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membrane-spanning domain (Hn) and a 50-KDa
C-terminal receptor-binding domain (Hc). After
that, they investigated the stability parameters of
isolated Lc and the binding domains Hc of BoNT
to mild agitation. They found that the recombinant
light chains of serotype A (LcA) rapidly lost
their secondary structures and that mild stirring
denatured Hc domains, but the addition of nonionic
detergents completely prevented denaturation. They
speculated that BoNT domains underwent surface
denaturation due to rapid exposure of hydrophobic
residues by mechanical agitation.
A recent experimental study evaluated the effect of
onabotulinumtoxinA after vigorous agitation (con-
tinuous inversion and straightening of the vial, 30
times per minute) for up to 6 weeks.
47
Eight mice
were each injected intraperitoneally with 1 U of the
agitated onabotulinumtoxinA on days 1, 3, 5, 7, 14,
21, 28, and 42. The main outcome measure was
death of the mice, demonstrating toxin efficacy.
At the end of the study, half of each group of mice
(4/8 mice) died within 48 hours of the injection
(range 16–48 hours), and the conclusion was that the
effect of onabotulinumtoxinA is maintained even
when it is agitated vigorously for up to 6 weeks.
47
In a clinical setting, Almeida and colleagues
48
compared muscle paralysis between the sides in a
split-face study in which they injected onabotuli-
numtoxinA gently reconstituted without foam for-
mation on one side of the face (periocular and
glabellar areas) and onabotulinumtoxinA rapidly
reconstituted with formation of as many bubbles as
possible on the other side. They concluded that the
presence of foaming during the reconstitution pro-
cess did not affect the potency or the short- or long-
term effects of the product. A consensus panel on
onabotulinumtoxinA held in 2004 concluded that
this report supports clinical experience, suggesting
that the fragility of BoNTA is not as problematic as
previously reported.
22
Kazim and colleagues
49
also studied the duration of
onabotulinumtoxinA when reconstituted vigorously.
They randomly selected seven patients who had half
of their forehead injected with onabotulinumtoxinA
reconstituted gently and the other half with
onabotulinumtoxinA reconstituted vigorously.
(The vial was placed on a vortex Touch mixer at
maximum speed for 30 seconds.) They concluded
that the effect and duration after 6 months was the
same on each forehead side.
The fact that the associated proteins inside the
complex of onabotulinumtoxinA might serve to
stabilize the neurotoxin molecule and protect it from
degradation may explain the different results found
between in vitro
46
and in vivo
47–49
studies.
Foreign Body
There have been no reports of injection of nonbio-
degradable material concomitantly with the injection
of BoNT, but there is a report of the presence of
rubber particles in a reconstituted onabotulinum-
toxinA vial. The authors and the manufacturer
recommend visual inspection of the product before
its use.
50
Storage
RimabotulinumtoxinB must be stored at 2
1
C to
8
1
C,
20
and according to Setler,
51
there is no signif-
icant loss of activity for up to 30 months under
refrigeration, although at room temperature (25
1
C),
it drops to at least 9 months.
Some controversy remains on the storage of recon-
stituted BoNTA vials. Manufacturers recommend
administration within 4 to 24 hours after reconsti-
tution,
15–19
storage at 2
1
C to 8
1
C (in the refrigera-
tor), and not freezing after reconstitution, but several
publications suggest that these recommendations
may be excessively strict.
Shelf Life After Reconstitution
A multicenter, double-blind study demonstrated in
88 patients that reconstitution with nonpreserved
saline up to 6 weeks before use did not reduce
DERMATOLOG I C SURGERY
HANDL I NG BOTUL I NUM TOX I NS