We have demonstrated that maneuvers capable of reducing Ca influx into cells have beneficial effects in dystrophic hamsters and Duchenne muscular dystrophy. Since dantrolene inhibits Ca release from the sarcoplasmic reticulum, its effects on DMD was studied in 7 patients of 6 to 13 years of age (mean 10.8 years). Patients were studied for 4 years with tri-monthly evaluations of manual muscle testing (MMT), functional activity, and serum CK and aldolase. During the first 2-year period, no medicines were given and served as control. In the second 2-year period, dantrolene 8 mglkgid was administered. No side effects were observed. In 1 patient, mild weakness occurred that disappeared when the dose was reduced to 6 mg/kg/d. The 95% confidence limit for the difference in slopes of regression lines from tri-monthly MMT was asymmetric in favor of dantrolene in 5 of 7 patients. Serum CK did not differ between the first and second year of the control and treatment periods, respectively. However, it fell significantly from the second year of control to the first year of treatment (P = 0.003). The fall during the first year of treatment was significantly greater (P < 0.01) than in age-matched natural history controls during the same length of observation. There was a 3-fold reduction in CK when the pooled values of the first and second year control vs. treatment periods were analyzed. No changes were observed in functional activity and serum aldolase. The data suggest that dantrolene reduces serum CK in DMD associated with a lessening trend in MMT deterioration. Key words: Duchenne muscular dystrophy dantrolene manual muscle testing functional activity serum creatine kinase MUSCLE & NERVE 14:503-507 1991

EFFECT OF DANTROLENE IN DUCHENNE MUSCULAR DYSTROPHY TULIO E. BERTORINI, MD, GENARO M.A. PALMIERI, MD, JUDY GRIFFIN, MS, MASANORI IGARASHI, MD, ABBIE HINTON, BA, and JAMES G. KARAS, MS

T h e r e is evidence of an abnormal accumulation of calcium in muscle in Duchenne muscular dystrophy (DMD),','9220which is already present in the prenatal stage.3 It is likely that this excessive calcium accumulation may have a pathogenic role

From the Departments of Neurology, Medicine and Physical Therapy, and the Clinical Research Center University of Tennessee at Memphis; the Muscular Dystrophy Association Clinic; and the Memphis Neuroscience Center, Memphis, Tennessee. Acknowledgments: The authors acknowledge the assistance of the staff of the Muscular Dystrophy Clinic at Baptist Memorial Hospital in Memphis, the support of the Memphis Neurosciences Center, and the Clinical Research Center of the University of Tennessee at Memphis We further acknowledge the assistance of Nowich Eaton Pharmaceuticals, Inc., Norwich, NY, for providing dantrolene capsules. The technical contribution of Linda H. Horner and the secretarial assistance of Renee Johnson are appreciated. Supported in part by a grant of the Muscular Dystrophy Association and the Clinical Research Center and CLINFO grant USPHS #RR00211 Address reprint requests to Tulio E Bertorini, MD, Department of Neurology, University of Tennessee, 956 Court Avenue, Memphis, TN 38163. Accepted for publication September 1, 1990 CCC 0148-639)(/911060503-05 $04.00 0 1991 John Wiley & Sons, Inc.

Effect of Dantrolene in DMD

in cell necrosis.'2325Previous work from our laboratories demonstrated that maneuvers capable of reducing Ca influx into muscle cells resulted in beneficial effects in animal and human muscular Cytosolic Ca can be modified also by affecting the release of Ca from intracellular stores such as the endoplasmic reticulum. Dantrolene is known to inhibit Ca release from the sarcoplasmic reticulum and is beneficial in malignant hyperthermia. 14,18 In malignant hyperthermia, there is a defective influx and efflux of Ca from the sarcoplasma ret i ~ u l u m . Since ~ , ~ ~ a malignant hyperthermia-like syndrome may occur in DMD patients during ane ~ t h e s i a ,w~e~ tested the hypothesis that dantrolene, by reducing cytosolic Ca, would be beneficial in DMD. The effect of dantrolene was evaluated in a pilot study on 7 children with DMD treated with this agent for a period of two years. The results of clinical assessment and serum CK concentration were compared with the results of evaluations from the preceding two years, when no medications were given. T h e first year of treat-

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ment with dantrolene was also compared with 7 DMD age-matched historical controls. SUBJECTS AND METHODS

Seven DMD children 6 to 13 years of age (mean 10.8 years) were evaluated every 3 months with assessment of functional activity and detailed manual muscle testing ( M M T ) as previously reThe same investigator performed all determinations of M M T and functional activity without prior knowledge of the values obtained in preceding evaluations. Serum CK and aldolase concentrations were also determined tri-monthly. The study took place during two, 2-year periods. During the first period (control) the patients received no medications. In the second, they received dantrolene, orally. For initiation of treatment, the patients were admitted to the Clinical Research Center of the University of Tennessee, with prior signed, informed consent by parents or guardians. Seven additional untreated DMD boys whose age matched the age of the children at the initiation of treatment with dantrolene, were followed for 1 year with identical techniques and served as historical controls. The protocol was approved by the Institutional Review Board of the University of Tennessee. The initial daily dose of dantrolene was 2 mglkg body weight given in 3 equal doses. Vital signs, electrocardiograms, and M M T were monitored daily. In the absence of subjective and objective increments in weakness or other side effects, the dose of dantrolene was increased by 2 mglkgld until reaching 8 mglkgld. T h e patients were then discharged from the hospital and followed with assessments of functional activity, detailed MMT, and determinations of serum CK and aldolase every 3 months. Statistical analysis of M M T scores was made by obtaining two slope estimates from simple linear

Table 1. Manual muscle testing scores Case 10 15 17 22 23 29 31

Control

Dantrolene

-0.03076 -0.07348 -0.09427 -0.04127 -0.01987 -0.06721 -0.03976

-0.00631 -0.01309 -0.02551 -0.04729 -0.00237 -0.04929 -0.03512

95% Confidence limits on difference of slope estimates (-0.01529, (+0.01524, (+0.02829, (-0.04767, (-0.04132, (-0.02919, (-0.03477,

0.06419) 0.10554) 0.10921 ) 0.05971) 0.07632) 0.06503) 0.04433)

Slopes of regression lines of tri-monthly determinations during 2 years of control and treatment periods

504

Effect of Dantrolene in DMD

regressions which spanned each phase: the control period of 2 years and the treatment period of 2 vears. This was implemented for each case. The 95% confidence interval was then constructed on the difference of these slope estimates (treatment phase- placebo phase). " A data reduction technique was implemented for analysis of serum CK values in which areas-under-the-curve over time were determined utilizing the trapezoidal rule.15 Comparison of these areas was made with the paired t-test in conjunction with the Bonferroni correction for multiple comparisons.13 RESULTS

Dantrolene was well tolerated, and no acute weakness was detected except for 1 child who developed a mild increment in muscle weakness when he reached the dose of 8 mglkgld that subsided when the dose was reduced to 6 mglkgld. He was maintained in this dose for the total treatment period. The remaining patients did not show side effects during the 2 years of observation on dantrolene 8 mglkgld. There was a gradual deterioration of M M T in all patients. The simple linear regression slopes and the 95% confidence limit for the difference of the control and treatment regression slopes are presented in Table 1. By examining these limits, it is noted that 5 of the 7 cases studied have intervals which encompass zero (0). For these 5 cases, there was no statistical difference in the deterioration between the control phase and the drug phase of the study. However 3 of these 5 cases depict an asymmetric shift at about the zero point (cases 10, 23, 29), thereby suggesting a trend toward lessening of muscle deterioration during the treatment phase. The remaining 2 cases have intervals that do not contain zero (0) and are located in the positive scale, thereby indicating a statistically significant ( P < 0.05) change in the slopes toward a lessening degree of muscle deterioration. Thus, a lessening trend in M M T deterioration during the treatment phase was observed in 5 of the 7 cases studied. The mean SE M M T slopes of the first year of treatment with dantrolene was -0.025 0.01 1, which was less steep but not statistically different from the slopes of 7 age-matched historical controls -0.034 0.014, during 1 year of observation.

Manual Muscle Testing.

*

*

*

There was, as expected, large individual variability in the levels of serum CK. Therefore, the data was analyzed as follows: the

Creatine Kinase.

v

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last measurement of the control period was considered to be the baseline value for the 2 years of control and treatment periods. Values were then transformed to percentages of the baseline for each subject and the area-under-the-curve was generated over months for each case for both periods. Table 2 shows the individual values for each patient during the first and second control and treatment periods as well as the means ? SE. There was a significant difference ( P = 0.003) when the second year of the control was compared with the first year of dantrolene treatment, and also an approximately 3-fold reduction in CK was observed when the pooled values of the first and second year control vs. treatment were analyzed ( P < 0.05). However, since 4 period comparisons are depicted in Table 2, the Bonferroni correction13 was applied, which resulted in a more restricted cut-off (alpha) level of 0.0125 for statistical significance to be declared. By this criterion, the only comparison that yielded significance was that of the second year of control vs. the first year of dantrolene treatment (2107.8 2 495.6, control; 990.2 -+ 287.6, dantrolene treatment) (Table 2). In other words, there was a nonsignificant 36% and 31% fall in CK between the first and second year of control and treatment periods respectively, that can be explained on the basis of the natural history of the disease. There was, however, a more significant 56% fall between the last year of control and the first year of dantrolene treatment. The marked fall in CK observed during the first year of treatment with dantrolene was also greater ( P = 0.01)

than in 7 untreated, age-matched historical controls (Table 3). Serum aldolase was elevated during the total length of the study. At the initiation of the control period, serum aldolase was 55.1 13.7, niU/mI,, mean -+ SE (normal range 1 to lo), and fell by 29.3 -+ 11.6 toward the end of the control period. Serum aldolase fell by 15.6 -+ 3.0 during treatment with dantrolene. The difference between both decrements was not significant. Although contractures developed as expected in the majority of the patients who required braces and/or wheelchairs, 3 boys aged 13 to 15 years did not develop noticeable muscle contracture during treatment with dantrolene. N o difference was observed in functional activity scores between the control and treatment periods.

*

DISCUSSION

The data demonstrate a significant reduction in serum CK in DMD children after initiation of treatment with dantrolene. There was no statistical difference between the CK of the first and second year of control and dantrolene periods, respectively. A marked fall of CK was observed between the second year of control and the first year on dantrolene, suggesting that in addition to the expected progressive fall in serum CK known to occur in this disease," dantrolene may have induced a steeper fall of CK in DMD. This conclusion is supported by the greater fall in serum CK observed during the first year of dantrolene treatment than in age-matched historical controls.

Table 2. Serum creatine kinase, areas-under-the-curve (% of baseline x months) Second year control

Pooled first and second years, control

First year dantrolene

Second year dantrolene

Pooled first and second years, dantrolene

866.69 4314.50 2010.70 1227.10 3553.40 1428.50 1353.80 2107.8 -C 495.6

2315.10 14305.80 2742.80 2641.96 9219.59 4222.65 2360.35 5401.2 -C 1750.6

389.38 2474.69 644.24 735.91 1577.72 545.20 564.12 990.2 287.6

515.39 998.98 390.47 276.10 1782.81 420.23 424.67 686.9 -e 202.6

904.76 3473.67 1034.71 1012.01 3360.53 965.43 988.79 1677.1 4 449.7

First year control

1448.40 9991.30 732.04 1414.90 5666.10 2794.20 1006.60 3293.4 2 1286.0

t

P

=

0.22

7

T

P = 0.003

t

P = 0.20

t

t P < 0.05

Effect of Dantrolene in DMD

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Table 3. Serum creatine kinase, areas-under-the-curve (% of baseline x months). First year of dantrolene treatment vs. age-matched historical controls. Age

Mean 'P

=

?

SE

Creatine kinase

Dantrolene

Control

Dantrolene

Control

9.3 14.4 8.6 12.8 13.4 10.2 15.0 11.96 ? 0.97

8.6 13.6 8.0 12.0 13.3 10.8 15.2 11.64 -+ 1.01

389.38 2474.69 644.24 735.91 1577.72 545.20 564.12 990.2 ? 287.6

895.33 2963.99 785.12 1129.89 4650.26 804.37 1032.15 1751.6 563.6*

*

0 006 using paired 1-test

T h e major potential risk of the chronic use of dantrolene is the accentuation of muscle weakness, Our data suggest that, on the contrary, the M M T scores showed a trend toward a lessening of muscle deterioration during dantrolene administration. Moreover, during the first year of treatment with dantrolene, when the CK fall was steeper, the M M T slopes were not different from those of historical controls. Thus, the dose of dantrolene used in this study does not accentuate muscle weakness in DMD. The dantrolene-induced CIC reduction, in the absence of an acceleration in the loss of muscle strength, may suggest that the dantrolene-induced fall in CK was due to a reduction in the rate of cell necrosis. It is of interest that dantrolene also lowers CK in MDX mice, an allelic animal model, which like DMD, lacks dystrophin.2' Because dantrolene is highly effective in controling muscle rigidity in malignant hyperthermia, and severe cramps in DMD,2 it is conceivable that this agent may play a beneficial role in DMD, particularly during the early stages of the disease when muscle necrosis is rampant. Moreover, the

recent discovery that dystrophin, absent in DMD muscle, l 7 may influence cellular Ca horneostasis,l6 adds relevance to the present findings. Perhaps, dantrolene, by reducing the extrusion of Ca from the sarcoplasmic reticulum, could partially compensate for the absence of dystrophin. Finally, the reduction of the progression of muscle contracture during dantrolene treatment strengthens the potential role of dantrolene in the treatment of DMD. The data collected in this lengthy but limited pilot study do not categorically demonstrate a beneficial effect of dantrolene in DMD. However, they do suggest the presence of a lessening trend in M M T deterioration, accompanied by a reduction in serum CK. Obviously, more extensive, double blind evaluations of this agent in DMD are needed. Since chronic diltiazem administration is beneficial in DMD,' the possibility that reduction of Ca influx by a Ca antagonist in combination with an inhibitor of Ca release from the sarcoplasmic reticulum, such as dantrolene, may result in additional benefit in DMD.

REFERENCES

1. Bertorini TE, Bhactacharya SK, Palmieri GMA, Chesney CM, Pifer D, Baker B: Muscle calcium and magnesium content in Duchenne muscular dystrophy. NeuroloLg 1982;32:1O88- 1092. 2. Bertorini T E, Palmieri GMA, Bhattacharya SK: Beneficial effects of Dantrolene sodium in exercise-induced muscle pains: Calcium mediated? Lancet 1982;1:616-617. 3. Bertorini TE, Cornelio F, Bhattacharya SK, Palmieri GMA, Dones I, Dworzak F, Brambati B: Calcium and magnesium content in fetuses at risk and prenecrotic Duchenne muscular dystrophy. Neurology 1984;34: 14361440.

4. Bertorini TE: Clinical trials in Duchenne muscular dystro-

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Effect of Dantrolene in DMD

phy: the experience in Memphis, Tennessee. Ilal J Neural Sci 1984;(suppl 1):153- 158. 5. Bertorini T E , Palmieri GMA, Griffin 1 , Chesney C, Pifer D, Verling L, Airozo D, Fox IH: Chronic allopurinol and adenine therapy in Duchenne muscular dystrophy: effects on muscle function, nucleotide degradation, and muscle ATP and ADP content. Npurolygy 1985;35:61-65. 6. Bertorini TE, Palmieri GMA, Griffin J W , Igarashi M , McGee J, Brown R, Nutting DF, Hinton AB, Karas JG: Effect of chronic treatment with the calcium antagonist diltiazem in Duchenne muscular dystrophy. Neurology 1988;38:609613. 7. Bhattacharya SK, Palmieri GMA, Bertorini TE, Nutting

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DF: T h e effect of diltiazem in dystrophic hamsters. Muscle Nerve 1982;5:73-78. 8 . Blanck TJJ, Gruener RP: Malignant hyperthermia. Biochem Pharmacol 1983;32:2287-2289. 9. Brooke MH, Griggs RC, Mendell JR, Fenichel GM, Shumate JB, Pellegrino RJ: Clinical trial in Duchenne dystrophy. I. T h e design of the protocol. Muscle Nerve 1981;4:186- 197. 10. Brooke MH: A Clinician’s View of Neuromwcuhr Diseases, 2nd ed. Baltimore, MD, Williams and Wilkins, 1986, p p 343-364. 11. Draper N, Smith H: Applied Regwssion Analysis, 2nd ed. New York, Wiley, 1981. 12. Ebashi S, Sugita H: T h e role of calcium in physiological and pathological processes of skeletal muscle, in Aguayo AJ, Karpati G (eds): I n Currenl Topics in Nerve and Muscle Research, Amsterdam, Excerpta Medica, ICS, 1979, no. 455, p p 73-84. 13. Fleiss JL: The Design and Analysis of Clinical Experiments, New York, Wiley, 1966. 14. Flewellen EH, Nelson TE: Dantrolene dose response in malignant hyperthermia susceptible (MHS) swine. Anesthe~ i o l o ~1 g9 80 ~ ;5 2 :303 - 30 8. 15. Hildebrand FB: Introduction to Numerical Analysis. New York, McCraw-Hill, 1956. 16. Hoffman EP, Knudson CM, Campbell KP, Kunkel LM: Subcellular fractionation of dystrophin to the triads of skeletal muscle. Nature 1987;330:754-758. 17. Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterson R, Brooke M, Specht L, Kupsky W, Chamberlain J , Caskey CT, Shapiro

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biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N Engl J Med 1988;318:13631368. Kolb ME, Horne ML, Martz R: Dantrolene in human malignant hyperthermia. Anesthesiology 1982;56:254-262. Maunder-Sewry CA, Gorodetsky R, Yarorn R, Dubowitz V: Element analysis of skeletal muscle in Duchenne muscular dystrophy using x-ray Huorescence spectrometry. Mwcle Nerve 1980;3:502-508. Oberc MA, Engel WK: Ultrastructural localization of calcium in normal and abnormal skeletal muscle. Lab Invest 1977;36:566-577. Palmieri GMA, Nutting DF, Bhattacharya SK, Bertorini TE, Williams JC: Parathyroid ablation in dystrophic hamsters: effects on calcium content and histology of heart, diaphragm and rectus femoris. J Clin Invest 1981;68:646654. Quilan JG, Johnson SR, Samaha FJ: Dantrolene normalizes serum creatinine kinase in MDX mice. Muscle Nerve 1990; 13:268-269. Willner J , Nakagawa M, Wood D: Drug-induced fiber necrosis in Duchenne’s dystrophy. Ital J Neurol Sci 1 9 8 4 ; 5 : ( ~ ~ p3):117pl 122. Wood DJ, Mozo A, Willner J: Malignant hyperthermia: T h e relation of sarcoplasmic reticulum dysfunction to the pathogenesis of the disease. Neurology 1979;29:557-558. Wrogemann K, Jacobson BE, Blanchaer MC: On the mechanism of a calcium-associated defect of oxidative phosphorylation in progressive muscular dystrophy. Arch Biochem Biophys 1973; 159:267-278.

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Effect of dantrolene in Duchenne muscular dystrophy.

We have demonstrated that maneuvers capable of reducing Ca influx into cells have beneficial effects in dystrophic hamsters and Duchenne muscular dyst...
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