Quy Trình Tiêm Botulinum Toxin Trong Da Liễu Thẩm Mỹ (Cosmetic Dermatology Series)

Botulinum
Toxin

Procedures in Cosmetic Dermatology
Series Editor; Jeffrey S. Dover MD, FRCPC, FRCP
Associate Editor: Murad Alam MD
For Elsevier
Content Strategist: Belinda Kuhn
Content Development Specialist: Martin Mellor Publishing Services Ltd
Project Manager: Sruthi Viswam
Design: Miles Hitchen
Illustration Manager: Jennifer Rose
Marketing Manager: Carla Holloway
Chemical Peels
Second edition
Rebecca C. Tung, MD and Mark G. Rubin MD
ISBN 978-1-4377-1924-6
Treatment of Leg Veins
Second edition
Murad Alam, MD and Sirunya Silapunt, MD
ISBN 978-1-4377-0739-7
Body Contouring
Bruse E Katz MD and
Neil S Sadick MD FAAD FAACS FACP FACPh
ISBN 978-1-4377-0739-7
Non Surgical Skin Tightening and Lifting
Murad Alam MD MSCI and Jeffrey S Dover MD RCPC FRCP
ISBN 978-1-4160-5960-8
Botulinum Toxin
Third Edition
Alastair Carruthers MA BM BCh FRCPC FRCP(Lon) and
Jean Carruthers MD FRCSC FRC(Ophth) FASOPRS
ISBN 978-1-4557-2781-0
Soft Tissue Augmentation
Third edition
Jean Carruthers MD FRCSC FRC(Ophth) FASOPRS and
Alastair Carruthers MA BM BCh FRCPC FRCP(Lon)
ISBN 978-1-4557-2782-7
Cosmeceuticals
Second edition
Zoe-Diana Draelos MD
ISBN 978-1-4160-5553-2
Lasers and Lights
Third edition
George Hruza MD and Mathew Avram MD
ISBN 978-1-4557-2783-4
Photodynamic Therapy
Second edition
Mitchel P. Goldman MD
ISBN 978-1-4160-4211-2
Liposuction
C. William Hanke MD MPH FACP and
Gerhard Sattler MD
ISBN 978-1-4160-2208-4
Scar Revision
Kenneth A Arndt MD
ISBN 978-1-4160-3131-4
Hair Transplantation
Robert S. Haber MD and
Dowling B Stough MD
ISBN 978-1-4160-3104-8
Blepharoplasty
Ronald L. Moy MD and
Edgar F Fincher MD
ISBN 978-1-4160-2996-0
Advanced Face Lifting
Ronald L. Moy MD and
Edgar F Fincher MD
ISBN 978-1-4160-2997-7

3rd
Edition
PROCEDURES IN COSMETIC DERMATOLOGY
Botulinum
Toxin
Edited by
Alastair Carruthers MA, BM, BCh, FRCPC, FRCP(Lon)
Clinical Professor, Department of Dermatology and Skin Science,
University of British Columbia, Vancouver, BC, Canada
Jean Carruthers MD, FRCSC, FRC (OPHTH), FASOPRS
Clinical Professor, Department of Ophthalmology and Visual Science,
University of British Columbia, Vancouver, BC, Canada
Series Editor
Jeffrey S. Dover MD, FRCPC, FRCP
Associate Professor of Clinical Dermatology, Yale University School of Medicine, New Haven, CT;
Adjunct Professor of Medicine (Dermatology), Dartmouth Medical School, Hanover, NH;
Adjunct Associate Professor of Dermatology, Brown Medical School, Providence, RI;
Director, SkinCare Physicians, Chestnut Hill, MA, USA
Associate Editor
Murad Alam MD
Professor of Dermatology, Otolaryngology, and Surgery;
Chief, Section of Cutaneous and Aesthetic Surgery,
Northwestern University, Chicago, IL, USA
For additional online content visit expertconsult.com
London  New York  Oxford  St Louis  Sydney  Toronto  2013

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© 2013, Elsevier Inc. All rights reserved.
First edition 2005
Second edition 2008
Third edition 2013
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This book and the individual contributions contained in it are protected under
copyright by the Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research
and experience broaden our understanding, changes in research methods, professional
practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowl­
edge in evaluating and using any information, methods, compounds, or experiments
described herein. In using such information or methods they should be mindful of
their own safety and the safety of others, including parties for whom they have a
professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised
to check the most current information provided (i) on procedures featured or (ii) by
the manufacturer of each product to be administered, to verify the recommended
dose or formula, the method and duration of administration, and contraindications.
It is the responsibility of practitioners, relying on their own experience and knowl­
edge of their patients, to make diagnoses, to determine dosages and the best treat­
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ISBN: 978-1-4557-2781-0
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CONTENTS










Series Preface
vii
Series Preface (First edition)
ix
Preface
xi
Contributors
xiii
Acknowledgments
xv
Dedication
xvii
  1 Therapeutic Uses of the Botulinum Toxins
1
Andrew Blitzer
  2 History of OnabotulinumtoxinA
Therapeutic
6
Mitchell F. Brin, Andrew Blitzer
  3 History of Cosmetic Botulinum Toxin
13
Alastair Carruthers, Jean Carruthers
  4 Basic Science: BOTOX® Cosmetic
18
Conor J. Gallagher
  5 Basic Science: Abobotulinum Toxin A
26
Gary D. Monheit, Andrew Pickett
  6 Basic Science: Xeomin®
35
Jürgen Frevert, Gerhard Sattler
  7 Basic Science: Myobloc®
43
Neil S. Sadick, Nils Krueger
  8 Medytoxin/Neuronox®
52
Kyle Koo-II Seo, Wooshun Lee
  9 Comparison of Botulinum Toxins
59
Chung-Yin Stanley Chan, Kathryn Kent,
Thomas E. Rohrer
10 Topical Neurotoxin
67
Jacob M. Waugh, Richard G. Glogau
11 Reconstitution / Dilution
72
Ada R. Trindade de Almeida,
Letícia Cardoso Secco
12 Glabella
77
Naissan O. Wesley, Derek H. Jones
13 Eyebrow Height / Shaping
85
Jason Sneath, Massimo Signorini
14 Frontalis and HFL
93
Austin Liu, David M. Ozog, Joel L. Cohen
15 Treatment of Crow’s Feet
100
Melanie Warycha, Jeffrey S. Dover,
Murad Alam
16 Infraorbital / Upper and Lower Eyelids
110
Shannon Humphrey, Steven Fagien
17 Treatment of the Mid-face with
Botulinum Toxin
117
Ian A. Maher, Timothy C. Flynn
18 Orbicularis Oris, Mentalis, Depressor
Anguli Oris
123
Fredric S. Brandt, Annelyse C. Ballin,
Jeremy B. Green, Joely Kaufman,
Alex Cazzaniga
19 Platysma and the Nefertiti Lift®
129
Phillip M. Levy
20 The Masseters and Their Treatment
with Botulinum Toxin
135
Greg J. Goodman
21 Darker Skin Types
142
Pearl E. Grimes, Andrew F. Alexis
22 Combination Treatment
151
Alastair Carruthers, Jean Carruthers
23 Focal Axillary Hyperhidrosis
160
Dee Anna Glaser, Adam R. Mattox
24 Palmo-Plantar Hyperhidrosis
169
Kavita Mariwalla, Nowell Solish
25 The Future of Botulinum Toxin
175
Alastair Carruthers, Jean Carruthers
Index
179

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Seven years ago we embarked on an effort to produce
Procedures in Cosmetic Dermatology, a series of high
quality, practical, up-to-date, illustrated manuals. Our
plan was to provide dermatologists, dermatologic sur­
geons, and others dedicated to the pursuit of functional
knowledge with detailed portable books accompanied by
high quality “how to” DVD’s containing all the informa­
tion they needed to master most, if not all, of the leading
edge cosmetic techniques. Thanks to the efforts of world
class volume editors, master chapter authors, and the tire­
less and extraordinary publishing staff at Elsevier, the
series has been more successful than any of us could have
imagined. Over the past seven years, 15 distinct volumes
have been introduced, and have been purchased by thou­
sands of physicians all over the world. Originally published
in English, many of the texts have been translated into
different languages including Italian, French, Spanish,
Chinese, Polish, Korean, Portuguese, and Russian.
Our commitment has always been to ensure that the
practical, easy to use information conveyed in the series
is also extremely up-to-date, incorporating all the latest
methods and materials. To that end, given the rapidly
changing nature of our subspecialty, the time has now
come to inaugurate the third edition. During the next
few years, refined, enlarged, and improved texts will be
released in a sequential manner. The most time-sensitive
books will be revised first, and others will follow.
This series is an ever evolving project. So in addition
to third editions of current books, we will be introducing
entirely new books to cover novel procedures that may
not have existed when the series began. Enjoy and keep
learning.
Jeffrey S. Dover MD FRCPC and
Murad Alam MD, MSCI
SERIES PREFACE TO THE THIRD EDITION

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While dermatologists have been procedurally inclined
since the beginning of the specialty, particularly rapid
change has occurred in the past quarter century. The
advent of frozen section technique and the golden age of
Mohs skin cancer surgery have led to the formal incorpo­
ration of surgery within the dermatology curriculum.
More recently technological breakthroughs in minimally
invasive procedural dermatology have offered an aging
population new options for improving the appearance of
damaged skin.
Procedures for rejuvenating the skin and adjacent
regions are actively sought by our patients. Significantly,
dermatologists have pioneered devices, technologies, and
medications, which have continued to evolve at a startling
pace. Numerous major advances including virtually all
cutaneous lasers and light-source-based procedures, botu­
linum exotoxin, soft tissue augmentation, dilute anesthe­
sia liposuction, leg vein treatments, chemical peels, and
hair transplants have been invented, or developed and
enhanced, by dermatologists. Dermatologists understand
procedures and we have special insight into the structure,
function, and working of skin. Cosmetic dermatologists
have made rejuvenation accessible to risk-averse patients
by emphasizing safety and reducing operative trauma. No
specialty is better positioned than dermatology to lead the
field of cutaneous surgery while meeting patient needs.
As dermatology grows as a specialty, an ever-increasing
proportion of dermatologists will become proficient in the
delivery of different procedures. Not all dermatologists
will perform all procedures, and some will perform very
few, but even the less procedurally directed amongst us
must be well versed in the details to be able to guide and
educate our patients. Whether you are a skilled dermato­
logic surgeon interested in further expanding your surgical
repertoire, a complete surgical novice wishing to learn a
few simple procedures, or somewhere in between, this
book and this series are for you.
The volume you are holding is one of a series entitled
‘Procedures in Cosmetic Dermatology’. The purpose of
each book is to serve as a practical primer on a major topic
area in procedural dermatology.
If you want to make sure you find the right book for
your needs, you may wish to know what this book is and
what it is not. It is not a comprehensive text grounded in
theoretical underpinnings. It is not exhaustively refer­
enced. It is not designed to be a completely unbiased
review of the world’s literature on the subject. At the
same time, it is not an overview of cosmetic procedures
that describes these in generalities without providing
enough specific information to actually permit someone
to perform the procedures. And, importantly, it is not so
heavy that it can serve as a doorstop or a shelf filler. What
this book and this series offer is a step-by-step, practical
guide to performing cutaneous surgical procedures. Each
volume in the series has been edited by a known authority
in that subfield. Each editor has recruited other equally
practical-minded, technically skilled, hands-on clinicians
to write the constituent chapters. Most chapters have two
authors to ensure that different approaches and a broad
range of opinions are incorporated. On the other hand,
the two authors and the editors also collectively provide
a consistency of tone. A uniform template has been used
within each chapter so that the reader will be easily able
to navigate all the books in the series. Within every
chapter, the authors succinctly tell it like they do it. The
emphasis is on therapeutic technique; treatment methods
are discussed with an eye to appropriate indications,
adverse events, and unusual cases. Finally, this book is
short and can be read in its entirety on a long plane ride.
We believe that brevity paradoxically results in greater
information transfer because cover-to-cover mastery is
practicable.
We hope you enjoy this book and the rest of the books
in the series and that you benefit from the many hours of
clinical wisdom that have been distilled to produce it.
Please keep it nearby, where you can reach for it when
you need it.
Jeffrey S. Dover MD FRCPC and Murad Alam MD
SERIES PREFACE FIRST EDITION

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Our authors join us in hoping that you find this volume
both instructive and entertaining. This is the third edition
of Botulinum Toxin in the Procedures in Cosmetic Der­
matology series, but do not think that this is the same old
material polished and updated! The topic has been com­
pletely rethought and reorganized and new authors
selected for almost all the chapters. We hope you appreci­
ate this new look and find it both more accessible and a
fresher presentation of all the material that has been gen­
erated since the last edition in 2008.
One of the important innovations relates to the differ­
ent commercially available neurotoxins. This is an area
where opinion may well be influenced by those commer­
cial interests and it is hard to make sure that all the
contributions are unbiased. We have therefore gone in the
other direction and have asked each of the companies to
produce a chapter about their approved product, avoiding
comparisons as much as possible. We hope that we have
achieved our aim of producing, overall, a result that bal­
ances the need to give accurate information with the
essential that it be fairly representative.
We are delighted to include historic chapters by the
pioneers of the therapeutic use of botulinum toxin, Drs
Blitzer and Brin, as well as a forward-looking chapter on
the topical use of the toxin by Drs Glogau and Waugh.
In general the book is divided between the background
information in the front half and the clinical, cosmetic
use in the second half. This clinical section looks at the
cosmetic use from a very current perspective and it is
fascinating to see how usage, even in the brow, has
changed in recent years. In particular, the clinical use of
the toxin in some of the smaller facial muscles and in the
lower face and neck, is much better understood and an
important part of facial cosmetic treatment. We have
concluded with a section on the treatment of excessive
sweating, which continues to be an important part of
clinical toxin use.
This volume and the attached video demonstrations
should give you an intensive view of leading experts in the
field worldwide about the cosmetic use of botulinum
toxin. We believe that we have covered the field broadly,
fairly, and accurately, and we hope that you will agree.
Alastair and Jean Carruthers
Vancouver, BC, Canada
April 2012
PREFACE

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Murad Alam MD
Professor of Dermatology, Otolaryngology, and Surgery; Chief,
Section of Cutaneous and Aesthetic Surgery, Northwestern
University, Chicago, IL, USA
Andrew F. Alexis MD
MPH Director, Skin of Color Center, Saint Luke’s Roosevelt
Hospital; Assistant Clinical Professor, Columbia University
College of Physicians & Surgeons, New York, NY, USA
Annelyse C. Ballin MD
Clinical Research, Dermatology Research Institute, Coral
Gables, FL, USA
Andrew Blitzer MD
DDS Professor of Clinical Otolaryngology, College of
Physicians and Surgeons, Columbia University; Director, New
York Center for Voice and Swallowing Disorders; Medical
Director, New York Center for Clinical Research, New York,
NY, USA
Fredric S. Brandt MD
Private Practice, Coral Gables, FL and New York; Dermatology
Research Institute, Coral Gables, FL, USA
Mitchell F. Brin MD
Senior Vice President, Global Drug Development, Chief
Scientific Officer, BOTOX®, Allergan, Inc., Irvine, CA;
Professor of Neurology, University of California, Irvine, CA,
USA
Letícia Cardoso Secco MD
Volunteer Dermatologist, Hospital do Servidor Público
Municipal de São Paulo, Brazil
Alex Cazzaniga BS, MBA
Director of Clinical Research, Dermatology Research Institute,
Coral Gables, FL, USA
Chung-Yin Stanley Chan MD
Procedural Dermatology Fellow, SkinCare Physicians, Chestnut
Hill, MA, USA
Joel L. Cohen MD
Director, AboutSkn Dermatology and DermSurgery,
Englewood; Associate Clinical Professor, Department of
Dermatology, University of Colorado, Denver, CO, USA
Jeffrey S. Dover MD, FRCPC, FRCP
Associate Professor of Clinical Dermatology, Yale University
School of Medicine, New Haven, CT; Adjunct Professor of
Medicine (Dermatology), Dartmouth Medical School, Hanover,
NH; Adjunct Associate Professor of Dermatology, Brown
Medical School, Providence, RI; Director, SkinCare Physicians,
Chestnut Hill, MA, USA
Steven Fagien MD, FACS
Private Practice, Aesthetic Eyelid Plastic Surgery, Boca Raton,
FL, USA
Timothy C. Flynn MD
Clinical Professor, Department of Dermatology, University of
North Carolina, Chapel Hill, NC; Medical Director, Cary Skin
Center, Cary, NC, USA
Jürgen Frevert PhD
Head of Botulinum Toxin Research, Merz Pharmaceuticals
GmbH, Potsdam, Germany
Conor J. Gallagher PhD
Director, Global Neurotoxin Science, Medical Affairs, Allergan
Inc., Irvine, CA, USA
Dee Anna Glaser MD
Professor and Vice Chairman; Director Cosmetic and Laser
Surgery, Department of Dermatology, Saint Louis University
School of Medicine, Saint Louis, MO, USA
Richard G. Glogau MD
Clinical Professor of Dermatology, University of California
Medical Center, San Francisco, CA, USA
Greg J. Goodman MBBS, FACD, MD
Associate Professor, Monash University; Chief of Surgery, Skin
and Cancer Foundation Inc, Carlton, VIC, Australia
Jeremy B. Green MD
Private Practice, Dr. Brandt Dermatology Associates, Coral
Gables, FL; Voluntary Assistant Professor, University of Miami
Department of Dermatology & Cutaneous Surgery, Miami, FL,
USA
Pearl E. Grimes MD
Director, Vitiligo and Pigmentation Institute of Southern
California; Clinical Professor, Division of Dermatology, David
Geffan School of Medicine, University of California, Los
Angeles, CA, USA
Shannon Humphrey MD, FRCPC, FAAD
Clinical Instructor, Department of Dermatology and Skin
Science, University of British Columbia, Vancouver, British
Columbia, Canada
Derek H. Jones MD
Associate Clinical Professor, Dermatology, David Geffan School
of Medicine, University of California, Los Angeles, CA, USA
Joely Kaufman MD
Private Practice, Coral Gables, FL; Voluntary Assistant
Professor of Clinical Dermatology, University of Miami Miller
School of Medicine; Co-Director of Cosmetic Medicine,
University of Miami, Miami, FL, USA
Kathryn Kent MD
Resident, Department of Dermatology, Boston University,
Boston, MA, USA
Nils Krueger PhD
Clinical Research Scientist, Sadick Research Group, New York,
NY, USA
CONTRIBUTORS

xiv
Contributors
Wooshun Lee MD
Medical Director, Medytox Inc, Seoul, Korea
Phillip M. Levy BA, MD, FMHDerm
Aesthetic Dermatologist, Private Practice, Geneva, Switzerland;
Lecturer, Faculty of Medicine and Pharmacology, University of
Franche-Comté, Besançon, France
Austin Liu MD
Chief Resident, Department of Dermatology, Henry Ford
Hospital, Detroit, MI, USA
Ian A. Maher MD, FAAD
Assistant Professor, Mohs Surgeon, Department of
Dermatology, Virginia Commonwealth University, Richmond,
VA, USA
Kavita Mariwalla MD
Assistant Clinical Professor, Department of Dermatology,
Columbia University, New York, NY, USA
Adam R. Mattox DO, MS
Physician Research Fellow, Department of Dermatology, Saint
Louis University School of Medicine, Saint Louis, MO, USA
Gary D. Monheit MD, FAAD, FACS
Private Practice, Total Skin and Beauty Dermatology Center,
PC; Associate Clinical Professor, Departments of Dermatology
and Ophthalmology, University of Alabama at Birmingham,
Birmingham, AL, USA
David M. Ozog MD, FAAD
Director of Cosmetic Dermatology; Vice-Chair of Operations,
Division of Mohs and Dermatological Surgery, Henry Ford
Hospital, Detroit, MI, USA
Andrew Pickett BSc, PhD
Director and Founder, Toxin Research Limited, Wrexham, UK;
Adjunct Professor, Botulinum Research Center, UMASS
Dartmouth, MA, USA
Thomas E. Rohrer MD
Clinical Associate Professor of Dermatology, The Warren Alpert
Medical School of Brown University, Providence, RI; Director
of Mohs Fellowship, SkinCare Physicians, Chestnut Hill, MA,
USA
Neil S. Sadick MD, FAAD, FAACS, FACP, FACPh
Clinical Professor, Weill Cornell Medical College, Cornell
University; Sadick Dermatology and Research Group, New
York, NY, USA
Gerhard Sattler MD
Fellow of Dermatology; Clinical Director of Rosenparkklinik,
Darmstadt, Germany
Kyle Koo-II Seo MD, PhD
Clinical Associate Professor, Dermatology, Seoul National
University College of Medicine; Director, Dermatology, Modelo
Clinic, Seoul, Korea
Massimo Signorini MD
Chief of Surgery, Istituto Dermatologico Europeo, Milan, Italy
Jason Sneath BSc, CPH, MD
Resident, Department of Dermatology and Skin Science,
University of British Columbia, Vancouver, British Columbia,
Canada
Nowell Solish MD, FRCPC
Assistant Professor of Dermatology, University of Toronto, ON,
Canada
Ada R. Trindade de Almeida MD
Dermatologist, Dermatologic Clinic, Hospital do Servidor
Público Municipal de São Paulo and private office, São Paulo,
Brazil
Melanie Warycha MD
Procedural Dermatology Fellow, Department of Dermatology,
Northwestern University Feinberg School of Medicine,
Chicago, IL, USA
Jacob M. Waugh MD
Chief Scientific Officer and Medical Director, Revance
Therapeutics, Newark, CA, USA
Naissan O. Wesley MD
Dermatologist and Dermatologic Surgeon, Skin Care and Laser
Physicians of Beverly Hills, Los Angeles, CA, USA

Looking back over the past 30 years of our involvement
in the clinical use of botulinum toxin there are very many
people who have supported and encouraged us. The first
to be mentioned, of course, is Alan Scott, MD, who has
been consistently generous with his educational efforts,
his clinical information, his research results and, with
his wife Ruth, hospitality. Sitting on their deck above
Mill Valley looking towards the Golden Gate Bridge we
have learned more about botulinum toxin than in any
lecture hall.
From the early days of oculinum and then botox we
have had fascinating and enlightening discussions with
Mitchell Brin, MD and Andrew Blitzer, MD both from
Columbia. The early group of us who formed a core set
of advisers for Allergan (our advice was not always well
received and usually was ahead of its time) consisted
of Andrew Blitzer, MD, Richard Glogau, MD, Nicholas
Lowe, MD, Arnold Klein, MD and the two of us. These
sessions and teleconferences set the stage for the future
education and development of the cosmetic use of botu­
linum toxin and were a major learning experience for us
all. The basic science which Roger Aoki, PhD, of Allergan
Inc., provided to us gave a solid basis for our understand­
ing of how the toxin works and the meaning of both basic
and clinical studies.
In more recent years we have had valuable assistance
from many individuals, many of whom are coauthors in
this book. We have greatly appreciated the contributions
of many of our international colleagues such as Ada Trin­
dade de Almeida, MD, Francisco Perez Atamoros, MD
and Koenraad de Boulle, MD. To all of the authors of this
text, we salute you! We are so grateful for the hard work
you have put into this volume and the tolerance you have
shown when faced with yet more unreasonable editorial
demands. The proof of your labours and dedication is
easily seen between the covers of this book.
Any list of acknowledgments would not be complete
without recognizing the people who actually do the work
to make it all work: our staff and our patients. Our patients
have been so very tolerant, especially in the beginning
when we did not fully understand the potential benefits
as well as the pitfalls of the cosmetic use of botulinum
toxin. Our staff have been consistently supportive and
helpful (finding photographs from the vaguest descrip­
tions, following up results good and bad, keeping things
running smoothly so we could go off and deliver another
lecture). We could not have done it without you!
Finally, our friends and family have accepted the fact
that we might or might not be in a given place at a given
time, that we spend more time thinking about them in
airports than actually talking to them, that our crazy idea
has generated a crazy life. Many thanks to you all!
Alastair and Jean Carruthers
ACKNOWLEDGMENTS

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We dedicate this volume to our children and their fami­
lies. Our sons were young when the botulinum toxin story
began and they have regarded the efforts of their parents
to cope with this accidental discovery with tolerance and
increasing pride over the years. We have appreciated the
support and encouragement they have given us (rather
than the other way round). The love they have given us
means we are indeed fortunate!
Alastair and Jean Carruthers
To the women in my life: my grandmothers, Bertha and
Lillian, my mother, Nina, my daughters, Sophie and Isabel
and especially to my wife, Tania. For their never-ending
encouragement, patience, support, love, and friendship
To my father, Mark – a great teacher and role model; my
mentor, Kenneth A. Arndt for his generosity, kindness,
sense of humor, joie de vivre, and above all else curiosity
and enthusiasm
Jeffrey S. Dover
Elsevier’s dedicated editorial staff has made possible the
continuing success of this ambitious project. The new
team led by Belinda Kuhn, Martin Mellor and the produc­
tion staff have refined the concept for the second edition
while maintaining the series’ reputation for quality and
cutting-edge relevance. In this, they have been ably sup­
ported by the graphics shop, which has created the signa­
ture high quality illustrations and layouts that are the
backbone of each book. We are also deeply grateful to
the volume editors, who have generously found time in
their schedules, cheerfully accepted our guidelines, and
recruited the most knowledgeable chapter authors. And
we especially thank the chapter contributors, without
whose work there would be no books at all. Finally, I
would also like to convey my debt to my teachers, Kenneth
Arndt, Jeffrey Dover, Michael Kaminer, Leonard Gold­
berg, and David Bickers, and my parents, Rahat and
Rehana Alam.
Murad Alam
DEDICATION

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thus weakening muscle strength by chemical denervation.
Brooks also reported the fact that botulinum toxin could
possibly be used therapeutically. The observation that
tetanus and botulinum toxins blocked the exocytosis of
acetylcholine was further refined with the discovery that
these toxins enzymatically degraded different portions of
the soluble N-ethyl maleimide-sensitive factor attach­
ment protein receptor (SNARE) proteins. Alan Scott’s
original work using botulinum toxin to weaken muscle for
the correction of strabismus, and then blepharospasm, led
others to begin to investigate the toxins for use with other
dystonias and hyperfunctional muscular disorders.
The original observations of Kerner that patients with
botulism had dry mouth and eyes suggested that the toxin
might be used to control hypersecretory states. With
the knowledge that the autonomic nervous system also
depended on acetylcholine as the neurotransmitter, it
seemed even more likely that botulinum toxin could be
used to control disorders of this system. Clinical trials
have shown efficacy in autonomic disorders such as hyper­
hidrosis, sialorrhea, and Frey syndrome.
A number of trials for hyperfunctional muscular disor­
ders such as cervical dystonia and spasticity showed a
dramatic reduction of pain, even greater than the reduc­
tion of muscle function. Our trials of toxin use for cos­
metic indications revealed a number of individuals whose
migraine headaches disappeared. These and other studies
revealed a pattern that seemed to indicate that the toxin
had a role in pain syndromes. Other studies then showed
that, even in cases of post-herpetic neuralgia, pain could
be decreased or eliminated. This clinical information led
to the discovery that inflammatory mediators such as cal­
citonin gene-related peptide (CGRP), substance P, gluta­
mate and others are also released by SNARE proteins. The
toxin will reduce or eliminate the release locally of inflam­
matory mediators that have the effect of lowering central
nervous system pain thresholds and thereby causing
central sensitization. Botulinum toxin was found to reverse
this effect. Many pain studies are in progress. Toxin treat­
ment of chronic migraine headaches was recently EU and
FDA approved after extensive clinical trials.
The newest change to potential toxin treatment has
been the ability to change the molecule. The binding site
of the molecule has been altered chemically to add spe­
cific ligands. This allows a toxin to be created that has an
affinity for sensory, but not motor neurons, autonomic
1 
Therapeutic uses of the
botulinum toxins
Andrew Blitzer
Introduction
Since the 1970s, when Alan Scott introduced botulinum
neurotoxin A as a therapeutic agent, the number of dif­
ferent uses for this drug has increased exponentially. In
the 1950s, Arnold Burgen and Vernon Brooks, at McGill,
discovered that botulinum toxin presynaptically blocked
the release of acetylcholine from motor nerve terminals,
Summary and Key Features
•
Botulinum toxins exzymatically affect the SNARE
proteins in neural endings
•
Botulinum toxins will reduce and prevent the
release of substances exocytosed by SNARE
proteins
•
Reduction of SNARE function may have
therapeutic implications for autonomic, sensory
and motor parts of the nervous system
•
Reduction of acetyl choline neurotransmitter can
reduce the hyperfunction of muscles in various
dystonias
•
Reduction of acetyl choline neurotransmitter can
reduce autonomic nervous system hyperfunction
such as hyperhidrosis and sialorrhea
•
Reduction of SNARE function can also reduce the
release of inflammatory mediators such as
substance P, CGRP, glutamate and others
•
Reduction of inflammatory mediators can have an
affect on pain syndromes
•
Pain syndromes such as post-herpetic neuralgia,
migraine headaches, TMD can be improved with
botulinum toxin
•
Inflammatory conditions such as rheumatoid
arthritis may be improved with intra-articular
injections of botulinum toxin
•
Bioengineered toxins may allow precise localization
and decrease of exocytosed proteins such as
hormones

2
Botulinum toxin
Pearl 1 
Botulinum toxins may be used for management of lines and
wrinkles of the face that are related to pleating of the skin
overlying the facial muscles of expression.
Box 1.1 
Therapeutic uses of botulinum toxins
I.
Hyperfunctional muscular use:
1. Dystonia
a. Cervical dystonia
b. Blepharospasm
c. Oromandibular dystonia
d. Spasmodic dysphonia
e. Occupational writer’s cramp
f. Foot dystonia
g. Musician’s dystonia
h. Facial dystonia
i. Meige syndrome
j. Tic disorders and stuttering
2. Hyperfunctional facial lines (cosmetic)
3. Hemifacial spasm / facial synkinesis
4. Strabismus
5. Esotropia / exotropia
6. Nystagmus
7. Post-stroke and cerebral palsy spasticity
8. Tremor disorders: limb, neck, vocal
9. Myoclonus
10. Other laryngeal disorders: puberophonia, vocalis
process granulomas
II. Autonomic nervous system use
1. Hyperhidrosis: axillary, palmar, plantar, and facial
2. Benign prostatic hypertrophy
3. Sialorrhea / sialocele / drooling
4. Rhinitis
5. UES / LES achalasia
6. Neurogenic hyperactive bladder
7. Frey syndrome
8. Anal fissures
9. Vaginismus / anismus
III. Afferent nervous system / pain
syndromes / anti-inflammatory:
1. Tension headaches
2. Migraine headaches
3. Temporomandibular disorders / bruxism
4. Myofascial pain
5. Post-herpetic neuralgia
6. Arthritis
7. Trigeminal neuralgia
8. Low back pain
IV. Glandular and secretory cell modulation
1. Growth hormone / acromegaly
neurons, and even certain excretory glands. Keith Foster,
of Syntaxin, recently described making a specific ligand
for growth hormone secretory cells in the pituitary gland.
Growth hormone is also secreted with a SNARE-related
exocytosis, and therefore can be modulated with targeted
specific botulinum toxin. This treatment could be used for
patients with acromegaly. Other glandular secretions
should be studied in this new paradigm for hypersecretory
control.
The following material will in greater depth describe
the use of botulinum toxins for the management of disor­
ders of efferent nerves and muscular hyperfunction; affer­
ent nerves, pain disorders, and inflammatory conditions;
autonomic nervous system disorders; and glandular hyper­
secretion. (See Box 1.1.)
Hyperfunctional muscular uses
Cervical dystonia (spasmodic torticollis)
Dystonia is a group of neurological disorders characterized
by muscular hyperfunction with abnormal movements
and / or postures often accompanied by pain. Cervical dys­
tonia, the most common of the dystonias (most often
lateral colli, but also retrocollis and anterocollis) com­
prises disorders with abnormal neck postures and pain.
Results of injecting the hyperfunctioning and painful
muscles with botulinum toxin in several randomized,
placebo-controlled or open-label studies have shown that
50–90% of patients receive benefit (improved function
and / or reduced pain).The results may vary with dose and
choice of muscles to be injected. The dosage range for CD
is 200–400 units of Botox®. Xeomin®, botulinum toxin A
(Merz Pharm.) has also been approved by the FDA for
CD. Myobloc®, botulinum toxin B (Solstice) has been
FDA approved for CD, particularly in patients who have
a resistance to type A toxin.
Blepharospasm is a focal dystonia affecting the orbicu­
laris oculi muscle producing excessive blinking or eye
closure. This was one of Alan Scott’s original studies, and
led to toxin use in other dystonias. Using botulinum toxin
A for management of blepharospasm has been shown in
several studies to produce significant improvement in
70–100% of patients. Toxin injections have become the
treatment of choice for this disorder. Scott’s original work
comprised studies of toxin use for eye muscle balancing
in cases of strabismus, and later esotropia, exotropia, and
nystagmus.
Scott extended the use of toxin to patients with hemi­
facial spasm. This is a condition in which the anterior
inferior cerebellar artery beats on and compresses the
facial nerve (VII), causing irritability and facial twitching.
The treatment can be a neurosurgical decompression of
the nerve, or injections of botulinum toxin into orbicularis
oculi, and may include the zygomaticus and levator labii
muscles to diminish the twitching and hyperfunctional
activity. The same approach may be used for patients with
post-traumatic or post-Bell’s palsy facial synkinesis. Aside
from treating the synkinetic side of the face, often toxin
is given to the contralateral face to balance the weakness
for symmetry. These facial studies led to a number of
investigators realizing the cosmetic benefit for functional
facial lines and opened the door for injections of toxin for
cosmesis.

3
1 
Therapeutic uses of the botulinum toxins
Extending the concept of reducing hyperfunctional
muscle activity with toxin injections, patients with tremor
disorders and myoclonus have been successfully treated.
This includes treatment of the limbs, neck, palate, and
vocal cords. Although the toxin doesn’t prevent the trem­
ulous activity it can decrease it, thereby decreasing the
patient’s symptoms.
Autonomic nervous system use
Based on observations of patients with botulism, many
have autonomic symptoms such as dry eyes and dry
mouth. Since the sweat glands are cholinergic, it became
obvious that toxin injections should be able to decrease
their function. Numerous studies have shown this and
led to FDA approval for axillary hyperhidrosis. Studies
have also shown efficacy on palmar, facial, and plantar
hyperhidrosis. The effects on these glands last longer than
in muscle perhaps owing to glandular atrophy. Frey syn­
drome or gustatory sweating is from aberrant regeneration
of the autonomic nerve supply of the salivary glands to
the sweat glands of the skin after trauma. Injecting the
facial skin with toxin can prevent the gustatory sweating
for 6–24 months. The salivary glands themselves can be
injected to decrease salivary production in patients who
have drooling, sialorrhea, and sialoceles. Recent studies are
extending the use of botulinum toxin to treat rhinitis with
rhinorrhea (also cholinergically induced).
Oromandibular dystonia is a focal dystonia affecting
the muscles of the jaw and may present most commonly
with closing spasms, making opening of the mouth for
eating and chewing very difficult. It may also present with
opening spasms, making closing of the mouth difficult, or
with lateral or protrusive spasms of the jaw. Some patients
may have writhing movements of the jaw making speaking
and eating difficult. In a number of cases the tongue is
also involved, producing not only a jaw opening but also
uncontrolled tongue protrusion. When this is combined
with other cranial dystonias (usually blepharospasm) it has
been termed ‘Meige syndrome’.
Our group successfully treated the first oromandibular
dystonia (OMD) with botulinum toxin in 1983. We
reported our series in a publication in 1989. Treatment of
the tongue with toxin is often ill advised in many of these
patients, since the production of a hypofunctional tongue
causes dysarthria and significant dysphagia. The successful
treatment of OMD led to the management of other
hyperfunctional disorders of the jaw including temporo­
mandibular disorders (TMD) and bruxism. The usual
muscles injected are the masseter, temporalis, and exter­
nal pterygoids.
Spasmodic dysphonia is a focal dystonia of the larynx.
Most patients have the adductor or closing type produc­
ing a strain-strangled voice type. We performed the first
injection of botulinum toxin injections of the larynx for
this condition in 1984. The other types are the abductor
or opening type that produces a whispering voice or voice
with breathy breaks. There are also patients with adduc­
tor respiratory spasms and a singer’s dystonia. All of these
can be managed with botulinum toxin injections, and
this has become the standard treatment for these disor­
ders. Laryngeal injections were extended to include
management of vocal tremor, vocalis process granulomas,
puberophonia, stuttering with glottal block, and other
hyperfunctional vocal disorders.
Occupational writer’s cramp (focal dystonia of the
hand) was also found to be a disorder in which toxin can
reduce spasm and return normal function. These injec­
tions are usually given with EMG guidance into the muscle
causing the abnormal postures or contractures. This treat­
ment also is for other functionally specific hand dystonias
including stenographer’s dystonia and musician’s dystonia.
This treatment was extended to include dystonias of the
feet. In cases of more generalized dystonia, the abnormal
limb postures can also be reduced and function increased
with toxin injections of the affected muscles. Treatment
has also been extended to patients with post-stroke spas­
ticity and cerebral palsy spasticity. Toxin treatment has
reduced the contractures and pain.
Pearl 2 
Botulinum toxin may be used to control hyperfunctional
muscular conditions such as dystonia, tremor, spasticity, and
synkinesis.
Pearl 3 
Acetylcholine release is also the neurotransmitter for the
parasympathetic nervous system, and blockade can thera­
peutically improve conditions such as hyperhidrosis, sialor­
rhea, sphincter spasms, and rhinitis.
Other autonomic indication began to evolve with the
success found with hyperhidrosis and salivary conditions.
Upper and lower esophageal sphincter achalasia or hyper­
activity and gastroparesis can be diminished with intra­
muscular injections of botulinum toxin. These are usually
performed endoscopically. Recently, the FDA has approved
Botox® for neurogenic hyperactive bladder function.
There are also several trials for the use of botulinum toxin
for benign prostatic hypertrophy. The initial data has
shown gland reduction for about 6 months with apoptosis
and sphincter weakness. Patients with painful, non-healing
anal fissures had been treated with surgical sphincterot­
omy. Not all of the patients had healing with this proce­
dure and some had permanent fecal incontinence. With
toxin injection, there is a temporary chemical sphincter­
otomy, improved blood flow with near-100% healing
of the fissures, and dramatic pain reduction. Anismus
and vaginismus management with toxin have also been
reported.

4
Botulinum toxin
Further reading
Aoki KR 2003 Evidence for antinociceptive activity of botulinum
toxin type A in pain management. Headache 43(suppl 1):
S9–S15
Arad-Cohen A, Blitzer A 2000 Botulinum toxin treatment for
symptomatic Frey’s syndrome. Otolaryngology, Head and Neck
Surgery 122:237–240
Binder WJ, Brin MF, Blitzer A, et al 2000 Botulinum toxin type A
(Botox) for the treatment of migraine headaches; an open-label
study. Otolaryngology, Head and Neck Surgery 123:669–676
Blasi J, Chapman ER, Link E, et al 1993 Botulinum neurotoxin A
selectively cleaves the synaptic protein SNAP-25. Nature
365:160–163
Blitzer A, Brin MF 1997 Use of botulinum toxin for diagnosis and
management of cricopharyngeal achalasia. Otolaryngology, Head
and Neck Surgery 116:328–330
Blitzer A, Brin MF, Greene PE, et al 1989 Botulinum toxin
injections for the treatment of oromandibular dystonia. Annals
of Otolaryngology, Rhinology and Laryngology 98:(2)93–97
Blitzer A, Brin MF, Stewart C 1998 Botulinum toxin management
of spasmodic dysphonia (laryngeal dystonia): a 12 year
experience in more than 900 patients. Laryngoscope
108:1435–1441
Blitzer A, Zalvan C, Gonzales-Yanes O, et al 2002 Botulinum toxin
injections for the management of the hyperfunctional larynx. In:
Brin MF, Jankovic J, Hallett M (eds) Scientific and therapeutic
aspects of botulinum toxin. Lippincott Williams & Wilkins,
Philadelphia, p 207–217
Carruthers JDA 1995 The treatment of congenital nystagmus with
Botox. Journal of Pediatric Ophthalmology and Strabismus
32:306–308
DeFazio G, Abbruzzese G, Girlanda P, et al 2002 Botulinum
toxin A treatment for primary hemifacial spasm: a 10 year
multicenter study. Archives of Neurology 59:418–420
Diener HC, Dodick DW, Aurora SK, et al 2010 PREEMPT 2
Chronic Migraine Study Group. OnabotulinumtoxinA for
treatment of chronic migraine: results from the double-blind,
randomized, placebo-controlled phase of the PREEMPT 2 trial.
Cephalalgia 30(7):804–814; Epub 17 Mar
Freund B, Schwartz M, Symington JM 2000 Botulinum toxin: new
treatment for temporomandibular disorders. British Journal of
Oral and Maxillofacial Surgery 38:466–471
Frucht SJ 2004 Focal task-specific dystonia in musicians. Advances
in Neurology 94:225–230
Gless R, Nauman M, Werner B, et al 2000 Injections of botulinum
toxin A into the salivary glands improve sialorrhea in amytropic
lateral sclerosis. Journal of Neurology, Neurosurgery, and
Psychiatry 69:121–123
Glogau RG 1998 Botulinum A neurotoxin for axillary hyperhidrosis.
Dermatologic Surgery 24:817–819
Gui D, Cassetta E, Anastasio G, et al 1994 Botulinum toxin for
chronic anal fissure. The Lancet 344(8930):1127–1128
Laskawi R, Damanez W, Roggenkamper P 1994 Botulinum toxin
treatment in patients with facial synkinesis. Archives of
Otolaryngology 4:S195–S199
Liu HT, Tsai SK, Kao MC, et al 2006 Botulinum toxin A relieved
neuropathic pain in a case of post-herpetic neuralgia. Pain
Medicine 7(1):89–91
Maria G, Brisinda G, Civello IM, et al 2003 Relief by botulinum
toxin of voiding dysfunction due to benign prostatic hyperplasia:
results of a randomized, placebo-controlled study. Urology
62:259–264
Meng J, Wang J, Lawrence G, et al 2007 Synatorbrevin I mediates
exocytosis of CGRP from sensory neurons and inhibition by
Botulinum toxin reflects their anti-nociceptive potential. Journal
of Cell Science 120:2864–2874
Pearl 4 
It has been found that botulinum toxin can bind and blockade
to the unmyelinated C-fibers and partially myelinated A-delta
fibers of the sensory nervous system.
Pearl 5 
Botulinum toxin has been found to also block the release
of inflammatory mediators such as CGRP, substance P,
glutamate, and others from its effect on SNARE proteins.
Pearl 6 
Pain syndromes such as chronic migraine, post-herpetic
neuralgia, temporomandibular disorders and others can be
treated with botulinum toxins.
Afferent nervous system / pain
syndromes / anti-inflammatory
A number of hyperfunctional muscular conditions had
pain associated with them, and it was thought that pain
reduction was related only to the reduced muscle contrac­
ture. Our group’s work with cosmetic injections led Bill
Binder to observe that several patients with a migraine
headache history ceased to have headaches. Patients with
post-herpetic neuralgia also could be injected with toxin
and have resolution of pain. These clinical observations led
basic scientists to discover that SNARE proteins also
release inflammatory mediators from afferent nerves. The
toxin can bind to C fibers and A-delta fibers, and reduce
or prevent release of the inflammatory mediators, thereby
reducing pain and allowing central thresholds to rise. The
management of chronic migraine headaches with Botox®
has recently been FDA approved. Numbers of other
studies of toxin in pain disorders have included trigeminal
neuralgia, myofascial pain, tension headaches, temporo­
mandibular disorders with pain, and low back pain. The
anti-inflammatory effects have also been studied in a small
series of patients with rheumatoid arthritis.
Glandular and secretory cell modulation
Foster and his group have shown that SNARE proteins are
also responsible for the exocytosis and release of many
hormones. Therefore, if ligands can be made to allow selec­
tive binding of the toxin to specific glandular cell proteins,
toxin can be released in these cells to cleave the SNARE
proteins and therefore prevent exocytosis of the hormone.
Foster reported reduction of growth hormone with a bio­
engineered toxin in animals. Much more work needs to be
done in this arena, but this exciting development opens up
brand new ways of managing many disorders.

5
1 
Therapeutic uses of the botulinum toxins
Pasricha P, Ravich W, Kalloo A 1993 Botulinum toxin for achalasia.
The Lancet 341:244–245
Phelan MW, Franks M, Somogyi GT, et al 2001 Botulinum toxin
urethral sphincter injection to restore bladder emptying in men
and women with voiding dysfunction. Journal of Urology
165:1107–1010
Scott AB 1981 Botulinum toxin injection of the eye muscles to
correct strabismus. Transactions of the American
Ophthalmologic Society 79:734–770
Shaari CM, Sanders I, Wu BL, et al 1995 Rhinorrhea is
decreased in dogs after nasal application of botulinum toxin.
Otolaryngology, Head and Neck Surgery 112:566–571
Smith SJ, Ellis E, White S, et al 2000 A double-blind, placebo
controlled study of botulinum toxin in upper limb spasticity
after stroke or head injury. Clinical Rehabilitation 14:5–13

Introduction
The development of botulinum toxin for therapeutic use
is marked by its cross-disciplinary use for varied medical
conditions. In particular, botulinum toxin has become a
valuable therapy for selected disorders, treated by mul­
tiple specialists, whereby symptoms were inadequately
managed with existing treatments.
In this chapter, we provide an overview of development
events, interspersed with personal observations of histori­
cal and human interest. Due to the structure and format
of this book, this chapter is not fully referenced. For full
reference citations to the events described here, the
reader is referred to the additional reading list at the end
of this document and the references therein and is encour­
aged to seek supplementary sources.
Currently unapproved uses are mentioned for historical
purposes, and health care providers should ascertain
product licensure information regarding locally approved
indications. In describing the formally developed uses of
botulinum toxin, we confine ourselves to those for which
Botox® (onabotulinumtoxinA) has been approved by regu­
latory agencies in the United States or Europe.
Identification, isolation, and characterization
The first well-described cases of medical illness after oral
ingestion of an apparent foodstuff that may have con­
tained some botulinum toxin were reported between
1817 and 1822 by the German physician Justinus Kerner.
Kerner noted that the active substance interrupted signals
from the motor nerves to muscles, but spared sensory
nerves and cognitive abilities. He also theorized that the
substance could possibly be used as therapy for medical
conditions when ingested orally.
The bacterial etiology of botulism was first noted by
the microbiologist Emile Pierre van Ermengen who docu­
mented his findings in 1897, identifying and naming the
responsible bacteria as Bacillus botulinus, which later
became Clostridium botulinum. In 1905, Tchitchikine
found that C. botulinum produced a substance that
affected neurotransmitter function. In 1919, Professor
Burke of Stanford University described an alphabetical
classification for the different serotypes of botulinum
toxin based on his toxin–antitoxin experiments.
2 
History of
onabotulinumtoxinA
therapeutic
Mitchell F. Brin, Andrew Blitzer
Summary and Key Features
•
Botulinum toxin has become a valuable therapy for
treating selected neurological and urological
disorders and managing the appearance of
glabellar facial lines
•
After identification of a muscle-relaxing substance
present in sausages, scientists attributed these
effects to a bacterium that became known as
Clostridium botulinum. They isolated and
characterized the neurotoxin, and described its
mechanism of action on nerve terminals
•
Dr Alan Scott, an ophthalmologist in San
Francisco, began studying botulinum toxin type A
(‘Oculinum’) in the 1960s and 1970s as a possible
treatment for patients with strabismus
•
Following Alan Scott’s successful studies in
strabismus patients, he and others, including our
group at Columbia University working under a
research protocol, examined botulinum toxin type
A in several neurological conditions, including
blepharospasm, cervical dystonia, and also
hyperfunctional facial lines, marked by the
overactivity of facial or neck muscles
•
Oculinum was approved for the treatment of
strabismus and blepharospasm by the United
Stated Food and Drug Administration in 1989
•
Alan Scott’s first manufactured research
therapeutic, Oculinum, was later acquired by
Allergan and renamed Botox®
•
Other botulinum toxin products have subsequently
been licensed, each having its own clinical profile
and dosing strategy; today these products have
unique non-proprietary names
•
As of 2011, onabotulinumtoxinA (Botox® / Botox®
Cosmetic) is approved for multiple indications
worldwide, including eight indications in the United
States
•
In providing a treatment option for several rare
neurological conditions, the clinical development of
onabotulinumtoxinA may have contributed to an
enhanced understanding of these disorders

7
2 
History of onabotulinumtoxinA therapeutic
conditions. He found that injecting minute amounts into
extraocular muscles of monkeys with electromyographic
(EMG) guidance produced long-lasting effects for the cor­
rection of strabismus and, at the doses used, without any
systemic effects. Scott published these preclinical results
in 1973, performed additional preclinical toxicology
studies to evaluate doses that produced systemic effects
in primates and obtained an Investigational New Drug
(IND) designation from the US Food and Drug Adminis­
tration (FDA) to conduct the first clinical trial for humans
with strabismus in 1977 by injecting minute amounts
locally into the extraocular muscles. He called his formu­
lation ‘Oculinum’ (Fig. 2.1). This clinical trial led to the
1980 publication of the first 19 patients, where efficacy
in treating strabismus was reported.
In the early 1980s, at the Columbia University Neuro­
logical Institute, we were funded by the Dystonia Medical
Research Foundation with the primary goal of identifying
effective treatments for dystonia, in addition to exploring
the disease’s genetic underpinnings. Stanley Fahn was the
principal investigator of the Center, Mitchell Brin was
a Movement Disorders Fellow and Center’s Program
Coordinator, and Andrew Blitzer was acting Chairman of
the Department of Otolaryngology. Dr Fahn learned
directly from Dr Scott when he attended a workshop in
which Dr Scott described his use of Oculinum. He sub­
mitted a protocol to the Columbia University Institutional
Review Board and also obtained an IND from the FDA to
evaluate the potential therapeutic use of Oculinum. The
Purification and crystallization of botulinum neuro­
toxin followed in the ensuing decades, eventually enabling
mechanism of action studies. Dr Carl Lamanna and col­
leagues observed dissociation between the hemagglutinat­
ing activity of botulinum toxin type A and its toxicity,
leading to further studies that identified both toxic and
non-toxic proteins in the crystalline toxin complex.
Lamanna also observed the extreme potency of botulinum
toxin, which would later be recognized as a major advan­
tage for a local, injectable therapy. In the late 1940s and
early 1950s, Arnold Burgen and Vernon Brooks at McGill
University discovered that botulinum toxin acted presyn­
aptically to block the release of acetylcholine from motor
nerve terminals. Brooks mentioned to a colleague, Edward
Schantz, that the toxin might be useful for weakening
hyperactive muscles.
Edward Schantz had worked in the Chemical Corps at
Fort Detrick purifying botulinum toxin using the method
established by Lamanna and Duff. Schantz then relocated
to the University of Wisconsin where he perfected the
purification and crystallization of botulinum toxin. In
1971, Daniel Drachman, with toxin purified and supplied
by Edward Schantz, showed that injecting minute amounts
of botulinum toxin type A in the hindlimbs of chicks
caused local denervation. In 1979, Lance Simpson
described the key elements of the mechanism of action:
binding, internalization, translocation, and interruption of
neurotransmitter release. Pamphlett clarified that there is
neither cell death nor axonal degeneration and the affected
nerve terminals do not degenerate. Although traditionally
called a neurotoxin because of its potential to cause gen­
eralized muscle weakness at exceedingly high doses, in
2004 our group reported that botulinum toxin type A is
not cytotoxic.
Exploration of clinical potential
In the 1960s and 1970s, Alan Scott, an ophthalmologist
in San Francisco, was seeking an alternative to surgery for
the correction of strabismus. Scott has described the
events that led to his clinical studies with botulinum toxin
type A in several articles, and readers are encouraged to
read his accounts. Scott and his colleague Carter Collins
were studying the forces and actions of eye muscles, and
they considered whether a long-acting muscle-weakening
agent could be used for treatment of strabismus. Based on
Daniel Drachman’s work, Scott thought that botulinum
toxin might be an option. Drachman directed Scott to
Edward Schantz, who provided him with botulinum toxin
for testing. It was noted that, historically, outbreaks of
type A botulism produced predominantly motor weak­
ness, whereas type B caused a predominance of autonomic
symptoms and thus type A was selected by Scott. Schantz
provided the crystallized botulinum toxin type A by
regular mail in a metal tube placed in another metal tube.
Scott took the crystalline toxin into his laboratory and
diluted it into small aliquots, buffered it with albumin
instead of gelatin, and developed testing and storage
Figure 2.1  Oculinum 100 units research vial.

8
Botulinum toxin
clinical protocol’s inclusion criteria enabled our broad
research program.
During our first year of study, we examined the poten­
tial use of Oculinum in treating patients with primary and
symptomatic inappropriate contractions, including those
of the facial muscles (e.g. blepharospasm, hemifacial
spasm, jaw (oromandibular dystonia, temporomandibular
joint dysfunction), facial synkinesis, palatal myoclonus,
tongue), neck (e.g. cervical dystonia), vocal cords (e.g.
spasmodic dysphonia), limbs (e.g. arm dystonia, writer’s
cramp, occupational / stenographer’s cramp, legs), various
tremors, etc. and reported our initial findings in 1985.
Many of our treatments were the first-described therapeu­
tic assessments of Dr Scott’s botulinum toxin type A, and
these were further elaborated upon in subsequent publica­
tions. Furthermore, the research contributed to the estab­
lishment of new areas for investigation.
In addition, we were interested in possible systemic
effects. In our initial studies, we tested vital capacity with
a hand-held spirometer and did not find an adverse effect
on pulmonary function. We also initiated single-fiber elec­
tromyography studies, identified subclinical distant muscle
effects, and reported our findings.
Nomenclature
Alan Scott called his manufactured research therapeutic
‘Oculinum’. The name Oculinum reflected the strabismus
use and was a combination of linking the ‘ocul’ for eyes,
with ‘lining’-up-the-eye and the Latinate ending ‘um’
added to legitimate drugs (personal communication, Alan
Scott). At Columbia University, we coined and published
the term ‘Botox’, which was the same product (Oculi­
num) manufactured by Dr Scott. This original formula­
tion, Oculinum, was licensed to Allergan with the first
approval for strabismus and blepharospasm in December
1989. Allergan subsequently acquired the product in 1991
as ‘Oculinum Injectable’, and then changed the trade
name to ‘Botox®; this name was accepted by the FDA
in 1992.
Although the finished, marketed botulinum neuro­
toxin products do not have the same properties, publica­
tions have discussed the neurotoxins without referring to
brand names. Historically, basic science colleagues used
the abbreviations BoNT, Botx, and BoTX and BoTx for
the research, raw material botulinum toxin, with BoNT/A
referring to serotype A. ‘BTX’ had also been used for
more than two decades to refer to finished products,
with BTX-A referring to serotype A. This nomenclature
should not be confused with Batrachotoxin (‘poison dart
frog’ poison), which has also been abbreviated as BTX.
In the early 2000s, ‘BTXA’ was used for the product
manufactured by the Lanzhou Institute of Biological
Products (LIBP) of the People’s Republic of China
(PRC). Consequently, most authors discontinued the use
of BTXA or BTX-A when referring generally to type A
botulinum toxin, and adopted the BoNTA or BoNT-A
terminology. However, in 2009, the FDA applied the
United States Adopted Names Council terminology to
botulinum toxins, with onabotulinumtoxinA referring to
Botox®, abobotulinumtoxinA to Dysport® and rimabotu­
linumtoxinB referring to Myobloc®, and subsequently,
incobotulinumtoxinA for Xeomin®, Merz’s formulation of
BoNTA.
When discussing different botulinum neurotoxin prod­
ucts, it is important to note their differences in clinical
dosing. Botulinum toxin doses are expressed in units of
biological activity, which differ for each product. For
instance, the units of onabotulinumtoxinA and abobotu­
linumtoxinA are different and in 1993 we published the
statement that ‘these products are distinct, and dosing is
significantly different so that if one administers the same
number of Dysport® units as when delivering Botox®,
serious side effects may occur.’ Regulatory agencies world­
wide have issued language stating that units are neither
interchangeable nor convertible between different botuli­
num products. Thus, unit doses of one product cannot be
used to generate doses of another.
Developed indications for
onabotulinumtoxinA
OnabotulinumtoxinA is approved for eight different indi­
cations in the United States, which are described in Table
2.1 along with their year of approval and the US FDA
indication. Table 2.1 also lists the approval years for these
indications in the European Union (EU).
Strabismus
As noted previously, the therapeutic use of botulinum
toxin type A began with strabismus, in 1978 when Alan
Scott used the toxin to treat the disorder in children after
successful experiments in monkeys. The rationale for
treating strabismus was that the toxin would create weak­
ness in the injected ocular muscles, causing them to relax
over time. This would enable the antagonist muscles con­
tract more effectively, achieving the desired result. Scott
noted in 1980 that, along with its efficacy in strabismus
correction, botulinum toxin type A was an appropriate
therapeutic agent because it had no known effects other
than muscle paralysis, had no antigenic effects at small
doses and, based on his experience at the time, did not
diffuse to muscles adjacent to those injected, produced
dose-dependent effects that could last for several weeks,
and, in the treatment of strabismus, did not show systemic
effects.
Scott’s work, including a combination of double-blind
and open-label studies under his original IND, led to the
initial FDA approval for botulinum toxin type A as Ocu­
linum in 1989 for the treatment of strabismus.
Blepharospasm
After his initial success with onabotulinumtoxinA for
the treatment of strabismus, Scott postulated that the

9
2 
History of onabotulinumtoxinA therapeutic
Table 2.1  Approved indications for onabotulinumtoxinA in the United States and Europe
Year of FDA
approval
Description
Indication statement approved by US FDA
Year of EU
approval*
1989
Strabismus
Deviation of the alignment of one eye in relation
to the other.
Treatment of strabismus and blepharospasm
associated with dystonia, including benign
essential blepharospasm or VII disorders in
patients ≥12 years of age and above
N/A
1989
Blepharospasm
A dystonic condition characterized by
involuntary, repetitive eyelid closure caused by
spasmodic or sustained contraction of the
orbicularis oculi muscles
1994
2000
Cervical dystonia
A focal dystonia characterized by involuntary
contractions of the neck and shoulder muscles
consisting of any combination of head tilting,
torsion, neck extension or flexion; frequently
accompanied by moderate to severe pain
Treatment of cervical dystonia in adult
patients, to reduce the severity of abnormal
head position and neck pain
1995
2002**
Glabellar lines
Vertical lines between the eyebrows caused by
contraction of the corrugator and procerus
muscles
For the temporary improvement in the
appearance of moderate to severe glabellar
lines associated with corrugator and / or
procerus muscle activity in adult patients
≤65 years of age
2003***
2004
Primary axillary hyperhidrosis
A condition of localized, excessive sweating of
the axillae
Treatment of severe axillary hyperhidrosis
that is inadequately managed by topical
agents in adult patients
2001
2010
Focal upper limb spasticity
A velocity-dependent increase in muscle tone of
the upper limbs with exaggerated tendon jerks
resulting from hyperexcitability of the stretch
reflex; occurs as one component of the upper
motoneuron syndrome
Treatment of upper limb spasticity in adult
patients, to decrease the severity of
increased muscle tone in elbow flexors
(biceps), wrist flexors (flexor carpi radialis
and flexor carpi ulnaris) and finger flexors
(flexor digitorum profundus and flexor
digitorum sublimis)
2001
2010
Chronic migraine
At least 15 headache dyas per month for >3
months, of which >8 headache days per month
are migraine and/or are treated and relieved by
triptan/ergot
Prophylaxis of headaches in adult patients
with chronic migraine (≥15 days per month
with headache lasting 4 hours a day or
longer)
2010
2011
Detrusor overactivity associated with a
neurological condition
Overactivity of the detrusor muscle (a smooth
muscle in the urinary bladder) caused by a
neurological condition that can result in urinary
incontinence
Treatment of urinary incontinence due to
detrusor overactivity associated with a
neurological condition [e.g. spinal cord injury
(SCI), multiple sclerosis (MS)] in adults who
have an inadequate response to or are
intolerant of an anticholinergic medication
2011
N/A
Juvenile cerebral palsy dynamic equinus foot
deformity
A neurological disorder that appears in infancy or
early childhood and permanently affects body
movement and muscle coordination but generally
does not worsen over time; equinus foot
deformity results from dynamic overactivity or
spasticity of the gastrocnemius–soleus muscle
complex alone, or in combination with other
ankle muscles, or from fixed muscle contractures
N/A
1997
EU = European Union, FDA = Food and Drug Administration, N/A = not applicable.
*First EU approval in United Kingdom or Ireland (Reference Member State of the Mutual Recognition Process). Specific indications
often vary from those licensed in the United States.
**As Botox® Cosmetic.
***Approved as VISTABEL® in France (Reference Member State of the Mutual Recognition Process).

10
Botulinum toxin
axillary hyperhidrosis. Two global pivotal studies were
conducted, with additional long-term follow-up exten­
sions. Whereas the benefit typically experienced by
patients treated with skeletal muscle disorders is approxi­
mately 3–4 months, the average duration of effect in
axillary hyperhidrosis is 6–7 months, with 28% of patients
experiencing benefit exceeding 1 year in one study. These
studies led to global registration of onabotulinumtoxinA
for the treatment of severe axillary hyperhidrosis, with
approval by the FDA in 2004.
Adult post-stroke spasticity
In 2009 Alan Scott reported treating the first patients
with thigh adductor spasms, ‘Rather large doses of 300
units in two patients with thigh adductor spasms showed
systemic safety…’. Many years after his initial treatments,
reports of possible systemic effects were described in
other patients.
In 1989, Das & Park of the Southend District Stroke
Unit of Rochford Hospital in Essex, United Kingdom
reported on the use of botulinum toxin type A to treat
eight post-stroke patients refractory to conventional
therapy and observed improvement in spasticity with an
acceptable side effect profile. Many open-label and several
double-blind trials followed and have confirmed these
findings.
Allergan initiated a global registration program focused
on treating patients with upper-limb post-stroke spastic­
ity. Approved in Europe in 2001, US approval was granted
in 2010.
Juvenile cerebral palsy
Most neurotoxin development programs of botulinum
toxin type A in children with cerebral palsy have focused
on equinus gait deformity, a common motor manifestation
of prolonged focal muscle spasticity in this population.
Approved for equinus gait deformity due to spasticity in
Europe in 1997 and currently in most of the world, ona­
botulinumtoxinA is not approved for this indication in the
United States, and development continues.
Chronic migraine
The migraine history, similar to that of cosmetic, was
serendipitous. Drs Blitzer and William Binder were resi­
dents at the Mount Sinai Hospital and together they pub­
lished their first manuscripts in 1978. In the early 1990s,
Dr Blitzer trained Dr Binder on the cosmetic treatment
approach, and he collaborated on our research and
co-authored our initial hyperfunctional facial lines manu­
scripts. Also in the 1990s, Dr Binder observed that, when
treating patients with onabotulinumtoxinA for cosmetic
enhancement, some of his patients with a history of
migraine headaches reported a reduction or elimination of
the number or intensity of their headaches concurrently
with the cosmetic effect. Having later contacted us, we
toxin could also be used to treat blepharospasm by
weakening the orbicularis oculi muscles. He proved his
theory correct when he injected 39 cases and observed
improvement in all of them. Several case series, open-label
and double-blind placebo-controlled (DBPC) studies,
including our own, confirmed the efficacy of onabotu­
linumtoxinA for blepharospasm. OnabotulinumtoxinA
was approved by the FDA in 1989, along with strabismus,
for the treatment of blepharospasm associated with dys­
tonia in patients at least 12 years of age.
Cervical dystonia
Alan Scott treated the first three cervical dystonia patients
with his product in 1983. Joseph Tsui et al at the Univer­
sity of British Columbia then extended this work and
published a pilot study in which 12 patients received
onabotulinumtoxinA injections into relevant neck muscles
for the treatment of spasmodic torticollis (cervical dysto­
nia). In both their pilot study and a follow-up DBPC
study, they observed that onabotulinumtoxinA not only
decreased abnormal movement but provided pain relief as
well; our studies demonstrated similar results.
Shortly after beginning our research with onabotu­
linumtoxinA, we were funded by the FDA’s Office of
Orphan Product Development to conduct a double-
blind study examining the efficacy of onabotulinumtox­
inA in the treatment of cervical dystonia, the results of
which were reported by Greene et al in 1990. Later,
based on an Allergan-sponsored, pivotal Phase III study,
onabotulinumtoxinA was approved by the FDA in 2000
for the treatment of cervical dystonia in adult patients,
to reduce the severity of abnormal head position and
neck pain.
Glabellar lines
The use of onabotulinumtoxinA for cosmetic purposes
dates back to the 1980s and the history of these investi­
gations is described in another chapter. Allergan initiated
a development program for the treatment of glabellar
lines that ultimately led to worldwide approvals of this
indication. OnabotulinumtoxinA was approved in several
countries in South America by 2000, as Botox® Cosmetic
in Canada in April 2001 and in the USA on 12 April
2002 for the treatment of moderate to severe glabellar
lines.
Primary focal hyperhidrosis
In the 1950s, Ambache reported on animal studies that
explored the potential blocking effect of botulinum toxin
on sweat glands. Human data were not available until
1994, when Bushara & Park reported areas of anhidrosis
around botulinum toxin type A injection sites in hemifa­
cial spasm patients.
Allergan initiated a series of registration clinical trials
examining onabotulinumtoxinA for the treatment of

11
2 
History of onabotulinumtoxinA therapeutic
botulinum toxin therapy has elevated the general aware­
ness of many conditions, particularly uncommon ones
where patients were enrolled into clinical research studies,
and also those approved by regulatory agencies for treat­
ment. This increased awareness afforded the medical
research community the opportunity to further character­
ize and study these disorders, often with remarkable
results. Historically, at the authors’ original Columbia Uni­
versity Center, the availability of onabotulinumtoxinA as
a research therapy brought patients into the clinic where
descriptive, genetic, pathophysiology, and other studies
were conducted. It seems possible that the increased
number of patient consultations, coupled with funding
from governmental, patient, and other organizations,
helped advance the original mission of the Dystonia
Medical Research Foundation and those of other organiza­
tions, and accelerated scientific progress.
None of this work could have been performed without
the broad contributions of so many patients, investigators,
scientists and clinicians.
Acknowledgments
The authors gratefully acknowledge the editorial assis­
tance of Mary Ann Chapman, PhD.
Further reading
Ambache N 1951 A further survey of the action of Clostridium
Botulinum toxin upon different types of autonomic nerve fibre.
Journal of Physiology (Lond) 113:1–17
Brin MF, Aoki KR, Dressler D 2004 Pharmacology of botulinum
toxin therapy. In: Brin MF, Comella C, Jankovic J (eds)
Dystonia: etiology, clinical features, and treatment. Lippincott,
Williams & Wilkins, Philadelphia, p 93–112
Brin MF, Blitzer A 1993 Botulinum toxin: dangerous terminology
errors [letter] [see comments]. Journal of the Royal Society of
Medicine 86:493–494
Brin MF, Fahn S, Moskowitz C, et al 1987 Localized injections of
botulinum toxin for the treatment of focal dystonia and
hemifacial spasm. Movement Disorders 2:237–254
Brooks VB 2001 Vernon Brooks. In: Squire LR (ed) The history
of neuroscience in autobiography. Academic Press, New York,
p 76–117
Burgen AS, Dickens F, Zatman LJ 1949 The action of botulinum
toxin on the neuro-muscular junction. Journal of Physiology
(Lond) 109:10–24
Burke GS 1919 Notes on Bacillus botulinus. Journal of Bacteriology
4:555–570
Bushara KO, Park DM 1994 Botulinum toxin and sweating. Journal
of Neurology, Neurosurgery and Psychiatry 57:1437–1438
Das TK, Park DM 1989 Effect of treatment with botulinum toxin
on spasticity. Postgraduate Medical Journal 65:208–210
Fahn S, List T, Moskowitz CB, et al 1985 Double-blind controlled
study of botulinum toxin for blepharospasm. Neurology
35(Suppl1):271
Greene P, Kang U, Fahn S, et al 1990 Double-blind, placebo-
controlled trial of botulinum toxin injections for the treatment
of spasmodic torticollis. Neurology 40:1213–1218
Kerner J 1822 Das Fettgift und die Fettsaure und ihre Wirkungen
auf den thierischen Organismus. Ein Beytrag zur Untersuchung
des in verdorbenen Wursten giftig wirkenden Stoffes. Cotta-
Verlag, Stuttgart, Tubingen
pooled our observations and reported our findings in 1998,
with a full manuscript published in 2000. Allergan initi­
ated a headache development program and explored dose,
treatment paradigm, and patient selection in multiple
studies. Two large Phase III studies were conducted and
onabotulinumtoxinA was approved for chronic migraine
in the UK and US in 2010.
Neurogenic detrusor overactivity
Schurch first reported the use of onabotulinumtoxinA to
treat detrusor overactivity in spinal cord-injured patients
in 2000. Allergan initiated a development program exam­
ining the effects of onabotulinumtoxinA treatment for
detrusor overactivity in patients with spinal cord injury
and multiple sclerosis. This program resulted in the issu­
ance of licenses for onabotulinumtoxinA for neurogenic
detrusor overactivity in North America, Europe and other
regions in 2011.
Cell-based potency assay
The mouse LD50 potency assay is required by global regu­
latory agencies for testing final commercial neurotoxin
product prior to release. This assay has been the standard
for potency and stability testing for all botulinum neuro­
toxins, and involves determining the median lethal dose
of botulinum neurotoxin in mice at 72 hours following
intraperitoneal injection. The ‘3R’ principles of refine­
ment, reduction and eventual replacement of laboratory
animals in manufacturing have been an objective of many
pharmaceutical companies.
Allergan addressed these principals for onabotulinum­
toxinA manufacturing, recognizing the hurdle to overcome
the technical challenges of establishing a non-animal assay
that: (1) reflected the four distinct mechanisms of neuro­
toxin action (binding, internalization, translocation, cleav­
age of SNAP-25); (2) established an appropriate level
of assay sensitivity; and (3) met quality standards for
commercial use and hurdles that regulatory agencies
established for approval of changes to product-related
procedures. In 2011, a cell-based potency assay (CBPA)
for onabotulinumtoxinA was approved by the FDA, and
subsequently additional regulatory agencies. The CBPA is
used, where approved, for stability and potency testing
with the goal of reducing the use of animal-based assay
testing.
Commentary
The seminal work of Dr Alan Scott has opened doors to
numerous patients and scientists, and botulinum toxin
therapy joins other biopharmaceuticals that have impacted
medical care by safely and effectively relieving a spectrum
of chronic and previously often ineffectively treated
human disorders. In the field of aesthetics, onabotulinum­
toxinA has advanced the science of facial rejuvenation. In
addition to the direct medical benefits for patients,

12
Botulinum toxin
Scott AB 2004 Development of botulinum toxin therapy.
Dermatologic Clinics 22:131–133, v
Simpson LL 1981 The origin, structure, and pharmacological
activity of botulinum toxin. Pharmacological Reviews
33:155–188
Snipe PT, Sommer H 1928 Studies on botulinus toxin. 3. Acid
precipitation of botulinus toxin. Journal of Infectious Diseases
43:152–160
Scott A 2009 Development of botulinum toxin (Foreword). In:
Jankovic J, Albanese A, Atassi MZ, et al (eds) Botulinum
toxin – therapeutic clinical practice and science. Saunders,
Philadelphia
Scott AB 1980 Botulinum toxin injection into extraocular muscles
as an alternative to strabismus surgery. Journal of Pediatric
Ophthalmology and Strabismus 17:21–25
Scott AB 1994 Preface. In: Jankovic J, Hallett M (eds)
Therapy with botulinum toxin. Marcel Dekker, New York,
p vii–ix

Scott of the Smith Kettlewell Institute of Visual Sciences,
San Francisco, CA) for the treatment of strabismus,
benign essential blepharospasm, and hemifacial spasm
(Smith Kettlewell 1990.) Many of these patients had
received multiple injections with no evidence of antibody
formation or systemic complications over 6 years of
continued use. In Vancouver, British Columbia, ophthal­
mologist Dr Jean Carruthers noticed a remarkable and
unexpected effect in the brow of a patient treated for
blepharospasm: a noticeable reduction in the appearance
of glabellar furrows, giving her a more serene, untroubled
expression. Dr Carruthers discussed the observation with
her dermatologist spouse, Dr Alastair Carruthers, who
was attempting to soften the forehead wrinkles of his
patients using soft-tissue-augmenting agents available in
the eighties.
3 
History of cosmetic
botulinum toxin
Alastair Carruthers, Jean Carruthers
Introduction
The discovery of ‘sausage poison’ and subsequent identi­
fication of Clostridium botulinum as the bacterium respon­
sible has had an enormous and lasting impact on the field
of cosmetic dermatology. As is often the case in medicine,
a series of serendipitous discoveries – coupled with astute
clinical observations – unlocked the potential of botuli­
num toxin (BoNT) and led to significant medical gains
(Tables 3.1 and 3.2). Once hailed as a promising break­
through for a handful of muscular disorders, BoNT has
since become a veritable mainstay of the cosmetic practi­
tioner, its popularity growing exponentially to become one
of the most requested procedures in facial rejuvenation.
Serendipitous discovery
By the late 1980s, nearly 10 000 patients had received
injections of BoNT type A (BoNT-A; then called ‘Oculi­
num’ and distributed to qualified injectors by Dr Alan
Summary and Key Features
•
In the mid-eighties, Dr Jean Carruthers noticed a
concomitant improvement in glabellar rhytides in a
patient treated with botulinum toxin (BoNT) for
blepharospasm
•
First trial involved 18 patients and was published in
1992
•
By 2002, open-label studies of more than 800
patients confirmed the efficacy and safety of BoNT
in the treatment of hyperfunctional wrinkles
•
In April 2002, the Food and Drug Administration
(FDA) approved BoNT for the non-surgical
reduction of glabellar rhytides
•
Cosmetic BoNT is now used for hyperkinetic lines
in the face, neck, and chest, for facial sculpting,
and as an adjunct to other rejuvenating modalities
•
The original formulation of BoNT has since gone
on to receive approval for 20 indications in more
than 75 countries
CASE STUDY 1 
Mrs. LW, 50-years-old, had suffered for 20 years from severe
bilateral spasms and twitches of her eyelids on both sides.
Despite her normal visual acuities, she was unable to drive
her car and apprehensive about crossing the street. Her
clerical job had become increasingly difficult to perform; her
eyelids would spasm and close without warning, worsening
toward the end of the working day. Treatment with 30 units
of onabotulinumtoxinA (Botox®) on each side resulted in
almost complete symptom relief for approximately 3 months.
In addition, she noticed that her brow and face appeared
more relaxed, and her friends commented about how ‘fresh’
and attractive she looked. The deep frown lines between her
brows appeared to efface, and her husband thought she
looked less intense and angry. At her next appointment, Mrs.
LW expressed concern when her doctor (JC) did not treat the
medial ends of her eyebrows. When her doctor explained that
she had not observed any spasm in those areas, Mrs. LW
said: ‘Every time you treat me there, I get this beautiful,
untroubled expression.’ It was the birth of the realization that
onabotulinumtoxinA could be helpful in other patients with
similar glabellar folds and medial brow ptosis.
Pearl 1 
Listen carefully to your patients – they often have valuable
insights to share!

14
Botulinum toxin
Current options for the aging face at the time included
surgery and soft-tissue augmentation, which either did not
address the underlying facial musculature contributing to
the emergence of hyperfunctional rhytides or were associ­
ated with considerable risk, questionable efficacy, and
inconvenience. Surgical procedures, such as rhytidectomy,
liposuction, brow lift, dermabrasion, and chemical peels,
constituted major procedures that were sometimes associ­
ated with significant side effects (including scarring and
abnormal facial movements) and long recovery periods.
Available soft-tissue augmenting agents included collagen,
silicone, or autologous fat, which have been reported to
produce embolic necrosis of the retina and blindness if
accidentally injected into the retinal circulation. Bovine
collagen was associated with allergic reactions, and con­
cerns about adjuvant disease were raised with the use of
silicone. The timing for a non-invasive and easy injectable
treatment that carried little risk of complication could not
have been more perfect, nor the aging population more
willing and eager.
Patient zero and the first clinical trials
Intrigued by the possibilities, the Carruthers injected a
small amount of Scott’s BoNT-A between the brows of
their assistant, Cathy Bickerton Swann – known as ‘patient
zero’ – and awaited the results (Figure 3.1A–D). Seventeen
more patients followed, aged 34 to 51, who would become
part of the first report on the efficacy of BoNT-A, pub­
lished in 1992. Subjects received injections directly into
the glabellar furrow (10–12.5 U/furrow), as well as one
or more subsequent injections into the corrugator muscles
(10–20 U/furrow or per corrugator) 3 to 4 months fol­
lowing previous injections. One patient did not respond
at all to injection, and one was lost to follow-up. The
remaining patients experienced varying degrees of brow
improvement, from complete line effacement (six of sev­
enteen) (Fig. 3.2) to discernible brow creases that simply
lessened in depth (eight subjects). The effects of the toxin
lasted from 4 to 11 months, depending on the number of
repeated injections and length of exposure. In general,
subjects who received treatment over a longer period of
time experienced a treatment persistence of 7–11 months.
Side effects included one case each of brow and lid ptosis
that resolved within 14 days, two cases of transient head­
ache, and one subject who experienced transient numb­
ness at the injection site. Although the authors conclude
by saying that BoNT-A is safe and effective, ‘We do not
believe at present that it is the treatment of choice’ except
Table 3.1  Timeline of clinical discovery
Late 1700s
Outbreaks of deadly illness from contaminated
foods sweeps across Europe
1793
Biggest outbreak in Wildebrad, Southern
Germany
1811
‘Prussic acid’ named as culprit in sausage
poisoning
1822
Dr Justinus Kerner publishes monograph of
‘sausage poison’ and accurately describes
botulism
1895
Professor Emile Pierre Van Ermengem
identifies Clostridium botulinum as causative
agent of botulism
1895–1915
Seven serotypes of toxins are recognized
1928
Dr Herman Sommer isolates most potent
serotype: BoNT-A
1946
Carl Lamanna and James Duff develop
concentration and crystallization techniques
subsequently used by Dr Edward J. Schantz at
Fort Detrick, Maryland, for possible biological
weapon
1972
Dr Schantz takes his research to the University
of Wisconsin, where he produces the large
batch of BoNT-A that remained in clinical use
until December 1997
Table 3.2  Timeline of therapeutic development and use
Late 1960s–early
1970s
Dr Alan Scott begins animal
experimentation with BoNT-A supplied
by Dr Schantz
1973
Dr Scott publishes the first report of
BoNT-A in primates
1978
FDA grants approval to begin testing
small amounts of the toxin (Oculinum)
in human volunteers
1980
Landmark paper demonstrating that
BoNT-A corrects gaze misalignment in
humans
1988
Allergan Inc. acquires rights to distribute
Dr Scott’s Oculinum in the United
States
1989–
FDA approves BoNT-A for the non-
surgical correction of strabismus,
blepharospasm, hemifacial spasm,
and Meige syndrome in adults
Clinical use expands to include
treatment of cervical dystonia and
spasmodic torticollis
Allergan purchases Dr Scott’s company
and renames the toxin Botox®
Pearl 2 
Keep an open mind about new treatments and indications
for old treatments. For example, long eyelashes were
reported as adverse events after treatment with bimatoprost.
Now, bimatoprost is an FDA-approved treatment to thicken,
darken, and lengthen eyelashes.

15
3 
History of cosmetic botulinum toxin
Figure 3.1  ‘Patient zero’ before (A, B) and after (C, D) injection of BoNT into the glabella: (A, C) resting, (B, D) frowning. From Carruthers JDA,
Carruthers JA 1992 Treatment of glabellar frown lines with C. botulinum-A exotoxin. Journal of Dermatologic Surgery and Oncology 18:17-21.
Reprinted with permission of Dermatologic Surgery.
A
B
C
D
Figure 3.2  The first clinical trial of BoNT in the glabellar furrow and corrugator muscles demonstrated varying degrees of improvement to the
appearance of the brow. (A) Pre-treatment. (B) Here, treatment led to complete line effacement.
A
B
for ‘those who are collagen-allergic or who are disinclined
to undergo surgery.’
The publication of their findings drew a flurry of inter­
est among other clinicians, some of whom had already
begun their own off-label experiments. Clark & Berris
restored symmetry in a post-surgical patient with unilat­
eral frontal nerve paralysis and one-sided frown lines and
brow elevation on animation by injecting BoNT-A into the
opposite functioning frontalis muscle. Blitzer and col­
leagues had used BoNT-A to treat focal dystonia since

16
Botulinum toxin
1984. By the late eighties, they had noticed a loss of
hyperfunctional lines in subjects treated with the toxin for
a number of disorders, including blepharospasm, Meige’s
syndrome, hemifacial spasm, and post-Bell’s palsy facial
synkinesis. In order to study the phenomenon further, 26
patients aged 3–84 years with hyperfunctional lines and
dystonia received aliquots of 1.2–10 U BoNT-A via elec­
tromyography (EMG) guidance into the frontalis and cor­
rugators muscles, nasolabial fold, lateral canthus, or
platysma. The effect of the BoNT-A occurred within the
first 24–72 hours, peaked at 2–3 weeks, when follow-up
injections were performed, if necessary, and lasted from
3 to 6 months. Minimal adverse effects included ptosis of
the eyelid and, in the case of nasolabial fold injections,
droop of the upper lip. All patients experienced substan­
tial benefit from treatment.
Recognizing the need for trials of longer duration, Keen
and colleagues devised the first double-blind, placebo-
controlled study to assess the efficacy and safety of BoNT-A
for the treatment of facial rhytides in a group of twelve
healthy subjects (one of whom was lost to follow-up) aged
from 32 to 62 years. Nine patients with hyperfunctional
forehead rhytides and two patients with prominent crow’s
feet received injections of either 0.2 mL normal saline or
BoNT-A (eight injection sites with 10 U in the forehead,
two sites with 5 U in the periorbital wrinkles) on both
sides of the face via EMG guidance. Before and after pho­
tographs documented the treatment effect (determined
by blind self-assessment and investigator grading) 2 and 6
weeks after injection, and patients were followed for a
minimum of 1 year. BoNT-A treatment led to a significant
(p <0.01) improvement in facial rhytides. All patients
requested the toxin after 6 weeks on the untreated side of
their face to ‘even out’ the results. There were no serious
complications; side effects included slight brow ptosis
(two patients), change in eyebrow shape (one), forehead
‘heaviness’ (one), and pain on injection (three). Ten of
eleven subjects requested further treatment after the
effect from the initial injections dissipated (4–6 months).
Pearl 4 
Performing treatments on yourself (and your staff) is often
the best way to demonstrate and explain the effects of said
treatments.
Pearl 3 
Pre- and post-treatment photography is a valuable
educational tool for both the patient and you (and your staff).
Lowe and colleagues found similar results in a double-
blind, placebo-controlled study of 30 subjects who received
10 U BoNT-A or normal saline into each corrugator muscle
under EMG guidance for the treatment of glabellar rhyt­
ides. Treatment with BoNT-A led to a statistically signifi­
cant reduction in glabellar frown line depth and length
compared to placebo control at 12 weeks post-injection.
Results of these trials indicated that BoNT-A was
indeed a novel – and promising – treatment for unsightly
facial rhytides. Clinicians began to investigate other poten­
tial cosmetic applications, such as crow’s feet, horizontal
forehead lines, and platysmal bands, all of which were
considered off-label, non-approved uses.
FDA approval
Good news spreads fast: between 1992 and 1997, the
popularity of off-label use of BoNT-A grew so rapidly that
Allergan’s supply temporarily ran out. By 2002, investiga­
tors had established an excellent efficacy and safety profile
for therapeutic doses of BoNT-A, which was used for a
host of disorders, including strabismus, blepharospasm,
hemifacial spasm, cervical dystonia, cerebral palsy, post­
stroke spasticity, hyperhidrosis, headache, and back pain.
Moreover, the safety and efficacy of BoNT-A in reducing
the appearance of hyperfunctional facial rhytides had been
demonstrated in numerous open-label studies totaling
more than 800 subjects. In the United States, the FDA
had approved BoNT-A for strabismus, blepharospasm,
hemifacial spasm, and cervical dystonia. Additional
approvals had been granted in the United Kingdom for
axillary hyperhidrosis, and in Canada for axillary hyperhi­
drosis, focal muscle spasticity, and for the cosmetic treat­
ment of glabellar wrinkles.
Two large, double-blind, placebo-controlled, random­
ized, multicenter clinical trials in 2002 and 2003 cemented
BoNT-A’s position as a safe and effective treatment for
facial rhytides and would pave the way for FDA approval
and future cosmetic applications. The first involved 264
patients with moderate-to-severe glabellar rhytides who
received 20 U BoNT-A (n = 203) or placebo (n = 61) into
five glabellar sites (one in the procerus muscle and two in
each corrugator supercilii) and were followed for 120 days
post-injection. Results were assessed by patient and inves­
tigator at days 7, 30, 60, 90, and 120 after treatment.
BoNT-A led to a significantly greater reduction in glabellar
line severity compared with placebo across all measures
at every follow-up visit (p <0.022). Response to treatment
was noted at first follow-up and peaked at day 30, although
clinical effect was still apparent in over half of the subjects
at day 90 and over one quarter at day 120. Treatment was
well tolerated; side effects included transient headache
(15.3%) and mostly unilateral blepharoptosis (5.4%) that
resolved by day 20 (in 8 cases) and day 40 (in 4 cases).
The second large trial was identical in design to the first
and comprised 273 patients injected with BoNT-A (n =
202) or placebo (n = 71). As in the first trial, response to
BoNT-A peaked at day 30 for both physician- and patient-
assessment and was significantly greater than for placebo
at every follow-up visit (p <0.001) (Fig. 3.3). No treatment-
related serious complications were reported; the most
common adverse events in the BoNT-A group were head­
ache (11.4%) and unilateral blepharoptosis (1%).
In April of 2002, the FDA approved BoNT-A for the
non-surgical reduction of glabellar furrows. The rest, as
they say, is history.

17
3 
History of cosmetic botulinum toxin
Figure 3.3  Response to BoNT peaked at day 30 and was
significantly greater than for placebo at every follow-up visit. Data
from Carruthers JA, Carruthers JDA, Lowe NJ, et al 2004 One year,
randomized, multicenter, two period study of the safety and efficacy
of repeated treatments with botulinum toxin type A in patients with
glabellar lines. Journal of Clinical Research 7:1–20.
7
*
*
*
*
*
30
60
Study day
BTX-A
Vehicle
*p<.001 vs vehicle
90
120
0
10
20
30
40
50
60
70
80
90
% Responders
The face of BoNT
The world of facial rejuvenation changed after the formal
FDA approval of BoNT, which is now used to treat a
variety of hyperkinetic facial lines in the upper face,
including crow’s feet, horizontal forehead lines, and gla­
bellar rhytides, as well as folds and lines in the lower face,
neck, and chest with a high level of efficacy and patient
satisfaction. Moreover, BoNT-A is used to sculpt and
shape the face, can prolong the effects of other cosmetic
modalities, particularly for the treatment of deeper, more
static rhytides and folds, and is used during surgery to
prolong or enhance the aesthetic results or as an aid in
wound healing and minimizing scars.
More tellingly, surveys and statistics paint a convincing
portrait of rising popularity: between 2005 and 2007, the
use of BoNT and soft-tissue-augmenting agents increased
by 130% and accounted for nearly half of all nonsurgical
cosmetic procedures in 2008. Other formulations of
BoNT-A have joined the original BOTOX, which has gone
on to receive approval for 20 indications in more than 75
countries.
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. BoNT has
made remarkable advances since its initial discovery as a
therapeutic agent over thirty years ago, becoming the
treatment of choice for smoothing hyperkinetic lines and
shaping the face, with an ever-expanding array of indica­
tions and formulations.
Further reading
Blitzer A, Brin MF, Keen MS, et al 1993 Botulinum toxin for the
treatment of hyperfunctional lines of the face. Archives of
Otolaryngology – Head and Neck Surgery 119:1018–1022
Blitzer A, Bonder WJ, Aviv JE, et al 1997 The management of
hyperfunctional facial lines with botulinum toxin. Archives of
Otolaryngology – Head and Neck Surgery 123:389–392
Carruthers A, Carruthers J 2001 Botulinum toxin type A: history
and current cosmetic use in the upper face. Seminars in
Cutaneous and Medical Surgery 20:71–84
Carruthers A, Carruthers JDA 1994 Botulinum toxin in the
treatment of glabellar frown lines and other facial wrinkles.
In: Jankovic J, Hallett M (eds) Therapy with botulinum toxin.
Marcel Dekker, New York, p 577–595
Carruthers J, Carruthers A 2004 Botulinum toxin A in the mid and
lower face and neck. Dermatologic Clinics 222:151–158
Carruthers J, Carruthers A 2007 The evolution of botulinum
neurotoxin type A for cosmetic applications. Journal of
Cosmetic and Laser Therapy 9:186–192
Carruthers JA, Lower NJ, Menter MA, et al 2002 A multicenter,
double-blind, randomized, placebo-controlled study of the
efficacy and safety of botulinum toxin type A in the treatment
of glabellar lines. Journal of the American Academy of
Dermatology 46:840–849
Carruthers JD, Lowe NJ, Menter AM, et al for the Botox Glabellar
Lines II Study Group 2003 Double-blind, placebo-controlled
study of the safety and efficacy of botulinum toxin type A for
patients with glabellar lines. Plastic and Reconstructive Surgery
112:1089–1098
Carruthers JDA, Carruthers JA 1992 Treatment of glabellar frown
lines with C. botulinum-A exotoxin. Journal of Dermatologic
Surgery and Oncology 18:17–21
Clark RP, Berris CE 1989 Botulinum toxin: A treatment for facial
asymmetry caused by facial nerve paralysis. Plastic and
Reconstructive Surgery 84:353–355
Fagien S, Carruthers JD 2008 A comprehensive review of
patient-reported satisfaction with botulinum toxin type a for
aesthetic procedures. Plastics and Reconstructive Surgery
122:1915–1925
Kane MA 1999 Nonsurgical treatment of platysmal bands with
injection of botulinum toxin A. Plastics and Reconstructive
Surgery 103:656–663
Keen M, Blitzer A, Aviv J, et al. 1994 Botulinum toxin A for
hyperkinetic facial lines: results of a double-blind, placebo-
controlled study. Plastics and Reconstructive Surgery 94:94–
99
Klein AW 2002 Treatment of wrinkles with Botox. Current
Problems in Dermatology 30:188–217
Kuczynski A PULSE; Drought Over, Botox Is Back. Retrieved April
13, 2011 from
pulse-drought-over-botox-is-back.html
Liew S, Dart A 2008 Nonsurgical reshaping of the lower face.
Aesthetic Surgery Journal 28:251–257
Lowe NJ, Maxwell A, Harper H 1996 Botulinum A exotoxin for
glabellar folds: a double-blind, vehicle-controlled study with an
electromyographic injection technique. Journal of the American
Academy of Dermatology 35:569–572
Lowe NJ, Yamauchi P 2004 Cosmetic uses of botulinum toxins for
lower aspects of the face and neck. Clinics in Dermatology
22:18–22
Pribitkin EA, Greco TM, Goode RL, Keane WM 1997 Patient
selection in the treatment of glabellar wrinkles with botulinum
toxin type A injection. Archives of Otolaryngology – Head and
Neck Surgery 123:321–326
Smith Kettlewell Institute of Visual Sciences 1990 Botulinum
toxin (Oculinum) study: IND-723. Patients treated as of 31
December 1989. Smith Kettlewell Institute of Visual Sciences,
San Francisco, CA.
The American Society for Dermatologic Surgery 2008 The
American Society for Dermatologic Surgery Releases New
Procedure Survey Data. Online. Available:
TheAmericanSocietyforDermatologicSurgeryReleasesNew
ProcedureSurveyData.aspx, 22 July 2010
The American Society for Aesthetic Plastic Surgery 2009 2008
Cosmetic Surgery National Data Bank Statistics

Introduction
The introduction of botulinum toxin type A into the field
of aesthetic dermatology has profoundly impacted the
clinical management of undesirable facial lines. Botulinum
toxins are injected into discrete facial muscles where they
act locally to reduce muscle contractions that produce
skin creases, either with facial animation or at rest. The
pattern of injections can be tailored to individual needs
and the results in glabella have been demonstrated by
Carruthers and colleagues in 2004 to last an average of
4 months.
This chapter discusses the basic science of Botox® Cos­
metic, also known in the United States and other coun­
tries by its non-proprietary name onabotulinumtoxinA.
Although other botulinum neurotoxin products are avail­
able in various countries worldwide, each contains a
unique protein drug substance manufactured using a pro­
prietary technology and containing a distinct formulation
of excipients. The biological activity units of each product,
and hence unit doses, are not interchangeable with those
of other products, as per guidelines issued by regulatory
agencies in all major countries throughout North America,
Europe, and Asia.
In line with the non-interchangeable nature of botuli­
num neurotoxins, this book includes separate chapters on
each of the main botulinum neurotoxin products available
for aesthetic use. In the following pages, we discuss the
basic properties of botulinum toxins, including their sero­
types, structure, and mechanism of action. In the sections
on manufacturing, formulation, immunology, and pharma­
cology in facial aesthetics, the focus is on onabotulinum­
toxinA and it should be noted that all dosing in this
chapter refers specifically to this product. (Due to the
structure and format of this book, this chapter is not fully
referenced; the reader is referred to the reading list at the
end of this chapter for further information.)
Serotypes and structure
Botulinum toxins are biological products produced by the
bacterium Clostridium botulinum. These neurotoxins have
been grouped into seven serotypes based on their immu­
nologic properties: types A, B, C1, D, E, F, and G.
All botulinum toxins are produced by bacteria as protein
complexes consisting of a core neurotoxin molecule with
4 
Basic science: BOTOX®
Cosmetic
Conor J. Gallagher
Summary and Key Features
•
The introduction of BOTOX® Cosmetic
(onabotulinumtoxinA) into aesthetic dermatology
has revolutionized the management of facial lines
•
Botulinum neurotoxins are biological products,
synthesized by bacteria, then purified, formulated,
and packaged into minute quantities for medical
use
•
Seven different serotypes of botulinum toxins
occur in nature (types A through G), although most
clinical products, including onabotulinumtoxinA,
are based on the A serotype
•
Botulinum toxin type A has a well-defined
mechanism of action; at the neuromuscular
junction, it reduces acetylcholine release from
motor nerves and inhibits muscular contractions
•
Clinical and preclinical data suggest that
onabotulinumtoxinA may also act on nociceptive
neurons
•
Each commercially available botulinum toxin
product is a unique biological therapeutic, with a
distinct structure, formulation, unit strength and
clinical profile
•
As biologics, the doses of botulinum neurotoxins
are expressed in units of biological activity that are
not interchangeable or convertible among different
products
•
Recent analyses demonstrate that the onset of
effect of onabotulinumtoxinA in the management of
glabellar lines occurs within 24 hours and that
benefits last at least 4 months
•
Although all botulinum toxin products may
stimulate antibody formation, the immunogenicity
profile of onabotulinumtoxinA is well characterized
•
The clinical efficacy and safety profile of
onabotulinumtoxinA in facial lines are well
understood by skilled practitioners

19
4 
Basic science: BOTOX® Cosmetic
resistance than the uncomplexed toxin to degradation by
pepsin, gastric juices, and pancreatic enzymes.
Although the physiological environment of the diges­
tive tract differs from that of skeletal muscle into which
therapeutic botulinum neurotoxins are typically injected,
the integrity of the protein complex is likely retained for
at least a period of time after injection. Muscle tissue
contains a plethora of extracellular enzymes that degrade
proteins. The effects of muscle proteases on botulinum
neurotoxins have not been investigated; however, it is
possible that NAPs may transiently protect the core neu­
rotoxin from enzymatic attack following intramuscular
injection.
Limited information is available regarding the role of
NAPs in the clinical use of botulinum neurotoxins.
However, multiple botulinum toxin type A products are
currently available for clinical use, and one of the most
prominent ways in which they differ is in the amount of
NAPs that they contain. Botox® (onabotulinumtoxinA;
Allergan, Inc., Irvine, California, USA) contains only the
purified form of a 900 kDa complex, according to the
company’s prescribing information. Another product,
Xeomin® (incobotulinumtoxin type A; Merz Pharmaceu­
ticals, Frankfurt am Main, Germany), contains only botu­
linum toxin type A molecules from which the NAPs have
been removed, according to the prescribing information.
Hambleton reported that Dysport® (abobotulinumtoxinA;
Ipsen Biopharm Ltd., Wrexham, UK) contains botulinum
toxin type A complexes of approximately 300–500 kDa.
In attempting to understand how these products behave
in the muscle following injection, the role of NAPs may
be an important area of future study.
Manufacturing
The production of botulinum toxin protein products
begins with fermentation of the clostridial bacteria in an
anaerobic environment, which results in the synthesis and
release of neurotoxin into the bacterial broth. In some
bacterial strains the 150 kDa polypeptide is ‘nicked’,
separating the protein into a 50 kDa light chain and a
100 kDa heavy chain, which remain closely associated via
a disulfide linkage.
Following the fermentation step, the neurotoxin is
purified from the bacterial broth. Each manufacturer has
its own proprietary methodology for purification of the
neurotoxin, excluding the bacterium, resulting in a unique
biological product. In the purification of onabotulinum­
toxinA, Allergan generally follows the methodology of
Schantz, which yields a highly purified crystalline neuro­
toxin with a homogenous molecular mass distribution of
approximately 900 kDa.
Formulation
In order to enhance the stability of botulinum toxins from
the point of manufacture to the point of injection into
patients, all products are formulated with excipients. For
a molecular mass of approximately 150 kDa and one or
more associated proteins. This protein contains three dis­
tinct functional domains. The binding domain is respon­
sible for the docking of the molecule with its specific cell
surface receptors, the translocation domain is critical
in allowing the catalytic domain to access the neuronal
cytosol, and the catalytic domain is responsible for the
enzymatic activity that ultimately interferes with neuro­
transmitter release (Fig. 4.1).
Associated with the core neurotoxin are one or more
additional proteins that are commonly known as accessory
proteins or neurotoxin-associated proteins (NAPs). For
type A botulinum toxins these consist, in part, of a
number of proteins that were identified initially by their
capacity for agglutination of blood, and thus are known as
hemagglutinin (HA) proteins. An additional protein that
is not agglutinating is always associated with the core
neurotoxin and this is referred to the NTNH or non-
toxic, non-hemagglutinin protein. Based on the serotype
a variety of neurotoxin complex sizes may be produced
by the bacterium. Type A-producing strains synthesize
complexes that are of 300 kDa, 500 kDa or 900 kDa in
molecular mass.
Role of NAPs
Given that botulinum toxins are always produced by the
bacteria as protein complexes, this assembly may have
some evolutionary advantage. NAPs serve a number of
critical roles in protecting the core neurotoxin protein
from harsh environmental conditions and events such
as proteolytic degradation, pH stress, and thermal stress.
In vitro studies by Chen and co-workers have confirmed
that the complex botulinum neurotoxin shows greater
Figure 4.1  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. Image courtesy of Lance E. Steward PhD, Allergan Inc.

20
Botulinum toxin
of the complex in muscle has never been studied, and
therefore the time taken for complete dissociation of the
neurotoxin complex in vivo is currently unknown.
Eisele and colleagues have recently speculated that dis­
sociation of the neurotoxin complex occurs in the vial
immediately upon reconstitution in clinical practice.
However, the pH of unbuffered normal saline (either
unpreserved or bacteriostatic) may range between 4 and
7, with a nominal pH of 5.5. This is very close to the
isoelectric point for the botulinum toxin type A complex,
at which the complex is most stable; thus dissociation in
the vial is highly unlikely. Indeed, the botulinum toxin
type A complex is stable at this pH.
Binding
The specific interaction between botulinum toxin A and
its target cell is facilitated by a two-receptor binding
mechanism. In the initial step, the C-terminus of the
heavy chain interacts with a cell-surface ganglioside on the
neuronal membrane. This binding maintains the botuli­
num neurotoxin in close proximity to the neuronal surface,
and perhaps increases the concentration of neurotoxin
molecules around the neuron, which facilitates the inter­
action of the heavy chain with its protein receptor, synap­
tic vesicle 2 (SV2). A review paper by Verderio et al
indicated that vesicular exocytosis exposes the lumen of
the synaptic vesicle to the extracellular milieu allowing
interaction between SV2 and the core neurotoxin. The
original studies were performed by Dong et al., 2006 and
Mahrhold et al., 2006.
This provides an elegant explanation for early physio­
logical experiments demonstrating an activity dependence
on the rate of onset of the effect of the neurotoxin.
Internalization and translocation
Once bound to SV2 the botulinum neurotoxin/receptor
complex is endocytosed in a manner similar to other
receptor ligand complexes. In this process a small portion
of cell membrane containing the neurotoxin / receptor
complex is pinched off to form a recycling endosome.
Following compartmentalization into the endosome,
botulinum neurotoxin is still isolated from the neuronal
cytosol, which it must reach in order to exert its activity.
Recycling endosomes are typically acidified to a pH of
approximately 5 through the action of proton pumps in
the endosomal wall. Koriazova & Montal proposed that
under these conditions the heavy chain may form a conduit
that allows the light chain to pass through the lipid bilayer
wall of the endosome and into the neuronal cytosol.
Cleavage
Once in the neuronal cytosol, the light chain of botulinum
neurotoxin interacts with its target substrates, the
proteins of the SNARE (soluble N-ethylmaleimide-sensi­
tive factor attachment protein receptor) complex, which
a 100 U vial of onabotulinumtoxinA, these excipients
include human serum albumin (0.5 mg) and NaCl
(0.9 mg). Human serum albumin acts as a stabilizer and
also enhances the recovery of the neurotoxin from the
glass vials. Albumin may also act as a cryoprotectant and
serve to prevent protein aggregation.
Mechanism of action
Following intramuscular injection, botulinum neurotoxins
inhibit the release of acetylcholine (ACh) from motor
nerve terminals, resulting in reduced muscular contrac­
tions. This inhibition occurs in multiple steps referred to
as binding, internalization, translocation, and cleavage.
Through a similar process, botulinum neurotoxins also
inhibit acetylcholine release from autonomic nerve termi­
nals that innervate smooth muscle or glands. Further
studies have found that botulinum toxin type A exerts
selective effects on the nociceptive system. These actions
are described in the following text, which for onabotu­
linumtoxinA begins with the dissociation of the 150 kDa
neurotoxin from the NAPs (Fig. 4.2).
Dissociation of NAPs from
the 150 kDa neurotoxin
For aesthetic use, onabotulinumtoxinA is injected into
muscle tissue where the neurotoxin protein complex is
disassembled and the 150 kDa core neurotoxin is released
to bind to its target cells. Dissociation of NAPs from the
150 kDa neurotoxin is a function of pH and ionic strength,
with exposure to more basic pH and increasing ionic
strength facilitating separation of the core from the sur­
rounding accessory proteins. The process of dissociation
Figure 4.2  Multiple steps in the mechanism of action of botulinum
toxin type A: (1) The 150 kDa core neurotoxin is internalized into the
neuron via a process of endocytosis. (2) The endosome is acidified,
which facilitates (3) the translocation of the light chain into the
neuronal cytosol. (4) Once in the neuronal cytosol the light chain of
type A botulinum toxin (blue) cleaves the membrane-associated
SNAP-25 (brown), one component of the SNARE complex. Another
neurotoxin serotype, type B, botulinum toxin cleaves a different
protein known as synaptobrevin or vesicle-associated membrane
protein (VAMP; green).
ACh
SV2
ACh
ACh
1. Endocytosis
2. Acidification
ACh
pH:5
pH:5
3. Translocation
of light chain
4. Cleavage of SNARE proteins

21
4 
Basic science: BOTOX® Cosmetic
substance P, and calcitonin gene-related peptide (CGRP).
Reduced release of these neurochemicals may reduce
peripheral sensitization and indirectly reduce central
sensitization.
Another potential antinociceptive effect of botulinum
toxin is on TRPV1. TRPV1 is an ion channel found on some
sensory neurons that is activated by capsaicin, protons, and
noxious heat, and is upregulated in tissues during chronic
pain and inflammation. Botulinum toxin type A was shown
by Morenilla-Palao and colleagues to reduce the expression
of TRPV1 in a number of cell and tissues types, suggesting
another mechanism by which botulinum toxin can poten­
tially reduce peripheral sensitization.
The actions of botulinum neurotoxin on the pain
system are selective, as this protein does not affect A-delta
sensory fibers, which mediate acute pain signals, or A-beta
fibers that mediate touch and pressure. These fibers do
not release neurochemicals from their peripheral termi­
nals and are therefore not affected by the protease activity
of botulinum toxin on SNAREs. As a result, botulinum
toxin does not induce cutaneous anesthesia or interfere
with the normal perception of acute pain.
Immunogenicity
Botulinum toxins are formulated biological products that,
like all foreign proteins, are capable of stimulating an
immune response and antibody formation. The onabotu­
linumtoxinA protein complex comprises the core neuro­
toxin and the protective accessory proteins, as discussed
previously, and Goschel and co-workers found that anti­
bodies may form against either component. Only those
antibodies that inhibit the function of the core neurotoxin
(neutralizing antibodies) are of clinical relevance: if selec­
tively targeted against the neurotoxin and present in suf­
ficient quantities, they may lead to clinical non-response.
In contrast, antibodies that may potentially form against
the NAPs do not affect the biological activity of botulinum
toxin type A.
The immunogenicity of onabotulinumtoxinA in the
management of glabellar lines has been studied in Allergan-
sponsored trials conducted for the purpose of product
registration. Based on a pooled population of 718 subjects
receiving a 20 U dose of onabotulinumtoxinA injected
into the glabella, only 2 (0.3%) seroconverted from
antibody-negative status at some time during these multi-
cycle trials analyzed by Naumann et al. Both subjects
who developed neutralizing antibodies retained an objec­
tive clinical response to treatment throughout the studies,
and were not positive at the end of the study. In a wider
analysis of onabotulinumtoxinA clinical trials by the same
authors, an overall rate of seroconversion of 0.5% was
observed, with more than 50% of neutralizing antibody-
positive patients retaining a clinical response. Importantly,
the development of neutralizing antibodies was not found
to be associated with skin reactions upon injection or
other immune-related adverse events in any clinical
indication.
normally enable calcium-mediated exocytosis of neuro­
transmitters. The light chain of botulinum toxins com­
prises proteases that selectively cleave one or more
SNARE proteins, rendering them inactive. Each botuli­
num toxin serotype has a distinct SNARE protein
substrate profile, with type A selectively cleaving a
membrane-associated protein known as SNAP-25 (synap­
tosomal associated protein, 25 kDa). This action tempo­
rarily blocks neuromuscular transmission.
Recovery of neurotransmitter release
The actions of botulinum neurotoxins are not permanent,
but rather reverse over time as nerve terminals recover
their normal function. In the first stage of recovery, axonal
sprouts arise from the silenced nerve in response to
growth factor signals from the inactive muscle. These
axonal sprouts make contact and form immature synapses
with the injected muscle. The new nerve terminals
are believed to account for the early phase of recovery
of function after botulinum neurotoxin injection. In
the latter phase, activity returns to the original nerve
terminal, resulting in a restoration of neuromuscular
transmission.
With the resumption of transmitter release at the neu­
romuscular junction, muscle activity returns. The duration
of clinical efficacy depends, at least in part, on neurotoxin
serotype and whether the tissue injected is innervated by
somatic or autonomic nerve terminals. In human skeletal
muscle, which is innervated by somatic nerve endings,
clinical weakening is generally observed for approximately
3–4 months. In contrast, in tissues innervated by the auto­
nomic nervous system such as the detrusor muscle of the
bladder and the sweat glands, studies by Dmochowski
et al and Lowe et al observed that the duration is typically
6 months or longer.
Potential actions on the sensory system
Borne from anecdotal observations in aesthetic applica­
tions, onabotulinumtoxinA has demonstrated efficacy,
and been approved by many regulatory agencies, for the
prophylactic treatment of chronic migraines. This effect
of onabotulinumtoxinA is supported by reports of pain
reduction following treatment in other neurological con­
ditions, which have been frequently reviewed. Although
the mechanism by which the neurotoxin reduces pain
in these conditions has not been definitively established,
a direct effect of the neurotoxin on sensory neurons
has been proposed and is supported by various lines of
evidence.
One sensory mechanism that has been proposed for
botulinum toxin type A is the inhibition of neuropeptide
and neurotransmitter release from peripheral terminals
of afferent neurons. Botulinum toxin type A was found
by several authors to inhibit the release of a number of
neurochemicals from sensory neurons that are known to
stimulate peripheral nociceptors, including glutamate,

22
Botulinum toxin
Onset of the clinical effect was not systematically
studied in the registration trials for onabotulinumtoxinA.
However, in a recent study by Beer and colleagues, more
than 90% of physicians and subjects observed onset within
3 days of injection and almost 50% of subjects reported
evidence for an onset of clinical benefit within 24 hours
of injection.
The safety of onabotulinumtoxinA in the management
of facial lines has been evaluated in a systematic meta-
analysis by Brin et al. published in 2009. This analysis was
based on nine manufacturer-sponsored clinical trials of
onabotulinumtoxinA (two on lateral canthal lines and
seven on glabellar lines), and included 1678 subjects who
received 3–18 U per side for lateral canthal lines or 10 U
or 20 U total dose for glabellar lines. In these studies, the
overall incidence of adverse events was not different
between the onabotulinumtoxinA- and placebo-treated
groups. Eyelid ptosis (1.8%) and eyelid sensory disorder
(characterized by feelings of tightness, pressure, or a
heaviness to the eyelids, 2.5%) were the only adverse
events that occurred at a significantly higher rate in the
onabotulinumtoxinA than in the placebo group, and this
was found only in the glabellar lines population. In the
assessment of treatment-related adverse events, eyelid
edema was also significantly more frequent in the ona­
botulinumtoxinA- than the placebo-treated group. As the
number of treatment cycles with onabotulinumtoxinA
increased, the incidence of all three of these adverse
events decreased.
The dose-response effects of onabotulinumtoxinA in
glabellar lines have been evaluated in randomized trials.
Clinical pharmacology of onabotulinumtoxinA
in aesthetics
The clinical profile of onabotulinumtoxinA in aesthetic
medicine has been studied intensively since the early
reports of its use for glabellar lines in the 1990s, and the
performance of this product is known to practitioners.
OnabotulinumtoxinA has been approved for 25 clinical
indications worldwide, with demonstrated safety and effi­
cacy in large muscle indications such as upper-limb spas­
ticity and cervical dystonia, as well as small muscle
indications such as strabismus, blepharospasm, and glabel­
lar rhytides. In addition, approvals in chronic migraine,
primary axillary hyperhidrosis, and neurogenic overactive
bladder demonstrate a breadth of clinical data support
and use unrivalled by any other botulinum neurotoxin
product.
The efficacy and safety of onabotulinumtoxinA in gla­
bellar lines has been studied in rigorous double-blind,
placebo-controlled registration trials in North America
by Carruthers et al and in Asia by Harii & Kawashima
and Wu et al. Responder rates in these trials were uni­
formly high, with more than 80% of subjects exhibiting
significant clinical efficacy on dynamic glabellar lines
with a 20 U dose of onabotulinumtoxinA, according to a
priori responder criteria (Fig. 4.3). Based on these trials,
a median duration of efficacy of 120 days or 4 months
may be expected in those exhibiting a clinical response.
The duration of efficacy on resting glabellar lines in these
trials was found to be longer than that on dynamic rhyt­
ides, with a median duration of 131 days (Fig. 4.4).
Figure 4.3  Repeat dosing efficacy of
onabotulinumtoxinA in US glabellar lines
trials: physician’s assessment of glabellar
line severity at maximum frown. Responder
rates (percentage of patients with a rating of
none or mild at follow-upon the 4-point
facial wrinkles scale with photonumeric
guide). Results from 258 patients receiving
all three botulinum toxin treatments are
illustrated. Data from Carruthers et al. 1994
J. Clinical Research 7:1–20.
Day
Physician's assessment
Time of treatment
*p<0.028 vs same time point for previous treatment
First
treatment
Second
treatment
Third
treatment
Responders to botulinum
toxin type A (%)
0
10
20
30
40
50
60
70
80
90
100
0
30
60
90 120/0 30
60
90 120/0 30
60
90 120

23
4 
Basic science: BOTOX® Cosmetic
female recipients, the labeled dose of 20 U would appear
to be the optimal dose for most patients.
Although anecdotal experience has suggested the
accrual of cumulative benefits in patients who receive
regular treatment with onabotulinumtoxinA in glabella,
this has only recently been confirmed in clinical trials by
Results of a 2005 study by Carruthers showed that in
females 20–40 U of onabotulinumtoxinA produce a longer
duration of action and lower relapse rate than 10 U of
onabotulinumtoxinA, but indicated no differences in effi­
cacy among the 20 U, 30 U, and 40 U doses. These find­
ings suggest that, when averaged across a broad group of
Figure 4.4  Duration of efficacy of onabotulinumtoxinA in glabellar lines: results of a pooled analysis of four onabotulinumtoxinA randomized
double-blind placebo-controlled trials. Panel A shows the proportion of responders demonstrating a clinical response on dynamic glabellar
lines through the course of the studies. A responder was defined as a subject with an improvement from moderate or severe glabellar lines to
mild or no glabellar lines. Panel B shows the proportion of responders demonstrating a clinical response on glabellar lines at rest through the
course of the studies. For this analysis a responder was defined as a subject with an improvement of 1 point on the 4-point facial wrinkle
scale with photonumeric guide.
Days
Percent responders
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90 100 110 120 130 140 150
Censored observations
Median duration of effect
A
Censored observations
Median duration of effect
Days
Percent responders
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90 100 110 120 130 140 150
B

24
Botulinum toxin
used to define a unit of onabotulinumtoxinA than when
tested in the Merz LD50 assay, which defines the unique
potency of incobotulinumtoxinA. These results illustrate
the differences between assays, which underlies the indi­
vidual potency of units of each botulinum toxin product.
Underscoring the differences in units between differ­
ent botulinum neurotoxin products, regulatory agencies
have determined that botulinum toxin products cannot
have generic substitutes, and furthermore there are
no biosimilars. The United States Food and Drug Admin­
istration (US FDA) and US Adopted Names Council
(USAN) review assigned all manufacturers of products
marketed in the US for therapeutic or aesthetic a non-
proprietary name to emphasize non-interchangeability and
reduce the potential risk for medication errors. As such,
Allergan – the manufacturers of Botox® Cosmetic –
adopted the non-proprietary name onabotulinumtoxinA
to refer to Botox®. Other non-proprietary names were
adopted by the manufacturers of other botulinum neuro­
toxin products. These names are now used extensively in
clinical practice and provide an important clarifying
dimension to the clinical and non-clinical literature.
Another important development related to the dif­
ferences in units among botulinum neurotoxin products
occurred in 2011. Historically, Allergan has employed
LD50 assays for batch release testing of onabotulinum­
toxinA; however, in July of 2011, the US FDA announced
the approval of the company’s breakthrough alternative
method of testing units, specific to onabotulinumtoxinA.
This method uses a cell line rather than animals for routine
batch release assays. As this assay gains regulatory approval
worldwide, it is anticipated that it will significantly reduce
the need for animal testing related to onabotulinumtox­
inA. The assay is highly sensitive and extensively cross-
validated against the LD50 assay.
Conclusion
OnabotulinumtoxinA is well established as a valuable
management strategy for upper facial lines with varying
facial regions approved in different countries. This biologi­
cal product is based on the A serotype of botulinum toxin,
derived from the Clostridium botulinum bacteria. The
900 kDa neurotoxin protein complex is isolated and puri­
fied from the bacteria and formulated into an injectable
therapy using state-of-the-art manufacturing methods.
Following injection into muscles, botulinum toxin type
A inhibits the release of acetylcholine at the neuromuscu­
lar junction, thereby reducing muscular contractions. Ben­
eficial effects of onabotulinumtoxinA on glabellar lines are
detectable within 24 hours and last a median of 4 months.
The safety of onabotulinumtoxinA in the treatment of
glabellar lines has been established in individual clinical
trials, as well as a meta-analysis in which the only adverse
events that occurred at a significantly higher rate than with
placebo were local effects in the eyelid region. Dose-
ranging studies have found that 20 U of onabotulinum­
toxinA is the optimal dose for most patients when treating
Dailey and co-workers. In this study, 50 botulinum toxin-
naïve subjects received treatment of glabellar lines at
regular 4-monthly intervals for 20 months. During this
period, their glabellar line severity never returned to
baseline and in fact improved. Upon cessation of treat­
ment (as part of the study protocol), the subjects con­
tinued to retain improvements over baseline out to
the final timepoint of 6 months following their last
treatment.
Approvals
The approvals for onabotulinumtoxinA in facial lines vary
based on country, with a broad indication of upper facial
lines (glabella, frontalis and crow’s feet) approved in some
regions, glabella and crow’s feet approved in others, and
only glabellar lines approved in some. Dose-ranging studies
enrolling over 200 subjects with onabotulinumtoxinA in
crow’s feet lines have demonstrated significant efficacy at
doses ranging from 6 to 18 U per side. Additional larger-
scale clinical trials for onabotulinumtoxinA in crow’s feet
lines will conclude in 2012.
Unique dosing of neuromodulators
As noted previously, botulinum neurotoxins are biological
products – proteins derived from bacteria. Biological prod­
ucts are distinguished from conventional drugs by a
number of properties, including their large size, complex
three-dimensional structure, and sensitivity to manufac­
turing methods. Regulatory agencies have determined that
botulinum toxin products cannot have generic substitutes
(ie, there are no biogenerics in this class), and furthermore
there are no biosimilars.
As biological products with an enzymatic mechanism
of action, doses of botulinum neurotoxins are measured
in units of biological activity. Unlike some other classes of
biologics, botulinum toxins do not have a universal refer­
ence preparation for standardizing unit potency. Typically,
the biologic activity of botulinum neurotoxin products is
determined in an LD50 bioassay, with each manufacturer
employing a different methodology, using different dilu­
ents and a unique reference standard. For this reason,
regulatory agencies are clear about the lack of comparabil­
ity of units between commercially available preparations.
Although it may seem convenient to assume that either
the units per vial or the labeled doses for particular indica­
tions suggest a conversion ratio between products, in fact,
per regulatory agency conclusions, unit doses are not inter­
changeable among different botulinum neurotoxin prod­
ucts even if products contain the same number of labeled
units per vial. This lack of unit interchangeability is dem­
onstrated by the results of a study by Hunt & Clarke that
evaluated one of the botulinum toxin type A products
(incobotulinumtoxinA) in the manufacturer’s assay used to
evaluate units of another product (onabotulinum­toxinA).
Results showed that incobotulinumtoxinA has different
biological activity when tested in the Allergan LD50 assay

25
4 
Basic science: BOTOX® Cosmetic
Eisele KH, Fink K, Vey M, et al 2011 Studies on the dissociation
of botulinum neurotoxin type A complexes. Toxicon
57(4):555–565
Goschel H, Wohlfarth K, Frevert J, et al 1997 Botulinum A toxin
therapy: neutralizing and nonneutralizing antibodies –
therapeutic consequences. Experimental Neurology
147(1):96–102
Grumelli C, Verderio C, Pozzi D, et al 2005 Internalization
and mechanism of action of clostridial toxins in neurons.
Neurotoxicology 26(5):761–767
Hambleton P 1992 Clostridium botulinum toxins: a general review
of involvement in disease, structure, mode of action and
preparation for clinical use. Journal of Neurology 239(1):16–20
Harii K, Kawashima M 2008 A double-blind, randomized,
placebo-controlled, two-dose comparative study of botulinum
toxin type A for treating glabellar lines in Japanese subjects.
Aesthetic Plastic Surgery 32(5):724–730
Hunt T, Clarke K 2009 Potency evaluation of a formulated drug
product containing 150-kd botulinum neurotoxin type A.
Clinical Neuropharmacology 32(1):28–31
Kawashima M, Harii K 2009 An open-label, randomized, 64-week
study repeating 10- and 20-U doses of botulinum toxin type A
for treatment of glabellar lines in Japanese subjects.
International Journal of Dermatology 48(7):768–776
Koriazova LK, Montal M 2003 Translocation of botulinum
neurotoxin light chain protease through the heavy chain channel.
Nature Structural Biology 10(1):13–18
Lowe NJ, Glaser DA, Eadie N, et al 2007 Botulinum toxin type A
in the treatment of primary axillary hyperhidrosis: a 52-week
multicenter double-blind, randomized, placebo-controlled study
of efficacy and safety. Journal of the American Academy of
Dermatology 56(4):604–611
Merz 2010 Xeomin(r) (incobotulinumtoxinA) 2010 Prescribing
information. Merz Pharmaceuticals, LLC
Morenilla-Palao C, Planells-Cases R, Garcia-Sanz N, et al 2004
Regulated exocytosis contributes to protein kinase C
potentiation of vanilloid receptor activity. Journal of Biological
Chemistry 279(24):25665–25672
Naumann M, Carruthers A, Carruthers J, et al 2010 Meta-analysis
of neutralizing antibody conversion with onabotulinumtoxinA
(BOTOX(R)) across multiple indications. Movement Disorders
25(13):2211–2218
Sakaguchi G, Kozaki SIO 1984 Structure and function of
botulinum toxins. In: AIouf JE (ed) Bacterial protein toxins.
Academic Press, London, p 435–443
Simpson LL, Maksymowych AB, et al 2005 The role of
exoproteases in governing intraneuronal metabolism of
botulinum toxin. Protein Journal 24(3):155–165
United States Food and Drug Administration Update of safety
review of onabotulinumtoxinA (marketed as Botox/Botox
Cosmetic), abobotulinumtoxinA (marketed as Dysport)
and rimabotulinumtoxinB (marketed as Myobloc). Online.
Available:
PostmarketDrugSafetyInformationforPatientsandProviders/
DrugSafetyInformationforHeathcareProfessionals/
ucm174959.htm, 5 February 2011
Verderio C, Rossetto O, Grumelli C, et al 2006 Entering neurons:
botulinum toxins and synaptic vesicle recycling. EMBO Reports
7(10):995–999
glabellar lines. This dose applies only to onabotulinum­
toxinA, as units are not interchangeable among botulinum
neurotoxin products.
The clinical efficacy and safety profile of onabotu­
linumtoxinA in facial lines is well understood by skilled
practitioners. Allergan is pursuing clinical studies evaluat­
ing additional uses of onabotulinumtoxinA as part of our
continued exploration into the properties of this biological
product that will likely be a useful tool for aesthetic der­
matologists and their patients for years to come.
Acknowledgments
The editorial assistance of Mary Ann Chapman, PhD is
gratefully acknowledged.
Further reading
Allergan, Inc. Corporate statement on animal testing. Online.
Available:
safety_and_animal_testing.htm, 6 February 2011
Allergan, Inc. 2011 Botox(r) (onabotulinumtoxinA) Prescribing
information. Allergan, Inc., Irvine, CA
Aoki KR 2005 Review of a proposed mechanism for the
antinociceptive action of botulinum toxin type A.
Neurotoxicology 26(5):785–793
Beer KR, Boyd C, Patel RK, et al 2011 Rapid onset of response and
patient-reported outcomes after onabotulinumtoxinA treatment
of moderate-to-severe glabellar lines. Journal of Drugs in
Dermatology 10(1):39–44
Brin MF, Boodhoo TI, Pogoda JM, et al 2009 Safety and tolerability
of onabotulinumtoxinA in the treatment of facial lines: a
meta-analysis of individual patient data from global clinical
registration studies in 1678 participants. Journal of the
American Academy of Dermatology 61(6):961–970
Carruthers A, Carruthers J, Lowe NJ, et al for the BOTOX(r)
Glabellar Lines I & II, Groups S 2004 One-year, randomised,
multicenter, two-period study of the safety and efficacy of
repeated treatments with botulinum toxin type A in patients
with glabellar lines. Journal of Clinical Research 7:20
Carruthers A, Carruthers J, Said S 2005 Dose-ranging study
of botulinum toxin type A in the treatment of glabellar
rhytids in females. Dermatol Surgery 31(4):414–422;
discussion 422
Chen F, Kuziemko GM, Stevens RC 1998 Biophysical
characterization of the stability of the 150-kilodalton botulinum
toxin, the nontoxic component, and the 900-kilodalton
botulinum toxin complex species. Infection and Immunity
66(6):2420–2425
Dailey RA, Philip A, Tardie G 2011 Long-term treatment of
glabellar rhytides using onabotulinumtoxina. Dermatol Surgery
37(7):918–928
Dmochowski R, Chapple C, Nitti VW, et al 2010 Efficacy and
safety of onabotulinumtoxinA for idiopathic overactive bladder:
a double-blind, placebo controlled, randomized, dose ranging
trial. Journal of Urology 184(6):2416–2422

Botulinum neurotoxin-A (BoNT-A) is the most com­
monly used serotype of BoNT for both clinical and aes­
thetic applications. Though all serotypes are synthesized
as a continuous 150 KDA protein, biological activity
requires post-translational proteolysis, or nicking, which
snips the BoNT polypeptide into two separate moieties of
approximately 100 kDA and 50 kDA. The heavy chain and
light chain remain bound together by a single disulfide
bridge although they carry out separate functions at the
nerve terminal. One part of the heavy chain is bound by
receptors on the presynaptic nerve terminals so that the
bonded molecule enters the nerve terminal by endocytosis.
The second part of the heavy chain then forms a channel
in the endosomal membrane as the disulfide bond is
reduced and the light chain travels to the cytosol where it
cleaves a portion of the protein receptor (SNARE) complex
thus blocking the release of acetylcholine and hence
nerve transmission. The molecular target for all BoNT-A
is SNAP-25, regardless of commercial preparation.
BoNT-A exists in nature as a complex with a shell of
surrounding protective proteins. These are present in
both onabotulinumtoxinA (onabotA) (Botox® / Vistabel®;
Allergan Inc., Irvine, CA) and abobotulinumtoxinA
(abobotA) (Dysport® / Azzalure®; Ipsen / Galderma). These
are known as neurotoxin-associated proteins (NAPS).
NAPS are composed of four distinct hemaglutinin proteins
and a non-toxic non-hemaglutinin protein. NAPS are also
synthesized by the clostridial bacterium. The purpose of
this protective protein coat is to shield the neurotoxin from
potential destruction by gastric acid, the first environment
the BoNT encounters when ingested from a contaminated
foodstuff. As clostridial culture conditions can vary, there
are three sizes of the progenitor complexes: 300 kDa,
500 kDa, and 900 kDa. Different methods of neurotoxin
isolation and purification have produced these different
molecular weight complexes.
Lietzow et al reported onabotA to be a 925 kDa
complex. The chromatography method of purifying
abobotA also produces a complex. Any molecular weight
differences are irrelevant since all the products are now
known to exist as free BoNT in the vial, before even
reconstitution. Whether any clinical differences occur due
to differences in the products is a point of debate between
the manufacturing companies. Previous theories that
certain product BoNT complexes confer a more rapid
diffusion in tissue have now been dispelled as a result of
5 
Basic science: Abobotulinum
toxin A
Gary D. Monheit, Andrew Pickett
With more than 11 million injections since 2001, botuli­
num toxin (BoNT) administration is by far the most
common cosmetic procedure performed in the United
States and has truly revolutionized the field of cosmetic
medicine. Since the FDA first approved its use for the
treatment of glabellar lines, many other aesthetic applica­
tions have been tried for aging skin of the face and neck.
Summary and Key Features
•
Abobotulinum toxin is a botulinum toxin-A with
very similar features to the other type A botulinum
toxins
•
The accessory proteins that surround the
neurotoxin protein are already dissociated in
the vial
•
Is ‘diffusion’ an issue? Clinical studies supporting
both issues are presented
•
Equivalency between abo- and obobotulinum
toxins is approximately 2.5; a true number is
impossible to define as the units – Speywood units
versus Botox units – are measured differently
•
Because the clinical products Dysport® and
Azzalure® are identical, except for the number of
Speywood units per vial, all clinical data for one
product can be considered applicable to the other
•
Clinical trials in the United States and Europe
confirm both efficacy and safety for glabellar
injection for frown lines
•
Other features evaluated include time of onset,
efficacy, and safety with re-injection and dose
equivalency to glabellar muscle mass
•
Reviews of the international consensus board for
dosage and treatment technique gave
recommendations for injections at both upper and
lower facial sites
•
Parameters for safe injections with abobotulinum
toxin are reviewed
•
Dosage and dilution are important determinant
points for efficacy, duration, and field of effect.

27
5 
Basic science: Abobotulinum toxin A
An additional issue emerged concerning the stability
of the neuroprotein as related to complex size. In 2009
Dr Eisele (of Merz Pharmaceuticals, the manufacturer
of incobotulinumtoxinA (incobotA), a non-complexed
150 kDa protein) using standard stability tests had found
no difference in any of the three commercially available
neurotoxins with respect to potency loss or shelf life.
Another myth surrounding botulinum neurotoxin
type-A formulations is that diffusion is product specific,
and that some products have a greater area of diffusion
throughout the target muscles than others. That one botu­
linum neurotoxin type-A product may diffuse to a greater
extent than another would again have safety implications.
A 2007 study by Trindade de Almeida and colleagues
compared the diffusion of 3 U per injection of Botox®
(total of four injections) with Dysport® given in dose
ratios of 1 : 2.5, 1 : 3, and 1 : 4 to randomized patients with
forehead hyperhidrosis. Dysport® was found to diffuse
further from the injection site than Botox® in a dose-
dependent manner. The so-called ‘diffusion differences’
seen between the products were, in fact, due simply to
dose differences. These studies on dose, not diffusion,
differences are supported by others including Wohlfarth
et al and Hexsel et al. Wohlfarth and co-workers reported
that diffusion of BoNT type-A beyond the target muscles
occurs with both Botox® and Dysport® when injected into
the feet, and that diffusion was associated with injection
volume – the greater the volume, the greater the diffusion
– and diffusion beyond the injection site is not an inherent
characteristic of botulinum neurotoxin type-A agents.
Likewise, the 2008 comparison of Botox® and Dysport®
by Hexsel and colleagues reported comparable action
halos for both products when used for the treatment of
forehead wrinkles. This study utilized a ration of 1 : 2.5 U
injected with the same volume and at the same depth, and
both products were found to be safe and predictable.
The most important differences between onabotA and
abobotA are the dosage or activity units defined by the
respective manufacturers: Botox units (BU) for onabotA
and Speywood units (s.U) for abobotA. Both use the LD50
test on mice to define a unit, but there are differences
in the experimental designs of the assays making the
units non-equivalent. The s.U (Speywood or Dysport unit
assay) has a greater sensitivity indicating less toxin needed
to kill a mouse. Indeed, in a small study by Hambleton &
Pickett, it was shown that, when tested in the Dysport®
assay, the LD50 of Botox® is achieved with 0.32 B.U (68%
less product than that required for LD50 in the Botox®
assay). Thus a Speywood unit corresponds to a smaller
quantity of active toxin than a Botox unit. There is no
direct conversion factor between units and this has been
recent biophysical data. At this point, there is no convinc­
ing evidence to support any argument of clinical differ­
ences due to product differences and in fact there is
scientific evidence to support the similarities in the per­
formance of the two complexed neuroproteins.
Pearl 3 
There are very few intrinsic differences between the abobot
and onabot botulinum toxins with respect to safety and
efficacy if units are adjusted accordingly.
Pearl 2 
There appears to be a faster onset of action for
abobotulinumtoxinA than for onabotulinumtoxinA, based on
recent clinical findings.
Pearl 1 
The neurotoxin of abobutulinum toxin performs the neuro­
muscular blockage in the same manner as the other BoNT-A
molecules.
Before the BoNT-A neuroprotein can become active,
the NAPS must release the active BoNT-A 150 kDa neu­
roprotein from the progenitor complex. This occurs with
a change in environment to a physiological pH. The issue
arises as to when this occurs. Earlier pilot in vitro studies
by Eisele & Taylor showed the timescale of release varied
from immediate with pH change to a delay from minutes
to hours, depending on the product serotype with BoNT
type B taking longest. A recent study by Merz Pharma­
ceuticals, reported by Eisele et al in 2011, found that the
naked neuroprotein was released from its associated
complex in less than 1 minute with a change to physiologi­
cal pH; this occurs with both onabotA and abobotA. The
investigation implied that release of the naked neuropro­
tein probably occurs in the vial during reconstitution, well
before injection and tissue spread. However, definitive
studies on complex dissociation by the same group were
also published in 2011 and clearly demonstrated that all
the BoNT type A products existed as free neurotoxin
within the vials even before reconstitution. In other
words, the manufacturing processes for the products all
released the free BoNT neurotoxin during manufacture.
These biochemical studies do have implications for
clinical usage in understanding efficacy and safety of each
product. With the neurotoxin protein of 150 kDa released
before injection, the active toxins are stoichiometrically
similar and one would not expect a difference in diffusion
since complex size is now shown to be irrelevant. It is the
authors’ opinion that the difference seen with the prod­
ucts has to do with dosage-unit differences and volume
reconstitution. The relative kinetics of dissociation versus
diffusion have implications for the safety profiles of the
various formulations of BoNT-A in current or future clini­
cal use, and therefore these remain contentious issues for
their respective manufacturers, as reported by Pickett
in 2009.

28
Botulinum toxin
According to prescribing information in the package
insert, the abobotulinumtoxin vial with 300 s.U of neuro­
toxin should be reconstituted with 2.5 mL of unpreserved
saline. The FDA clinical studies, which had 500 s.U per
vial, were reconstituted with 2.5 mL s.U, and the equiva­
lency would be 1.5 mL per 300 s.U vial. Other dilutions
used are 3.0 mL per 300 s.U vial. Though the package
insert recommends non-preserved saline, most injectors
prefer preserved saline, which has been shown by both
Alam et al and Allen et al to have equal efficacy with
less pain.
Dilution (the reconstitution volume) is one important
factor in evaluating field of effect. Does a greater recon­
stitution volume create a greater field of effect? Probably
it does, up to a dilution of 3 mL per vial, but not a great
effect. There is a dilution factor that will decrease the
dosage as well, especially if the dilution is greater than
3.0 mL per 300 s.U vial. The author (GM) prefers the
3.0 mL dilution thus making it easy to calibrate the unit
dosage in the 1 mL syringes used. At a 3 mL dilution for
a 300 s.U vial a concentration of one unit per 0.01 mL
can be drawn up corresponding to the 1 unit gradation on
an insulin syringe. This also corresponds to the concentra­
tion of onabotA giving a similar 1 unit gradation on the
insulin syringe when diluted at 1 mL per 100 BU vial. But
this practice of similarity is not to be recommended
simply because the practitioner will then have to clearly
identify and label which BoNT product will be in which
syringe!
discouraged by the manufacturers. Thus we do not have
a direct conversion factor between the two toxins. Nev­
ertheless, practitioners have sought to define a conversion
factor to guide the novice injector when changing from
one toxin to the other for a given application.
A number of attempts have been made to define a
conversion number. A summary of recent dosage studies
places the ratio between 1 : 2 and 1 : 2.5. Multiple other
studies – both therapeutic and cosmetic – have suggested
ratios of 1 : 2.5, 1 : 3, and 1 : 4 for bioequivalence. An
earlier review by Sampaio and colleagues concluded that
a 1 : 4 ratio was too high, and a 1 : 3 ratio approached
bioequivalence although the included studies suggested
that an even lower ratio might be more appropriate. An
independently funded, double-blind study by Karsai and
co-workers of Dysport® versus Botox® for the treatment
of glabellar lines found a longer duration of action as
assessed by electromyographic studies with Dysport® uti­
lized at a 1 : 3 ratio. This led the authors to conclude the
bioequivalence ratio was less than 1 : 3. Though these clini­
cal trials for efficacy and safety were performed at a ratio
of 1 : 2.5, other recent studies by Nettar et al and Kerscher
et al have suggested a ratio of 1 : 3 provides abobotA with
a greater longevity and equivalent safety to Ona-A. At a
lower dosage (1 : 25) the study by Lowe and colleagues
found a greater longevity to onabotA for glabellar lines.
Thus we can see that dosage is really a determining factor
in efficacy. The dosage should be determined by physio­
logical response using the individual units rather than by
comparing product dosage units.
Pearl 5 
Dilution, dosage, and injection points are most important for
proper usage of abobotulinum toxin.
Pearl 4 
Units of potency – Botox versus Speywood units – are
measured differently, thus there is not an exact conversion
factor. The dosage difference is 2.0–2.5 Botox versus
Speywood units.
If we re-examine the safety studies previously pre­
sented one can see that the halos of diffusion (called the
fields of effect) found by both Almeida and Hexsel are
related more to the dosage equivalents than to intrinsic
differences in the products.
As we correlate this data with clinical practice, we
must realize there are subtle differences in the properties
of the BoNT-A products. At this point, exact data on
clinical composition, diffusion properties. and potencies
are not fully known. Until we have a more complete
understanding of these differences the clinician should
think and treat each of these products independently and
avoid relying on conversions factors. The authors recom­
mend a conversion factor of 1 : 2.5, which has become the
most commonly quoted unit dose ratio among experi­
enced injectors. The multiple studies that underpinned
the FDA approved dosages for glabellar lines (50 s.U of
Dysport® and 20 B.U of Botox®) demonstrated compa­
rable efficacy with the two BoNT-A products, further
supporting the 1 : 2.5 ratio as a starting point for aesthetic
applications.
Clinical studies performed for
abobotulinumtoxinA
Aesthetic studies for abobotulinum toxin (Dysport®) by
Rzany et al and Ascher et al followed a similar pattern to
those performed for Botox® by Allergan Inc. a few years
earlier (reported by Carruthers et al in 2004). The first
European studies for aesthetic usage of abobotulinum
toxin (Dysport®) were performed by Ascher and col­
leagues in the later 90s, and these were followed by
Ascher and Rzany in 2004 and 2006. A ‘dose-finding’
study by Vandenbergh & Lison was first performed on 119
patients using placebo, 25, 50, and 75 s.U in a double-
blinded control. The subjects were measured for efficacy
as a responder at 1 month and then assessed for safety
and duration. A responder rate of over 80% was found for
all three groups and at 6 months two-thirds of the treated
patients were still responders. There was a favorable
safety profile of 7% mild adverse events, with headache
the most common. There were no reported cases of bleph­
aroptosis or diplopia. The result suggested 50 units was
the optimal dosage for the glabella with 10 units injected
into each of five glabellar sites.

29
5 
Basic science: Abobotulinum toxin A
One factor reported by the subjects was an onset of
action within 1–2 days. Subsequent studies by Monheit &
Cohen have included a diary that the patient recorded
when an onset of effect was first noted. In three of the
studies the onset was recorded; 50% of subjects noted an
onset within 2 days and 80% noted it within 3 days.
Re-injection treatment studies were performed to ensure
that repeated exposure to the toxin did not influence
efficacy or duration. But later data now available indicate
a very fast response for abobotA: within a day for the initial
onset as studied in a frontalis model by Nestor & Ablon.
The repeat administration studies involved 768 indi­
viduals from Phase III clinical trials who received up to
six repeated treatments over 17 months. A patient was
re-injected when glabellar wrinkle lines returned to base­
line. The patients were followed for efficacy and safety
including adverse effects and assessment of serum-
neutralizing antibodies to abobotA. Results confirmed
continued effectiveness throughout the study with no
increase of active events and no patients developing neu­
tralizing antibodies.
As the data of these initial trials were reviewed it was
noted that efficacy and duration of action for men was less
than that for women on a 50 s.U dosage for treating the
glabella. This observation stimulated a variable-dose study
which stratified patients by race / ethnicity, sex and ran­
domized by muscle mass. The muscle mass groups were
to receive a single treatment of various doses of abobotA,
which was administered as a single dose of 50, 60, or 70
s.U for women and 60, 70, or 80 s.U for men based on
glabellar muscle mass. This is based on procerus / corruga­
tor muscle mass – small, medium, or large – and was
determined by the observation of an active frown, noted
individual muscle bulging, length of the infrabrow space,
and brow depression. Efficacy and duration were evalu­
ated during a 5-month period. The results, reported by
Rubin et al and Kane et al, indicated that 87% of men and
women had full efficacy at 30 days with a mean duration
of 109 days for both men and women with no difference
in ethnicity or gender. Duration of action was found to be
increased with the greater doses for larger muscle mass.
Though clinicians have individualized abobotA glabellar
treatment as to sex, ethnicity, and muscle mass in clinical
practice, this is the first controlled clinical study to verify
this common practice.
AbobotA has been used worldwide for aesthetic needs
for over 10 years. Both glabella and crow’s feet have
been well studied by Ascher with results comparable to
onabotA. AbobotA has been used in clinical cosmetic
practice worldwide over the last 10 years with similar
injection points and techniques as onabotA, but with dif­
ferent dosages. An international consensus conference
held in Paris in January 2009 provided general guidelines
for effective and safe use of abobotA on the generally
useful yet off-label sites for injection, as reported by
Ascher and colleagues. These have included the common
upper face sites: the glabella, forehead, brow, crows’ feet,
and eyelid; and other less common facial sites including:
This was followed by the US studies beginning in
2003 and extending to the present day. The Inamed,
Ipsen, and Medicis studies included Phase II dose-ranging
and Phase III single and repeat dosage studies with a total
of 2300 patients assessed for efficacy and safety. All
studies evaluated efficacy and safety of glabellar frown
lines with rating scales at rest and at maximal frown. The
US studies used the same injection points as the US
onabotA trials and were evaluated at maximal frown as
the end points. In Europe, end points for response were
determined at rest for efficacy and the lateral corrugator
injection points were placed 12 centimeter more medially.
The results, though, for efficacy and safety were similar
for both studies.
Phase II trials involved a dosage-ranging study including
placebo, 20 units, 50 units, and 75 units. There was a 90%
responder rate at both 50 s.U and 75 s.U. All doses were
well tolerated with only minor side effects including head­
ache, needle pricks, and bruising, but blepharoptosis was
observed in only three patients. Of those reported cases,
only one demonstrated to the investigator a true clinical
ptosis. Ptosis had been reported in other product studies
but not in other studies involving abobotA (as reported by
Kessler et al).
Neutralizing antibody production has always been of
concern with clinical usage of BoNT-A. This has been
considered a main cause for ‘non-response’ in patients
after injection. In 2003 this was studied by Carruthers and
colleagues in patients with cervical dystonia. None of the
patients in this study showed any evidence of neutralizing
antibodies either at baseline or on follow-up evaluations.
From these observations, the 50-unit dosage was recom­
mended as the optimal dose for safety and efficacy. The
lower dosage of 50 units was chosen for all of the Phase
III trials, as reported by Monheit and colleagues. Later,
the large Phase III trials by Lawrence & Moy for abobotA
examined the potential for antibody generation in patients
from up to 9 injection cycles; none was detected in 1200
patients. At present, the rate of antibody formation in
patients treated aesthetically during many years and mul­
tiple cycles is estimated to be less than 1 in a million
patients, based upon all case reports for all such patients
and for all products worldwide since aesthetic treatments
began (Pickett, data on file).
In 2004 the initial single-dosage, placebo-controlled,
double-blinded, randomized trial by Carruthers and col­
leagues, of glabellar frown lines for FDA Phase III studies,
included over 400 subjects followed for 150 days. The
patients were then enrolled in a repeat dosage trial for 23
months including four cycles of repeat dosage when the
patient’s frown lines returned to baseline. The patients
were evaluated for efficacy (i.e. number of responders at
30 days and duration of response) and safety. In 2009
Ascher and co-workers reported that over 90% responder
rate was recorded at 30 days, at a duration of 4 months
there were 40% responders, and at 5 months 25% were
still full responders. This produced a very similar efficacy
case for responders and duration as onabotA.

30
Botulinum toxin
dose range is from 50 s.U to 80 s.U depending on muscle
mass.
Forehead lines
For the off-label usage of forehead lines, BoNT-A provides
excellent smoothing of forehead rhytides. However, the
more severe forms of forehead wrinkles cannot be cor­
rected by BoNT-A denervation alone and may need soft
tissue augmentation or surgery.
The goal for the treatment of forehead wrinkles is to
soften the undesirable lines without causing brow ptosis
or eliminating all expressiveness on the upper face. A
conservative approach is preferred, as most patients do
not want a ‘frozen face’. The patient is asked to forcefully
raise his / her eyebrows and the strength of the frontalis is
assessed. Any discrepancy in brow position at baseline
and at maximal contraction is noted and brought to the
attention of the patient. It is especially important to note
bunny lines, depressor anguli oris, orbicularis oris, men­
talis, and platysma. These recommendations gave guide­
lines to beginning dosage and range for each site (see Table
5.1) as well as injection points and technique.
Treatment of the upper face
The site-specific dosage recommendations for the upper
face are shown in Table 5.1.
Glabellar lines
Injection of the glabella is the original and by far most
common cosmetic usage of BoNT-A. The standard
5-injection-point approach is appropriate for most patients
with two injections in each corrugator and one injection
in the procerus as shown in Figure 5.1. The procerus and
medial corrugators are injected deeply and directly into
the bodies of the muscles, which are easily identified at
maximal frown in most patients. The lateral corrugators
and orbicularis oculi muscle fibers are injected more
superficially as it inserts into the dermis medial to the
mid-pupillary line. All injections should be 1.0 cm
(approximately one finger-breadth) above the orbital rim
to limit the risk of eyelid ptosis from the spread of toxin
(at the time of injection) to the levator palpebrae muscle.
The measurement should be made from the orbital rim
and not from the brow itself as a dropped brow may
mislead the physician to inject the toxin below the orbital
rim. Injecting too high on the forehead – above 1 cm. at
the lateral glabellar site – can also cause ptosis in those
individual who recruit eyelid and eyebrow elevation from
the frontalis muscle.
The standard total dose of abobotA has been shown to
be 50 s.U in women and those with small to medium
muscle mass, divided evenly among the five injection
points. As always, doses must be adjusted for the strength
of individual muscles and the patient’s desired outcome.
The author (GM) does use a higher dose of toxin in
men and may add two additional injection points at the
mid-pupillary line in those with bulky corrugators. The
Table 5.1  Recommended Dysport® dosages for the upper face
Indications
Total usual dose (Dysport® / Speywood units)
Dose range (Dysport® / Speywood units)
Glabella
50
30–70
Forehead
40–50
40–70
Crows’ feet
30 × 2
20–50 × 2
Lateral eyebrow lift
20 × 2
20–40 × 2
Glabella and forehead
90–100
70–140
Glabella and lateral eyebrow lift
90
50–110
Complete upper third face
150
110–240
Figure 5.1  Injection sites for glabellar line treatment: blue dots =
corrugator insertion / orbicularis oculi fibers; yellow dots = corrugator
body; green dot = procerus.

31
5 
Basic science: Abobotulinum toxin A
chemical brow lift, the author typically injects 10 s.U of
abobotA intradermally at the lateral tail of the eyebrow
5–7 mm superolateral to the orbital rim (Fig. 5.3A). If
performed only for brow lift and not to correct frown
lines, an additional 5–10 s.U are injected into each cor­
rugator body. In combination with medial and central
frontalis denervation, which tends to lower the medial
brow, a pleasingly arched female pattern eyebrow can be
shaped (Fig. 5.3B).
Crow’s feet
Lateral periorbital wrinkling is effectively treated with
neurotoxin. Crow’s feet are typically treated with three
equal injections of 10–15 s.U evenly spaced along an arc
at the external orbital rim (Fig. 5.4A, B). The middle injec­
tion is placed in line with the lateral canthus. Injections
Figure 5.2  Multiple steps in the mechanism of action of botulinum
toxin type A: (1) The 150 kDa core neurotoxin is internalized into the
neuron via a process of endocytosis. (2) The endosome is acidified,
which facilitates (3) the translocation of the light chain into the
neuronal cytosol. (4) Once in the neuronal cytosol the light chain of
type A botulinum toxin (yellow) cleaves the membrane-associated
SNAP-25 (brown), one component of the SNARE complex. Another
neurotoxin serotype, type B, botulinum toxin cleaves a different
protein known as synaptobrevin or vesicle-associated membrane
protein (VAMP; green).
ACh
SV2
ACh
ACh
1. Endocytosis
2. Acidification
ACh
pH:5
pH:5
3. Translocation
of light chain
4. Cleavage of SNARE proteins
Figure 5.3  The chemical brow lift: (A) Injection sites for a chemical
brow lift if performed alone are shown (5–10 s.U or 3–5 B.U per
injection site). This patient had a male pattern eyebrow without
perceptible arching. She received abobotA treatment to the lateral tail
of each eyebrow (10 s.U per side) and into the corrugator bodies
(total 20 s.U) (blue dots), and to the frontalis (20 s.U). (B) Post-
treatment photo at 33 days reveals an elevated and laterally arched
feminine brow.
A
B
brow asymmetry, photograph it and bring it to the patient’s
attention prior to treatment.
It is important to evaluate the significance of brow
position in relationship to the frontalis, a brow elevator.
Very conservative forehead treatment is given to those
who use the frontalis to maintain normal brow position.
The author commonly uses a V-shaped configuration
for injections in women (Fig. 5.2). While often desirable
in females, this should be avoided in male patients. This
approach can also produce an excessively arched brow
(the mephisto sign or ‘Mr Spock look’). This is prevented
with a high lateral forehead injection above the tail of
the brow. If an excessive arched brow occurs, it can be
corrected with a small dose of additional toxin 1–2 cm
superior to the apex of the arched brow.
Unless no significant glabellar lines are present, the
author commonly injects the glabella simultaneously
whenever treating forehead wrinkles. This generally pro­
duces better overall aesthetic results and the concomitant
paralysis of the brow depressors reduces the incidence of
brow ptosis.
Lateral eyebrow lift
Brow position is influenced by the elevator muscles (fore­
head) and depressor muscles (glabellar complex and orbi­
cularis). The paresis of muscles of facial expression not
only smoothes dynamic wrinkles but can also influence the
resting position of various facial elements. This property
has been successfully exploited to lift the eyebrow, cor­
recting mild brow ptosis, restoring a youthful brow arch,
and giving the eye a more ‘open’ appearance.
The vertical fibers of the lateral orbicularis oculi func­
tion to depress the lateral brow. When injected at the tail
of the brow the lateral brow will be elevated. For this