ORIGINAL ARTICLES

Ahsonidazole Newopathy: A Chcal, Electrophysiological, and IEbstological Study

B. Melgaard, MD,* H. Sand Hansen, MD,$ 2. Kamieniecka, MDJ 0. B. Paulson, MD," A. Gersel Pedersen, MD,5 Xiaofu Tang, MD,j-$lI and W. Trojaborg, MDj-

~ ~

We studied eight patients with carcinoma of the pharynx and larynx (five cases) or lungs (three cases) who, during treatment with the radiosensitizing drug misonidazole, developed peripheral neuropathy dominated by severe sen- sory symptoms and signs mainly localized to the lower extremities. The symptoms partially subsided within months after cessation of therapy. Electrophysiological and histological findings indicated an axonal neuropathy with loss of large fibers and secondary demyelination. The neurotoxic property of misonidazole limits its therapeutic use.

Melgaard B, Hansen HS, Kamieniecka Z , Paulson OB, Pedersen AG, Tang X, Trojaborg W: Misonidazole neuropathy: a clinical, electrophysiological, and histological study. Ann Neurol 12: 10-17, 1982

Misonidazole is a 2-nitroimidazole which has given promising results as an adjuvant to radiotherapy. Clinical testing of this compound as well as of the related compound metronidazole in humans has re- vealed troublesome neurotoxic side effects: periph- eral neuropathy as well as encephalopathies have been reported [6, 12, 15, 23-25, 28, 291. As there are few detailed investigations 125, 28, 291, we report eight patients in whom misonidazole treat- ment provoked a severe subacute sensory neurop- athy and present the clinical, neurophysiological, and morphological findings.

Material and Methods Five men and three women aged 54 to 72 years, all treated with misonidazole, were examined because of symptoms of polyneuropathy. All had previously been well. Two pa- tients had consumed 80 to 160 gm of alcohol daily for sev- eral years without social or physical complications, and in particular had never experienced symptoms of peripheral neuropathy. Otherwise, apart from the malignant disease, no factors contributory to the development of a peripheral neuropathy were known.

Five patients (Nos. 1, 2, 3, 5 , and 8) were referred for radiation treatment because of carcinoma of the pharynx or larynx. Three patients had inoperable squamous cell car- cinoma of the lung. No remote secondary growth was demonstrated in any case. The patients received miso- nidazole as an adjuvant to radiotherapy for three to five weeks according to the schedule given in Table 1.

PLASMA MISONIDAZOLE. The plasma peak concentration and half-life were measured from heparinized blood sam- ples taken 4 , 8, and 24 hours after the first dose of misonidazole. Plasma concentrations were determined by reverse-phase high-performance liquid chromatography using a modification of the method described by Workman et a1 [30].

ELECTROPHYSIOLOGICAL STUDIES. Electrodiagnostic tests were performed according to the usual routine of the Laboratory of Clinical Neurophysiology [7-91. The an- terior tibial muscle was examined electromyographically in all patients, and conduction studies were performed along median, peroneal, tibial, and sural nerves.

HISTOLOGICAL STUDIES. Sural nerve biopsy was per- formed in three patients (Nos. 5 , 7, and 8). About 30 mm of the sural nerve was resected in toto at the level of the lateral malleolus under local anesthesia. The samples were fixed in 2% buffered glutaraldehyde, postfixed in 1% os- mium tetroxide, dehydrated, and embedded in Epon-812 [ I , 31. Cross sections 3 pm thick were stained with 1% paraphenylenediamine.

The transverse endoneurial area was measured on mi- crographs. The total number of myelinated fibers, their size distribution, and a count of "clusters" (three or more closely packed regenerating rnyelinated :,.,en) were de- termined from a sampled area of all fascicics [3 ,4] . Eighty to 100 fibers from each sural nerve were isolated in 60% glycerol over a length of about 10 mm. In 20 to 50 fibers, the internode lengths were measured on micrographs and plotted against the diameter of the longest internodal seg-

From the *Department of Neurology and the thboratory of Clinical Neurophysiology, Rigshospitalet, the ZInscitute of publication Sept 21, 1981.

Received July 15, 1981, and in revised form Sept 14. Accepted for

Neurophysiology, University of Copenhagen, and Che seninstituttet, Copenhagen, Denmark, and the "Department of Neurology, Capital Hospital, Peking, China.

Address reprint requests to Dr Trojaborg, Laboratory of Clinical Neurophysioiogy, Rigshospitalet, Blegdmsvej 9, DK 2 100 Copenhagen 0, Denmark.

10 0364-5 134/82/070010-08$01.25 @ 1081 by the American Neurological Association

Table 1 . Schedule of Misonidazole Treatment and Plasma Concentration in Eight Patients with Neuropathy

Dose per Fraction Total Dose of Period of Peak Plasma Plasma Patient Misonidazole Treatment No. of Total Fraction Concentration Half-Life No. (gm) (days) Fractions (mg) (mdm’) (Pdml) (hr) 1 18.7 22 7 2,700 1,377 42.3 10.5 2 18.0 32 20 700 5 08 20.6 7.7 3 22.0 32 20 1,100 608 . . . . . . 4 17.0 33 10 1,700 1,200 68.3 . . .

6 22.0 32 10 2,200 1,220 75.6 . . . 7 22.0 34 10 2,200 1,220 77.4 . . . 8 20.8 24 8 2.600 1.368 52.6 11.0

5 20.0 2 5 8 2,500 1,377 51.7 11.7

ment [ 171. In addition, we determined the number of fibers with (1) abnormal myelin sheaths (swelling, fragmentation, balls and ovoids), (2) paranodal demyelination, (3) solitary intercalated internodes, and (4 ) remyelinated segments (two or more short segments between segments of normal length) and shorter internodes than in normal fibers of similar diameter (regenerating fibers [2]).

In Patient 5, a biopsy was obtained from the anterior tibia1 muscle. Fresh-frozen sections were stained with hematoxylin and eosin, modified van Gieson [16], and modified Gomori trichrome [ 181 and for various enzymes. The fibers were classified as types 1, 2a, and 2b by their reactions with adenosine triphosphatases at pH 7.4 and 4.6 [171.

Results Clinical Findings At the end of the misonidazole treatment or shortly thereafter, all eight patients complained of painful paresthesias first occurring in the feet. All later de- veloped paresthesias in the fingers, whereas the sen- sory disturbances remained confined to the feet in one patient. Paresthesias were moderate to severe in six patients and mild in two. Four patients had severe difficulty walking because of pain and loss of deep

sensation. Within one to two weeks the symptoms were accompanied by slightly reduced strength in the feet in two patients; the remaining six had no motor symptoms.

Clinical examination showed that cranial nerves were normal in all patients. Two patients had slight muscle weakness without atrophy distally in the legs. Tendon reflexes were preserved in all patients. Touch and pain sensation was severely impaired in the lower extremities of six patients. In the hands it was markedly impaired in four, slightly so in two, and normal in the two remaining patients. Vibration sen- sation in the feet was impaired in six patients and normal in two. Position sensation was impaired in the feet in all but one patient. In the hands, vibration and position sensation was impaired in one patient. Thus, three patients (Nos. 1, 3, and 8) had a severe sensory polyneuropathy, four (Patients 2, 5 , 6, and 7) a moderate, and one (Patient 4 ) a mild sensory polyneuropathy (Table 2).

Three to five months later three patients were dead. Of the remaining five, one was unchanged and recovery or improvement had occurred in four, though symptoms and signs were still severe in Pa-

Table 2. Clinical Features of Patients with Misonidazole Neuropatby

Impairment of:a Patient No., Age (yr), Site of Painful Vibra- Posi- Tendon and Sex Carcinoma Paresthesias” Weaknessa Touch tion tion Pain Reflexes

1. 53, M Pharynx + +/+ +/N ++/+ +/N +/N ++/+ N 2. 53, M Pharynx + +/+ NIN + /N N +/N ++/+ N 3. 58, M Larynx + +/+ N/N ++I+ +/N ++/N ++/+ N 4. 63, F Lung + +/+ N/N +/N N N +/N N 5 . 65, M Pharynx + +/+ N/N ++/+ +/N +/N +/+ N 6. 67, M Lung +/+ N/N ++/+ +/N +/N ++/+ N

8. 71, F Larynx + +/+ +/N ++ ++/+ ++/+ +/+ N 7. 71, F Lung +I+ N/N ++/+ +/N +/N ++IN N

aFindings are given for lower extremitieshpper extremities. N = normal; + = mildly affected; ++ = moderate to severe abnormalities

Melgaard et al: Misonidazole Neuropathy 11

Table 3. Quantitated Electroniyogruphic Findings in Tibiulis Anterior Muscle

Patient No. ~

Fi n d ing 1 2 3 4 5 6 7 8

Pattern at full effort R N R N N R R N Amplitude at full effort N N N N N R N N Sites with denervation 0 0 7 0 0 9 0 0

Mean M U P duration N N N N N N N N Mean M U P amplitude (% increased) 366 118 119 75 190 79 220 173 Polyphasic 14 20 32 15 19 28 25 6

No of MUPs measured 28 35 22 26 36 29 36 35

potentialsa

potentials (%)

aFibrillation potentials, positive sharp waves, or both N = normal, R = reduced, MUP = motor unit potential

Table 4 Conh i t ion Veloiitiri and Atnplztudt of Eiokei i Patentiali 111 tbr Lfediati and Sural r' leiiri

Median Nerve Sural

Sensory Motor Nerve

Wrist-Abd. Lat. Digit I-Wrist Digit IIILWrisr Poll. Br. Malleolus-Sura

Patient cv Ampl CV Amp1 Latency Ampl CV Ampl No. (rniscc- ) ( p V ) (m/sec) (FV) (mseci ( m V ) (m/sec) (PV)

1 44 1.5 45 0.3 4.0 2 0 47 0.3 2 44 10.0 48 10.0 4.0 11 48 L. 1 7 I

3 49 16.0 51 4.5 3.2 16 43 0.7 4 51 60.0 57 15.0 3.3 11 51 2 .2 5 49 10.0 54 4.0 3.8 2 2 5 5 0.25 6 48 3.0 53 1 .0 3.8 1 5 43 0.12 7 5 2 20.0 54 14.0 3.8 16 48 0.25 8 49 3 1.0 56 10.0 3.4 14 56 5.0

Normal values 12'1 5 0 23.0 56 10.0 3.1 14 53 9.0

959; lower limir 41 10.0 47 4.0 3.8 6 46 3.5 (52-72 yr)

Abd. poll. br. = abductor pollicis brevis muscle; CV = conduction velocity; Ampl = amplitude.

tient 8. All patients still complained of paresthesias, particularly in the feet, but these were less painful than tbrmerl y. Clinical examination revealed that sensory impairment was less pronounced and was confined to the feet in three patients (Nos. 3, 6, and 7) but included the hands in one. One patient was not reexamined. Tendon reflexes were still preserved in all patients.

Electropbysiological Findings The force of the anterior tibia1 muscle was normal on electromyographic testing, and atrophy was not con- spicuous. Half of the patients had a reduced recruit- ment pattern of the electrical muscle activity at full effort, the amplitude being reduced in only one pa- tient (Table 3 ) . Two of the muscles with reduced re- cruitment pattern also showed abundant fibrillation

potentials and positive sharp waves (Patients 3 and 6). The mean duration of motor unit potentials was normal, whereas the mean amplitude was increased more than 100% in all but two patients (Nos. 4 and 6). There was also an increased incidence of polyphasic potentials (14 to 32%) in all but one (Pa- tient 8), the upper limit in normal subjects being 12% [lo].

The sensory conduction velocity along the distal part of the median nerve was normal in all but Patient 1 (Table 4). In contrast, the amplitude of the sensory action potential evoked by stimulation of the first and third digits was severely reduced in two patients (Nos. 1 and 6) and at the lower limit of normal in two others (Patients 2 and 5 ) . The latency to the abductor pollicis brevis muscle was increased in Patients 1 and 2, borderline in Patients 5 to 7 , and normal in the

12 Annals of Neurology Vol 12 No 1 July 1982

Table 5 . Conduction Velocities and Amplitude of Evoked Potentials in the Peroneal Nerve

Motor

Sensory Extensor Dig. Brevis Tibialis Ant. Peroneus Longus

Patient cv Ampl CV Latency Ampl Latency Ampl Latency Amp1 No. (misec) (yv) (dsec) (msec) (mV) (msec) (mV) (msec) (mV)

1 . . . 38 5.9 5.4 2 47 1.0 Abolished 4.4 5.0 4.0 6.0 3 . . . 4 54 0.8 50 5.0 6.0 3.0 9.0 2.8 15.0 5 43 0.9 40 5.4 10.0 4.2 11.0 3.5 7.5 6 40 0.25 39 6.4 4.0 . . . . . . . . . . . . 7 56 0.4 44 5.6 6.6 3.6 6.0 2.8 8.4 8 53 0.4 43 4.8 2.7 3.6 3.3 3.3 3.0 Normal values [27] 54 3.5 50 4.1 10.0 3.2 15.0 3.2 15.0

95% lower limit 47 1.2 43 5.0 3.0 3.9 6.0 3.9 6.0

. . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . .

(52-72 yr)

CV = conduction velocity; Amp1 = amplitude.

other three patients. The amplitude of the motor re- sponse was normal in all. The conduction velocity along the sural nerve was normal in all but two pa- tients (Nos. 3 and 6), but the amplitude of the sen- sory potential was reduced 70 to 979% in all but one (Patient 8) compared to the normal mean value for their respective ages.

The sural nerve was examined immediately before the onset of misonidazole treatment in Patient 4. The amplitude of the sensory potential then was 17 pV, compared to 2.2 pV eleven weeks later when she had developed symptoms and signs of neuropathy.

Motor conduction velocity along the peroneal nerve was determined in seven patients and was mildly slowed in three (Patients 1, 5 , and 6), while no potential could be obtained in one (Patient 2). The distal latency was slightly increased in Patients 1 ,2 , 5 , 6, and 7: to the extensor digitorum brevis muscle in four of them, the tibialis anterior in two, and the peroneus longus in one. The sensory conduction ve- locity along the superficial peroneal nerve was deter- mined in six patients and was slightly decreased in two (Patients 5 and 6). The amplitude of the sensory action potential recorded at the capitulum fibulae was reduced in all six tested (Table 5) .

The tibial nerve was examined in two patients (Nos. 1 and 3). The sensory conduction velocity was normal in Patient 1, but the amplitude of the sensory potential evoked by stimulation of the big toe was only one-fifth of the normal value. In Patient 3, a sen- sory potential could not be discriminated from noise and the patient was unable to recognize a stimulus of 50 mA applied to the big toe. The distal latency to the flexor hallucis muscle was slightly increased in Patient 1 and normal in Patient 3.

In summary, electromyography revealed changes

in two or more motor unit variables in all but two patients (Nos. 4 and 8), compatible with mild loss of axons accompanied by peripheral sprouting. Con- duction studies revealed evidence suggestive of axonal damage, more pronounced in the lower than in the upper limbs and affecting sensory more than motor fibers, in all patients but least so in Patients 4 and 8.

Biopsy Findings Nerve biopsies in cross section showed similar findings in the three patients studied, the nerve from Patient 7 being most severely affected and that from Patient 8 least so. The total number of myelinated fibers was diminished (32 to 83%), and there was a high occurrence of myelin debris and clumps of dis- organized myelin. The number of large-caliber fibers (>7 pm) was especially diminished (Figs 1, 2), whereas the number of fibers less than 3.5 prn in diameter was increased (20 to 3396, compared with 129% in normal nerve). The incidence of clusters of regenerating myelinated fibers was increased, espe- cially in Patient 8.

A high incidence of abnormalities was observed among teased fibers (Table 6, Figs 3, 4) , the most conspicuous being fragmentation of myelin into balls and ovoids (Fig 4 ) . Regenerated fibers and remy- elinated segments occurred in all three nerves and intercalated fibers in two (Patients 5 and 8), but none of these findings exceeded the upper limit of normal. Paranodal demyelination over a length of up to 300 pm was seen in all nerves.

In muscle biopsies, atrophic fibers were seen as a small group surrounded by fibers rather large in diameter (70 to 75 pm). Twenty percent of the fibers contained internal nuclei, and “splitting complexes”

Melgaard et al: Misonidazole Neuropathy 13

NUMBER OF FIBRES

tJm

3500-

X X

3000- s(

2500- xx

.-x--

X

2000-

X

1500-

1000-

500-

P 0-

10000

9000 1: 8000 ; 70 oo-- -x - -

YXX

6000-

5000- x x

L000-

3000-

2000-

0

8

1000- A

0-

7 000-

6000-

5000-

cooo-

3000-

2000-

1 000-

tJm

X

X --x--

; 0;

8

A

0-

80

6 0

L O

2 0

0

0

A 8

X

X X

xx

I(X)

--.

P F i g 2. Number of m.yelinatedjihevs (a1ljibers;jibers smaller than 7 pm in diameter: undfi'bevs largeif than 7 pm in diameter) and of clusters of regeneratingjibers. Data shown for Patient 7 (W), Patient 8 (O) , Patient 5 (A), and 10 c0ntrol.r ( x ). Dushed lines denote mean 5alues. (Data for controls are from Behse et al [.3 I.)

were numerous. Necrotic fibers and inflammatory infiltrates were absent. Connective tissue was not in- creased and blood vessels appeared normal. His- tochemical preparation showed type grouping of typr 1 fibers and smaller clusters of type 2 fibers in some sections, while others had a normal pattern of fiber types.

Discussion The present findings illustrate the neurotoxic effect of the radiosensitizer misonidazole. All our patients developed signs and symptoms of a mainly sensory neuropathy, similar to that previously reported [ 15, 25, 28, 291. The close relationship between the de- velopment of symptoms after misonidazole therapy,

C F i g 1. Myelinatedjihers in sural newe biopsies of Patient 7 iA), Patient 8 iB) , and Patient 5 (C). (Left) Cross section of each sural nerve show niyelin debris (A, C) and decreased numbers of ni.yelinatedjibers. (Right) Distribution ofjiber diameters (dashed line indicates distribution in normal sural neives according t o data of Behse et ul [? 1).

Table 6. Patholo~ii-ul FindrnR.\ in Teaed Fiber.,

Abnormal Myelin Sheath Paranodal Solitary

Fragmen- Balls and Regener- Remyelin- Demyelin- Intercalated Normal Total Swelling IdtlOn Ovoids atiun atiun ation Internodes Fibers

No. Ci No. (7 No. (7 No. '$ No. 57 Patient No. o f N o Fibers '; NO " i No. ci

5 86 it 4.8 5 5.8 30 34.9 4 4.8 10 11.6 15 17.4 2 2.3 22 25.6

8 09 1 1 22 22.2 26 26.2 14 14.1 14 14.1 5 5 1 I 18 18.2 82 8 9 8 10 16.6 26 31.' 12 14.6 6 7.3 4 4 .9 0 0 0 0

14 Annals of Neurology Vol 12 No 1 July 1982

% 100

LO

20

NORM.FIBRE!

Q% 100

80

2 0

ABN.t+ ZLI N

% % 50

1 0

O x x x

SEGM.DEMYEL

5 0

L O

1 0

O x x x

PARANOD.

0

RE MY EL.SE GM.

F i g 3 . Incidence of abnormalities in teasedfibers of each sural nerve compared u i t h control nerves from seven normal subjects. (Data for controls are from Behse et al 13 1. Symbols are as in Figure 2.)

I

F i g 4. Sural nerue biopsy from Patient 8 showing four con- secutice segnzentj. o f two reuJedjibei*.r: one,hberJ 9 p m thick, with normal internode lengths, the other with abnormal myeliri sheath (fragmentations, balls and ovoids). The arrows mavk nodes of Ranvier.

particularly the reduction in amplitude of the sensory potential after treatment in Patient 4, suggests that misonidazole was responsible for the neuropathy. The occurrence and severity of the disorder appear to be related to both the total dose and the fraction- ing of individual doses.

Electrophysiologically, the neuropathy was clas- sified as a primary axonal degeneration since the amplitudes of sensory action potentials were severely reduced and sensory conduction velocities were normal or only mildly decreased. The mild slowing of conduction along motor fibers distally could be ac- counted for by assuming loss of large-diameter fibers. It might more likely be attributed to a reversible de- fect of membrane function analogous to findings in animal experiments. In these, misonidazole treat- ment results in a significant reduction of motor con- duction velocity, which, however, returns to normal after withdrawal [2 11. The preservation of tendon re- flexes in all patients who had only minor motor symptoms or none suggests sparing of the afferent limb of the reflex arch.

The only available morphological report on a human case of misonidazole neuropathy has shown axonal degeneration and some segmental demyelina- tion [29]. The histological findings in our three sural biopsies are compatible with an axonal neuropathy affecting primarily large fibers.

In some cases of toxic neuropathies the toxic sub- stance acts mainly on the perikaryon [ 5 , 111, while in others it acts directly on the axon; in both circum- stances it produces an axonal degeneration. Similarly, we found wallerian-like degeneration in teased fibers. It is possible that misonidazole also affects sensory ganglia and that the morphological changes are the combined result of the ganglionopathy and a distal axonopathy. However, the clinical pattern in our pa-

Melgaard et al: Misonidazole Neuropathy 1 5

tients differs from that of a subacute sensory neurop- 2. Behse F, Buchthal F: Peroneal muscular atrophy and related

athy in which tendon reflexes are absent [22]. Motor fibers are involved to a lesser degree, as evidenced by the electrophysiological studies and muscle biopsy findings (type grouping).

Animal experiments have shown distal axonal de- generation of intramuscular nerve branches and edema of the dorsal root ganglion. Distinctive ul- trastructural changes were not seen in axons prior to the appearance of wallerian-like degeneration [20]. Significant increases in both P-glucuronidase and P-galactosidase have been found in the trigeminal ganglion of rats exposed to misonidazole, providing biochemical evidence consistent with degeneration [26]. It is surprising in view of the histological findings that the symptoms of neuropathy remitted in most cases several months after discontinuation of misonidazole therapy [ 2 5 ] . This does not support the hypothesis of a necrotizing ganglionopathy such as is also seen experimentally in adriamycin-treated rats [ 111. Improvement occurred in our patients after ces- sation of therapy, but no patient was symptom free at reexamination. This might be explained by attribut- ing the clinical features partly to the membrane dys- function caused by the toxic substance, whereas persisting symptoms and signs are due to axonal degeneration.

The first clinical reports of misonidazole therapy recommended a single dose of 5 gm per square meter of body surface area [ 151; later this was reduced to 4 gm/m2 for single doses or 6 gm/m2 weekly due to the neurological side effects. A maximum total dose of 11 gm/m2 has been recommended [ 131.

Because of drug accumulation, neurotoxicity ap- pears to increase not only when the total dose is raised, but also when the drug is administrated daily [13]. In a recent study [14], 12 gm/m2 given over a 17-day period was suggested as acceptable since the incidence of peripheral neuropathy was markedly re- duced. Experience from the present study has led us to reduce the maximum dose to 11 gm/m2 and to monitor the plasma concentrations of the agent in patients with laryngeal and pharyngeal cancers. It may thus be possible to develop a therapeutic regi- men that reduces the prevalence and severity of neu- ropathy.

High-performance liquid chromatography to determine plasma concentrations of misonidazole was supported by Grant 5/80 from the Danish Cancer Society and by the Grosserer Sigurd Abraham- son og hustru, Addie Abrahamsons mindelegar.

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Melgaard et al: Misonidazole Neuropathy 17