(Investigative Ophthalmology and Visual Science. 2001;42:1254-1257.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Doxil-Induced Chemomyectomy: Effectiveness for Permanent Removal of Orbicularis Oculi Muscle in Monkey Eyelid
Linda K. McLoon1,2 and
Jonathan D. Wirtschafter1,3,4
1 From the Departments of Ophthalmology,
2 Neuroscience,
3 Neurology, and
4 Neurosurgery, University of Minnesota, Minneapolis.
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Abstract
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PURPOSE. To test the safety and effectiveness of Doxil chemomyectomy in monkey
eyelids using treatment schedules and doses similar to those proposed
for the human blepharospasm patients in Phase I and II trials.
METHODS. Bupivacaine/hyaluronidase and Doxil were injected sequentially into the
eyelids of five Cynomolgus monkeys. Eyelids received 1, 2, or 3 sets of
injections. The monkeys were euthanatized at a minimum of 2 months and
a maximum of 12 months after the final treatment. The eyelids were
prepared for histologic examination, and muscle fiber loss was
quantified.
RESULTS. All Doxil injections resulted in a significant loss of myofibers. No
bruising, ulceration, or other skin injuries occurred, even after a
third injection regimen within a single treated eyelid. Two-day
preinjury with a bupivacaine/hyaluronidase mixture had a significant
adjuvant effect.
CONCLUSIONS. Doxil chemomyectomy is an effective protocol to permanently remove
muscle from injected eyelids in nonhuman primates. Serial injections
over the course of several months using the preinjury protocol combined
with Doxil treatment significantly increased Doxil’s myotoxic effects.
Additionally, the injection of the liposome-encapsulated form of
doxorubicin did not result in skin injury or ulceration. Species
differences demonstrated the importance of testing these drugs in
nonhuman primates. Thus, repeated doses of Doxil may prove to be as
clinically effective as free doxorubicin injections in reducing muscle
spasms in blepharospasm patients but with increased safety to the skin
and tissue around the injection site.
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Introduction
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Currently doxorubicin chemomyectomy is the only
nonsurgical, permanent treatment option for blepharospasm and
hemifacial spasm patients.1
Repeated injections of 1 to
1.5 mg free doxorubicin have proven to be permanent and extremely
effective for reducing both muscle volume and muscle spasms in
patients.2
However, the eyelids were sore to the touch
after the doxorubicin injections. In eyelids that received two or three
injections, the chance of developing skin ulceration and scabbing over
the injection site increased. Although these side effects slowly
resolved in both the laboratory studies in rabbits and in the human
patients, they were a deterrent to patient and physician acceptance of
the procedure.
In an effort to develop a protocol that would reduce or eliminate the
risk of skin injury and hyperalgesia over the injection site, we
examined the efficacy of using doxorubicin in its liposome-encapsulated
form in rabbit eyelid.3
Injection of liposome-encapsulated
doxorubicin resulted in significant muscle loss after treatment. No
skin injury was seen in any of the treated rabbits. This represented a
significant improvement over the use of free doxorubicin. Previous
studies also demonstrated that preinjury of the orbicularis oculi
muscle with bupivacaine resulted in an amplification of the myotoxic
effect of subsequent doxorubicin or Doxil injections.3
4
5
Thus, these studies showed that pretreatment with local anesthetic
would be desirable in Doxil injection protocols to ensure the maximum
chemomyectomy effect of each injection series.
In human patients, the single-dose limit for free doxorubicin treatment
was lower than was used in the rabbit dose-ranging
experiments.1
Effective treatment would necessitate
multiple injections over time. These observations were incorporated
into the protocol for a Phase I dose-ranging trial for the use of
liposome-encapsulated doxorubicin in blepharospasm and hemifacial spasm
patients. In an effort to determine potential efficacy and possible
negative sequellae of this multiple-injection protocol before treatment
of patients, the present study in nonhuman primates was initiated. Five
monkeys received injections of Doxil, a commercially available
liposome-encapsulated form of doxorubicin, following the same treatment
schedule and doses proposed for the human blepharospasm patients. After
each set of eyelid injections, the monkeys were examined daily for up
to 1 month for inflammatory changes or changes in the skin over the
injection site. Previous studies demonstrated that the chemomyectomy
effect of both doxorubicin and Doxil on skeletal muscle is both
rapid6
and permanent.2
At a minimum of 2
months and a maximum of 12 months after the termination of the full
sequence of treatments, the orbicularis oculi muscles of these monkeys
were examined for permanent muscle loss using histologic and
morphometric procedures.
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Materials and Methods
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All animal studies were approved by the University of Minnesota
Institutional Animal Care and Use Committee. Animal studies were
performed in accordance to the published guidelines of the NIH on the
use of animals in research and with adherence to the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. Five Cynomolgus
monkeys were treated with Doxil (Sequus Pharmaceuticals, Menlo Park,
CA) using the following set of injection parameters (Table 1)
. Doxil was injected at a dose of 0.5, 1, or 2 mg in a volume
of 1 ml saline into the eyelid region. As in our other
studies,7
care was taken to ensure that the injection
covered the length of the eyelid from medial to lateral canthus. The
injection was placed within the palpebral region of the muscle. Eight
lids were subjected to one course of treatment, and 12 eyelids were
subjected to three courses of treatment. Upper and lower eyelids were
never injected simultaneously in an individual monkey. Rather, upper
and lower eyelid injections were alternated at a minimum interval of 2
months between injection sessions. Two distinct preinjury treatment
paradigms were tested to maximize the chemomyectomy effect of the
liposome-encapsulated doxorubicin.4
5
Approximately 50%
of the monkey eyelids received an injection of a mixture of 0.75%
bupivacaine (Sensorcaine; Astra Pharmaceuticals, Westborough, MA)
containing 1:200,000 epinephrine and hyaluronidase (150 units, Wydase;
Wyeth Laboratories, Philadelphia, PA) in 1 ml 20 minutes before
administration of Doxil into that eyelid. The second half of the
eyelids was also treated with bupivacaine with epinephrine and
hyaluronidase 2 days before injection of Doxil into that eyelid. All
eyelids were monitored daily for the first month after injection. In
all reinjection series, a minimum of 2 months was allowed between
subsequent injections of Doxil. Thus, the postinjection survival times
were a minimum of 2 months for a subset of the treated lids, a minimum
of 4 months for the other set of lids on the same monkey, and in
several specimens, the monkeys were euthanatized 6, 8, or 10 months
after the last injection. In a final monkey,7
two eyelids
received no injections, and two eyelids received saline only to serve
as a control. A statistical analysis was done using the Least
Significance Difference for All Pairwise Comparisons. For each dose of
Doxil (0.5, 1, or 2 mg), no significant differences were seen in the
myofiber counts when muscle loss for the different postinjury survival
times was compared statistically.
At the end of the sequence of injections for each monkey, the animals
were euthanatized with an overdose of barbiturate anesthesia. Eyelid
samples were removed and immediately frozen in methylbutane chilled to
a slurry over liquid nitrogen. Sections were prepared at 12 µm on a
cryostat, and the tissue was stained using the histochemical myosin
ATPase procedure to visualize the myofibers. Myofiber number was
determined using a computer-aided morphometry program (Bioquant; R & M
Biometrics, Nashville, TN). Statistical significance was determined
using a Student’s t-test, with assistance from the Prism
statistical software (Graphpad, San Diego, CA).
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Results
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The eyelids were monitored daily after the Doxil injections, and
all eyelids appeared to be completely normal. No skin injury or overt
swelling of the lids was seen in any of the days after the initial
injection at any of the Doxil doses administered. Some redness often
was present within the treated eyelid, but usually dissipated within 2
to 3 days. The Doxil itself is red, and thus this redness was thought
to be Doxil remaining within the injected eyelids. However, we did not
observe or recognize the erythematosis and erythrodysesthesia that can
occur as a side effect of Doxil extravasation in
humans.8
9
Some of the redness we observed could be due to
the development of a short-lived erythema in the treated eyelids. In
any event, this reaction was in dramatic contrast to the skin redness,
inflammation, and injury we observed after injection of free
doxorubicin.7
Thus, injection of liposome-encapsulated
doxorubicin, or Doxil, completely eliminated these undesirable side
effects. No observable changes were seen in blink, nor were any
irregularities seen in the cornea. The same was true for the many
studies using either free doxorubicin5
or
Doxil3
in rabbits and in the human blepharospasm and
hemifacial spasm patients treated with either free
doxorubicin1
or Doxil.
All the Doxil-treated eyelids were analyzed morphometrically for muscle
loss. Muscle loss was significant for all doses of Doxil tested. As in
previous studies,9
muscle loss was greatest in the
preseptal regions of the orbicularis oculi, although muscle loss was
seen in the pretarsal region as well (Fig. 1)
. There was a slight dose-related effect on muscle loss after single
injections of Doxil at doses of 0.5, 1, and 2 mg averaging 63%, 70%,
and 79%, respectively (Fig. 2A
). Muscle loss was examined in the upper eyelids and compared with that
seen for a comparable dose and treatment protocol in the lower eyelids.
No significant difference was seen in the myotoxic effect of Doxil
injections, based on the eyelid injected (Fig. 2B)
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Figure 1. (A) The pretarsal region of a saline-injected control
eyelid. (B) The pretarsal region of an eyelid 2 months after
the last of a series of three injections, spaced over the course of 10
months, of 2 mg Doxil 2 days after preinjury with bupivacaine and
hyaluronidase. (C) The preseptal region of a saline-injected
control eyelid. (D) The preseptal region of an eyelid 2
months after a one time injection of 2 mg Doxil 2 days after preinjury
with bupivacaine and hyaluronidase. Arrows indicate
individual myofibers. In all photomicrographs the skin side is at the
top. Scale bar, 100 µm.
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Figure 2. (A) All doses of Doxil injected in the eyelids of monkeys
significantly reduced the number of muscle fibers in the treated
eyelids compared with controls. A small dose-related effect was seen
when Doxil was combined with a preinjury protocol using bupivacaine
treatment 20 minutes before injection of Doxil, with 2 mg being the
most effective. There was a significant difference only between 0.5-
and 2-mg doses. (B) After either 0.5 or 1 mg Doxil
injections, there was no significant difference in the amount of muscle
loss in the upper lid compared with the lower lid.
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The range of muscle loss in the palpebral portion of the orbicularis
oculi resulting from Doxil injections was from 64% to 94% (Fig. 3)
. Although all muscle was significantly decreased compared with
controls, only when a protocol was followed that included a 2-day
preinjury protocol with multiple injections of Doxil was muscle loss
significantly increased compared with other Doxil treatments (Fig. 2)
.
Thus, the maximal muscle loss was seen when the orbicularis oculi was
preinjured with the local anesthetic bupivacaine followed 2 days later
by injection of the Doxil and repeated two or three times in the same
lid (Fig. 2)
. It should be noted that in no instance was skin injury or
signs of a localized inflammatory reaction present, even after a third
exposure to the Doxil.
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Figure 3. All doses of Doxil injected in the eyelids of monkeys significantly
reduced the number of muscle fibers in the treated eyelids compared
with controls. Only a treatment protocol that involved preinjury
(preinj) of the orbicularis oculi 2 days before Doxil exposure,
repeated at least two times per lid, significantly increased the muscle
loss over a single injection of Doxil at the same dose. *Significantly
different from control; **significantly different from the same dose
given in a single injection protocol or given after a 20-minute
preinjury protocol.
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Discussion
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These results demonstrate that Doxil injections into monkey
eyelids are a safe and effective strategy to permanently remove muscle
from within the eyelid. Previous studies have shown that even 2 years
after an injection sequence of free doxorubicin, the muscle that had
been destroyed did not regenerate.2
The additional benefit
of using Doxil compared with free doxorubicin is that the skin and
eyelid tissue are not injured beyond the appearance of mild redness.
This represents a major improvement to the doxorubicin chemomyectomy
protocol.
There was a species difference between the relative effectiveness of
chemomyectomy as a result of the injection of free doxorubicin compared
with liposome-encapsulated doxorubicin. In rabbit studies, a single
injection of Doxil produced less of a chemomyectomy effect than a
single injection of free doxorubicin.3
A single dose of 2
mg Doxil resulted in a 60% loss of myofibers compared with a loss of
87% of the myofibers after a single injection of 2 mg
doxorubicin.3
In the monkey the average muscle loss in the
palpebral portion (i.e., the pretarsal and preseptal portions combined)
of the orbicularis oculi muscle from a single 1- or 2-mg injection of
Doxil was 74.5%. This is compared with an average myofiber loss of
70% after a single eyelid injection of 1 or 2 mg free
doxorubicin.7
Thus, in monkey, the use of doxorubicin in
its liposome-encapsulated form was equally as effective as the use of
free doxorubicin.
In the rabbit we have found that preinjury of the orbicularis oculi
with bupivacaine 2 days before Doxil treatment was useful in increasing
the chemomyectomy effect. These studies confirmed in nonhuman primates
that preinjury of the orbicularis oculi with bupivacaine 2 days
before Doxil treatment also significantly increased its chemomyectomy
effect. We have previously hypothesized that this phenomenon can be
correlated with the time when muscle satellite cells are maximally
activated by the local anesthetic injury4
5
and that the
doxorubicin treatment arrives at a time of increased susceptibility to
injury.
Within the palpebral portion of the eyelid, the pretarsal portion of
the orbicularis oculi muscle has proven to be very recalcitrant to
injury both in monkeys and in rabbits.7
This study
demonstrates that it is indeed possible for Doxil to be effective in
the pretarsal region, but it can take a minimum of three injections to
do so. It may be related to the ability of the drug to spread within
the injected eyelids, because rabbit eyelids are noticeably thicker,
with denser connective tissue, than either nonhuman primate or human
eyelids. This finding underscores the importance of testing treatments
destined for human patients in nonhuman primates before proceeding with
a human trial.
All studies thus far have shown that direct, local injection of
doxorubicin, either free in solution or in liposome-encapsulated form,
results in permanent muscle loss.2
7
This study confirms
the apparent permanence of this effect for liposome-encapsulated
doxorubicin in nonhuman primate lids, with the maximum postinjection
interval being 12 months. We have previously demonstrated that muscle
loss after doxorubicin administration is fairly rapid and relatively
complete within 7 days.6
Histologic examination of biopsy
material removed from an eyelid 2 years after a final doxorubicin
treatment of a blepharospasm patient demonstrated that muscle killed by
doxorubicin did not regenerate; the treated eyelids remained
essentially muscle free.2
Long-term follow up of
blepharospasm patients treated with free doxorubicin also confirm that
the muscle loss is permanent.1
Blepharospasm patients
treated with free doxorubicin have now gone more than 9 years without
requiring further botulinum toxin treatment.1
The most significant finding in this study is the complete absence of
skin ulceration and injury after any of the Doxil injections, even
after three separate injection regimens in the same lid. This is in
marked contrast to the skin reaction after injection of free
doxorubicin, where each additional exposure increased the risk of skin
ulceration.1
Although the side-effects to the skin over
the injection site caused by free doxorubicin slowly resolved in both
the laboratory studies and in the human patients, they were a deterrent
to patient and physician acceptance of the procedure. The absence of
significant injury to the overlying skin after local Doxil injections
correlates with the distinctly different classification of these two
drugs pharmacologically. Free doxorubicin is considered a vesicant, and
this drug has a long history of causing skin ulcers with sufficient
exposure to the skin after an extravasation from IV drug therapy for
cancer treatment.10
Doxil, however, is classified as an
irritant. The only skin-related side effect from its use in high
systemic doses in cancer patients has been the development of
palmar-plantar erythematosis or erythrodysesthesia.8
9
Although some redness over the injection site was seen in the treated
monkeys, it may have been due to the Doxil itself, which is red. This
redness always resolved within 2 to 3 days in the injected monkey
eyelids.
We have not studied functional changes in blink frequency with any of
the various protocols in either the animals or the patients. It is
possible to measure blink squeezing strength semiquantitatively in
humans, which is reduced at the end of the free doxorubicin treatment
cycle. However, we have not seen excessive weakness of the eyelid,
paralytic ectropion, corneal dryness, or impaired lacrimal drainage,
which occasionally occur in patients treated with botulinum
injections.11
As a result of this study, the human Doxil Blepharospasm Treatment
Trial was initiated (www.med.umn.edu/ophthalmology/dbnews.html). Two
blepharospasm patients have been treated thus far in a Phase I clinical
trial (Wirtschafter JD, McLoon LK, unpublished results, 2000).
The Doxil injections have proved to be very effective in providing
spasm relief in these patients. Both patients have developed much of
the desired weakness in their orbicularis oculi muscles. Both patients
developed marked erythematosis and mild swelling of the eyelids,
primarily after the first and second injections. After subsequent
injections the skin was much less red and swollen. There was only mild
discomfort when the eyelids were swollen. An unanticipated positive
effect was a mild peeling of the skin that restored the eyelids to a
"younger" appearance that was pleasing to both patients. It is
apparent, however, that species differences between young adult monkeys
and older adult humans indicate that human clinical trials remain a
necessary measure of safety and efficacy.
Direct injections of Doxil for the treatment of blepharospasm and
hemifacial spasm represent a major improvement over the injection of
free doxorubicin. Even multiple exposures to the Doxil in the monkey
eyelids did not result in any skin ulceration or injury. By following
the protocol proposed in the human blepharospasm Doxil trial, these
studies in nonhuman primates support its use as safe and effective for
patients.
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Footnotes
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Supported by the Benign Essential Blepharospasm Research Foundation,
National Eye Institute Grants EY07935 and EY11375, the Minnesota Lions
and Lionesses, the Frank E. Burch chair (JDW), and an unrestricted
grant to the Department of Ophthalmology from Research to Prevent
Blindness, Inc.
Submitted for publication October 16, 2000; revised January 16, 2001;
accepted February 7, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Linda K. McLoon, Department of Ophthalmology,
University of Minnesota, Room 374 Lions Research Building, 2001 6th
Street SE, Minneapolis, MN 55455.
mcloo001{at}tc.umn.edu
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References
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Wirtschafter, JD, McLoon, LK (1998) Long-term efficacy of local doxorubicin chemomyectomy in patients with blepharospasm and hemifacial spasm Ophthalmology 105,342-346[Medline][Order article via Infotrieve]
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McLoon, LK, Wirtschafter, J, Cameron, JD (1993) Muscle loss from doxorubicin injections into the eyelids of a patient with blepharospasm Am J Ophthalmol 116,646-648[Medline][Order article via Infotrieve]
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McLoon, LK, Wirtschafter, JD (1999) Direct injection of liposome-encapsulated doxorubicin optimizes chemomyectomy in rabbit eyelid Invest Ophthalmol Ophthal Vis Sci 40,2561-2567
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McLoon, LK, Nguyen, LT, Wirtschafter, JD (1998) Time course of the regenerative response in bupivacaine injured orbicularis oculi muscle Cell Tissue Res 294,439-447[Medline][Order article via Infotrieve]
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Nguyen, LT, McLoon, LK, Wirtschafter, JD (1998) Doxorubicin chemomyectomy is enhanced when performed two days following bupivacaine injections: the effect coincides with the peak of muscle satellite cell division Invest Ophthalmol Vis Sci 39,203-206[Abstract/Free Full Text]
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McLoon, LK, Luo, XX, Wirtschafter, J. (1993) Acute morphologic changes in orbicularis oculi muscle after doxorubicin injection into the eyelid Muscle Nerve 16,737-743[Medline][Order article via Infotrieve]
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McLoon, LK, Bauer, G, Wirtschafter, J. (1991) Quantification of muscle loss in the doxorubicin-treated orbicularis oculi of the monkey Invest Ophthalmol Vis Sci 32,1667-1673[Abstract/Free Full Text]
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Amantea, M, Newman, MS, Sullivan, TM, Forrest, A. (1999) Working PK. Relationship of dose intensity to the induction of palmar-plantar erythrodysesthesia by pegylated liposomal doxorubicin in dogs Hum Exp Toxicol 18,17-26[Abstract/Free Full Text]
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Lopez, AM, Wallace, L, Dorr, RT, Koff, M, Hersh, EM, Alberts, DS (1999) Topical DMSO treatment for pegylated liposomal doxorubicin-induced palmar-plantar erythrodysesthesia Cancer Chemother Pharmacol 44,303-306[Medline][Order article via Infotrieve]
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Bowers, DG, Lynch, JB (1978) Adriamycin extravasation Plast Recontr Surg 61,86-92
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Patrinely, JR, Whiting, A, Anderson, RL (1989) Local side effects of botulinum toxin injections Adv Neurol 49,493-499
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Abstract
Introduction
