Abstract
purpose. The immunotoxin, ricin-mAb 35, composed of ricin conjugated to a
monoclonal antibody against the nicotinic acetylcholine receptor of
skeletal muscle, has been proposed as a potential new agent for
treatment of focal muscle dystonias. It has been demonstrated that
direct injection of ricin-mAb 35 into rabbit extraocular muscle (EOM)
results in significant muscle loss within 1 week. In this study, the
long-term myopathic effects of ricin-mAb 35 on extraocular muscle were
investigated.
methods. Rabbit superior rectus muscles were injected with ricin-mAb 35 at a
dose of 0.2 μg/kg, with the contralateral superior rectus muscle
serving as the control. After 56 days, 105 days, and 1 year, the
superior rectus muscles were removed and prepared for light or electron
microscopy. Postinjection changes in muscle fiber morphometry and
ultrastructure were examined. Immunohistochemical markers were used to
identify inflammatory cellular infiltrate and myosin heavy chain (MHC)
isoform expression.
results. Despite evidence of ongoing regeneration, treated muscles continued to
show a decrease in both myofiber number and in total cross-sectional
area 56 and 105 days after injection. Individual myofiber
cross-sectional areas were markedly heterogeneous at 56 days. Myofiber
number and muscle cross-sectional area returned to normal 1 year after
injection, but pronounced heterogeneity of myofiber size remained. The
most significant changes in myosin heavy chain (MHC) isoform expression
occurred in the orbital layer, where, at 56 and 105 days, there were
increased numbers of fast and neonatal myofibers and decreased numbers
of slow myofibers. In the global layer, after both 105 days and 1 year,
there was a decrease in myofibers that were positive for slow,
neonatal, and developmental MHC expression.
conclusions. EOM injection with ricin-mAb 35 results in a sustained decrease in
muscle mass at 105 days after injection, with subtler morphometric
changes persisting even to 1 year. Changes in muscle force development
as a result of ricin-mAb 35 injection are currently under
investigation. This novel immunotoxin may be useful in the treatment of
strabismus if these studies show sustained weakness in treated
muscles.
Ricin is a cytotoxic protein composed of a
ribosome-inactivating enzyme (A chain), linked by a disulfide bond to a
galactose-
N-acetylgalactosamine–binding lectin (B chain).
Ricin, by irreversibly inactivating ribosomes, inhibits protein
synthesis and kills affected cells. A newly developed immunotoxin,
ricin-mAb 35, consists of ricin chemically linked to a monoclonal
antibody against nicotinic acetylcholine receptors.
1 This
skeletal muscle–specific immunotoxin appears to cause long-lasting
weakness in limb muscle as a consequence of local direct myotoxicity
and has been suggested to be a potential treatment for focal muscle
dystonias.
1 The antibody to the nicotinic acetylcholine
receptor specifically targets the ricin to mature myofibers that
express that receptor and appears to spare other cells, including
myoblasts.
1 This permits the muscle to regenerate after
the injury induced by ricin-mAb 35 injection.
If the effects of ricin-mAb 35 are similar in extraocular muscle, it
may also be useful in the treatment of strabismus or nystagmus, by
permitting long-term, dose-related adjustments in the force generation
of specific extraocular muscles that could, in turn, result in changes
in eye alignment. It may also have some advantages over currently
available modalities. Incisional surgery, for example, compromises
normal muscle dynamics by altering the arc of contact of the muscle
with the globe, the intrinsic elasticity of the surgically altered
muscles, the resting tension on the agonist–antagonist
pair
2 and generated twitch tension.
3 In
addition, surgery unavoidably induces scarring and often disrupts
muscle relationships with soft-tissue pulleys that could further alter
extraocular muscle function.
4
Botulinum toxin, approved for clinical use more than a decade ago, has
been used effectively in both childhood and adult
strabismus.
5 6 However, the treatment of congenital
strabismus with botulinum toxin often yields inconsistent results,
particularly when the initial deviation is large.
7 8 Reinjections are commonly necessary to achieve a stable result. The
principle limitation of botulinum toxin injection has been its
relatively short duration of action. The possibility that ricin-mAb 35,
a targeted myotoxic agent, may have long-term effects on muscle
strength prompted this investigation.
Initial studies of the acute response of rabbit extraocular muscles to
direct injection with ricin-mAb 35 demonstrated that there was a
dose-related focal injury to the muscles, with a self-limited
inflammatory component and significant muscle fiber loss.
7 In this study, we examined the long-term effects of ricin-mAb 35 on
extraocular muscle morphometry, ultrastructure, and
immunohistochemistry.
New Zealand White rabbits were obtained from Birchwood Farms
(Red Wing, MN) and housed with Research Animal Resources at the
University of Minnesota. All experimental procedures conformed to the
NIH guidelines for use of animals in research and the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research.
Ricin-mAb 35 was prepared and purified as previously
described.
1 It is free of unbound ricin as indicated by
HPLC data
(Fig. 1A) . The HPLC procedure separates ricin-mAb 35 from 99.99% of free ricin
and 80% of the excess antibody. The ricin-mAb 35 fractions from the
HPLC column were examined by Western blot analysis. The construct band
is indicated by the arrow
(Fig. 1B) . Further purification was achieved
with an affinity column
1 that removes essentially all the
remaining free antibody, as demonstrated by the Coomassie-stained gel
(
Fig. 1C ; arrow).
Toxicity testing by Hott et al.
1 showed that the maximum
tolerated dose (MTD) of ricin-mAb 35, the highest dose at which all
treated animals survived, is 2 μg/kg for mice. Unbound ricin,
however, is far more toxic. In rabbits, the MTD for free ricin is 0.22μ
g/kg. The minimum lethal dose of free ricin in rabbits is 0.44 μg
/kg, which results in a precipitous decline in both systolic and
diastolic blood pressure.
9 Our rabbits remained healthy
and active after the ricin-mAb 35 injections, again suggesting minimal
exposure to free ricin. In previously published work, we found that to
achieve a consistent histologic effect, an injected dose of
1/10 of the mouse MTD was required in rabbit extraocular
muscle.
10 All animals in the present study received an
injection of ricin-mAb 35 at a dose of 1/10 MTD, or 0.2 μg/kg, in one
superior rectus muscle.
Each rabbit was anesthetized with an intramuscular injection of
ketamine (10 mg/kg) and xylazine (2 mg/kg). Proparacaine solution was
placed in the conjunctival cul-de-sac of both eyes to reduce the blink
reflex. Treatment and control eyes were randomized before surgery. The
superior rectus muscles were exposed through a conjunctival peritomy,
using aseptic technique. These muscles were chosen because of their
proximity to the limbus and ease of surgical exposure. Under direct
observation, one superior rectus muscle of each rabbit was injected
with ricin-mAb 35, diluted with sterile isotonic saline to a dose of
1/10-MTD in a volume of 100 μL. Injections were made slowly
through a 30-gauge needle that was directed posteriorly into the muscle
belly. The needle was left in place for 30 seconds after completion of
injection to reduce leakage into the orbit. The control (contralateral)
superior rectus muscle was injected with 100 μL sterile isotonic
saline alone. As in both previous studies involving the ricin-mAb 35
construct, no signs of systemic toxicity were seen. No animals ever
appeared to be sick or lethargic. All appeared to be completely
healthy. No animals died as a result of the ricin-mAb 35 injections.
The animals were killed with an overdose of barbiturate anesthesia 56
days (
n = 2), 105 days (
n = 3), or 1 year
(
n = 2) after the ricin-mAb 35 injections. The injected
superior rectus muscles were excised in their entirety. Each muscle was
embedded in either optimal cutting temperature compound (OCT) or
tragacanth gum, frozen on 2-methylbutane chilled to a slurry in liquid
nitrogen, and sectioned at 12 μm on a cryostat. The muscle sections
were stained immunohistochemically with antibodies to fast, slow,
developmental, and neonatal myosin heavy chain (MHC) isoforms
(NovoCastra-Vector Laboratories, Burlingame, CA). Immunohistochemistry
was performed without fixation or quenching on frozen sections.
Sections were rinsed in phosphate-buffered saline (PBS; pH 7.4),
incubated in normal horse serum for 15 minutes, and then incubated in
the appropriate primary antibody for 1 hour at room temperature.
Antibodies were diluted 1:40 for fast and slow MHC isoforms and 1:20
for developmental and neonatal MHC antibodies. The sections were rinsed
in PBS and incubated using an avidin-biotin peroxidase kit (Vectastain
ABC; Vector Laboratories). The peroxidase was visualized by incubation
with diaminobenzidine and heavy metals. Sections were immunostained for
the presence of cd11b-positive neutrophils, lymphocytes, and
macrophages by methods described previously.
10
The long-term effects on MHC isoform expression were examined by
counting contiguous fibers in three to four random fields in each of
the three layers of the superior rectus cross sections. The number of
myofibers that were positive or negative for the expression of the four
MHC isoforms was determined, and calculations were performed to
determine the percentage of fibers positive for each of the isoforms.
All muscles were analyzed using an image analysis system (Bioquant; R
and M Biometrics, Nashville, TN). Muscle loss after ricin-mAb 35
treatment was determined by comparing means for total muscle
cross-sectional area in square millimeters and total myofiber number
measured in three cross-sections from the midbelly of treated and
control muscles at each postinjection interval. Individual myofiber
cross-sectional areas were determined under bright-field microscopy by
manual tracing of three to four representative fields in each of the
three layers of the muscles (outer orbital, inner orbital, and global)
of all muscles. Histograms were prepared of individual control- and
ricin-mAb 35–treated muscles at each of the three postinjection
intervals. The cross-sectional areas were divided into bins at 200-μm
increments, and the histograms were constructed using biostatistics
software (Prism and Statmate; Graphpad, San Diego, CA). Although only
single muscles of each age are shown, the results for identically
treated muscles were similar. Statistical significance is defined as P < 0.05. An F test was used to indicate that the
variances of the control and injected groups were not statistically
different.
One pair of ricin-mAb 35–injected superior rectus muscles treated with
a dose of 1/10 MTD was prepared for electron microscopic examination of
the long-term effects of ricin-mAb 35 on the treated muscle. After a
postinjection interval of 105 days, the rabbit was killed with an
overdose of barbiturate and perfused through the heart with 1%
paraformaldehyde-1.25% glutaraldehyde in 0.1M phosphate buffer (pH
7.4). The superior rectus muscles were dissected from the orbits,
trimmed, and postfixed in 1% osmium tetroxide in phosphate buffer. The
muscles were dehydrated in a graded series of alcohols, embedded in
Epon, and sectioned in both 1-μm and ultrathin sections for
examination under the electron microscope.
Injection of ricin-mAb 35 appears to have long-lasting, discrete,
sustained, and well-tolerated myotoxic effects on extraocular muscles
of rabbits. The toxic effects of the ricin-mAb 35 appeared to be
directed at muscle only. It did not appear to spread to neighboring
structures within the orbit. This decreases the likelihood of unwanted
myotoxic effects on nearby extraocular muscles or of nonspecific
toxicity to other orbital tissues. Ricin-mAb 35 did not appear to be
acutely toxic to peripheral nerves or capillaries within the treated
muscles
10 nor at the long postinjection intervals in the
present study
(Fig. 5C) . The muscle-specific binding property of the
ricin-mAb 35 molecules presumably plays an important role in the
containment of the toxin after a direct muscle injection.
The myotoxic effects of ricin-mAb 35 injection are not likely to be
caused by contamination with free ricin for two reasons. First, the
purification method used in the production of ricin-mAb 35 ensures that
the injected material is essentially devoid of free ricin. Second, free
ricin exerts its toxic effects systemically. Direct injection of free
ricin into leg muscles, even at lethal doses, does not result in muscle
injury (Youle, unpublished data, 1999). An important benefit of
the ricin-mAb 35 conjugate appears to be the specific targeting of the
toxin to myofibers, which permits myotoxicity without appreciable
systemic toxicity.
Several studies have shown that ricin conjugated to antibody molecules,
such as CD22 or CD19, targets the toxicity of ricin to specific cancer
or immune system cells,
11 reducing the risk to patients of
systemic toxicity.
12 13 Several clinical trials using
immunotoxin therapy in patients with brain cancer are in progress,
testing either ricin A or a genetic mutant of diphtheria toxin
conjugated to transferrin. These clinical trials have been very
successful, and the patients have shown no evidence of systemic
toxicity with these targeted immunotoxins.
14 15 Thus, the
strategy of linking a potent toxin to a targeting antibody or molecule
is a successful approach for the direct, pharmacologic treatment of a
number of diseases.
In contrast to botulinum toxin, with which functional recovery occurs
by means of axonal sprouting and formation of new neuromuscular
junctions,
16 muscle recovery after injection of ricin-mAb
35 occurred slowly, by regeneration of the muscle. The initial work by
Hott et al.
1 showed that ricin-mAb 35 was selectively
myotoxic to myotubes and myofibers, sparing myoblasts and other cells
in vitro that did not express the nicotinic acetylcholine receptor. The
regeneration that occurred over the course of this experiment suggests
that ricin-mAb 35 also spares the satellite cells within treated
extraocular muscles. At 105 days after toxin treatment, myofiber number
and overall muscle cross-sectional areas were still reduced, compared
with control muscles. In addition, ultrastructural abnormalities in
treated muscle persisted at 105 days including myofibers with central
nuclei that are characteristic of regenerating muscle. There was a slow
return, over the course of 1 year, to the normal number of myofibers in
the treated muscles. Even at 1 year, however, there was a more
heterogeneous range of myofiber cross-sectional areas than that seen in
control muscles.
Muscle regeneration is normally a slow process. Previous studies have
shown that even in muscles with an apparently normal morphologic
appearance 90 days after a muscle injury, the regenerated muscles still
produce tetanic tensions that are below normal.
17 18 This
suggests that the long-lasting morphologic alterations in extraocular
muscles injected with ricin-mAb 35 could result in long-term
physiological changes in the treated EOM as well. Future work will test
this hypothesis.
Although the clinical use of botulinum toxin is now well
established,
19 the development of new pharmacologic agents
for the treatment of strabismus has not been pursued, despite the
potential advantages of ease of administration, limitation of
postoperative scarring, and preservation of normal extraocular
muscle–globe mechanical dynamics. There are other myotoxins that
produce localized muscle weakness that have been investigated as
potential treatments for focal muscle dystonias and contracture. None
seems satisfactory for treatment of strabismus, however. For example,
in animals with passive immunity to tetanus toxin, injections of
tetanus toxin into orbicularis oculi resulted in weakness in the
treated muscles.
20 However, local spread of this toxin
resulted in unwanted muscle weakness in neighboring muscles.
Doxorubicin, another potent myotoxin, has permanent effects on muscle,
and muscle loss is profound.
21 Ideally, injected agents
should allow titratable adjustment of extraocular muscle force
generation so that, in the presence of abnormal efferent motor signals,
binocular alignment can be achieved. These effects must last
sufficiently long that sensory and motor adaptation can occur to create
a permanent change in the rotational position of the globe. The
treatment must not create significant inflammation or cause unwanted
collateral damage to adjacent extraocular muscles or other orbital
structures.
Our studies suggest that ricin-mAb 35 may have some advantages over
other pharmacologic approaches currently in clinical use or under
investigation in laboratory studies. It appears to have a long-lasting
and muscle-specific effect, presumably because it is specifically
targeted to mature myofibers. It is important to note that treatment
with ricin-mAb 35 does not result in permanent muscle loss. This,
combined with the self-limited and short-lived inflammatory response,
suggests that ricin-mAb 35 may be a potent and well-tolerated new
treatment for strabismus.
Supported by The Minnesota Lions and Lionesses and an unrestricted
grant to the Department of Ophthalmology from Research to Prevent
Blindness.
Submitted for publication January 31, 2001; revised September 6, 2001;
accepted November 19, 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 McLoon, Department of Ophthalmology,
University of Minnesota, Room 374 LRB, 2001 6th Street SE, Minneapolis,
MN 55455;
[email protected].
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