Multicenter, Double-Blind Study of the Efficacy of

Injections With Botulinum Toxin Type A Reconstituted

Up to Six Consecutive Weeks Before Application
D ORIS M ARIA H EXSEL , MD, n A DA T RINDADE DE A LMEIDA , MD, w M ARCIO R UTOWITSCH , P H D, n
I N Ê S A LENCAR DE C ASTRO , MD, z V ERA L Ú CIA B AUER S ILVEIRA , MD, n D ANIELA O RSO G OBATTO ,
MD, z M Ô NICA Z ECHMEISTER , MD, n R OSEMARI M AZZUCO , MD, n AND D Ê BORA Z ECHMEISTER ,
P HARM n
n
Private Clinic, zHospital dos Servidores do Estado (HSE-RJ/MS), and wHospital do Servidor Publico Municipal de São
Paulo, Brazil

BACKGROUND. It is recommended that botulinum toxin be
used immediately or within 2 weeks after its reconstitution
because its efficacy might be compromised by prolonged
storage.
OBJECTIVES. To evaluate the efficacy of botulinum toxin type A
(BTX-A) reconstituted over 6 consecutive weeks for the
treatment of glabellar frown lines.
METHODS. Four vials of BTX-A were reconstituted each of 7
days over a period of 6 weeks, totaling 28 vials, corresponding
to seven reconstitution dates. During this period, the BTX-A
was stored according to the manufacturer’s instructions. On the
day after the last reconstitution, all of the reconstituted vials
were injected in patients from four dermatologic centers taking
part in this study. A total of 88 patients were treated on the
same day and were followed every 2 weeks for 4 months. All
patients were photographed at all stages. A number of
professionals assessed the efficacy of reconstituted BTX-A
based on the reduction of the maximum frowning capacity of
the treated muscles.
RESULTS. Of the 88 patients who were selected, 3 were
excluded. Three forms of evaluation were applied, and no
statistically significant differences were found in the results
presented.
CONCLUSION. BTX-A may be applied up to 6 weeks after
reconstitution without losing its effectiveness. Other factors,
which are probably individual, may influence the response to
BTX-A injections.

DRS. DORIS HEXSEL AND ADA TRINDADE DE ALMEIDA ARE CONSULTANTS OF ALLERGAN.

THE BOTULINUM toxin type A (BTX-A; Botox)
produced by Allergan has proven therapeutic action in
the treatment of glabellar frown lines1–8 and has been
approved by the Food and Drug Administration for
this indication. The glabellar region has frequently
been used for research when assessing the efficacy and
safety of BTX-A.3,5,9–11
Some precautions should be taken in order to achieve
effective, long-lasting results from BTX-A application.
Among them, the recommendations for reconstitution
stand out. BTX-A should be reconstituted in sterile
0.9% saline solution without preservatives.2,3,12–18 It is
recommended, among other things, to avoid shaking,
bubbling, and storing the vial for a long period of
time.12,13,17,19 The manufacturers of the commercially
available BTX-A products recommend that they should
Address correspondence and reprint requests to: Doris Hexsel, MD,
Plinio Brazil Milano Avenue 476, Porto Alegre, RS, Brazil, or e-mail:
dohexsel@zaz.com.br.
be used within the first 4 hours after reconstitution
when using the BTX-A produced by Allergan (Botox)17
or within 1 hour after reconstitution when using the
BTX-A produced by Speywood (Dysport).18 Using the
reconstituted BTX-A after a period of time longer than
that recommended may jeopardize the efficacy of the
products. However, studies have shown that Botox does
not have its potency altered even when it is refrigerated
or refrozen for up to 1 week20 or for up to 2 weeks after
reconstitution.15,21,22 Klein23 has reported that some
investigators use BTX-A for 7 to 30 days after its
reconstitution, which is also reported by Khawaja and
Hernandez-Perez.24 Alam et al.25 in their study,
mention that Aoki, a researcher working for Allergan,
has reported that reconstituted botulinum toxin pre-
served in saline and kept in refrigeration is stable for up
to 5 weeks. However, Fagien and Brandt26 observed a
subtle, although significant, decrease in the potency of
botulinum toxin that had been reconstituted 48 hours
previously.

r 2003 by the American Society for Dermatologic Surgery, Inc.
Published by Blackwell Publishing, Inc.
ISSN: 1076-0512/03/$15.00/0
Dermatol Surg 2003;29:523–529
524 HEXSEL ET AL.: EFFICACY AFTER RECONSTITUTION
This study was carried out in four dermatologic
centers using BTX-A produced by Allergan (Botox).
All of the toxin used in this study was reconstituted
over a period of 6 weeks and was applied into the
glabellar region of individuals of different genders and
ages. The aim of the study was to assess the efficacy of
BTX-A reconstituted beyond the time limits recom-
mended by the manufacturers and until now found in
the literature.
Methods
In the first stage of this study, representing 6 weeks,
a single professional (V.L.B.S.) was responsible
for reconstituting, labeling, and storing the vials of
BTX-A. This professional did not participate in
the other stages. Each 7 days, four vials of BTX-A
were reconstituted over a 6-week period, resulting in
seven predetermined reconstitution dates: 43, 36, 29,
22, 15, 8, and 1 day before the application day. On
each date, the four reconstituted vials were labeled
with color-coded tags to indicate the date of recon-
stitution. The reconstituted vials were stored in a
refrigerator at a constant temperature of 41C.2,12 The
nonreconstituted vials were stored in the freezer
according to the manufacturer’s instructions.17 The
color code for the reconstitution dates was known to
only the single professional responsible and was
maintained confidential until the end of the study.
Seven different colored-labeled vials were sent to each
of the four study centers 1 day before the application
date.
In the second stage of this study, representing 16
weeks, 88 patients of both genders and ranging from
19 to 79 years of age (mean age 46.41) were selected in
four participating centers. These exclusion criteria
were considered: pregnant women; people with
muscular, rheumatic, neurologic, or psychiatric dis-
eases; patients having taken aminoglycoside antibiotics
and anti-inflammatory agents within the last 4 weeks;
and those with known hypersensitivity to human
albumin or botulinum toxin.2,24,26–28 Three patients
were excluded. The remaining 85 patients signed a free
postinformed consent form.4,28
The BTX-A used in the study was produced by
Allergan (Botox), a 100 U/vial, reconstituted in 1 mL
of sterile 0.9% saline solution without preservatives,17
which was applied with 3-mL insulin syringes (Becton-
Dickinson Canada Inc., Mississauga, Ontario, Cana-
da) and 21-gauge needles2,12,13,29 and resulting in a
concentration of 100 U per 1 mL.2,12,13,26 All vials
containing the product belonged to the same manu-
facturer’s batch (C373) and were valid up to March
2005. Bubbling and excessive handling of the vials
were avoided, as recommended.2,12,30
Dermatol Surg 29:5:May 2003
A single professional from each of the four
participating dermatologic centers was responsible
for injecting the BTX-A from the seven color-coded
labeled vials on the application day, 1 day after the last
vials were reconstituted. On the application day, all
patients were photographed at rest and during max-
imum contraction of the procerus and corrugator
supercilii muscles, which are responsible for the
glabellar frown lines. The vial to be used for
application was randomly selected and was recorded
in a specific register.
BTX-A was applied into three sites according to the
study protocol: one at each corrugator muscle, one at
the point in the brow area along a vertical line
originating from the inner eye canthus, and one at
the procerus muscle, in the middle of an imaginary
‘‘X’’ between the brows and the inner eye canthus
(Figure 1). Male patients received 9 U into each site,
totaling 27 U, and female patients received 8 U at each
site, totaling 24 U. For the application, a Becton-
Dickinson Ultra Fine II 0.3-cc syringe with a short
needle was used.14,29
Patients were followed every 2 weeks over a period
of 4 months, and the results were recorded in the
following three forms: (1) clinical and photographic
evaluation, with Kodak-Ektachrome ISO 100 film, at
maximum voluntary frowning (frequency: at each
visit); (2) evaluating physician’s and patient’s percep-
tions (frequency: at each visit); and (3) blind and
independent analysis of the photographs by the
participating physicians as well as by two other invited
physicians who were not involved in the other study
phases (at separate sessions).
In the third stage of the study, 3 months after the
end of the second stage, the results obtained from the
Figure 1. Location of application sites at the glabella.
Dermatol Surg 29:5:May 2003
Figure 2. Comparative photographic sequence of the same patient. (A) Forty-three days of reconstitution. (B) Day 1 of reconstitution.
blind analysis were evaluated by all of the investigators
and two other collaborators at a joint session.
A simultaneous exhibition using two Kodak 4400
carousel projector was carried out. First, the initial
photographs (Day 1) at rest were exhibited in order
to evaluate the presence of wrinkles at rest
and then those during maximum voluntary contrac-
tion of the muscles in the glabella were exhibited
HEXSEL ET AL.: EFFICACY AFTER RECONSTITUTION 525
in order to evaluate muscle contraction capacity.
Immediately afterward, the photograph with the
maximum contraction was kept on the left, and
the control photographs were exhibited on the
right in a decreasing chronological order of visits,
beginning from the comparative control at 120 days
and successively (105, 90, 75, etc.), as illustrated in
Figure 2.
526 HEXSEL ET AL.: EFFICACY AFTER RECONSTITUTION
Evaluators filled out an evaluation report according
to the following criteria: (1) the presence or the
absence of wrinkles or furrows on the glabella,
resulting from contraction of the corrugator supercilii
and procerus muscles at rest, on the first day of the
study, and the classification of the wrinkle that is
formed during contraction of the muscles in the
glabella, before botulinum toxin application, as small,
medium, or large; and (2) motility degree of the
muscles in the glabella at all visits compared with Day
1 of the study in four categories: unaltered (0), slightly
reduced (1), moderately reduced (2), and greatly
reduced (3).
It should be reiterated here that the none of the
participants in this stage were aware of the significance
of the color-coded labels.
Statistical Data Analysis
Several statistical tests were applied with the goal of
identifying possible relationships between the different
variables. The Kruskal-Wallis test was employed in the
statistical analysis of the data obtained in the study
because it permits the comparison of more than one
variable in just one treatment. The critical a value (P
value) used was lower than or equal to 0.05. Data
were loaded in a Microsoft Excel 7.0 spreadsheet for
Windows 98, transferred to a database, and were
subsequently assessed using the software for statistical
analysis SPSS 8.0.
Results
Of the 88 patients in the study, 3 were excluded. Of
the remaining patients, 13 were included in group 1
(received BTX-A reconstituted 1 day before), 14 in
group 2 (received BTX-A reconstituted 8 days before),
12 in group 3 (received BTX-A reconstituted 15 days
before), 12 in group 4 (received BTX-A reconstituted
22 days before), 12 in group 5 (received BTX-A
reconstituted 29 days before), 10 in group 6 (received
BTX-A reconstituted 36 days before), and 12 in group
7 (received BTX-A reconstituted 43 days before), with
a mean of 12.14 patients per group. Eighty-one
patients attended the first evaluation at 15 days
Table 1. Significance Values, Critical a (P), of the Different Ascertainments, on the Evaluation Dates, Based on the Kruskal–
Wallis Test (Po0.05)
Day 15 Day 30
Motility as evaluated by the observer 0.549 0.332
Motility as evaluated by the patient 0.221 0.295
Motility as jointly evaluated 0.082 0.132
Dermatol Surg 29:5:May 2003
(95.2%), and 65 patients completed the evaluation at
120 days (76.4%). Considering losses, only those
patients that missed the last evaluation or more than
two consecutive evaluations were considered as such.
There was no statistically significant difference in loss
between the groups; the highest loss was in group 5,
with six patients, and the lowest losses were in groups
2 and 3, with one patient.
As for the genders, 11 (12.9%) male patients and 74
(87.1%) female patients were included. Fitzpatrick
standards for skin phototypes were used, and patients
were classified as follows: 2 patients (3.2%) belonging
to phototype I, 22 (34.9%) belonging to phototype II,
23 (36.5%) belonging to phototype III, 15 (23.8%)
belonging to phototype IV, and 1 patient belonging to
phototype V.
Five patients had minor bleeding during the
application. One patient reported erythema at the
application site. One patient experienced worsening of
pre-existing unilateral eyelid ptosis, which resolved
spontaneously within approximately 30 days. One
patient reported a decrease in visual acuity in the first 3
days.
Three distinct evaluations of the responses to these
applications of Botox were analyzed: (1) The percep-
tion of the physician who performed the application
was evaluated at 15, 30, 45, 60, 75, 90, 105, and 120
days after injection when the muscle motility at the
injected area was verified; (2) the perception of the
patient regarding motility at the injected area, on those
same evaluation days; and (3) the average of each one
of the six evaluators’ perceptions, in a joint session at
the end of the study, with regard the presence of
wrinkles and the intensity of maximum voluntary
contraction through the photographs taken on the
evaluation days, compared with maximum voluntary
frowning at the first day.
The Kruskal–Wallis statistical analysis test was
employed. No statistically significant difference,
in terms of motility and efficacy, was found in the
results of the three ascertainments cited previously
here. This can be observed in accordance with
the significance value, statistic a (P), expressed in
Table 1 and also with the results obtained with Botox
from different dates of reconstitution, showed in the
Figure 2A,B.
Day 45 Day 60 Day 75 Day 90 Day 105 Day 120
0.616 0.815 0.891 0.97 0.884 0.53
0.481 0.485 0.697 0.471 0.88 0.844
0.431 0.928 0.237 0.792 0.699 0.643
Dermatol Surg 29:5:May 2003
Figure 3. Perception of the evaluating physician who performed the application about the muscle motility at the injected area at 15, 30, 45, 60, 75,
90, 105, and 120 days after injection.
Figure 4. Perception of the patient who was submitted to the procedure about the muscle motility at the treated area at 15, 30, 45, 60, 75, 90,
105, and 120 days after injection.
Figures 3–5 show the comparison of the averages
of the evaluating physician’s perceptions, the patient’s
perceptions, and the analysis at a joint session.
Discussion
This study evaluated an 85-patient sample, with mean
age of 46.41 years, homogeneous regarding the
phototype, and predominantly female. This sample is
coherent with that of people who go to practices in
order to make use of Botox for cosmetic purposes.
Each patient has particularities regarding their
facial muscles, and they need individualized cosmetic
treatment. However, each of the muscles in the glabella
HEXSEL ET AL.: EFFICACY AFTER RECONSTITUTION 527
in different individuals may require more than one
application site and different doses to reach the same
efficacy on paralysis.31,32 However, a comparative
study such as this requires standardization of some
items. Standardization of application sites for all of the
patients and the doses according to the gender was
preferred, which was higher for male patients. It is
consensual that men have more potent muscles and
thus need higher doses for treatment.5,33,34
Typically, the dose predicted for the glabella is
25 U——distributed into five sites2,3,13,35——or 20 or
25 U——distributed into five or seven sites (depending
on the wrinkle type and intensity) for women, and
35 U into seven sites for men.5,33
528 HEXSEL ET AL.: EFFICACY AFTER RECONSTITUTION
Figure 5. Average of the perceptions of the six evaluators in the study, at a joint session, about the presence of wrinkles in the photographs taken
on the evaluation days (15, 30, 45, 60, 75, 90, 105, and 120 days after injection).
Although Gartland and Hoffman found in an in
vitro study a 69.8% loss in potency when Botox was
reconstituted, immediately frozen, and then assayed 2
weeks later and a statistically significant degradation
in potency after refrigerator storage for 12 hours,36 in
this study, it has been demonstrated that with correct
sterile handling and storage under refrigeration12,13,17
using Botox within up to 6 weeks after reconstitution
does not significantly alter the detectable clinical
response, whether from the patient’s or the observer’s
point of view. The relevance of this lies in the fact that
it is possible to fractionate doses from the same Botox
vial, which may be stored for a longer period of time;
this facilitates the clinical practice of physicians who
work in specialties that use very low doses.37,38 The
reduction of Botox activity, with motility and wrinkles
returning over the study time, was clear and may be
evidenced in the three forms of evaluation illustrated
in Figures 1–3. Such a reduction is well known in
clinical practice and in the studies of efficacy of the
product2,3,13 and could be considered as a suggestive
indirect indication that the forms of evaluation used
showed enough sensitivity to detect alterations in
motility and in the pattern of wrinkles. It should be
highlighted that the methods used for the evaluation of
results are subjective. To obtain objective results, the
use of electromyographic recording,13,39 an invasive
procedure that requires some additional financial
investment and that is painful for the patient, thus
diminishing the compliance of patients with the study,
would be necessary.
However, as for the reduction of motility in the
treated areas, compared with the initial motility, no
statistically significant differences were found. These
results may be observed in Table 1, where Po0.05 is
considered. This assessment proves that vials of
Dermatol Surg 29:5:May 2003
reconstituted BTX-A can be preserved in refrigeration
and stored in optimal conditions for up to 6 weeks.
Few complications or adverse effects have been
reported to date.7,40 This study verified the occurrence
of few unwanted effects.
Some differences in the duration of effect were
observed for some patients who received BTX-A from
the same vial, reconstituted on the same date, and
applied with the same dose. From this, we could infer
that there is the possibility that individual factors may
influence the response to BTX-A application.31,32,41,42
Because this was not the objective of the study,
comparisons to ascertain this fact have not been
performed.
Conclusion
This study showed that BTX-A applied up to 43 days
after reconstitution does not present significant differ-
ences in terms of efficacy, in accordance with the used
methods. Differences observed in the responses of
patients who received the same BTX-A, reconstituted
on the same date, show that other, perhaps individual
factors may interfere with the response.
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supports our using BTX-A for periods longer than those on the
label.
ALASTAIR CARRUTHERS, MD
JEAN CARRUTHERS, MD
Vancouver, Canada
References
1. Garcia A, Fulton JE. Cosmetic denervation of the muscles of facial
expression with botulinum toxin: a dose–response study. Dermatol
Surg Oncol 1996;22:39–43.