital and palpebral (preseptal and pretarsal)
segments. Redrawn based on an original drawing by Margaret Ditr´e.
The orbicularis oculi muscle possesses not
only critical functional aspects to the eye
including eye closure, but also has significant
aesthetic influence on the upper one-third of
the face. The muscle is divided into orbital
and palpebral portions (Figure 2.8). The
palpebral portion is then divided into the
preseptal and pretarsal segments–named
for the muscle fibers that lie over the orbital
septum and tarsal plate respectively. This
portion is responsible for the gentle closure
of the palpebral fissure of the eye. The orbital
portion originates from three areas, which
include the medial palpebral ligament, nasal
portion of the frontal bone and frontal
process of the maxilla. The orbital portion
extends beyond the orbital rim to the cheeks,
forehead and temples and interdigitates with
surrounding muscles, including the frontalis
and
corrugator
supercilli
muscles.
Care
should be taken when injecting the lower
crow’s-feet rhytids so as not to weaken the
zygomatic group and cause ptosis to the oral
commissure. It is important to recognize
that the orbital portion of the orbicularis
oculi is considered an accessory muscle
to smiling, and is responsible for forceful
closure of the palpebral fissure of the eye.
The orbital portion of the orbicularis oculi
muscle is also responsible for the creation
of lateral canthal rhytids (crow’s-feet), and
depressing the medial and lateral aspects of
the brow (Figure 2.9) as well as adducting it.
Its influence on depressing the lateral brow
owes to the fact that the orbital fibers extend
more laterally than the most lateral aspect of
the frontalis muscle, thus creating an unop-
posed downward force vector on the tail of
the brow. Chemodenervation of the vertical
fibers of the orbital portion of the orbicularis
oculi results in a lateral brow lift, as well as
improvement in the crow’s feet.Persistent
adduction of the glabella following neuro-
toxin injections may occur from recruitment
of the orbital portion of the orbicularis oculi.
The palpebral segment of the orbicularis
oculi is composed of preseptal and pretarsal
components. The preseptal portion overlies
the orbital septum and the pretarsal portion
overlies the tarsal plate as mentioned earlier.
2 Anatomy and Aesthetic Principles
(a)
(b)
Figure .(a) Contraction of the orbital portion of the orbicularis oculi resulting in (b) creation of lateral
canthal rhytids and depression of tail of brow.
The medial attachments of the preseptal
and pretarsal portions onto the anterior and
posterior lacrimal crests form a muscle sling
surrounding the lacrimal sac – which allows
for the pumping mechanism of tears into the
canalicular system. Injection of botulinum
toxin into the medial eyelid may interfere
with this pumping mechanism. Diffusion of
neurotoxin onto the medial orbicularis oculi
muscle may weaken lid tone and cause punc-
tal eversion and epiphora (tearing). Hyper-
trophy of the pretarsal orbicularis muscle
may be responsible for creating a bulge
underneath the ciliary margin of the lower
eyelid commonly referred to as the “jellyroll”
deformity, as well as a decreased palpebral
fissure (Figure 2.10). Unilateral decrease of
the palpebral fissure with a hypertonic pre-
tarsal orbicularis oculi muscle is responsible
for “Large eye/Small eye syndrome.” Appli-
cation of botulinum toxin to the pretarsal
Figure .Contraction of pretarsal orbicularis oculi
creating subtle roll of skin under lower eyelid lashes.
orbicularis in the mid papillary line 3mm
below the ciliary margin can improve this
appearance along with improving rhytids
of the lower eyelid, as well as providing
a subtle widening of the palpebral fissure
with smiling. Before injecting the pretarsal
orbicularis, a snap test or lid distraction test
should be performed to evaluate adequate
lid tone. Also, closely examining preinjection
photographs to rule out preexisting scleral
show is important in patient selection.
Patients with impending pseudo-herniation
of the lower lid orbital fat should be treated
with caution, since relaxation of the preseptal
orbicularis oculi may exacerbate this hernia-
tion along with interference of the lymphatic
drainage system of the lower eyelid. This
may also worsen malar festoons which are
defined as skin protrusion on the lateral
aspect of the malar region caused by aging
and inflammation. This injection should also
be cautiously considered in patients with dry
eye syndrome.
The dynamic antagonists of the orbicularis
oculi (lid depressor) in the eyelids are the lev-
ator palpebral superioris and Mueller’s mus-
cle (lid elevators). The levator palpebral supe-
rioris is responsible for the active opening of
the eye and has its origin on the lesser wing
of the sphenoid. In the region of the superior
orbital rim, a ligamentous structure known as
Whitnall’s ligament acts to convert the ante-
rior posterior vector force to a superior infe-
rior direction, thus providing the appropriate
mechanics for opening the eye [21]. The
Botulinum Toxins
levator palpebral superioris muscle converts
into a fibrous aponeurosis before inserting
onto the superior border of the upper tarsal
plate. Titration of botulinum toxin onto
the levator muscle may result in lid ptosis.
The orbital septum is a delicate fascial layer
that connects the arcus marginalis with the
inferior border of the tarsal plate. It acts as
a mechanical barrier to the spread of neu-
rotoxin to the levator palpebrae superioris.
Patients who have had previous blepharo-
plasty may have had this septum encroached
to remove eyelid fat, and therefore may be
theoretically more predisposed to spread
of toxin due to comprise of this anatomic
barrier. Mueller’s muscle runs underneath
the levator aponeurosis and has sympathetic
innervation. It is also an upper lid elevator
that can be stimulated pharmacologically to
improve lid ptosis. It provides 2mm of eleva-
tion to the upper eyelid and is responsible for
the correction of mild ptosis associated with
neurotoxin when pharmacologically stimu-
lated with a sympathetic agent (prescription
iopidine or even naphazoline hydrochloride).
The anatomic and mechanical relation-
ship of the frontalis, corrugator supercilli,
and procerus and orbicularis oculi create
a unique antagonistic/protagonistic relation-
ship which determines brow position and
shape (Figure 2.11). Thoughtful and careful
placement of neurotoxin can result in brow
elevation and brow reshaping by selectively
altering this relationship.
The Midface
The primary muscle associated with the nose
is the nasalis muscle (Table 2.2). This mus-
cle contains both an upper transverse por-
tion (which compresses the cartilage frame-
work of the nose including the upper lateral
cartilages and the lower lateral crura), and an
alar portion, the dilator naris (which dilates
the nares). Both are innervated by the buc-
cal branch of the facial nerve. The transverse
portion originates on the maxilla and inserts
with the opposite muscle via an aponeuro-
sis in the midline of the dorsum of the nose.
The dilator portion originates in the region of
the lateral incisor of the maxilla and inserts
onto the lower lateral crura. The dilator por-
tion, along with the medial part of the levator
labii superioris alaeque nasi muscle, widens
or dilates the nostril and can be injected for
those with excessive nasal flare (Figure 2.12).
The transverse portion is responsible for the
oblique expression wrinkles found over the
bony dorsum of the nose. It produces the so
called “bunny lines,” which are exaggerated
with scrunching up of the nose (Figure 2.13).
Neurotoxins can help soften these lines and
may help to improve the recruitment phe-
nomenon of the nasalis – which is sometimes
Frontalis
Procerus
Corrugator supercilli
Orbicularis oculi
Brow Depressors
Brow Elevator
Figure .Antagonist and protagonist relationship of musculature of upper one-third of face. Redrawn based
on an original drawing by Margaret Ditr´e.
2 Anatomy and Aesthetic Principles
Table .Additional facial musculature important in neurotoxin injection.
Muscle
Origin
Insertion
Action
Cosmetic consequence
Other
Nasalis
– transverse
portion
Maxilla
Midline of
dorsum of nose
Lifts nares
“Bunny lines”-oblique
expression wrinkles
over bony dorsum of
nose
– dilator
portion
Lateral incisor of
maxilla
Lower lateral
crura
Dilates nares
Flaring of nostrils
Orbicularis
oris
Maxilla and mandible
Skin and mucosa
of lips
Puckers lips;
brings lips
together
Vertical lip rhytids
Masseter
Zygomatic arch
Lateral surface of
the ramus and
angle of the
mandible
Mastication
Hypertrophy can result
in masculinizing of
lower face
Depressor
nasi septi
Type 1: orbicularis
oris
Type II: periosteum
of the premaxilla
below nasal spine
Type III: no
discernable muscle
Medial crural
footplates of the
lower lateral
cartilages
Aesthetics of
nasal tip and
upper lip with
smiling
Decreased nasal tip
projection, shortening
of upper lip, Animation
ptosis
Temporalis
Temporal fossa
Coronoid process
and ramus of
mandible
Primary muscle
of mastication
Hypertrophy can result
in an unpleasing bulge
with mastication in
lateral forehead region
seen when the corrugator and procerus mus-
cles are injected for glabellar frown lines.
The depressor nasi septi (DNS) muscle is
a delicate paired muscle that can have pro-
found effects on the aesthetics of the nasal
tip and upper lip with smiling (Figure 2.14).
Drooping of the nasal tip with smiling (ani-
mation ptosis), decreased tip projection and
shortening of the upper lip are the pri-
mary effects of this muscle on the nasal tip
(Table 2.2). Rohrich has described three vari-
ations of this muscle [22]. Type I originates
(a)
(b)
Figure .Dilation of the nares caused by stimulation of dilator naris and the dilator portion of the levator
labii superioris alaeque nasi.
Botulinum Toxins
Figure .Contraction of transverse portion of
nasalis muscle creating “bunny lines”.
and interdigitates with the orbicularis oris.
Type II originates on the periosteum of the
premaxilla below the nasal spine and has no
discernable relationship with the orbicularis
oris. Type III involves little if any discernable
muscle. The insertion of the DNS is on the
medial crural footplates of the lower lateral
cartilages. Injection of the depressor septi
muscle can at best result in a subtle upward
nasal tip rotation in repose. This is usually
performed in the midline of the vertex of the
nasolabial angle in the region of the ante-
rior nasal spine. With animation and smiling
there is less downward rotation of the nasal
tip with loss of tip projection when the DNS
is injected. In addition, there is less shorten-
ing of the upper lip with smiling.
The primary muscle group in the midface
is the elevators of the upper lip (Table 2.3).
Their insertions into the orbicularis oris par-
tially contribute to the formation of the
nasolabial fold. These muscles are primar-
ily innervated by the buccal branches of the
facial nerve. These muscles all insert into
the orbicularis oris, and primarily provide an
upward vector force to the upper lip and com-
missure (Figure 2.15) with the risorius draw-
ing the commissure more posteriorly.
The zygomaticus major muscle arises on
the zygoma and courses inferiorly and medi-
ally to insert onto the skin and mucosa of the
oral commissure in the region of the modi-
olus (a muscular condensation comprised of
multiple muscular insertions) as well as the
orbicularis oris muscle sphincter. It is a super-
ficial muscle that can be seen frequently on
raising of the skin flap during facelift. It, along
with the zygomaticus minor, lifts the oral
commissure superiorally and laterally. The
zygomaticus minor originates medial to the
zygomaticus major on the zygoma, and runs
anterior and medial to its larger counterpart
and inserts onto the skin, mucosa and orbicu-
laris oris muscle just medial to the zygomati-
cus major.
The levator labii superiorus originates
on the superior aspect of the maxilla just
below the margin of the infraorbital rim and
above the infraorbital foramen. It courses
medially and inferiorly to insert on the lat-
eral aspect of the orbicularis oris, skin and
mucosa of the lateral upper lip. As the name
(a)
(b)
Figure .Contraction of
depressor septi muscle with
resultant derotation (animation
ptosis) of nasal tip, decreased
tip projection and shortening of
the upper lip.
2 Anatomy and Aesthetic Principles
Table .Elevators and depressors of the lip.
Muscle
Origin
Insertion
Action
Cosmetic
Consequence
Lip Elevators
Levator labii
superiorus
Superior aspect of
maxilla below
infraorbital rim
margin and above
infraorbital
foramen
Lateral aspect of
orbicularis oris,
skin and mucosa
of lateral upper lip
Primary elevator
of lip
Contributes to the
smile and
nasolabial fold
formation
Levator anguli
oris
Canine fossa
Corner of mouth
Elevates angle of
mouth
Contributes to the
smile and
nasolabial fold
formation
Levator labii
superioris alaeque
nasi
Medial aspect of
maxilla
Skin, mucosa and
orbicularis oris of
medial upper lip
Primary elevator
of central upper
lip
Contributes to the
smile and
nasolabial fold
formation
Zygomaticus
major
Lateral zygoma
Corner of mouth
(modiolus)
Elevates oral
commisure
superiorly and
laterally
Contributes to the
smile and
nasolabial fold
formation
Zygomaticus
minor
Medial zygoma
Corner of mouth
(modiolus) just
medial to
zygomaticus
major
Elevates oral
commisure
superiorly and
laterally
Contributes to the
smile and
nasolabial fold
formation
Lip Depressors
Depressor anguli
oris
Body of mandible
between canine
and first molar
Skin, mucosa and
orbicularis oris at
oral commisure
(modiolus)
Primary
depressor of oral
commissure
Marionette folds,
parentheses
rhytids, prejowl
sulcus
Depressor labii
inferioris
Anterior aspect of
oblique line of
mandible
Skin, orbicularis
oris and mucosa
of lower lip
Depressor of
tubercle of lower
lip
Lower incisior
show
Mentalis
Mandible near
lateral incisor
Skin of chin
Protrudes lower
lip
Cobblestoning,
peau d’ orange
Platysma
Deltoid and
pectoral fascia
Mandible and
depressors of
lower lip
Depressor of lip
and corners of
mouth
Vertical bands,
transverse rhytids
of neck (necklace
lines)
implies, the levator labii superioris is the pri-
mary elevator of the upper lip.
The levator anguli oris originates in the
region of the canine fossa. It runs deep to
the other levators in the area, including the
zygomaticus group and levator labii superi-
orus. It inserts on the corner of the mouth
in the region of the modiolus and primar-
ily elevates this structure. The levator labii
superioris alaeque nasi muscle is a major
elevator of the central upper lip. It origi-
nates on the medial aspect of the maxilla
just below the infraorbital rim and medial to
the origin of levator labii superiorus. It runs
Botulinum Toxins
Levator labii superioris
alaeque nasi
Levator labii
superioris
Zygomaticus minor
Zygomaticus major
Levator anguli oris
Lip Elevators
Figure .Elevators of
the upper lip. Redrawn
based on an original
drawing by Margaret
Ditr´e.
Figure .Mona Lisa smile secondary to majority
of activity of zygomaticus major resulting in
elevation of corners of mouth.
inferiorly along the pyriform aperture. The
insertion is into the skin, mucosa and orbic-
ularis oris muscle of the medial upper lip.
In 1974, Rubin characterized the nature
of smiles into three types [23]. The first type
involves a majority of activity from the zygo-
maticus major muscle resulting in noticeable
elevation of the corners of the mouth and is
known as the “Mona Lisa smile” (Figure 2.16).
The second smile involves a significant com-
ponent of the levator labii superioris and
levator labii superioris alaeque nasi resulting
in elevation of the central upper lip. This is
known as the “canine smile” (Figure 2.17).
The third type involves the contraction
of both the lip elevators and depressors
resulting in display of the upper and lower
dentition. This smile is referred to as the “full
denture smile” (Figure 2.18).
Kane found the canine smile group, espe-
cially the gummy smile subset he calls the
“extreme canine smile,” were the patients
who benefited the most from neurotoxin
Figure .Canine smile caused by significant
activity of levator labii superioris and levator labii
superioris alaeque nasi.
injection, since relaxation of the levator labii
superioris alaeque nasi resulted in significant
improvement of the gummy display [24]. It
also helped improve the heaviness or sharp-
ness of the medial nasolabial fold. The injec-
tion technique involved one injection at the
superior aspect of the nasolabial fold in the
region of the pyriform aperture. Polo found
significant improvement in the gummy smile
with injection of the levator labii superioris,
Figure .Full denture smile resulting from
contraction of both lip elevators and depressors.
2 Anatomy and Aesthetic Principles
levator labii superioris alequae nasi, the over-
lap areas of the levator labii superioris and
zygomaticus minor muscles, as well as injec-
tion of the depressor septi nasi at the origin
of the orbicularis oris muscle [25]. All these
injections were performed with low doses
(1.25 units of onabotulinum toxin A, Botox).
The risorius plays no significant role in the
aesthetic application of neurotoxins. Unlike
the bony origin of other muscles in this
region, it arises from the layer of tissue above
the parotid gland and inserts onto the cor-
ner of the mouth and draws the corner of
the mouth laterally. The buccinator also plays
no significant role in the formation of hyper-
functional rhytids but does play a signifi-
cant role in mastication. Proper mastication
is achieved by the transfer of a bolus of food
over the molars, and this is aided by the
action of the buccinator muscle. The buccina-
tor muscle is also important in those patients
who play wind instruments, since it is respon-
sible for the mouth orientation necessary to
generate the force needed to play such instru-
ments. It originates from the posterior aspect
of the superior alveolar process and ptery-
gomandibular raphe and passes anteriorly to
insert onto the upper and lower portions of
the orbicularis oris muscle, skin, and mucosa
of the lateral aspects of the mouth.
The masseter muscle is not a muscle of
facial expression, but of mastication itself
(Table 2). It is derived from the first branchial
arch mesoderm as are all the muscles of mas-
tication and is innervated by the trigeminal
nerve. However, hypertrophy of this mus-
cle can have a profound effect on the shape
of the lower face, particularly masculinizing
the lower face in women by making the jaw-
line more “square.” The Asian face, particu-
larly Korean, has a greater propensity for this
appearance. This condition can also be asso-
ciated with temporomandibular joint (TMJ)
disease and bruxism (grinding of teeth at
night). The muscle originates on the zygo-
matic arch and angles posteriorly to insert
onto the lateral surface of the ramus and angle
of the mandible. This muscle has two parts –
which include a larger superficial belly and
a smaller deeper belly. The muscle is inner-
vated by the third division of the trigemi-
nal nerve and acts primarily to close the jaw
and also aids in adducting the jaw to the
contralateral side. Both actions are critical to
mastication.
The lower lip depressors play an integral
role in the function and aesthetics of the
lower one-third of the face (Table 3). Despite
the fact that fillers and volumization are pri-
marily the means of treatment in this area,
neurotoxins play a meaningful role in the
rejuvenation of the lower face. The key to suc-
cessful injection of neurotoxins into the lower
face is dependent upon a keen understand-
ing of the facial musculature in this region.
There is an intimacy and precision of action
of these muscles that is not seen in the upper
one-third of the face. This explains why accu-
rate injections of low doses of neurotoxins
are of paramount importance in the lower
face.
The depressor anguli oris is the primary
depressor of the oral commissure and con-
tributes to the dynamic component of the
marionette fold (Figure 2.19). This muscle
along with the depressor labii inferioris con-
tributes to the antagonistic action on the oral
commissure by depressing it (Figure 2.20).
The zygomaticus major, zygomatic minor and
levator anguli oris are the primary protag-
onists of the oral commissure. When the
antagonistic force vector is negated with neu-
rotoxins, the marionette fold improves in
Figure .Down turning of oral commissures with
formation of marionette folds by contraction of
depressor anguli oris. Notice also the formation of
the prejowl sulcus.
Botulinum Toxins
Depressor labii inferioris
Depressor anguli oris
Mentalis
Platysma
Figure .Depressors
of the lower lip. Redrawn
based on an original
drawing by Margaret
Ditr´e.
appearance and there is a conservative upturn
to the corner of the mouth due to a net posi-
tive protagonistic effect of the elevators of the
commissure. This is enhanced with the addi-
tion of fillers into the marionette fold. This
is the second example of where antagonist/
protagonist actions are critical to determin-
ing the shape and position of aesthetic struc-
tures of the face with the first example being
the brows.
The depressor anguli oris is a triangular
shaped muscle with its base originating from
the body of the mandible and between the
canine and first molar. It runs superiorly and
inserts onto the skin, mucosa and orbicularis
oris muscle in the region of the modiolus at
the oral commissure. It has intimacy with
the platysma muscle laterally and depressor
labii inferioris medially. This relationship
is important when injecting the depressor
anguli oris in that it is important to stay lat-
eral and posterior when injecting this muscle
to avoid diffusion of neurotoxin toward the
more medial depressor labii inferioris. This
diffusion into the region of the depressor
labii inferioris can create an upward posture
of the lower lip on smiling. This appear-
ance is similar to that seen when there is a
marginal paresis – a potential complication
of a face-lift procedure. The depressor anguli
oris contributes to the formation of the
marionette fold, parentheses rhytids found
midway between the oral commissure and the
margin of the mandible, and the formation
of the prejowl sulcus. It is easy to under-
stand why neurotoxin treatments of this
muscle along with fillers in this region can
play a significant role in perioral rejuvenation.
The depressor labii inferioris is the second
major depressor of the lower lip. Its action
is more medial depressing the tubercle of
the lower lip resulting in lower incisor show
(Figure 2.21). It originates on the anterior
aspect in the region of the oblique line of
the mandible and runs deep to medial fibers
of the depressor anguli oris. It inserts onto
the skin, the orbicularis oris, and mucosa
of the lower lip in the region of the tuber-
cle. Medial fibers may insert onto the cor-
responding depressor labii inferioris muscle
on the contralateral side. As mentioned, this
muscle may be therapeutically injected with
neurotoxin if the opposite depressor labii
inferioris has been weakened by a malposi-
tioned injection of the depressor anguli oris
on the ipsilateral side. This can help to create
symmetry with smiling. Also, in patients who
Figure .Contraction of depressor labii inferioris
resulting in depression of lower lip tubercle with
mandibular dental show.
2 Anatomy and Aesthetic Principles
Figure .Contraction of mentalis muscle with
cobblestoning appearance to skin of chin and
protrusion of lower lip.
experience marginal paresis after face lift-
ing, a temporary injection of neurotoxin into
the contralateral depressor labii inferioris and
depressor anguli oris can produce symme-
try with smiling until the paresis hopefully
resolves.
The mentalis muscle is responsible for
protruding the lower lip by elevating the
skin of the chin upward. It originates on the
mandible in the region of the lateral incisor.
It runs inferiorly and medially deep to the
depressor labii inferioris and inserts onto the
skin of the chin. It is responsible for “cobble-
stoning” or “peau d’orange” appearance of the
chin seen when this muscle contracts (Fig-
ure 2.22). Injection of neurotoxin can amelio-
rate this appearance by relaxing the mentalis.
Orbicularis oris
Figure .Sphincteric fibers of the orbicularis oris.
Redrawn based on an original drawing by Margaret
Ditr´e.
These depressors of the lower lip are all
innervated by the marginal branch of the
facial nerve.
The orbicularis oris muscle, due to the
circular nature of its fibers, is a sphincteric
muscle which creates a puckering or pro-
truding effect of the lips (Figure 2.23). It also
brings the lips together and approximates
the corners of the mouth with contraction
(Table 2.2). Although the majority of the mus-
cle originates and inserts upon itself because
it is circular in nature, deeper fibers originate
from the maxilla and mandible to insert
onto the deeper aspects of the sphincter.
Therefore, the sphincter has both superficial
and deep functional units. The levators
and depressors of the lips also contribute
muscle slips to this sphincter. The buccal
and marginal nerves are responsible for its
innervation.
The complex and diverse movements of
the lips are primarily the result of the com-
plicated interplay between the elevators and
depressors of the lips – which insert into
the orbicularis oris as well as the skin and
mucosa of the lips. These muscles provide a
radial vector to the oral sphincter whereas
the orbicularis oris provides the circumfer-
ential vector. It is the fine interplay of these
radial and circumferential forces that create
a precision in the perioral area that is really
unmatched in the human body. This is what
allows for fine movements seen with articu-
lation and mastication. The skin of the lips is
closely adherent to the orbicularis oris, and
therefore the pursing action of this muscle
can readily transmit its force to the skin over
the muscle. This leads to the creation of ver-
tical rhytids in the region of the lip (Fig-
ure 2.24). Injection of neurotoxin into the
superficial aspect of the sphincteric fibers of
the orbicularis oris relaxes and softens these
rhytids. This effect can be complemented
with the use of fillers. The deeper fibers of
the orbicularis oris are primarily responsi-
ble for maintaining oral competence. It is for
this reason that injections for the fine peri-
oral rhytids must be kept superficial to pre-
vent deep diffusion into these muscles, which
Botulinum Toxins
Figure .Perioral rhytids caused by contraction of
the orbicularis oris.
results in incompetence of the oral sphincter,
i.e. drooling.
The Platysma
The primary superficial muscle of the neck
is the platysma muscle. It originates on the
deltoid and pectoral fascia to insert onto
the mandible and the depressors of the
lower lip. Three separate parts have been
described, the pars modiolaris (most lateral
and superior inserting into modolaris), the
Figure .Contraction of the platysma with
depression of the lower lip and corners of mouth.
pars labialis (inserting into lower lip), and the
pars mandibularis (most medial and inferior
inserting into chin) (Figure 2.25).
A recent study examining the platysma
muscle during deep plane rhytidectomies
shows a significant superior extent of the
platysma muscle into the midface [26]. It is
innervated by the cervical branch of the sev-
enth cranial nerve and is contiguous with
the superficial musculoaponeurotic system
(SMAS) of the face. It has varying degrees of
decussation in relation to the hyoid bone. Its
primary action is a depressor of the lower lip
and corners of the mouth (Figure 2.26). As
this muscle ages, a natural dehiscence may
occur centrally. This in combination with the
Pars modiolaris
Pars labialis
Pars mandibularis
Platysma
ars labialis
ars labialis
Pars labialis
bialis
Pa
l b
ars mo
Pa
Pars
ars m
s mo
mo
Pa
Pars
ars m
s mo
mo
mo
P
bi li
bi li
bi li
odiola
odiola
odiola
odiol
odiola
ol
odiol
di
ma
Platysm
ulari
s
ris
ris
ris
ri
is
Pars modiolaris
Pars labialis
Pars mandibularis
Platysma
Figure .Platysma: Pars modiolaris, labialis, and mandibularis.
2 Anatomy and Aesthetic Principles
hypercontraction of the medial borders of the
platysma muscle can create vertical cords or
bands. Contractile bands may also be present
laterally just below the level of the jowl. Injec-
tion of neurotoxin into both sets of bands as
well as along the margin of the mandible can
improve the appearance of the aging neck and
jaw line. This effect occurs due to a subtle
upward repositioning of the soft tissues of the
jaw line secondary to relaxation of the lower
extent of the platysma and persistent contrac-
tion of the superior extent. This is the premise
behind the “Nefertiti lift” [27] to aesthetically
improve the appearance of the jawline. Along
with the vertical bands, transverse rhytids of
the neck (necklace lines) are also caused by
a contraction of the platysma muscle. These
lines may be conservatively improved with
injection of neurotoxin into the muscle in
addition to the use of a low-viscosity filler.
The Craniofacial Skeleton
The craniofacial skeleton and the changes
that occur as it ages are critical to under-
standing the art and science of chemoden-
ervation of the muscles of the face since
the majority of these muscles take origin
here. The primary bones of the facial skele-
ton include the frontal bone, nasal bones,
zygoma, maxilla and mandible. The frontal
bone is composed of the vertical aspect of
the squama. This is responsible for the shape
and extent of the forehead. The caudal por-
tions include the supraorbital rim – with
its extensions medially articulating with the
paired nasal bones and the extensions later-
ally articulating with the frontal process of the
zygoma. The zygoma forms the major por-
tion of the lateral orbital rim and a portion of
the lateral inferior orbital rim where it artic-
ulates with the maxilla. The zygoma extends
posteriorly as the temple process and articu-
lates with the zygomatic process of the tem-
poral bone. The apex of the zygoma forms the
maler eminence (height of the cheek bone).
This union of the two processes forms the
zygomatic arch – which is where the mas-
seter muscle originates. Running underneath
the arch is the temporalis muscle, a primary
muscle of mastication that is innervated by
the third division of the trigeminal nerve
(Table 2). It originates in the temporal fossa
and inserts onto the coronoid process and
ramus of the mandible. It, too, can exhibit
hypertrophy, particularly in patients with
bruxism, and can contribute not only to TMJ
symptoms but also create an aesthetically
unpleasing bulge with mastication in the lat-
eral forehead region. The maxilla is the pri-
mary skeleton of the midface. It is the origin
of the majority of the levators of the upper
lip. It forms the majority of the inferior and
medial orbital rims and articulates with the
zygoma and frontal bones respectively. The
nasal process of the maxilla articulates with
the nasal bones medially and the lacrimal
bone laterally. It also forms the lateral and
inferior aspects of the pyriform aperture with
the superior aspect of the aperture being
formed by the inferior borders of the nasal
bones. The maxilla houses the superior alve-
olar ridge, which contains the maxillary den-
tition. The mandible is the only true mobile
bone of the facial skeleton and is critical to the
aesthetics of the lower one-third of the face.
It is also the origin of the muscles which pri-
marily depress the lip complex. It articulates
with the maxilla through the mandibular den-
tition anteriorly. The relationship of the max-
illary dentition to the mandibular dentition
defines the concept of occlusion. Angle’s clas-
sification describes three primary occlusal
relationships. Class I is a normal or orthog-
nathic occlusion, with Class II being retrog-
nathic and Class III being prognathic. This
occlusal alignment is critical to the aesthet-
ics of the lower one-third of the face. Ret-
rognathic patients have a weak/recessed chin
and this relationship not only has a profound
effect on the profilimetry of the face but also
on the aesthetic relationship of the upper and
lower lips, with the upper lip projecting more
anteriorly than is seen in the normal occlu-
sion. The prognathic patient has a retrusive
maxilla in relation to the mandible and results
in over projection of the lower lip on pro-
file. The mandible articulates with the tempo-
ral bone posteriorly in the mandibular fossa
Botulinum Toxins
via the mandibular condyle thus forming the
temporomandibular joint.
The changes seen in the aging facial skele-
ton are profoundly affected by the condition
of the mandibular and maxillary dentition.
Loss of dentition in either region results
in decrease of bony volume as a result of
demineralization due to an increase in the
osteoclastic activity of the bone, resulting
from reduction in transmitted forces to the
maxilla and mandible. This is also known as
contraction of the facial skeleton. Another
significant change in the aging craniofa-
cial skeleton which profoundly affects the
appearance of the soft tissues, is the clock-
wise rotation of the midfacial skeleton in
relation to the cranial base as proposed by
Lambros and confirmed by Pessa [28]. They
showed that there was a continual growth and
remodeling of the aging craniofacial skeleton
with a resultant clockwise rotation. This was
confirmed by measuring the key angles of the
face (glabellar, orbital, pyriform, and maxil-
lary) and showing that these angles became
more acute with age. These bony changes
help define the nature of the ptotic soft tissue
changes occurring in the midface and perioral
area secondary to loss of structural support of
the midface.
Shaw and Kahn confirmed the decrease in
maxillary and glabella angles as seen by Pessa
[29]. However, they also showed an increase
in the surface area of the pyriform aperture
with aging, thus signifying that aging has a
deteriorating and contracting effect on the
maxilla as opposed to a growth and remod-
eling effect.
Pessa and Chen showed that the nature of
the aging orbital aperture is such that curve
distortion occurs in the inferior lateral and
superior medial regions due to bony remod-
eling of the orbit with aging [30]. This skeletal
change of the orbit is reflected to the eyelids
resulting in scleral show and the tear trough
deformity seen in the aging eye.
All of the above craniofacial skeleton
changes have a profound secondary effect
on the overlying soft tissues. Several exam-
ples include: soft tissue repositioning which
is seen with descent of the cheek fat pad;
increased facial muscle tone resulting in
greater incidence of hyperfunctional rhytids
and aging facial reshaping; and laxity of the
key ligaments of the face. This increased
facial muscle tone may explain why typically
acceptable neurotoxin dosages have less of an
effect on older patients in their seventh and
eighth decades.
Acknowledgements
The authors would like to thank Joanne
Scarpulla and Kristen Whitney D.O., for their
tireless effort in the preparation of this chap-
ter. Also, many thanks to Maggie Ditr´e (Class
of 2014, Yale University) for her illustrations
for this chapter.
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Botulinum Toxin: From Molecule to Medicine
Conor J. Gallagher, PhD1 and Alan Ackerman, PhD2
1Executive Director Medical Affairs, Facial Aesthetics, Allergan plc, Irvine, USA
2Master Medical Scientific Liaison, Retired, Ackerman LLC, Greeley, USA
Introduction
Exocytosis of neurotransmitters forms the
physiological basis for linking our sensory
inputs and cognitive processes to mus-
cle contraction. This mechanism underlies
our ability to voluntarily move and inter-
act with our environment, to express facial
emotions and in neurally-initiated invol-
untary muscle or glandular activity The
exocytotic processes that accomplish the
conversion of sensory/environmental inputs
to actions are highly conserved across mul-
tiple species and constitute the mechanism
by which neurotransmitter-containing vesi-
cles are induced to release their contents
into the synaptic cleft, triggering muscle
contraction [1]. The bacterium Clostridium
botulinum produces a neurotoxin that specif-
ically disrupts this vital process, specifically
SNAP-25 (synaptosomal associated protein,
25 kDa) mediated exocytosis. In deciphering
the mechanism by which botulinum toxin
acts, scientists have unlocked a useful tool for
the management of clinical disorders of mus-
cle hyperactivity [2]. Many biological pro-
cesses besides muscle contraction rely on
neurotransmitter release and, through scien-
tific experimentation, many additional clini-
cal conditions amenable to botulinum toxin
therapy have been reported [3–5].
This chapter will review the state of our
knowledge on the mechanism of action of
botulinum toxin that underlies its clinical use
in therapeutic and aesthetic indications.
Botulinum Toxin
Clostridium botulinum is ubiquitous in soil
where it typically lies dormant as a spore.
Under optimal conditions, including low oxy-
gen, the bacterial spores germinate and pro-
duce neurotoxin [6]. Seven immunologically
distinct serotypes of botulinum toxin have
been identified, categorized as A–G [7]. Each
serotype has distinct pharmacological char-
acteristics [8,9], and only products containing
type A or type B botulinum toxin have ever
been approved for human use.
In nature, botulinum toxins are produced
by the bacteria as protein complexes, con-
sisting of two elements: the core neurotoxin
that contains the protease moiety responsi-
ble for the intracellular biological activity of
the neurotoxin, and a number of nontoxin
accessory proteins [10, 11]. The core neuro-
toxin molecule always has a molecular weight
of approximately 150 kDa, and while there is
only approximately 30–40% protein homol-
ogy [12] between serotypes, a high degree of
structural similarity has been observed based
on crystal structures [13].
The number and identity of the accessory
proteins can vary depending on the strain of
bacteria, the serotype of neurotoxin, and the
Botulinum Toxins: Cosmetic and Clinical Applications, First Edition. Edited by Joel L. Cohen and David M. Ozog.
© 2017 John Wiley & Sons Ltd. Published 2017 by John Wiley & Sons Ltd.
Companion Website:
Botulinum Toxins
Figure .Molecular structure of the
botulinum toxin type A 150 kDa core
neurotoxin. The three functional
domains are color coded: Yellow: light
chain catalytic domain with a zinc
atom shown in blue; Red: heavy chain
translocation domain; Green: heavy
chain binding domain. Source: Image
courtesy of Lance E. Steward (Allergan
plc.).
growth conditions. In general, three neuro-
toxin “complex” (core neurotoxin plus acces-
sory proteins) sizes have been identified with
molecular weights of approximately 300, 500
and 900 kDa respectively [14]. Each complex
consists of the 150 kDa neurotoxin with the
balance of the molecular weight made up of
the accessory proteins. In nature, the acces-
sory proteins are known to have a protective
role, helping to preserve the activity of the
neurotoxin against exposure to proteolytic
enzymes and extremes of pH and tempera-
ture [11, 15–19]. This is particularly impor-
tant in the oral route of intoxication, as the
neurotoxin is exposed to both changes in pH
and proteolytic enzymes. It has been shown
that when given orally the type A neurotoxin
complex is 350 times more potent than the
unprotected 150 kDa neurotoxin alone due to
the protective actions of the accessory pro-
teins [20] (Figure 3.1).
Mechanism of Action
In the therapeutic or aesthetic application
of botulinum toxin products, minute doses
(picograms to nanograms) are administered
to induce a focal weakening of the injected
muscle leading to a reduction of tone in a
hypertonic muscle or to reduce neuronally
evoked secretions at exocrine glands. The
exquisitely specific action of botulinum toxin
at the presynaptic nerve terminal is a func-
tion of the 150 kD protein we refer to as the
core neurotoxin: a protein in which struc-
ture and function are intimately and elegantly
linked [2].
The structure and modular organization of
the core neurotoxin is generally conserved
among the different serotypes; however, small
differences in several regions lead to the dif-
ferent receptor and substrate specificities of
each of these proteins [21, 22]. The core