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Misonidazole neurotoxicity in the mouse: Evaluation of functional, pharmacokinetic, electrophysiologic and morphologic parameters
Int. .I. Radiation Oncology Bid. Q Pergamon
Press Inc.. 1979
Phys., Vol.
Printed
5, pp. 983.991
0360.3016/79/0701-0983/$02.00/O
in the U.S.A.
l Rapid Communication MISONIDAZOLE FUNCTIONAL,
NEUROTOXICITY IN THE MOUSE:* EVALUATION OF PHARMACOKINETIC, ELECTROPHYSIOLOGIC AND MORPHOLOGIC PARAMETERS
PETER J. CONROY,** Ph.D., RUDY VON BuRG,J~ Ph.D., PASSALACQUA, ** B.S. ROBERT
M.
DAVID P. PENNEY,??
SUTHERLAND,**
WILLIAM
Ph.D.,
Ph.D.
University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester,
NY 14642, U.S.A.
The neurotoxic effects of chronic administration of misonidazole (0.3 mg/g/day, 5 times weekly) were investigated in Balb/cKa mice over 12 weeks; a variety of measurements were used, including functional and clinical performance, morphologic, electrophysiologic and pharmacokinetic parameters. The half life of drug for a single dose was greater in brain (3 hrs) compared to serum (1.2 hrs); these values decreased to 1.9 hrs and 1.0 hrs respectively after 3 weeks. Misonidazole induced a peripheral lesion after three weeks with a total administered dose of 13.5 g/m* or exposure dose of 57 to 75 mM X hrs, which is similar to the doses that cause neuropathy in humans. There was some suggestion of a central neurological deficit related to locomotor control and balance; however, no gross morphological damage was found in the brain. The sequence of effects demonstrated began at 3-4 weeks and included: 1) morphologic damage to peripheral nerves; 2) hyperactivity and listlessness; 3) a decrease in rotarod retention time which reached a value 50% of that of saline injected control mice at 8-10 weeks; 4) walking on tiptoes with a slightly hunched back (4-6 weeks); and 5) an increase in hind foot splay (6-7 weeks). The morphologic damage primarily involved the more distal portions of the nerves supplying the interosseous muscles and footpads of the hind limbs. The damage was more severe and progressed more rapidly with time in these distal areas compared to the more proximal regions of the nerves. No marked changes were found in nerve conduction velocity although neuropathy produced by acrylamide produced significant decreases. The changes in neurological parameters reported here may be useful in the further evaluation of hypoxic cell radiosensitizers. Misonidazole,
Neurotoxicity,
Mouse, Electrophysiology,Pharmacokinetics.
INTRODUCTION
incidence of neuropathy to an acceptable level. Present clinical protocols have established a maximum tolerated dose of 12 g/m2 over 3 weeks or I5 g/m2 in 6 weeks, irrespective of the dose fractionation of misonidazole. 7.‘7.20It appears that patients who have received the greatest exposure doses (product of half life and serum plateau level of the drug) may be at greatest risk.7 Electron microscopy of a sural nerve biopsy from a 54 year old male patient who received 24 g of misonidazole (approximately 18 g/m’) revealed residual of previous distal axonal degeneration, with some segmental demyelination and remyelination.” R. Urtasun (written communication, October 1978) has also recently reported on the drug related death of an 85 year old male patient who had no evidence of disseminated disease other than local regional recurrence. This patient had received a total dose of 16 g in 8 fractions over 3 weeks (10.7 g/m’).
The nitroimidazole, misonidazole (Ro-07-0582), has been shown to selectively sensitize hypoxic mammalian cells in vitro’ and in viva’ to ionizing radiation at concentrations below those toxic for aerobic cells. Considerable interest has developed to assess the use of misonidazole in combination with radiation therapy in human solid tumors. Preliminary studies with misonidazole given in single doses of l-4 g to normal human volunteers did not indicate any toxic side effects.” However, several phase I studies have now reported contraindicative side effects such as convulsions at high doses and peripheral sensory neuropathy in cancer patients who received lower multiple doses of misonidazole.‘.6,8,‘7 The incidence and severity of peripheral neuropathy is related primarily to the total dose of misonidazole administered. In general, dose schedules have been revised downwards to reduce the
*This work was supported by National Institutes (NIH) Grants CA-11051, CA-11198, ES-01247, Contract I-NOl-CM87175. **Multimodalities Research ter Cancer Center.
Section,
University
of Health and NIH
tvisiting Scientist, Environmental Health Sciences. j’tuniversity of Rochester Cancer Center Experimental Pathology-Ultrastructure Research Facility. Accepted for publication 13 April 1979.
of Roches983
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The preliminary interpretation of brain sections taken at autopsy is that the findings are consistent with a probable toxic etiology of a demyelinating necrotic type. In man, a significant neurological deficit following misonidazole or other nitroaromatic compounds4,‘3 can be reported by the patient. At present no comparable satisfactory parameter has been identified in experimental animals in order to study mechanisms and to compare effects of different radiosensitizing agents. Since most radiobiological investigations of the effectiveness of hypoxic cell sensitizers have used tumor systems in mice, it was considered important to identify a satisfactory neurological parameter in the same species. It has been reported that the nerve conduction velocity (NCV) of mice was altered by administration of a single i.p. dose of I mg/gm misonidazole.‘2 However in a similar study we have shown no significant decrease in NCV or other electrophysiological parameters following a single dose of drug.19 The present study was initiated to determine the effect on the nervous system of chronic administration of misonidazole in mice using pharmacokinetic, clinical, functional, electrophysiologic and morphologic measurements. Acrylamide was used as a positive control in these studies since acrylamide neuropathy has been produced in all mammals studied by repetitive doses of the compound.‘5 During this investigation Griffin? (personal communication) in an independent study found morphologic damage and symptoms of neurological deficit following chronic administration of misonidazole in rats. After 3-4 weeks there was distal axonal degeneration of a Wallerian type associated with marked spasticity and abnormal righting reflexes. The overall aim of the present investigation was to identify neurological parameters for misonidazole toxicity in the mouse using an integrated approach. METHODS
AND MATERIALS
Six to eight week old female Balb/cKa mice (18-20 g)? and 7-9 week old male CBA/J mice (21-24 g) were used in these studies.+ The number of animals used in each experiment is indicated in the corresponding figure or figure legend. Misonidazofe toxicity: The LD,, for a single i.p dose of misonidazole in female Balb/cKa mice is 1.82 mg/g (range 1.7551.92) computed by the method of Litchfield and Wilcoxon.’ A marked short-term soporific effect was noted at all doses in excess of 0.3 mg/g. Misonidazole doses in excess of the LD,, value generally resulted in convulsions, clonic-tonic spasms and death within 2-4 hours. In a preliminary study, no significant weight changes were observed in mice that were treated 5 times weekly (total weekly dose 4.5 g/m2) for 2 weeks with 0.3 mg/g/day misonidazole (0.9 g/m2/day). This dose
tJ. Griffin, Dept. of Neurology, Johns Hopkins Medical School, Baltimore, Md., as reported at the NCI Radiation Sensitizer Workshop, Washington, D.C., Dec., 1978.
July 1979, Volume 5, Number
7
regime was chosen since it would give a total administered dose of 18 g/m2 within 4 weeks. Acrylamide toxicity: The LD,, in rodents for a single dose of acrylamide is 0.17 mg/g regardless of the route of administration.15 Clinical symptoms of peripheral neuropathy are generally evident in 2 weeks within the dose range 0.01-0.05 mg/g/day. Animals that receive amounts of acrylamide smaller than 0.01 mg/g/day develop symptoms later which progress insidiously. We chose a dose of 0.01 mg/g administered by i.p. injection 3 times a week since preliminary data indicated that a severe deficit would occur within 334 weeks. Misonidazole pharmacokinetics: The half life and other pharmacokinetic parameters of misonidazole in serum, brain and liver of Balb/cKa mice were determined at the beginning and after 3 weeks of chronic administration of misonidazole (0.3 mg/g/day, total dose 13.5 g/m’). The method involved a spectrophotometric assay described previously.” The drug was dissolved in phosphate buffered saline (pH 7.4) at a concentration of 20 mg/ml (100 mM) and passed through a 0.45 P filter prior to use. It was injected intraperitoneally into recipient mice at a dose of 0.3 mg/g body weight (0.30 ml of drug solution per 20 g mouse). Blood samples (0.3 ml) were taken from groups of 3 or 6 mice by cardiac puncture following ether anaesthesia at 5,10,15,30 and 45 minutes and 1,2,4,8 and 24 hours after injection. 100 mg samples of brain and liver were also removed at the times given above. Control samples, taken from groups of mice that received buffered saline, were used as blanks in the spectrophotometric determinations. In general, the method can detect misonidazole equivalents down to a concentration of 2 yg/ml. Clinical observations and functional tests: In the group of 90 Balb/cKa mice receiving misonidazole, IO animals were selected for clinical observation and testing. The responses of these mice were compared to those of 10 saline treated control animals. A similar procedure was followed for the group of 45 CBA/J mice treated with acrylamide. The animals in these groups were weighed twice weekly and observed daily for gross clinical changes such as irritability or listlessness and defects in gait and coordination. In addition, two types of quantitative measurements were made to determine the degree of neurological disability of the mice treated with misonidazole or acrylamide. In general, each test was separated from the other by several hours and performed before the daily dose of drug. Locomotor coordination and balance (rotarod performance): The rotarod method was developed by Dunham and Miya’ and involves the ability of an animal to maintain the locomotor coordination and balance on a rotating rod. The test has the potential advantage of
IBiobreeding Laboratories, Ontario, $Jackson Laboratories, Bar Harbor,
Canada. Maine.
Misonidazole
neurotoxicity
in the mouse 0 P. J. CONROY ef al.
being easily quantifiable and objective, but it involves factors other than motor performance and coordination, notably memory. Rodents must be conditioned either by repeated exposure to the rotarod or by being punished for failure. In our experiments we employed a Splace rotarod? equipped with a variable speed motor$. The initial rotarod speed was set at 10 rpm thereafter increasing linearly over the next 3 minutes to a final speed of 40 rpm. Each mouse in the treatment protocol was exposed to the rotarod for a period of 5 days before the beginning of the experiment. At each experiment observation, the mouse was tested 3 times over a 2 hour period to allow rest periods. The mean retention time on the rotarod was recorded. Any animal that fell off the rotarod within 20 seconds three times in succession was arbitrarily scored as having zero retention time. This occurred only in the mice that were treated with acrylamide. Proprioceptive dejicit and hind limb splay (drop test): This was a modification of the method of Edwards and Parker” who observed that hind limb splaying occurred early in the course of acrylamide neuropathy in rats. This would be expected to be associated with sensory deficits of proprioceptors and with neuromuscular dysfunction. They found this method to be more sensitive than the measurement of gait parameters and the ability of rats to remain on an inclined slope. Each mouse was held in a horizontal position with the dorsal side uppermost and the tip of the nose 32 cm from a cushioned flat surface. Each foot was lightly marked with non-toxic food coloring; the mouse was dropped and the position of the fourth digit in each hind limb on landing was marked. The distance between the two marks was measured and the mean of 5 measurements was calculated. Mean control footspread was 4.22 k 0.11 cm (1 standard deviation from the mean). Electrophysiofogy: Calculations for nerve conduction velocity (NCV) were made directly from photographs of the oscilloscope trace and were based on the image of the oscilloscope graticule, the recorded time base setting and the msec delay between the onset of the stimulus artifact and the highest point of the observed waveform. Our estimate of the overall error from all sources in any one determination is approximately 10%. Sensory and motor NCV measurements in vitro: The procedure employed has been described previously.” These determinations were made after 0,2,3,4,8 and 12 weeks of misonidazole treatment and 0 and 3 weeks for acrylamide. Sensory NCV measurements in situ: The conduction velocity of the sural-sciatic nerve was determined by the latency difference in action potentials recorded at the level of the pelvic girdle by stimulation of the distal segments of the foot and a stimulus applied across the sural nerve just prior to its penetration into deeper nerves of the ankle. Dessication of the exposed tissues was IStoeleing
Corp.,
Chicago,
Illinois.
985
prevented by a layer of mineral oil warmed to 37°C. Temperature of the preparation was controlled by the use of an infra-red heat lamp which held the ambient temperature of 37” f 1.5%. Nerve distances were estimated to the nearest 0.25 mm with the aide of screw adjustable dividers and a ruler. Measurements were made after 0,3,4,8 and 12 weeks of misonidazole dosing and 0 and 3 weeks for acrylamide. Motor NCV measurements in situ: Motor NCV measurements employed the same temperature and dessication controls as described above and dissection was limited to the thigh area and sciatic nerve. However, this determination employed a 3 pronged “J” stainless steel stimulating electrode. Distances between the anode and 1st cathode was approximately 1 mm. The 2nd cathode was located 6.1 * 1 .O mm from the first. Cathodal distance was measured by a micromanipulator micrometer. Muscle action potentials were recorded from the tarsal adductor muscles of the foot by means of implanted fine wire electrodes.3 Conduction velocities were calculated on the basis of the time delay between muscle action potentials evoked at the 2 different stimulus points and the distance between the stimulating electrodes. These measurements were made at the beginning and the end of the treatment protocols for misonidazole and acrylamide.
Morphologic
evaluation
All perfusion solutions were maintained at 37’ * 1°C. Perfusion pressure was controlled at 75-85 mm Hg with a manostat. Mice were pretreated with (a) 0.125 mg/g propranalol 15 minutes and (b) 200 units of heparin 5 minutes before administration of 0.1 mg/g of pentobarbital anesthetic. Following the induction of anesthesia, the animal was taped down on an inclined metal tray maintained at 38°-400C; the thoracic cavity was exposed and a polyethylene catheter terminating in a 20G needle was inserted into the left ventricle. After a small incision was made in the right auricle, the mouse was serially perfused with (a) 10 ml of phosphate buffered saline (2 ml/min) (b) 15 ml 50% strength Karnofsky’s solution (1 ml/min) and (c) 30 ml of 100% Karnofsky’s solution (1 ml/min). The perfused mouse was carefully skinned down to the level of the wrist and ankle and the entire fore and hind limbs were removed at their points of insertion into the pectoral and pelvic girdles respectively. The carcass was decapitated, the brain and spinal column removed and placed along with the limbs in full strength Karnofsky’s fixative for a further l-2 hours and then stored in 0.1 M phosphate buffer, pH 7.2. The anterior tibia1 and a 5 mm x 4 mm section of the gastrocnemius muscle at the level of the ankle were dissected free from the hind limb, the footpad carefully peeled back and the flexor and adductor digitorum $B. B. Motor Control
Corp.,
New
York,
N.Y.
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July 1979, Volume 5. Number
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muscles exposed and removed into 0.1 M phosphate buffer, pH 7.2. The various nerves, together with their muscle attachments, were post-fixed in 1% 0~0, for one hour, dehydrated in increasing concentrations of ethanol and embedded in epoxy resin.lh Thick (0.551 .O pm) sections. cut on a microtome* were stained with toluidine blue for histologic observations. Thin sections (40-80 nm), stained with lead citrate and uranyl acetate, were observed and photographed using an electron microscope.? RESULTS Misonidazole pharmacokinetics: Measurements of drug serum concentration indicate a peak serum level (C,,,,,) of approximately 400 pg/ml (2 mM) was obtained 0.5 hours (T,,,,,) following a single i.p. dose of misonidazole at 0.3 mg/g with a half life (tr/l) of I .2 hours (Figure I, panel A). The total exposure dose, computed over a 24 hour period, was 5.0 mM x hours. After 3 weeks of dosing, the serum C,,,,, had declined to 300 pg/ml (1.5 mM) with a tr/~ of I .O hours. The corresponding exposure dose was 3.8 mM x hours, a reduction of approximately 25%. Brain misonidazole levels of approximately 160 pg/g (0.8 mM) were observed I .O hour after a single i.p. dose with a t’/x of 3.0 hours and an exposure dose equivalent to 4.2 mM x hours. The brain half life declined to 1.9 hours after 3 weeks of dosing with a corresponding reduction in the exposure dose to 3.1 mM x hours (approximately 25%). Measureable amounts of drug were present in brain tissue 24 hours later (Figure I, Panel B). The tr/l of misonidazole equivalents in liver declined from 2.4 hours for a single i.p. dose (10.8 mM x hours) to I .5 hours (6.5 mM x hours) after 3 weeks of dosing. The 25% reduction of the misonidazole exposure dose for brain tissue seen after 3 weeks of chronic administration reflects that (i) less drug is delivered to the tissue per unit time and (ii) the t$ in the brain has declined, presumably as a result of enzyme induction. Clinical observations and functional tests: No deaths or overall weight changes were observed in the misonidazole treated mice over the I2 week course of the experiment. However the control mice showed a weight increase of approximately 10% over this period. The soporific effect of misonidazolc declined from an initial value of 25 minutes to approximately IO minutes at I2 weeks. The misonidazole treated mice alternated between hyperactivity and listlessness after 3-4 weeks of dosing. This continued for the full I2 weeks but did not appear to progress. The mice began walking on tip-toes with a slightly hunched back after 446 weeks. This symptom also did not appear to progress after this time. Although no gross disturbances of gait or righting reflexes were observed in the misonidazole treated animals, their rotarod performance began to decline at 3
*LKB Ultratome
III.
B.
l.330
I
BRAIN
Fig. I. Semilog plot of serum (pg/ml, Panel A) and brain (&g, Panel B) concentrations with time (hours) in Balb/cKa mice following a single i.p. injection of misonidazole at 0.3 0, 6 mice per plotted point) compared to mg/g (0 animals receiving the same dose after chronic administration of the drug for 3 weeks (5 days/week, 0.3 mg/g/day) (0- . - . -0; 3 mice per plotted point). The error bars represent r I standard deviation of the mean.
weeks (total administered dose 13.5 g/m’) (Figure 2, upper panel). The fall in rotarod retention time became statistically significant at 4 weeks (p < 0.05, student t-test) and reached a value 50% lower than the control mice between weeks 8 and I2 (p < 0.001). In addition to peripheral neurological damage, (see Morphology section), this result may indicate a central lesion related
SZeiss IOA
Misonidazole
neurotoxicity
in the mouse
ROTARODPERFOWANCE
l
P. J. CONROY et al
987
MISONIDAZOLE
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DROP TEST PERFORNANCE
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to control of locomotion and balance. The hind foot splay of the misonidazole treated animals did not begin to increase until weeks 6-7, it approached statistical significance at 8-10 weeks and became significant at lo-12 weeks (p < 0.05) (Figure 2, lower panel). In contrast, the acrylamide treated mice had suffered a 20-25% weight loss, had marked hind limb spasticity, muscle wasting and poor locomotor coordination by the second week. This group of mice failed completely on the rotarod at this time and could not be assessed on the drop test since the hind limbs remained crossed with the paws clenched. Electrophysiology: Figure 3 (upper panel) illustrates that no significant reduction in the conduction velocity of the sciatic-sural nerve measured in situ occurred in mice
-_ (6)
WEEKS OF DOSINS
Fig. 2. Upper Panel: Mean rotarod retention time (seconds) with time (weeks) in a group of IO Balb/cKa mice receiving 0.3 mg/g/day misonidazole (0 0) compared to a group of 10 mice receiving an equivalent volume of saline (0). Each mouse was observed 3 times within a 2 hour period at each determination. Lower Panel: Hind foot splay (drop test) performance of 10 misonidazole treated Balb/cKa mice (expressed as a % of that of 10 saline treated control animals) (Cl 0) with time (weeks). Each mouse was tested 5 times within a 2 hour period at each determination. The dashed lines represent the standard deviation of the mean of the pooled control data obtained over 12 weeks. The error bars represent t I standard deviation of the mean.
I
20
I 12
(6)
10
0
T 't!EEK
Fig. 3. Upper Panel: Sural-sciatic sensory conduction velocity (m/set) measured in situ in Balb/cKa mice treated with misonidazole for 3 or 12 weeks at 0.3 mg/g/day, 5 times weekly, total dose 4.5 g/m2/week). Error bars represent + 1 standard deviation of the mean. The number of animals in each determination appears in parentheses within the approximate bar. Note that we have chosen to show only the 2 extremes of the period over which measurements were taken with misonidazole. Other measurements taken at 4 and 8 weeks with 5-8 animals/time also showed no significant reduction in conduction velocity. Lower Panel: Sural-sciatic sensory conduction velocity (m/set) measured in situ in CBA/J mice treated with acrylamide for 3 weeks at 0.01 mg/g/3 times weekly, total dose 0.09 g/m2/week).
that were treated for 3 or 12 weeks with misonidazole. Values obtained at intermediate time points (see Methods) on 5-8 animals did not differ from the control values by more than 10%. The in situ measurements of motor nerve
conduction
velocity
after
12
weeks
of
dosing
988
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confirm this result. The data obtained for the in vitro measurement of NCV in isolated sural (sensory) and tibia1 (motor) nerves is in comparable agreement (data not shown). In addition we found no significant reduction in conduction velocity in isolated sural or tibia1 nerves taken from mice that were exposed to a total dose of 5 mg/g (15 g/m*) misonidazole administered over 10 days either as 0.5 mg/g/day or 1.O mg/g on alternate days. By contrast the acrylamide treated mice showed a significant reduction in sensory conduction velocity of approximately 35% (p < 0.001) after 3 weeks (lower panel, Figure 3). This reduction correlates with the functional and clinical observations outlined above. It may be argued that if misonidazole induced damage is confined to the most distal segment of the affected nerve, then we may not expect to see a reduction of overall conduction velocity until a late stage. We have attempted to test this possibility by measuring the sensory conduction velocity in situ of the most distal segment of the sensory nerves which supply the hind limb phalanges. We examined the difference between the time taken for an impulse to pass over the total length of the sciatic nerve from the stimulus point at the most distal segment of the foot to the point of entry of the nerve into the spinal cord and that for a stimulus point located at the level of the ankle. There were no significant differences in the conduction velocity of an impulse passing over the entire length of the nerve and that of its proximal or distal segments. The morphologic changes seen 3-9 weeks after misonidazole treatment in the sural and tibia1 nerves (see next section) indicate that the damage induced by misonidazole may not necessarily express itself in a reduction in the overall conduction velocity of a majority of the fibres. However, some reduction in the amplitude of the action potential could be expected at later times. Some of the measurements of NCV at 0.3 mg/gm/day of misonidazole showed a variable decrease in this parameter at late times (9912 weeks). Morphology: Misonidazole treated mice were taken for histologic and electron microscopic examination at 0,2,3,4,6,9,10, and 12 weeks. After 3 weeks there were demonstrable morphological changes. At this time, the more distal portions of the nerves supplying the hind limbs were primarily affected. The damage was more severe and progressed more rapidly with time in these areas compared to the more proximal regions of the sural and tibia1 nerves. After 6-9 weeks, the peripheral nerve components, particularly in the footpad and in flexor muscles were severely damaged. When compared to nerves from untreated control animals (Figure 4), the fine structural changes attributable to the radiosensitizer involved both the neuronal processes and the accompanying Schwann cells. The myelin sheath of the distal segments of the nerves supplying the interosseous muscles and the footpads of misonidazole treated mice exhibited an edematous sepa-
July 1979, Volume 5, Number
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Fig. 4. Adductor nerve from untreated control animal. The integrity of the Schwann cells, myelin sheaths, axoplasm and its organelles, unmyelinated fibers, and epineurium is maintained (x 15,400). ration of the lamellar leaflets by 3 weeks (Figure 5) which progressed with time so that at 9 weeks there were marked separations of the neuronal processes from the sheath (Figure 6). Myelin sheath degeneration, vacuolation and the inclusion of a finely granular component within the myelin lamellae tended to precede changes to axoplasmic organelles such as mitochondria where enlargement and loss of internal membranous cristae were observed by 9 weeks. No evidence of damage to axonal microfilaments was seen in the misonidazole treated mice. This differs from the response of rodents treated with acrylamide” where such changes were clearly evident at an early stage. In general, the morphologic changes seen in the neuronal processes following
Fig. 5. Adductor nerve from mouse treated with misonidazole for 3 weeks (0.3 mg/g/day, 5 days/wk). Leaflets of myelin sheath are distended, and Schwann cell appears vaeuolated (x 13,900).
Misonidazole
neurotoxicity
in the mouse
Fig. 6. Flexor nerve from mouse treated with misonidazole for 9 weeks. The myelin sheath exhibits granularity and focal separations. The mitochondria of the Schwann cell and neuron were distended and exhibited a loss of internal membranous cristae. In addition to cytoplasmic vacuolation, the Schwann cell endoplasmic reticulum was also distended (x 13,900).
misonidazole development cells. At
3 weeks,
administration were less rapid in their than those in the surrounding Schwann the
Schwann
cell
cytoplasm
exhibited
swelling and dilatation of the endoplasmic (Figure 5). Progressive vacuolation and the internal structure of cytoplasmic organelles at 6 weeks and became marked by 9 weeks. In Schwann cell nuclear heterochromatin had more prominent (Figure 6), suggestive of pykno-
mitochondrial
reticulum loss of occurred addition, become sis. Histologic observations of several nerves from misonidazole treated mice revealed no marked endoneurial or perineurial edema. At the later stages (6-9 weeks) the morphological changes described above using the electron microscope were observable histologically at the light microscope level. Morphologic evaluation of the brain tissue of misonidazole treated mice is in progress. At the dose level employed in this study (0.3 mg/g/day, 4.5 g/m2/week) we have observed no gross lesions in the brain at the histological level at 3 or 9 weeks which would correlate with the central lesion implied by the decline in the rotarod performance of the misonidazole treated mice which began at 3-4 weeks. More detailed studies at the electron microscope level of brain tissue taken at other times at this and higher doses are required and are presently underway. DISCUSSION
The morphological evidence obtained in the present study indicates that chronic administration of misonidazole (0.3 mg/g/day, 5 times weekly) in mice results in a *J. Griffin, Dept. of Neurology, Johns Hopkins Medical School, Baltimore, Md., as reported at the NC1 Radiation
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peripheral neurological lesion after 3 weeks (total admmistered dose 13.5 g/m*). At this time, the more distal portions of the nerves supplying the interosseous muscles and footpads of the hind limbs are primarily affected. The misonidazole induced damage is more severe and progresses more rapidly with time in these distal areas compared to the more proximal regions of the sural and tibia1 nerves. Histological evidence at the light microscope level of the peripheral morphological changes was apparent after 6-9 weeks of drug administration (total dose 27-40.5 g/m2 misonidazole). In the present study we have not been able to demonstrate any marked change in nerve conduction velocity indicating that at the dose level employed it did not predict or indicate any significant neurological deficit induced by misonidazole. The overall evaluation of the effects of chronic administration of misonidazole at this dose level (0.3 mg/g/day) is that of a mixed peripheral and central neurological deficit. There was no gross morphological damage in the brain but further histological investigation at this and other doses and schedules is required since some of the data is consistent with central involvement related to locomotor control and balance. Such information is also necessary in order to obtain dose-response relationships for the effects demonstrated. Griffin* (personal communication) has evaluated morphological changes and clinical symptoms in the rat treated with misonidazole at 0.3 mg/g/day (5 times weekly). He noted endoneurial edema and a marked loss of sensory endings on muscle spindle cells in the peripherary of the misonidazole treated rats within 2-3 weeks. Peripheral denervation, endoneurial edema, and distal axonal degeneration were observed in the nerves supplying the interosseous muscles of the foot. Although we have not observed any endoneurial edema and our examination at the EM level of the sensory endings on muscle spindle cells are not extensive enough to confirm those observations, the morphologic evidence of drug induced damage in the distal areas of the hind limbs indicate that the effects are, in general, very similar in both the mouse and the rat. The clinical symptoms of hyperactivity and listlessness and walking on tip toes following 2-3 weeks of misonidazole were also similar in both studies. However, rats begin to show a deficit in righting reflexes after 3-4 weeks of misonidazole administration at 0.3 mg/g/day and died if the drug treatment continued beyond this point. We did not observe such a gross deficit in our study in the mouse, nor did we see any marked weight losses or deaths in the misonidazole treated mice for up to 12 weeks. This shows that at this dose level, the mouse responds in some respects differently from the rat to chronic administration of misonidazole. The pharmacokinetic data obtained in the present Sensitizer
Workshop,
Washington,
D.C., Dec., 1978.
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study indicates that the first dose of misonidazole at 0.3 mg/g is not equivalent to the same dose given 3 weeks after chronic administration of the drug (4.5 g/m2/week, total administered dose 13.5 g/m’). In general, a 25% reduction in the misonidazole exposure dose for both serum and brain was observed after 3 weeks. This response may be significant when attempting to correlate the total dose administered (g/m’), total exposure dose (mM x hours) and a neurotoxicity endpoint in mice treated with multiple doses of misonidazole. We have reported that in mice, a single i.p. injection of 1 mg/g misonidazole produced a marked decrease in body temperature which persisted for at least two hours following the elimination of the drug from the serum.‘9 Hirst et al. have reported that a single dose of I mg/g misonidazole decreased the nerve conduction velocity of mice.12 This reduction could be correlated to be uptake and excretion profile of the drug in the blood serum. However our previous studies” have shown no significant decrease in nerve conduction velocity or other electrophysiological parameters following a single i.p. injection of misonidazole at the same dose. Since nerve conduction velocity is strongly dependent on temperature, and the study of Von Burg et aIt9 was conducted at a fixed temperature of 36.8OC, the two reports may not be contradictory. Therefore, the mouse may be able to accommodate to the toxic insult of misonidazole treatment by two mechanisms, a fall in body temperature and a gradual reduction of the size of the exposure dose with time probably resulting from the induction of enzymes. If other rodents or mammals do not show this effect, then it is likely that the drug effects at the same total dose would be more severe. The evidence available in the human studies involving misonidazole’,h,7.R,‘7 indicate that the compound gives a serum exposure dose of approximately 8.7 mM x hrs per
July 1979, Volume 5, Number 7
2.0 g/m2 dose. Since the established maximum tolerated dose is 12 g/m’ in 3 weeks or I5 g/m2 in 6 weeks irrespective of the dose fractionation, then we could reasonably expect a total misonidazole exposure dose of between 52 and 65 mM x hrs would express itself in terms of peripheral neuropathy. In the present study on the mouse, the morphologic and behavioral evidence indicates that following a total administered dose of 13.5 g/m2 at 3 weeks and a total exposure dose of between 57 to 75 mM x hrs, depending on how soon (O-3 weeks) the animal accommodates to the dose, peripheral nerve damage is seen with the suggestion of some central involvement. Thus, both the total administered dose and the total integrated exposure dose which produce neurotoxicity are similar at these dose levels in the mouse and human. Further studies are clearly required to define the kinetics and dose response relationship of misonidazole induced neurotoxicity in the mouse to clarify the usefulness of the neurological parameters identified in this study. ACKNOWLEDGEMENTS Misonidazole (Ro-07-0582, NSC #26 1037) was synthesized by National Cancer Institute Contractor and supplied by the National Institutes of Health, Bethesda, Maryland. Technical assistance of Mrs. Karen Jensen and the staff of the Experimental Pathology-Ultrastructure Research Facility and the American College of Radiology Service Facility, University of Rochester Cancer Center provided the photomicrographs and the initial histologic assessment of the material presented in the current report. Dan Henshaw, a summer student at the University of Rochester Cancer Center, assisted in the development of the functional tests of behavior in this study. Mrs. Laura Nelson typed the manuscript.
REFERENCES 1. Adams, G.E., Hypoxic
Fowler,
cell sensitizers
J.F.
and Wardman,
in radiobiology
P. (Eds.)
In:
and radiotherapy.
Proceedings of the 8th L.H. Gray Conference. Cambridge, England. Sept., 1977. Br. J. Cancer 37 (Suppl. III) 276,281,286,3 18, 1978. 2. Asquith, J.C., Watts, M.E., Patel, K., Smithen, C.W. and Adams, G.E.: Electron-affinic sensitization. V. Radiosensitization of hypoxic bacterial and mammalian cells in vitro by some nitroimidazoles and nitropyrazoles. Radiation Res. 60: 10881 18, 1974. 3. Basmajlan, J.V. and Stecko, G.: A new bipolar electrode for electrophysiology. J. Appl. Physiol. 17: 849-856, 1962. 4. Coxon, A. and Pallis, CA.: Metronidazole neuropathy. J. Neurol.
Neurosurg.
Physchiat.
39: 403-405,
1976.
5. Denekamp,
tumours
J. and Fowler, J.F.: Radiosensitization of solid by nitroimidazoles. Int. J. Radiation Oncol. Biol.
Phys. 4: 143-151, 6. Dische,
1978.
S., Saunders, M.I., Anderson, P., Urtasun, R.C., Karcher, K.H., Kogelnik, H.D., Bleehan, H.D., Bleehan, N., Phillips, T.L. and Wasserman, T.H.: The neurotoxicity of misonidazole. The pooling of data from 5 centers. Brit. J. Radiol. 51: 102331024, 1978. 7. Dische, S., Saunders, M.I., Flockhart, I.R., Lee, M.E. and Anderson, P.: Misonidazole, a drug for trial in radiotherapy
and oncology.
Int. J. Radiat.
Oncol. Biol. Phys.
(in press)
1979. 8. Dische,
S., Saunders, M.I., Lee, M.E., Adams, G.E. and I.R.: Clinical testing of the radiosensitizer Flockhart, Ro-07-0582: experience with multiple doses. Br. J. Cancer
35: 567-579, 1977. 9. Dunham, N.W. and
Miya, T.S.: A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Amer. Pharmacol. Assoc. 46: 208-209, 1957. IO. Edwards, P.M. and Parker, V.H.: Short Communication. A simple, sensitive, and objective method for early assessment of acrylamide neuropathy in rats. Toxicol. Appl. Pharmaco/. 40: 589-591,
1977.
I 1. Gray, A.J., Dische, S., Adams,
G.E., Flockhart, I.R. and Foster. J.L.: Clinical testing of the radiosensitizer Ro07-0582. I. Dose tolerance, serum and tumour concentration. Clin. Radiol. 27: 151-157, 1976. 12. Hirst, D.E., Vojnovic, B., Stratford, I.J. and Travis, E.L.: The effect of the radiosensitizer misonidazole on motor nerve conduction velocity in the mouse. Br. J. Cancer 37: (Suppl. III) 2733281, 1978. 13. LeQuesne, P.M.: Neuropathy due to drugs. In: Peripheral neuropathy. (Eds.) Dyke, P.J., Thomas, P.K. and Lambert, E.H. Philadelphia, Saunders, 1272-1273, 1975.
Misonidazole
neurotoxicity
14. Litchfield, J.T. and Wilcoxan, F.: A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96: 99%113,1949. 15. Spencer, P.S. and Schaumberg, H.H.: A review of acrylamide neurotoxicity, Part II. Experimental animal neurotoxicity and pathologic mechanisms. Can. J. Neurol. Sci. 1: 152-169, 1974. 16. Spurr, A.R.: A new low viscoscity epoxy resin embedding material for electron microscopy. J. Ultrastruct. Res. 26: 31-43, 1969. 17. Urtasun, R.C., Band, P.R., Chapman, J.D., Rabin, H., Wilson, A.F. and Freyer, C.G.: Clinical phase I study of the hypoxic cell radiosensitizer Ro-07-0582, a 2-nitroimidazole derivative. Radiology 122: 8OlL804, 1977.
in the mouse l
P. J. CONROY e-2al.
991
18. Urtasun, R.C., Chapman, J.D., Feldstein, M.L., Band, P.R., Rabin, H.R., Wilson, A.F., Marynowski, B., Starreveld, E. and Shnitka, T.: Peripheral neuropathy related to misonidazole: incidence and pathology. Br. J. Cancer 37: (Suppl. III) 271-275, 1978. 19. Von Burg, R., Conroy, P.J. and Passalacqua, W.: Peripheral electrophysiological parameters in mice treated with Ro-07-0582. Br. J. Cancer (in press) 1979. 20. Wasserman, T.H., Phillips, T.L., Johnson, R.J., Gomer, C.J., Lawrence, A.G., Sadee, W., Marques, R.A., Levitt, V.A. and Van Raalte, G.: Initial clinical and pharmacologic evaluation of misonidazole (Ro-07-0582), a hypoxic cell radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. (in press) 1979.
Press Inc.. 1979
Phys., Vol.
Printed
5, pp. 983.991
0360.3016/79/0701-0983/$02.00/O
in the U.S.A.
l Rapid Communication MISONIDAZOLE FUNCTIONAL,
NEUROTOXICITY IN THE MOUSE:* EVALUATION OF PHARMACOKINETIC, ELECTROPHYSIOLOGIC AND MORPHOLOGIC PARAMETERS
PETER J. CONROY,** Ph.D., RUDY VON BuRG,J~ Ph.D., PASSALACQUA, ** B.S. ROBERT
M.
DAVID P. PENNEY,??
SUTHERLAND,**
WILLIAM
Ph.D.,
Ph.D.
University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester,
NY 14642, U.S.A.
The neurotoxic effects of chronic administration of misonidazole (0.3 mg/g/day, 5 times weekly) were investigated in Balb/cKa mice over 12 weeks; a variety of measurements were used, including functional and clinical performance, morphologic, electrophysiologic and pharmacokinetic parameters. The half life of drug for a single dose was greater in brain (3 hrs) compared to serum (1.2 hrs); these values decreased to 1.9 hrs and 1.0 hrs respectively after 3 weeks. Misonidazole induced a peripheral lesion after three weeks with a total administered dose of 13.5 g/m* or exposure dose of 57 to 75 mM X hrs, which is similar to the doses that cause neuropathy in humans. There was some suggestion of a central neurological deficit related to locomotor control and balance; however, no gross morphological damage was found in the brain. The sequence of effects demonstrated began at 3-4 weeks and included: 1) morphologic damage to peripheral nerves; 2) hyperactivity and listlessness; 3) a decrease in rotarod retention time which reached a value 50% of that of saline injected control mice at 8-10 weeks; 4) walking on tiptoes with a slightly hunched back (4-6 weeks); and 5) an increase in hind foot splay (6-7 weeks). The morphologic damage primarily involved the more distal portions of the nerves supplying the interosseous muscles and footpads of the hind limbs. The damage was more severe and progressed more rapidly with time in these distal areas compared to the more proximal regions of the nerves. No marked changes were found in nerve conduction velocity although neuropathy produced by acrylamide produced significant decreases. The changes in neurological parameters reported here may be useful in the further evaluation of hypoxic cell radiosensitizers. Misonidazole,
Neurotoxicity,
Mouse, Electrophysiology,Pharmacokinetics.
INTRODUCTION
incidence of neuropathy to an acceptable level. Present clinical protocols have established a maximum tolerated dose of 12 g/m2 over 3 weeks or I5 g/m2 in 6 weeks, irrespective of the dose fractionation of misonidazole. 7.‘7.20It appears that patients who have received the greatest exposure doses (product of half life and serum plateau level of the drug) may be at greatest risk.7 Electron microscopy of a sural nerve biopsy from a 54 year old male patient who received 24 g of misonidazole (approximately 18 g/m’) revealed residual of previous distal axonal degeneration, with some segmental demyelination and remyelination.” R. Urtasun (written communication, October 1978) has also recently reported on the drug related death of an 85 year old male patient who had no evidence of disseminated disease other than local regional recurrence. This patient had received a total dose of 16 g in 8 fractions over 3 weeks (10.7 g/m’).
The nitroimidazole, misonidazole (Ro-07-0582), has been shown to selectively sensitize hypoxic mammalian cells in vitro’ and in viva’ to ionizing radiation at concentrations below those toxic for aerobic cells. Considerable interest has developed to assess the use of misonidazole in combination with radiation therapy in human solid tumors. Preliminary studies with misonidazole given in single doses of l-4 g to normal human volunteers did not indicate any toxic side effects.” However, several phase I studies have now reported contraindicative side effects such as convulsions at high doses and peripheral sensory neuropathy in cancer patients who received lower multiple doses of misonidazole.‘.6,8,‘7 The incidence and severity of peripheral neuropathy is related primarily to the total dose of misonidazole administered. In general, dose schedules have been revised downwards to reduce the
*This work was supported by National Institutes (NIH) Grants CA-11051, CA-11198, ES-01247, Contract I-NOl-CM87175. **Multimodalities Research ter Cancer Center.
Section,
University
of Health and NIH
tvisiting Scientist, Environmental Health Sciences. j’tuniversity of Rochester Cancer Center Experimental Pathology-Ultrastructure Research Facility. Accepted for publication 13 April 1979.
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The preliminary interpretation of brain sections taken at autopsy is that the findings are consistent with a probable toxic etiology of a demyelinating necrotic type. In man, a significant neurological deficit following misonidazole or other nitroaromatic compounds4,‘3 can be reported by the patient. At present no comparable satisfactory parameter has been identified in experimental animals in order to study mechanisms and to compare effects of different radiosensitizing agents. Since most radiobiological investigations of the effectiveness of hypoxic cell sensitizers have used tumor systems in mice, it was considered important to identify a satisfactory neurological parameter in the same species. It has been reported that the nerve conduction velocity (NCV) of mice was altered by administration of a single i.p. dose of I mg/gm misonidazole.‘2 However in a similar study we have shown no significant decrease in NCV or other electrophysiological parameters following a single dose of drug.19 The present study was initiated to determine the effect on the nervous system of chronic administration of misonidazole in mice using pharmacokinetic, clinical, functional, electrophysiologic and morphologic measurements. Acrylamide was used as a positive control in these studies since acrylamide neuropathy has been produced in all mammals studied by repetitive doses of the compound.‘5 During this investigation Griffin? (personal communication) in an independent study found morphologic damage and symptoms of neurological deficit following chronic administration of misonidazole in rats. After 3-4 weeks there was distal axonal degeneration of a Wallerian type associated with marked spasticity and abnormal righting reflexes. The overall aim of the present investigation was to identify neurological parameters for misonidazole toxicity in the mouse using an integrated approach. METHODS
AND MATERIALS
Six to eight week old female Balb/cKa mice (18-20 g)? and 7-9 week old male CBA/J mice (21-24 g) were used in these studies.+ The number of animals used in each experiment is indicated in the corresponding figure or figure legend. Misonidazofe toxicity: The LD,, for a single i.p dose of misonidazole in female Balb/cKa mice is 1.82 mg/g (range 1.7551.92) computed by the method of Litchfield and Wilcoxon.’ A marked short-term soporific effect was noted at all doses in excess of 0.3 mg/g. Misonidazole doses in excess of the LD,, value generally resulted in convulsions, clonic-tonic spasms and death within 2-4 hours. In a preliminary study, no significant weight changes were observed in mice that were treated 5 times weekly (total weekly dose 4.5 g/m2) for 2 weeks with 0.3 mg/g/day misonidazole (0.9 g/m2/day). This dose
tJ. Griffin, Dept. of Neurology, Johns Hopkins Medical School, Baltimore, Md., as reported at the NCI Radiation Sensitizer Workshop, Washington, D.C., Dec., 1978.
July 1979, Volume 5, Number
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regime was chosen since it would give a total administered dose of 18 g/m2 within 4 weeks. Acrylamide toxicity: The LD,, in rodents for a single dose of acrylamide is 0.17 mg/g regardless of the route of administration.15 Clinical symptoms of peripheral neuropathy are generally evident in 2 weeks within the dose range 0.01-0.05 mg/g/day. Animals that receive amounts of acrylamide smaller than 0.01 mg/g/day develop symptoms later which progress insidiously. We chose a dose of 0.01 mg/g administered by i.p. injection 3 times a week since preliminary data indicated that a severe deficit would occur within 334 weeks. Misonidazole pharmacokinetics: The half life and other pharmacokinetic parameters of misonidazole in serum, brain and liver of Balb/cKa mice were determined at the beginning and after 3 weeks of chronic administration of misonidazole (0.3 mg/g/day, total dose 13.5 g/m’). The method involved a spectrophotometric assay described previously.” The drug was dissolved in phosphate buffered saline (pH 7.4) at a concentration of 20 mg/ml (100 mM) and passed through a 0.45 P filter prior to use. It was injected intraperitoneally into recipient mice at a dose of 0.3 mg/g body weight (0.30 ml of drug solution per 20 g mouse). Blood samples (0.3 ml) were taken from groups of 3 or 6 mice by cardiac puncture following ether anaesthesia at 5,10,15,30 and 45 minutes and 1,2,4,8 and 24 hours after injection. 100 mg samples of brain and liver were also removed at the times given above. Control samples, taken from groups of mice that received buffered saline, were used as blanks in the spectrophotometric determinations. In general, the method can detect misonidazole equivalents down to a concentration of 2 yg/ml. Clinical observations and functional tests: In the group of 90 Balb/cKa mice receiving misonidazole, IO animals were selected for clinical observation and testing. The responses of these mice were compared to those of 10 saline treated control animals. A similar procedure was followed for the group of 45 CBA/J mice treated with acrylamide. The animals in these groups were weighed twice weekly and observed daily for gross clinical changes such as irritability or listlessness and defects in gait and coordination. In addition, two types of quantitative measurements were made to determine the degree of neurological disability of the mice treated with misonidazole or acrylamide. In general, each test was separated from the other by several hours and performed before the daily dose of drug. Locomotor coordination and balance (rotarod performance): The rotarod method was developed by Dunham and Miya’ and involves the ability of an animal to maintain the locomotor coordination and balance on a rotating rod. The test has the potential advantage of
IBiobreeding Laboratories, Ontario, $Jackson Laboratories, Bar Harbor,
Canada. Maine.
Misonidazole
neurotoxicity
in the mouse 0 P. J. CONROY ef al.
being easily quantifiable and objective, but it involves factors other than motor performance and coordination, notably memory. Rodents must be conditioned either by repeated exposure to the rotarod or by being punished for failure. In our experiments we employed a Splace rotarod? equipped with a variable speed motor$. The initial rotarod speed was set at 10 rpm thereafter increasing linearly over the next 3 minutes to a final speed of 40 rpm. Each mouse in the treatment protocol was exposed to the rotarod for a period of 5 days before the beginning of the experiment. At each experiment observation, the mouse was tested 3 times over a 2 hour period to allow rest periods. The mean retention time on the rotarod was recorded. Any animal that fell off the rotarod within 20 seconds three times in succession was arbitrarily scored as having zero retention time. This occurred only in the mice that were treated with acrylamide. Proprioceptive dejicit and hind limb splay (drop test): This was a modification of the method of Edwards and Parker” who observed that hind limb splaying occurred early in the course of acrylamide neuropathy in rats. This would be expected to be associated with sensory deficits of proprioceptors and with neuromuscular dysfunction. They found this method to be more sensitive than the measurement of gait parameters and the ability of rats to remain on an inclined slope. Each mouse was held in a horizontal position with the dorsal side uppermost and the tip of the nose 32 cm from a cushioned flat surface. Each foot was lightly marked with non-toxic food coloring; the mouse was dropped and the position of the fourth digit in each hind limb on landing was marked. The distance between the two marks was measured and the mean of 5 measurements was calculated. Mean control footspread was 4.22 k 0.11 cm (1 standard deviation from the mean). Electrophysiofogy: Calculations for nerve conduction velocity (NCV) were made directly from photographs of the oscilloscope trace and were based on the image of the oscilloscope graticule, the recorded time base setting and the msec delay between the onset of the stimulus artifact and the highest point of the observed waveform. Our estimate of the overall error from all sources in any one determination is approximately 10%. Sensory and motor NCV measurements in vitro: The procedure employed has been described previously.” These determinations were made after 0,2,3,4,8 and 12 weeks of misonidazole treatment and 0 and 3 weeks for acrylamide. Sensory NCV measurements in situ: The conduction velocity of the sural-sciatic nerve was determined by the latency difference in action potentials recorded at the level of the pelvic girdle by stimulation of the distal segments of the foot and a stimulus applied across the sural nerve just prior to its penetration into deeper nerves of the ankle. Dessication of the exposed tissues was IStoeleing
Corp.,
Chicago,
Illinois.
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prevented by a layer of mineral oil warmed to 37°C. Temperature of the preparation was controlled by the use of an infra-red heat lamp which held the ambient temperature of 37” f 1.5%. Nerve distances were estimated to the nearest 0.25 mm with the aide of screw adjustable dividers and a ruler. Measurements were made after 0,3,4,8 and 12 weeks of misonidazole dosing and 0 and 3 weeks for acrylamide. Motor NCV measurements in situ: Motor NCV measurements employed the same temperature and dessication controls as described above and dissection was limited to the thigh area and sciatic nerve. However, this determination employed a 3 pronged “J” stainless steel stimulating electrode. Distances between the anode and 1st cathode was approximately 1 mm. The 2nd cathode was located 6.1 * 1 .O mm from the first. Cathodal distance was measured by a micromanipulator micrometer. Muscle action potentials were recorded from the tarsal adductor muscles of the foot by means of implanted fine wire electrodes.3 Conduction velocities were calculated on the basis of the time delay between muscle action potentials evoked at the 2 different stimulus points and the distance between the stimulating electrodes. These measurements were made at the beginning and the end of the treatment protocols for misonidazole and acrylamide.
Morphologic
evaluation
All perfusion solutions were maintained at 37’ * 1°C. Perfusion pressure was controlled at 75-85 mm Hg with a manostat. Mice were pretreated with (a) 0.125 mg/g propranalol 15 minutes and (b) 200 units of heparin 5 minutes before administration of 0.1 mg/g of pentobarbital anesthetic. Following the induction of anesthesia, the animal was taped down on an inclined metal tray maintained at 38°-400C; the thoracic cavity was exposed and a polyethylene catheter terminating in a 20G needle was inserted into the left ventricle. After a small incision was made in the right auricle, the mouse was serially perfused with (a) 10 ml of phosphate buffered saline (2 ml/min) (b) 15 ml 50% strength Karnofsky’s solution (1 ml/min) and (c) 30 ml of 100% Karnofsky’s solution (1 ml/min). The perfused mouse was carefully skinned down to the level of the wrist and ankle and the entire fore and hind limbs were removed at their points of insertion into the pectoral and pelvic girdles respectively. The carcass was decapitated, the brain and spinal column removed and placed along with the limbs in full strength Karnofsky’s fixative for a further l-2 hours and then stored in 0.1 M phosphate buffer, pH 7.2. The anterior tibia1 and a 5 mm x 4 mm section of the gastrocnemius muscle at the level of the ankle were dissected free from the hind limb, the footpad carefully peeled back and the flexor and adductor digitorum $B. B. Motor Control
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N.Y.
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muscles exposed and removed into 0.1 M phosphate buffer, pH 7.2. The various nerves, together with their muscle attachments, were post-fixed in 1% 0~0, for one hour, dehydrated in increasing concentrations of ethanol and embedded in epoxy resin.lh Thick (0.551 .O pm) sections. cut on a microtome* were stained with toluidine blue for histologic observations. Thin sections (40-80 nm), stained with lead citrate and uranyl acetate, were observed and photographed using an electron microscope.? RESULTS Misonidazole pharmacokinetics: Measurements of drug serum concentration indicate a peak serum level (C,,,,,) of approximately 400 pg/ml (2 mM) was obtained 0.5 hours (T,,,,,) following a single i.p. dose of misonidazole at 0.3 mg/g with a half life (tr/l) of I .2 hours (Figure I, panel A). The total exposure dose, computed over a 24 hour period, was 5.0 mM x hours. After 3 weeks of dosing, the serum C,,,,, had declined to 300 pg/ml (1.5 mM) with a tr/~ of I .O hours. The corresponding exposure dose was 3.8 mM x hours, a reduction of approximately 25%. Brain misonidazole levels of approximately 160 pg/g (0.8 mM) were observed I .O hour after a single i.p. dose with a t’/x of 3.0 hours and an exposure dose equivalent to 4.2 mM x hours. The brain half life declined to 1.9 hours after 3 weeks of dosing with a corresponding reduction in the exposure dose to 3.1 mM x hours (approximately 25%). Measureable amounts of drug were present in brain tissue 24 hours later (Figure I, Panel B). The tr/l of misonidazole equivalents in liver declined from 2.4 hours for a single i.p. dose (10.8 mM x hours) to I .5 hours (6.5 mM x hours) after 3 weeks of dosing. The 25% reduction of the misonidazole exposure dose for brain tissue seen after 3 weeks of chronic administration reflects that (i) less drug is delivered to the tissue per unit time and (ii) the t$ in the brain has declined, presumably as a result of enzyme induction. Clinical observations and functional tests: No deaths or overall weight changes were observed in the misonidazole treated mice over the I2 week course of the experiment. However the control mice showed a weight increase of approximately 10% over this period. The soporific effect of misonidazolc declined from an initial value of 25 minutes to approximately IO minutes at I2 weeks. The misonidazole treated mice alternated between hyperactivity and listlessness after 3-4 weeks of dosing. This continued for the full I2 weeks but did not appear to progress. The mice began walking on tip-toes with a slightly hunched back after 446 weeks. This symptom also did not appear to progress after this time. Although no gross disturbances of gait or righting reflexes were observed in the misonidazole treated animals, their rotarod performance began to decline at 3
*LKB Ultratome
III.
B.
l.330
I
BRAIN
Fig. I. Semilog plot of serum (pg/ml, Panel A) and brain (&g, Panel B) concentrations with time (hours) in Balb/cKa mice following a single i.p. injection of misonidazole at 0.3 0, 6 mice per plotted point) compared to mg/g (0 animals receiving the same dose after chronic administration of the drug for 3 weeks (5 days/week, 0.3 mg/g/day) (0- . - . -0; 3 mice per plotted point). The error bars represent r I standard deviation of the mean.
weeks (total administered dose 13.5 g/m’) (Figure 2, upper panel). The fall in rotarod retention time became statistically significant at 4 weeks (p < 0.05, student t-test) and reached a value 50% lower than the control mice between weeks 8 and I2 (p < 0.001). In addition to peripheral neurological damage, (see Morphology section), this result may indicate a central lesion related
SZeiss IOA
Misonidazole
neurotoxicity
in the mouse
ROTARODPERFOWANCE
l
P. J. CONROY et al
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MISONIDAZOLE
40
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DROP TEST PERFORNANCE
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to control of locomotion and balance. The hind foot splay of the misonidazole treated animals did not begin to increase until weeks 6-7, it approached statistical significance at 8-10 weeks and became significant at lo-12 weeks (p < 0.05) (Figure 2, lower panel). In contrast, the acrylamide treated mice had suffered a 20-25% weight loss, had marked hind limb spasticity, muscle wasting and poor locomotor coordination by the second week. This group of mice failed completely on the rotarod at this time and could not be assessed on the drop test since the hind limbs remained crossed with the paws clenched. Electrophysiology: Figure 3 (upper panel) illustrates that no significant reduction in the conduction velocity of the sciatic-sural nerve measured in situ occurred in mice
-_ (6)
WEEKS OF DOSINS
Fig. 2. Upper Panel: Mean rotarod retention time (seconds) with time (weeks) in a group of IO Balb/cKa mice receiving 0.3 mg/g/day misonidazole (0 0) compared to a group of 10 mice receiving an equivalent volume of saline (0). Each mouse was observed 3 times within a 2 hour period at each determination. Lower Panel: Hind foot splay (drop test) performance of 10 misonidazole treated Balb/cKa mice (expressed as a % of that of 10 saline treated control animals) (Cl 0) with time (weeks). Each mouse was tested 5 times within a 2 hour period at each determination. The dashed lines represent the standard deviation of the mean of the pooled control data obtained over 12 weeks. The error bars represent t I standard deviation of the mean.
I
20
I 12
(6)
10
0
T 't!EEK
Fig. 3. Upper Panel: Sural-sciatic sensory conduction velocity (m/set) measured in situ in Balb/cKa mice treated with misonidazole for 3 or 12 weeks at 0.3 mg/g/day, 5 times weekly, total dose 4.5 g/m2/week). Error bars represent + 1 standard deviation of the mean. The number of animals in each determination appears in parentheses within the approximate bar. Note that we have chosen to show only the 2 extremes of the period over which measurements were taken with misonidazole. Other measurements taken at 4 and 8 weeks with 5-8 animals/time also showed no significant reduction in conduction velocity. Lower Panel: Sural-sciatic sensory conduction velocity (m/set) measured in situ in CBA/J mice treated with acrylamide for 3 weeks at 0.01 mg/g/3 times weekly, total dose 0.09 g/m2/week).
that were treated for 3 or 12 weeks with misonidazole. Values obtained at intermediate time points (see Methods) on 5-8 animals did not differ from the control values by more than 10%. The in situ measurements of motor nerve
conduction
velocity
after
12
weeks
of
dosing
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confirm this result. The data obtained for the in vitro measurement of NCV in isolated sural (sensory) and tibia1 (motor) nerves is in comparable agreement (data not shown). In addition we found no significant reduction in conduction velocity in isolated sural or tibia1 nerves taken from mice that were exposed to a total dose of 5 mg/g (15 g/m*) misonidazole administered over 10 days either as 0.5 mg/g/day or 1.O mg/g on alternate days. By contrast the acrylamide treated mice showed a significant reduction in sensory conduction velocity of approximately 35% (p < 0.001) after 3 weeks (lower panel, Figure 3). This reduction correlates with the functional and clinical observations outlined above. It may be argued that if misonidazole induced damage is confined to the most distal segment of the affected nerve, then we may not expect to see a reduction of overall conduction velocity until a late stage. We have attempted to test this possibility by measuring the sensory conduction velocity in situ of the most distal segment of the sensory nerves which supply the hind limb phalanges. We examined the difference between the time taken for an impulse to pass over the total length of the sciatic nerve from the stimulus point at the most distal segment of the foot to the point of entry of the nerve into the spinal cord and that for a stimulus point located at the level of the ankle. There were no significant differences in the conduction velocity of an impulse passing over the entire length of the nerve and that of its proximal or distal segments. The morphologic changes seen 3-9 weeks after misonidazole treatment in the sural and tibia1 nerves (see next section) indicate that the damage induced by misonidazole may not necessarily express itself in a reduction in the overall conduction velocity of a majority of the fibres. However, some reduction in the amplitude of the action potential could be expected at later times. Some of the measurements of NCV at 0.3 mg/gm/day of misonidazole showed a variable decrease in this parameter at late times (9912 weeks). Morphology: Misonidazole treated mice were taken for histologic and electron microscopic examination at 0,2,3,4,6,9,10, and 12 weeks. After 3 weeks there were demonstrable morphological changes. At this time, the more distal portions of the nerves supplying the hind limbs were primarily affected. The damage was more severe and progressed more rapidly with time in these areas compared to the more proximal regions of the sural and tibia1 nerves. After 6-9 weeks, the peripheral nerve components, particularly in the footpad and in flexor muscles were severely damaged. When compared to nerves from untreated control animals (Figure 4), the fine structural changes attributable to the radiosensitizer involved both the neuronal processes and the accompanying Schwann cells. The myelin sheath of the distal segments of the nerves supplying the interosseous muscles and the footpads of misonidazole treated mice exhibited an edematous sepa-
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Fig. 4. Adductor nerve from untreated control animal. The integrity of the Schwann cells, myelin sheaths, axoplasm and its organelles, unmyelinated fibers, and epineurium is maintained (x 15,400). ration of the lamellar leaflets by 3 weeks (Figure 5) which progressed with time so that at 9 weeks there were marked separations of the neuronal processes from the sheath (Figure 6). Myelin sheath degeneration, vacuolation and the inclusion of a finely granular component within the myelin lamellae tended to precede changes to axoplasmic organelles such as mitochondria where enlargement and loss of internal membranous cristae were observed by 9 weeks. No evidence of damage to axonal microfilaments was seen in the misonidazole treated mice. This differs from the response of rodents treated with acrylamide” where such changes were clearly evident at an early stage. In general, the morphologic changes seen in the neuronal processes following
Fig. 5. Adductor nerve from mouse treated with misonidazole for 3 weeks (0.3 mg/g/day, 5 days/wk). Leaflets of myelin sheath are distended, and Schwann cell appears vaeuolated (x 13,900).
Misonidazole
neurotoxicity
in the mouse
Fig. 6. Flexor nerve from mouse treated with misonidazole for 9 weeks. The myelin sheath exhibits granularity and focal separations. The mitochondria of the Schwann cell and neuron were distended and exhibited a loss of internal membranous cristae. In addition to cytoplasmic vacuolation, the Schwann cell endoplasmic reticulum was also distended (x 13,900).
misonidazole development cells. At
3 weeks,
administration were less rapid in their than those in the surrounding Schwann the
Schwann
cell
cytoplasm
exhibited
swelling and dilatation of the endoplasmic (Figure 5). Progressive vacuolation and the internal structure of cytoplasmic organelles at 6 weeks and became marked by 9 weeks. In Schwann cell nuclear heterochromatin had more prominent (Figure 6), suggestive of pykno-
mitochondrial
reticulum loss of occurred addition, become sis. Histologic observations of several nerves from misonidazole treated mice revealed no marked endoneurial or perineurial edema. At the later stages (6-9 weeks) the morphological changes described above using the electron microscope were observable histologically at the light microscope level. Morphologic evaluation of the brain tissue of misonidazole treated mice is in progress. At the dose level employed in this study (0.3 mg/g/day, 4.5 g/m2/week) we have observed no gross lesions in the brain at the histological level at 3 or 9 weeks which would correlate with the central lesion implied by the decline in the rotarod performance of the misonidazole treated mice which began at 3-4 weeks. More detailed studies at the electron microscope level of brain tissue taken at other times at this and higher doses are required and are presently underway. DISCUSSION
The morphological evidence obtained in the present study indicates that chronic administration of misonidazole (0.3 mg/g/day, 5 times weekly) in mice results in a *J. Griffin, Dept. of Neurology, Johns Hopkins Medical School, Baltimore, Md., as reported at the NC1 Radiation
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peripheral neurological lesion after 3 weeks (total admmistered dose 13.5 g/m*). At this time, the more distal portions of the nerves supplying the interosseous muscles and footpads of the hind limbs are primarily affected. The misonidazole induced damage is more severe and progresses more rapidly with time in these distal areas compared to the more proximal regions of the sural and tibia1 nerves. Histological evidence at the light microscope level of the peripheral morphological changes was apparent after 6-9 weeks of drug administration (total dose 27-40.5 g/m2 misonidazole). In the present study we have not been able to demonstrate any marked change in nerve conduction velocity indicating that at the dose level employed it did not predict or indicate any significant neurological deficit induced by misonidazole. The overall evaluation of the effects of chronic administration of misonidazole at this dose level (0.3 mg/g/day) is that of a mixed peripheral and central neurological deficit. There was no gross morphological damage in the brain but further histological investigation at this and other doses and schedules is required since some of the data is consistent with central involvement related to locomotor control and balance. Such information is also necessary in order to obtain dose-response relationships for the effects demonstrated. Griffin* (personal communication) has evaluated morphological changes and clinical symptoms in the rat treated with misonidazole at 0.3 mg/g/day (5 times weekly). He noted endoneurial edema and a marked loss of sensory endings on muscle spindle cells in the peripherary of the misonidazole treated rats within 2-3 weeks. Peripheral denervation, endoneurial edema, and distal axonal degeneration were observed in the nerves supplying the interosseous muscles of the foot. Although we have not observed any endoneurial edema and our examination at the EM level of the sensory endings on muscle spindle cells are not extensive enough to confirm those observations, the morphologic evidence of drug induced damage in the distal areas of the hind limbs indicate that the effects are, in general, very similar in both the mouse and the rat. The clinical symptoms of hyperactivity and listlessness and walking on tip toes following 2-3 weeks of misonidazole were also similar in both studies. However, rats begin to show a deficit in righting reflexes after 3-4 weeks of misonidazole administration at 0.3 mg/g/day and died if the drug treatment continued beyond this point. We did not observe such a gross deficit in our study in the mouse, nor did we see any marked weight losses or deaths in the misonidazole treated mice for up to 12 weeks. This shows that at this dose level, the mouse responds in some respects differently from the rat to chronic administration of misonidazole. The pharmacokinetic data obtained in the present Sensitizer
Workshop,
Washington,
D.C., Dec., 1978.
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study indicates that the first dose of misonidazole at 0.3 mg/g is not equivalent to the same dose given 3 weeks after chronic administration of the drug (4.5 g/m2/week, total administered dose 13.5 g/m’). In general, a 25% reduction in the misonidazole exposure dose for both serum and brain was observed after 3 weeks. This response may be significant when attempting to correlate the total dose administered (g/m’), total exposure dose (mM x hours) and a neurotoxicity endpoint in mice treated with multiple doses of misonidazole. We have reported that in mice, a single i.p. injection of 1 mg/g misonidazole produced a marked decrease in body temperature which persisted for at least two hours following the elimination of the drug from the serum.‘9 Hirst et al. have reported that a single dose of I mg/g misonidazole decreased the nerve conduction velocity of mice.12 This reduction could be correlated to be uptake and excretion profile of the drug in the blood serum. However our previous studies” have shown no significant decrease in nerve conduction velocity or other electrophysiological parameters following a single i.p. injection of misonidazole at the same dose. Since nerve conduction velocity is strongly dependent on temperature, and the study of Von Burg et aIt9 was conducted at a fixed temperature of 36.8OC, the two reports may not be contradictory. Therefore, the mouse may be able to accommodate to the toxic insult of misonidazole treatment by two mechanisms, a fall in body temperature and a gradual reduction of the size of the exposure dose with time probably resulting from the induction of enzymes. If other rodents or mammals do not show this effect, then it is likely that the drug effects at the same total dose would be more severe. The evidence available in the human studies involving misonidazole’,h,7.R,‘7 indicate that the compound gives a serum exposure dose of approximately 8.7 mM x hrs per
July 1979, Volume 5, Number 7
2.0 g/m2 dose. Since the established maximum tolerated dose is 12 g/m’ in 3 weeks or I5 g/m2 in 6 weeks irrespective of the dose fractionation, then we could reasonably expect a total misonidazole exposure dose of between 52 and 65 mM x hrs would express itself in terms of peripheral neuropathy. In the present study on the mouse, the morphologic and behavioral evidence indicates that following a total administered dose of 13.5 g/m2 at 3 weeks and a total exposure dose of between 57 to 75 mM x hrs, depending on how soon (O-3 weeks) the animal accommodates to the dose, peripheral nerve damage is seen with the suggestion of some central involvement. Thus, both the total administered dose and the total integrated exposure dose which produce neurotoxicity are similar at these dose levels in the mouse and human. Further studies are clearly required to define the kinetics and dose response relationship of misonidazole induced neurotoxicity in the mouse to clarify the usefulness of the neurological parameters identified in this study. ACKNOWLEDGEMENTS Misonidazole (Ro-07-0582, NSC #26 1037) was synthesized by National Cancer Institute Contractor and supplied by the National Institutes of Health, Bethesda, Maryland. Technical assistance of Mrs. Karen Jensen and the staff of the Experimental Pathology-Ultrastructure Research Facility and the American College of Radiology Service Facility, University of Rochester Cancer Center provided the photomicrographs and the initial histologic assessment of the material presented in the current report. Dan Henshaw, a summer student at the University of Rochester Cancer Center, assisted in the development of the functional tests of behavior in this study. Mrs. Laura Nelson typed the manuscript.
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Proceedings of the 8th L.H. Gray Conference. Cambridge, England. Sept., 1977. Br. J. Cancer 37 (Suppl. III) 276,281,286,3 18, 1978. 2. Asquith, J.C., Watts, M.E., Patel, K., Smithen, C.W. and Adams, G.E.: Electron-affinic sensitization. V. Radiosensitization of hypoxic bacterial and mammalian cells in vitro by some nitroimidazoles and nitropyrazoles. Radiation Res. 60: 10881 18, 1974. 3. Basmajlan, J.V. and Stecko, G.: A new bipolar electrode for electrophysiology. J. Appl. Physiol. 17: 849-856, 1962. 4. Coxon, A. and Pallis, CA.: Metronidazole neuropathy. J. Neurol.
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Miya, T.S.: A note on a simple apparatus for detecting neurological deficit in rats and mice. J. Amer. Pharmacol. Assoc. 46: 208-209, 1957. IO. Edwards, P.M. and Parker, V.H.: Short Communication. A simple, sensitive, and objective method for early assessment of acrylamide neuropathy in rats. Toxicol. Appl. Pharmaco/. 40: 589-591,
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G.E., Flockhart, I.R. and Foster. J.L.: Clinical testing of the radiosensitizer Ro07-0582. I. Dose tolerance, serum and tumour concentration. Clin. Radiol. 27: 151-157, 1976. 12. Hirst, D.E., Vojnovic, B., Stratford, I.J. and Travis, E.L.: The effect of the radiosensitizer misonidazole on motor nerve conduction velocity in the mouse. Br. J. Cancer 37: (Suppl. III) 2733281, 1978. 13. LeQuesne, P.M.: Neuropathy due to drugs. In: Peripheral neuropathy. (Eds.) Dyke, P.J., Thomas, P.K. and Lambert, E.H. Philadelphia, Saunders, 1272-1273, 1975.
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14. Litchfield, J.T. and Wilcoxan, F.: A simplified method of evaluating dose-effect experiments. J. Pharm. Exp. Ther. 96: 99%113,1949. 15. Spencer, P.S. and Schaumberg, H.H.: A review of acrylamide neurotoxicity, Part II. Experimental animal neurotoxicity and pathologic mechanisms. Can. J. Neurol. Sci. 1: 152-169, 1974. 16. Spurr, A.R.: A new low viscoscity epoxy resin embedding material for electron microscopy. J. Ultrastruct. Res. 26: 31-43, 1969. 17. Urtasun, R.C., Band, P.R., Chapman, J.D., Rabin, H., Wilson, A.F. and Freyer, C.G.: Clinical phase I study of the hypoxic cell radiosensitizer Ro-07-0582, a 2-nitroimidazole derivative. Radiology 122: 8OlL804, 1977.
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18. Urtasun, R.C., Chapman, J.D., Feldstein, M.L., Band, P.R., Rabin, H.R., Wilson, A.F., Marynowski, B., Starreveld, E. and Shnitka, T.: Peripheral neuropathy related to misonidazole: incidence and pathology. Br. J. Cancer 37: (Suppl. III) 271-275, 1978. 19. Von Burg, R., Conroy, P.J. and Passalacqua, W.: Peripheral electrophysiological parameters in mice treated with Ro-07-0582. Br. J. Cancer (in press) 1979. 20. Wasserman, T.H., Phillips, T.L., Johnson, R.J., Gomer, C.J., Lawrence, A.G., Sadee, W., Marques, R.A., Levitt, V.A. and Van Raalte, G.: Initial clinical and pharmacologic evaluation of misonidazole (Ro-07-0582), a hypoxic cell radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. (in press) 1979.