Medical Hypotheses A&dial IYy@w Group

e Longman

(1992) 38.13~15.4 UK Ltd 1992

The Dystrophin Connection -

ATP?

CA. BONSETT and A. RUDMAN Department of Neurology, Indiana University School of Medicine, Emerson Hall 725, 545 Barnhill Drive, Indianapolis, IN 46223, USA (Reprint requests to CB)

Abstract - Clinical evidence is presented supporting the hypothesis that the metabolic abnormality in the dystrophindefective muscular dystrophies (DMD and BMD) involves the ATP pathway. Objective laboratory data show corrective trends in the abnormal values of parameters relating to creatine and calcium metabolism (ATP) by use of glucagon-stimulated c-AMP and by use of synthetically produced adenylosuccinic acid (ASA). Disease accelerating mechanisms as suggested by analysis of the clinical features, and the therapeutic potential of ASA are discussed.

Introduction Duchenne muscular dystrophy (DMD) is a sexlinked, recessive genetic disease whose inherent protein transcription error results in a quantitative deficiency of the sarcolemmal structural element dystrophin (1). Beckermusculardystrophy (BMD) whose clinical course is much more benign has the identical characterization except that the dystrophin abnormality is qualitative (2). At the cellular level, the pathological sequence of events is characterized by necrosis and regeneration of muscle cells, the former prevailing (3). The principal mechanism for necrosis is calcium accumulation within the fiber (4,5,6), but how this occurs and how dystrophin relates to the process has yet to be clarified. It is evident that a metabolic malfunction exists. Is its dystrophin link chemical in nature or is it Date received 20 June 199 1 Date accepted 2 January 1992

simply a physical process, a ‘leak’ whose dimensions become increasingly exaggerated by the accumulated physical stresses of muscle contraction and use (7.8)? In the present paper we give clinical evidence supporting the hypothesis that the basic metabolic defect involves the adenosine triphosphate (ATP) pathway, that inadequate availability of energy phosphate in the working muscle precludes its enduring ability for normal sustained contraction, active transport of calcium, and other functions, failure of which leads to muscle fiber loss. The following five statements outline the basis of the present study: 1. All muscles am genetically abnormal in these dystrophies and, therefore, should be vulnerable to the metabolic and physical factors condusive to clinical weakness and muscle necrosis. 139

140

MEDICAL HYPOTHESES

2. Not all muscles demonstrate clinical abnormality in these diseases, not even terminally (e.g. SERUM CREATININE VALUES IN NORMAL SOYS AND THOSE extraocular muscles). WITH OUCHENNE’S MUSCULAR DYSTROPHY 3. Such ‘spared’ muscles demonstrate minimal, if any, of the pathological features leading to ne,.. (MQ /DI.) crosis, but they do, at least, reveal a propenst ity for hyperlipidosis, a microscopical manii ;; ANoR*.. festation of metabolic dysfunction (9,lO). 4. Tissue culture responses suggest the hyperlipidosis to result from low adenosine diphosph‘. ate (ADP) concentration at isocitrate dehyd---._ 0.2 -- - - - - rogenase, a lipid control point in the citric ’ 1 acid cycle (11, 12) (i.e. the mitochondrial 2466 IO 12 14 16 AGE IN YEARS matrix). 5. Correction is effected in vitro by addition to the cells nutrient medium of adenylosuccinic Ffg. 1 Graph showing the nahval history of DMD in terms of acid (ASA, ADSA), the immediate precursor serum cmatinine. (Normal values are from Schwartz, et al. (10). DMD values from IUMCMuscularDystrophy Clink.) The value of adenosine monophosphate (AMP) (12) or is age related by addition of cyclic-AMP (c-AMP) (unpub lished) (ASA or c-AMP + AMP _j ADP + ATP). Method (1: ,y

I I

These in vitro observations suggest the hypothesis: that muscle necrosis in these dystrophies results from inadequate maintenance of the AS A + AMP -+ ADP + ATPreserve in the working muscle. If this is correct, then objective evidence supporting the concept should be demonstrable in vivo in terms of the effect of muscle-added c-AMP or ASA on the parameters of creatine metabolism; and to the extent that the deficiency can be corrected by use of replacement metabolite. the muscle necrosis-muscle regeneration ratio should be improved.

AGE

IN

-.

DUCHEHHE

4

1

I

I

I

I

OYSTROPHY

I

In order to test this hypothesis, the following graphs were prepared in the late 1970s from data collected with informed consent from the DMD patient population of the Indiana University Medical Center (IUh4C) Muscular Dystrophy Clinic. Data were collected over a 5 year interval from approximately 30 Dh4D patients, all of whom had creatine kinase (CK, CPK) values 25-100 times normal, serum creatinine values of 0.5 mg/ml or less, 24 h urine creatinine-creatine (Cr/C) ratios below 1.O.andmuscle biopsy with diagnostic DMD features; and most, but not all, had a family history

NORMAL

w

OYSTAOPHY

m

YEARS

Fig. 2 Natural history of DMD in terms of the 24 h urine WC ratio. The value is age related and remains below 1.O in DMD, slightly above in other forms of girdle dystrophy

141

THE DYSTROPHtN CONNECPION - ATP?

DUCHENNE CPK

x

*

VALUES

MUSCULAR

DURING

THE

DYSTROPHY

FIRST

YEARS

OF LIFE

150

s! __

=

MAXIMUM

-

MINIMUM

OUT-PATIENT

=

MINIMUM

IN-PATIENT

IN-PATIENT

VALUE

(P.M.)

3 : ”

x 100

6 MONTHS

(P.M.)

3 MONTHS

(P.M.)

BIRTH (CORD

x 10 NORMAL

PATIENT

VALUE (A.M.)

VALUE (A.M.)

BLOOD)

‘A’

PATIENT

“B’

AGE 2 YEARS

Fig. 3 Graph showing the elevation of CK (CPK) values during the fmt 2 years of life in DMD. Increase in value is associated with increase in muscle activity with development, time of day that the test was done and nature of the environment (home usually fostering more physical activity than a hospital crib)

of DMD. Figures l-6 graph the natural history of DMD in terms of parameters relating to creatine metabolism (i.e. ATP utilization). DIJCHENNE

MUSCULAR

CPK

VALUES WITH

DYSTROPHY

CORRELATED lNACTl”lTY

AND WITH EXERCISE

1

2

3

4

5

DAYOF

CONSECUTlVE DAYSOF ,NACT,VlTY EXERCISE

These graphs provide a means for comparative measure in determining the effect, if any, of replacement metabolite. Figure 1, serum creatinine is self-explanatory. Normal values are from Schwartz et al (13). Note the normal values are age-related. Figure 2 is the 24 h urine Cr/C ratio. Note that this is also age-related in the normal but remains below 1.Oin DMD regardless of age (14). Figure 3 demonstrates the gradual elevation of the CK value during the first 2 years of life as demonstrated in 2 DMD infants, both of whom had a family history of DMD. The graph demonstrates that the value trends upward with the increased physical activity associated with growth, that the Fig.4 (Left) The CK (CPK) value has a diurnal cycle falling with the inactivity associated with sleep, elevating with muscular activity. The drop is slow. It takes 4-5 daysofcontinuous inactivity to achieve the lowest value possible. The elevation with exercise is prompt Graph shows daily early AM values for 6 consecutive days of continuous bed rest. On the sixth day the patients participated in a progmm of vigorous exercise prior to the day’s second testing. Values from two 5-year-old DMD patients

142

MEDICAL

250 x NORMAL

DUCHENNE EARLY SIGNS

200 x NORMAL

l

150 x_ NORMAL

.’

:

A. a *a

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LATE AMBULATORY SIGNS

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DYSTROPHY

CLINICAL CORRELATIONS WITH CK VALUES

.

.

MUSCULAR

HlTvmEsEs

0%.

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. a

l

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L, 1

2

3

4

5

6

7

8

AGE

9

10

11 12 13

14

15

16 17

18 19 20

IN YEARS

Fig. 5 Range of CK values from birth to death in DMD, correlated with gmss clinical events

morning value is lower than the evening value, and thatthe values tend to be higher as an outpatient than with the more restricted physical activity associated with the inpatient status. Clinically, both patients

appeared normal at this stage. Figure 4 shows the CK value to be diurnal. 4-5 days of continuous bed rest are necessary to lower the outpatient AM value to the new inpatient AM

OUCHENNE

X 250 NORMAL-

MUSCULAR

MAXIMUM-MINIMUM

DYSTROPHY

CK VALUES

x 200 NORMALi.? 2

x 150_ NORMAL

ACTIVE

VALUES

(P.M.1

3 RESTING

::

VALUES

(A.M.) - - - --

/ / x lo_ NORMAL _ 1

2

3

4

5

6

7

8

AGE

9

10

11 12 13

14 15

16

17

18 19 20

IN YEARS

Fig. 6 Natural history of DMD in terms of high and low points of the daily CK cycle (inpatient 07.00 resting value vs 17.00 value following an exercise period). Note that the analysis of CK behavior is more complex than noting high values to be associated only with the early years. Rather there is a period of elevation, a peak and a decline. The clinical features unfold more rapidly once the peak is reached

THE DYSTROPHIN

3.5

8

d

1

CONNFKTION

-

143

All”!

Duchenne Muscular Dystrophy Glucagon Effect on Urine Creatinine-Creatine Ratio

2.0 -

0

1

2

3 Day of h-Patient

4

5

6

Study

Fig. 7 Graph showing effect of 0.5 cc subcutaneous glucagon injection on the urine WC ratio in a 5-year-old DMD patient

plateau. The value begins its daily climb with the morning’s arousal and elevates promptly with muscular activity. In order to determine any given patient’s lowest possible CK value, an early AM blood sample after 4-5 days of continuous bed rest is necessary. Figure 5 shows the range of values from day of birth by elective cesarean section to day of death in DMD, correlated with gross clinical events. The value peaks during the fourth to fifth year. Figure 6 utilizes the same data source as Figure 5 but is limited to inpatient, same patient-same day minimum and maximum (after exercise) values. This demonstrates the diurnal range and limits.

Our next effort was c-AMP. This metabolite was not commercially available for a clinical trial. We postulated, however, that since c-AMP functions as the ‘second messenger’ in mediating the action of a number of water soluble hormones and since it is produced intra-cellularly from ATF through action of the membrane-bound enzyme, adenyl cyclase, that an initial flush of hormone-stimulated c-AMP might escape the membrane to enter the circulation. If so, then ATP from the liver, say, could become a source of ATF’ for skeletal muscle in the form of circulating c-AMP. Muscle contains the enzyme, phosphodiestemse for converting c-AMP to AMP. (DMD muscle is reported to be deficient in the membrane-bound enzyme adenyl cyclase (1%) To test this concept (with informed DMD parental consent) we injected a fractional dose (O.w.7 ml) of glucagon subcutaneously while monitoring the urine for the CrIC ratio. A prompt ratio elevation occurred (Fig. 7). We than graphed responses from a series of patients representative of the total course of the disease and noted the highest ratios with the youngest patients (over 100.0) and almost no response from those with advanced disease (less than 2.0). Following the glucagon injection, a refractory period of 3-4 days ensued during which time a second injection was without affect on reproducing the phenomenon. There was no

Glucagon on Serum

600

C-AMP

At the time we commenced clinical trials in the late 1970s. AMP was available as a prescription drug, manufactured by Dome Laboratories (West Haven, CT, USA) under the trade name ‘My-B-Den’. It produced no detectable, beneficial effect either in tissue culture or clinically (or in terms of the laboratory parameters of creatine metabolism). It apparently could not pass membrane barriers.

-

-2

5 m

700 -

z 5

2; L-

Results

Effect c-AMP

.

500 “, 2

400

!z

300

.

z

g 200 fioo!

.;

looiv 0

’

;\I

.

1

0

1

2

3

4

5

Time I” Hours

Fig. 8 Graph showing effect of glucagon injection on se-rum C-AMP level. (Thanks to Dr Mike Schmidt and the Eli Lilly Company)

144 GLUCAGON EFFECT ON URINE CREATINE AND CREATININE

60

Limb-Girdle

Muscular Dystrophy

50

.o z a

40

._z 5 0’

30

h, .-c .c i! b

20

10

0 0

10

20

30

50

40

60

Hours

Fig. 9 Gmph showing glucagon response of limb-girdle muscular dystrophy.

Increase in Cc/C ratio results from decrease in urinary

excretion of creatine and increase in creatinke

beneficial or adverse clinical effect noted with glucagon. (Glucagon per se has no effect on skeletal muscle (16)) Figure 8 graphs the semm c-AMP flush response to glucagon. Figure 9 shows in more detail that creatine excretion is actually lowered, that creatinine excretion actually increases, and that the peak Cr/C ratio occurs in response to glucagon injection. These results indicate increased efficiency in intramuscular phosphorylation of creatine and are consistent with the postulated concept of resultant increase in available intramuscular ATE. (Injection of glucagon diluent alone produced no effect.) Note in Figure 9 the subject is a patient with limb-girdle dystrophy. The glucagon-Cr/C phenomenon is also demonstratable in patients with BMD and Em’s dystrophy. In these other forms of girdle dystrophy, the natural Cr/C ratio is above 1.0 and, except for DMD toddlers, the glucagon-evoked ratios tend to be greater.

ASA (ADSA) ASA was not available as a clinical or experimental drug. We requested and were granted approval by the US Department of Health and Human Services, Food and Drug Administration, to have the metabolite custom-synthesized for a limited phase I (toxicity) and phase II (efficacy) evaluation (IND 17848). The metabolite was custom-produced for this study by Sigma Chemical Company of St Louis, Missourl, USA. Institutional review was provided by appropriate committees of IUMC and Community Hospital, Indianapolis, Indiana, USA. Written, informed consent was obtained from the families of all patients tested. The program commenced in 1981 and terminated in 1991. The program commenced with an approved maximum dosage of lOmg/kg/24h. We knew the normal daily ATP utilization to be measured in terms of kg per 24 h (17), possibly at least one-third

THE DYSTROPHIN

CONNECTION

-

145

ATP?

Serum Creatine & Creatinine Mg./ DI. 0.8

Urine Creatinine Creatine Ratio

Serum CK

1.8 1.4

0.8

1.2 1.0

0.4

0.8 0.8

0.2

0.4 0.2

0

0 CaCREATlNE CR: CREATININE

on cmatine metabolism; DMD patient representative of the slow end of tk disease spectrum, tested in the basal state at time of lowest value of the 24 h CK cycle

Fig. 10 Effect of ASA (ADS-A)

0 4 Inject

4

8 Hours

after

16

24

irijection

Ng. 11 Effect of ASA on 24 h mine WC ratio. Ratio usually well below 1.0 in DMD. urine sample

being derived de novo. We did not know the DMD replacement need but suspected from the high CK values that 10 mg/kg would be grossly inadequate.

Maximum dmg effect is found in the 4-8 h

A major problem was how to detect positive objective data with this dosage limitation. The DMD volunteer for the initial trial was the

146 available patient having the slowest clinical course (hence, most apt to be responsive to low ATP replacement). He was still ambulatory at age 12 years. All muscle stretch reflexes were absent He was unable to arise from the floor unassisted but could stand from sitting in a straight-back chair by rocking forward and then using his hands on his legs. Muscle biopsy was diagnostic of DMD. CK was over 100 times normal (outpatient PM value). Urine 24 h Cr/C ratio was less than 1.O. The patient was admitted to the hospital for continuous bed rest so as to minimize his ATP need. Urine was collected at intervals then frozen for Cr/C testing throughout the hospital stay. On the morning of the fourth hospital day, time of achieving the lowest possible AM CK value, blood was drawn for serum creature, creatinine and CK. He was then given ASA subcutaneously at lOmg/kg (250 mg) and blood tests were repeated 1 h later. Precautions were observed to maintain the basic metabolic state. The results are shown in the graphs Figures 10 and 11. These show lowered values for creatine and CK; elevated values for creatinine and the Cr/C ratio. The results are consistent with the hypothesis that a significant metabolic malfunction is linked to the inborn sarcolemmal structural defect of DMD (and BMD) and must relate to the ASA + AMP + ADP + ATP pathway. Further, of practical significance, ASA might be effective as replacement therapy. In this regard, the next question to be answered was: To what extent can the ASA _j AMP + ADP -+ ATP deficiency be corrected by replacement ASA? During the past 10 years, we have been conducting a very limited phase I and phase II study of ASA, testing a total of 17 patients (15 DMD and 2 BMD), usually a total of 5 at any given time. We have been authorized to increase the dosage over the years from 1 mglkg/24h to 1 gfkgL24h. We have periodically monitored the blood count, the urine, the serum bilirubin, blood urea nitrogen, uric acid (for possible drug overdose), serum creatinine, and CK. There have been no signs of toxic effect and no adverse drug-related problem of any kind. We have covered the total course of DMD with representative examples from toddler to terminal, gaining months of experience with each, (Use of ASA diluent alone produced no detectable effect.) A technical problem has been encountered, however, with drug delivery. We initiated the program using the subcutaneous route with

MEDICAL. HYPoTHEsEs

tuberculin syringes, replacing this with a miniature insulin pump containing a 3 cc syringe. These worked very well up to a dosage of 200 mg/kg. We then went to the intraperitoneal route with an implanted catheter. We attempted this method with a total of 11 patients, all of whom ultimately had to have theprogramtexminated because of a variety of inflammatory responses (none of which were serious) or because the catheters became completely obstructed with fibrous tissue. This obstruction usually occurred within a matter of months - in some cases, a matter of weeks. 2 patients only had prolonged unintermpted programs measured in terms of years thus permitting long-term evaluation while dosage was gradually being increased as permitted (300 mg/kg for BMD and 600 mg/kg for DMD). (Success in the latter case is attributed to the fact that the daily drug care was attended by the father, a gastroenterologist who knew and practiced all possible precautions and techniques.) The records of these 2 patients provide the next evidence. Dh4D example (Dystrophin content, second biopsy less than 3% Dystrophin quantity and quality testing by Genica Pharmaceutical Corporation.) The patient was 2.5-years-old at the time of entering the program. His calves were enlarged. He could neither hop nor jump. He fell frequently and had very limited endurance. A paravertebral muscle biopsy was done before starting the drug. Pathology Report ‘...them clearly is an increase in connective tissue fairly diffusely through this muscle, both perimysial and endomysial...there are occasional inflammatory infiltrates; they are clearly related to muscle fibers undergoing necrosis ’ . ‘The muscle fibers am for the most part reduced in size. The majority are rounded in outline... There are no remnants of closely packed polyhedral muscle fibers anywhere. Average sizes of these better looking fibers are in the 35-50 micron range. It is very difficult to fiid larger fibers. Occasional internal nuclei are seen in these fibers but they remain the exception... Although occasionally small fibers are interspersed between these larger fibers,

THE DYSTROPHIN CONNECtION -

ATP?

Fig. 12A DMD patient, age 25years. Wravertebral extensor M. Before ASA. Degenerative featum are more pxwninent than those of regeneration. (Cryostat, H and E x 25)

usually the smaller fibers occur in clusters or groups; many of these groups show smaller, very basophilic fibers with large nuclei, prominent nucleoli, sometimes increase in number of nuclei and internal displacement of such nuclei. Such clusters on occasion show some necrotic fibers and have attracted au inflammatory following...’ ‘This is already a reasonablyadvancedcase despite the early age of the patient.' (Authors’ italics) (Pig. 12A) ASA was commenced in 1985 at a dosage of 25 mg/kg/24h, subcutaneously with aminiature insulin pump. A prompt increase occurred in energy, stamina, and endurance and was maintained. The dosage was increased at intervals over the next 4.5 years. A catheter was placed in the peritoneum in

147 1987 permitting the dosage to increase to 333 mg/kg by 1988 and to 500 mg/Icg in 1989 at which time a second biopsy was done. The dosage later increased to 600 mg/kg with a goal of ultimately achieving a dosage of 1 g/kg/24h. ‘Ihe catheter, however, became obstructed with fibrous tissue at 600 rug/kg and had to be removed. A second catheter was implanted but this one promptly obstructed also. The intraperitoneaI program was discontinued at that time. The program continues using the oral route which is less efficient. When the program commenced at age 2.5 years, the outpatient PM CK value was in excess of 200 times normal. Semm creatinine was 0.2 mg/% (normal for age: 0.4-0.5 mg/%; usual DMD this age: O&O.3 mg/%). At time of the second biopsy at age 7 years, posture was erect, patient walked well, flat on his heels without falling. He could cover 37 m in 32 s. He was unable to hop on one foot but could jump multiple times (two feet) with the feet completely clearing the floor. He arose from the prone position on the floor to the standing position without use of his hands in 11 s. He could climb and descend five, 7 inch steps in 12 s using one hand on the rail. Serumcmatinine was 0.4-0.5 mgl% (normal for age OS-O.6 mg/%; usual DMD for this age: 0.1-0.3 mgNb). Outpatient PM CIC value was 50 times normal. (It is of interest that the patient’s DMD maternal uncle had become chair-bound by age 6 years.) The second biopsy was obtained from the identical muscle and approximate surgical site as the original biopsy. After a lapse of 4.5 years and considering the natural history of the disease, extensive fatty replacement would be expected. Instead PathologyReport ‘There is clearly an increase in connective tissue. The increase in the septa is modest..in the major septa, there am no intIammatoxy iDfiltIXk%.

Muscle fibers are still reasonably closely packed in their fascicles even if there is increased connective tissue. The majority, however, are rounded in outline. Maximum size is about 45 microns (most fibers are smaller). Occasional internal nuclei are seen. The major change is the fact that in some sharply circumscribed areas, many of the fibers become much smaller, have undergone

Ng. 12B DMD patient, age 7 ye=. Same muscle, same biopsy site 4.5 years later. On drug ahnost continuously and during this time the dosage has increased slowly at intervals from 25 mgkd24h to 500 m@g/24h. Note that fibers anz now mom closely compacted. The degenerative vs regenerative features aremoreinbalancethaninFigue12A. (Cryostat,HandEx25)

necrosis with some$imes inflammatory response, and on the other side are in the process of regeneration, very basophilic cytoplasm, numerous large nuclei. The inflammatory infiltrates are mononuclear only.’ Compared with the previous biopsy, the disease is, ifanything, less developed. (Authors

italics) (Fig. 12B). The ratio of regeneration improved.

to necrosis has

BMD example

(Mild to moderate BMD), (dystrophin quantity

Fig. 13A BMD patient, age 3.5 years. Vastus lateralis M. Before ASA. Some fibers tend to be rounded rather than polygonal in contour. Anow indicates site of necrosis marked by presence of macmphages. (Cryostat, H and E x 100)

normal: molecular weight 350 kD vs 400 normal dystrophin quantity and quality testing by Genica Pharmaceutical Corporation.) The dystrophin aspect of DMD and BMD was not discovered until our study was several years underway. The dystrophin determination of these two patients was done with their second biopsies. The BMD volunteer selected for this study had been diagnosed at age 3 years on the basis of CK in excess of 100 times normal and frommuscle biopsy. Serum creatinine was 0.5 rngl% (normal for age). His only complaint was cramping associated with vigorous exercise, this being limited to leg muscles only. This led to medical evaluation which demonstrated no objective clinical sign of muscular dystrophy but did prompt a diagnostic muscle biopsy.

149

THEDYSTFtOPHINCONNEClTON-ATP?

uphill. At that time, he was studied on an inpatient basis at complete bed rest (to minimize ATP demand). ASA was then commenced at 30 mg/kg/ 24h, increasing to 100 mg/kg/24h, and then to 200 mg/kg/24h (Fig. 14). CK dropped with each dosage increase and then plateaued until the next dosage increase. The drug was discontinued and the patient discharged. Within the next 2 weeks the CK had increased to a value 70 times normal. The experiment was repeated on an outpatient basis and the identical dose-related CK drop again obtained. Becker dystrophy was found not to be as sensitive to the need for absolute bed rest in order to demonstrate the drug-related drop in CK value. Because of an extremely limited drug supply, the patient was given just enough ASA to keep the CK level below 50 times normal (Fig. 15), a value below which no complaint of muscle cramping occurred and no clinical sign of dystrophy developed. A dosage of 200 mg/kg/24h was sufficient to drop the CK well within the normal range on a program of modest physical activity. The patient was receiving

BECKER EFFECT CREATINE

DYSTROPHY

OF ADSA ON SERUM KINASE AND URIC AClD MWKWS4HRS

Fig. 13B BMD patient, age 10 years. Same muscle. same biopsy site 7 years later. ASA was commenced at age 6 years at a dosage of 30 mg/kg/24h and, except for a brief inpatient trial during which time a dosage of 200 mg/k&?4h was permitted and again briefly as an outpatient, the dosage had increased gradually over the years to 150 mg/kg/24h at the time of this biopsy which shows minimal signs of abnormality (see text). (Cryostat, H and E x 100)

Pathology Report, original biopsy at age 3 years ‘...there is minimal increase in the endomysial connective tissue... Through the entire biopsy, there are groups of small basophilic fibers with large vesicular nuclei and prominent nucleoli. The majority of these fibers appear to be regenerating fibers. In a few focal areas there am fibers undergoing frank necrosis marked by the presence of numerous macrophages’ (Fig. 13A).

In 1985 at the age of 6 years he first became aware of possible thigh weakness when riding his bicycle

ADS-A

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I

,i

-3 ,,_-4’

(‘, .(,C

-1

’

/

I

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Fig. 14 Becker mtr&ar dystrophy. JZtTectof inmase in daily ASA (ADS-A) dosage on CK value (solid line) with patient maintained in the bed rest (i.e. low energy demand) state. Note that an exercise program was initiated on day 10. Broken line is uric acid value, monitored for detection of ASA overdose

150

BECKER MUSCULAR

DYSTROPHY

RELATIONSHIP OF DAILY ADSA DOSAQE TO SERUM CK VALUES

.

ADSA

mglkgl24

hours

Fig. 15 Effect of adenylosuccinate (ASA, ADSA) on creatine kinase in childhood Becker dystrophy. Total outpatient 4-year CK experience

a daily dosage of 150 mg/kg/24h when his second biopsy was done. (The goal was to double this dose.) His CK value at that time was 10 times normal and serum creatinine was 0.8 mgl% which is normal. His original biopsy scar was opened and a second biopsy obtained from the original biopsy site. PathologyReport,second biopsy ‘The muscle fibers within their fascicles still appear to be closely packed and more or less polyhedral in outline. The average sizes are in the 40-50 micron range but occasionally smaller, more rounded fibers are seen down to about 30 microns. Here and them arc small foci in these fascicles where the picture drastically alters. These are clusters of smaIl, usually rounded fibers, sometimes with internal nuclei. Sarcoplasmic net is quite coarse, fibers are overall basophilic staining, nuclei are large and have large nucleoli as well. It is clear that some of these fibers are degenerating because there is a small accompaniment of inflammatory infiltrate. all mononuclear. Only a few such clusters are seen...’

‘The slides are compared toa biopsyobtained in 1982. The disease is similar but, ifanything, is of lesser severitynow than it was in 1982.’ (Authors’ italics) (Fig. 13B). The ratio of regeneration to necrosis has improved. The patient’s CK value dropped well into the normal range before his peritoneal catheter became plugged and his program terminated (Fig. 15). CK value then elevated to its former range and leg muscle cramping has recurred with muscular activity. These results are objective. One anecdotal aspect of the study deserves mention however. Each patient in this study developed an increase in energy, stamina, and endurance while receiving ASA. Some, but not all, demonstrated increase in hand muscle strength as measured with a pinch meter. None of the DMD patients achieved appropriate replacement dosage, and only one BMD patient did. When the metabolite is discontinued in DMD, energy, stamina, and endurance decrease immediately and the disease then proceeds at its usual, more rapid pace. When the metabolite is discontinued in BMD, symptoms recur weeks to

THE DYSTROPHIN CONNECI’ION -

ATP?

months later. The CK value increases promptly. The question arises whether this BMD patient’s muscle problem is identical to the familial x-linked myalgia and cramps cases described by Gospe et al (18). These were myopathies with qualitative dystrophin abnormality, no indication of muscle weakness, but presence of cramps not limited to the leg muscles as in our example, but also in the hands and arms, The question cannot be answered, nor is it important. Our intent was to study a qualitatively deticient dystrophin patient with minimal objective signs of myopathy. This we did. The question is moot. Gospe’s cases would be expected to respond to a similar ASA dosage as our own.

Discussion These results show that exogenous AMP-precursor (c-AMP and ASA) pass sarcolemmal and mitochondrial membrane barriers to enter productively into the purine nucleotide cycle. By so doing, the metabolism of creatine is improved indicating increase in phosphorylation by ATP. It has long been known that creatine metabolism is abnormal in DMD (14, 19, 20). Creatine normally is transported to muscle from the liver, to pass freely in either direction through the sarcolemmal membrane until phosphorylated (a function of CK and ATP). Once phosphorylation has occurred, the entrapped phosphocreatine can only be expended in muscle contraction, and the unrecyclable waste product, creatinine, eliminated. In DMD and BIND, creatine accumulates (apparently because of inadequate phosphorylation due to ATP deficiency) and creatinine production thereby diminishes, hence the grossly abnormal urine Cr/C ratio.

In our example of BMD, the degree of metabolic and clinical abnormality is minimal. Impression is gained from the clinical course and from the initial biopsy that prior to ASA therapy, muscle regeneration is almost abreast of muscle necrosis. The second biopsy was obtained before the replacement dosage was sufficient to return the CK value to a normal level. The clinical impression and second biopsy suggest improvement in the regenerationnecrosis ratio. Circumstances of very limited amount of available replacement metabolite prevented a long-term, full-dosage program with this patient. However, no objective clinical sign of disease developed during the years he was receiving medication. He probably represents the dystrophin

151

abnormality with minimal degree of ATP replacement need (i.e. 200-300 m@g/24h.) The dosage received, however, was sufficient to keep the rate of muscle regeneration ahead of degeneration in the area biopsied, and to prevent or delay overt clinical expression of disease. The DMD patient on the other hand is probably representative of the maximum degree of detlciency. At a dosage of 600 mgkgl24h, he was markedly improved but had not yet achieved optimum dosage. It is clear by his serum creatinine and CK values (which are outpatient PM values, i.e. maximum diurnal) and also tiom his second biopsy, that the regeneration-necrosis ratio improved considerably. It is to be emphasized that the second biopsies were obtained from the sites of the original biopsies. Considering that a period of 7, and 4.5 years, respectively, separated these biopsies in time, it would be expected that without improvement in the regeneration-necrosis ratio both would show significant progressive deterioration and demonstrate some degree of fatty replacement in the second biopsy. Them should not be a lesser degree of necrosis if calcium metabolism had not improved. Calcium influx into the muscle fiber occurs normally only with the transient altered permeability associated with the nerve-impulse-musclemembrane depolarization which initiates muscle contraction. Unless there were then available sufftcient sarcoplasmic ATP to energize the active transport of calcium from the sarcoplasm back into the sarcoplasmic reticulum, the relaxation mechanism would become impeded and calcium would collect within the sarcoplasm to initiate its pathological mischief. Such appears to be the mechanism with these dystrophies during sustained contraction demand. There appears to be a damming to the normal metabolic flow of calcium and creatine. The impediment to both appears to be an insufficient availability of metabolic energy (i.e. ATP). The effect of the genetic abnormality in DMD and BMD is to impart a muscle membrane structural defect. How then does that defect cause the distinctive sequence of clinical events which characterize the diseases’ natural history? Does calcium cross the dystrophin defect passively to accumulate within the cytoplasm to cause necrosis? If such were true, then why don’t all DMD muscles deteriorate more-or-less uniformly? Why ate the extraocular and other muscles ‘spared’? Why do the gluteal muscles disappear relatively early with copious fatty replacement? Why does the lower portion of the pectoralismajor disappear selectively

152 with essentially no fatty replacement? It appears that the genetic abnormality alone can account for neither the clinical manifestation nor progression of disease. All DMD (and BMD) muscles are genetically abnormal. They do not respond uniformly to the disease. Why not? If we accept the observation that hyperlipidosis is a manifestation of DMD’s metabolic malfunction, then we must accept the fact that all skeletal muscle is metabolically abnormal (10) and that the combination of genetic abnormality linked to its associated metabolic connection cau not rogether account for clinical expression or progression of disease. There must be an additional factor or factors involved. DMD, except for rate of progression, unfolds clinically in a unique predictable sequence of events. If we look for the metabolic and diseasedynamic information latent in this pattern, then the various muscle behaviors mentioned above become comprehensible. Our studies to date identify 3 disease accelerating or muscle destructive mechanisms of which calcium accumulation, through sustained muscle contraction (and associated calcium pump failure), is but one. It is the most important, however (i.e. inadequate availability of ASA + AMP + ADP + ATP) and if controlled at birth would probably control the total disease. This first, and most important mechanism, sets the stage for the second and the third. The fust is determined by the work destiny of the muscle. Those muscles which secure posture and stability in the standing position axe the most vulnerable (10). In the normal infant and in the DMD infant, postural function is maintained primarily by the physical property of skeletal muscle elasticity. This property disappears in Dh4D skeletal muscles (as evidenced by loss of muscle stretch reflexes) (lo), the pelvis rotates forward, muscles get stretched and active muscle contraction increasingly but ineffectively attempts to compensate for postural loss. It is here with sustained contraction activity that the calcium necrosis mechanism plays its role. The most worked fibers become necrotic and become the first replaced by adipose tissue (10) (e.g. the gluteal muscles). This disease mechanism appears to be most active during the elevating phase of the CK maximum value (i.e. during the first 4-5 years of life). A second mechanism is associated with muscle inactivity. When the DMD youngster loses his ability to walk he can still elevate his arms overhead

MEDICAL HYPOTHESES

without difficulty. Within 12-18 months of being chair-bound, he is limited to being barely able to lift his forearms against the force of gravity, and pronation aud supiaation at the wrists is impeded. Histologic evaluation of a forearm extensor at this stage of the disease demonstrates the primary mode of muscle fiber extinction to be adipocytosis (Fig. 16). a very passive mechanism in terms of muscle function; a very active biological mechanism in temrs of adipocyte activity. (We postulate this aspect of the disease to be related to ADP deficiency.) The third mechanism is traumatic muscle stretch. It is related to a) anatomical location and b) loss of protection against such activity with Ioss of the muscle stretch reflex. Such a phenomenon is readily demonstrated in a clinical feature first described by Gowers (21) and later emphasized by Bramwell (22), namely the selective wasting of the sterno-

Fig. 16 Dh4D skeletal mucle forearm extensor. (Cqostat, H and E x 250) Demonatratcl compression of muscle in various stages of destmction (A, B aad C) by proliferating and growing intmfascicular adipose cells. l’%is mechanism of muscle dcstmction appears to be independent of calcium metabolism

153

THEDYSTROPHINCONNECllON-ATP?

of the pectoralis major muscles in contrast to the relative sparing of the clavicular origins. The lower border of the stemocostal head disappears progressively as the pelvis rotates forward and the rib cage tilts posterially (10). The stretch force brought to bear on the lower pectorals by this deformity is tremendous. Muscle stretch necrosis appears as Zenkers hyaline degeneration and on routine stain is not different in appearance from calcium necrosis (7). Not mentioned above am the muscles which are clinically spared in this disease. They are not spared genetically or metabolically. They are spared from the disease accelerating mechanisms. What is the nature of the dystrophin-ATP link? The reason for the ASA -_) AMP + ADP -+ ATP malfunction with sustained energy demand is not evident. The lipid manifestation is demonstrable in the myocyte. (11). This apparently does not interfere with the muscles’ development or with the developed muscles’ participation in modest physical activity. As energy demand increases. however, there appears to be a point of no return where the sustained energy demand exceeds the affected muscles’ compensatory and recuperative abilities. If a chemical link could be found and successfully treated, it could greatly reduce if not eliminate the need of ASA replacement. If such a link exists, it may well be found in relation to the loss of the muscle’s physical property of elasticity. It is not clear whether this loss is a biochemical phenomenon or, if as seems more probable, results from physical attrition to the muscle fiber and its defective sarcolemma by sustained muscle contraction. In this regard, the ATP connection may well be an ATP defect in modulation of a calcium-dependent or other channel (23). If such should prove to be the case, then ASA replacement therapy would need to be initiated as early in life as possible before further structural damage to the muscle fiber or its membrane could occur (24). To do this, however, a method for delivering the daily metabolic need for a lifetime would have to be devised (e.g. an intraperitoneal catheter with a disposable and easily-replaceable sterile inner liner). In closing, a word about glucagon and about AS A. We conceive the use of glucagon as a method for evaluating creatine chemistry in these dystrophies, not as a means for therapy. With regard to ASA, it is mgretable that this metabolite is not yet available and at reasonable cost for clinical trial. The glucagon Cr/C response of the various girdle

costal origins

dystrophies suggests that ATP deficiency may be common to all, although the genetic mechanism to explain the deficiency will be different. Further, ragged red muscle fiber disease and other disorders (e.g. Liebers optic atrophy) characterized by mitochondrial (the ATP source) degeneration merit a therapeutic ASA trial. Our limited phase I-phase II studies with ASA suggest the metabolite to be safe to use. The importance of ATP as the principal source of chemical energy in all living things is well recognized. The fact that ASA can pass membrane barriers to enhance this energy source merits verification and utilization.

Acknowledgements Support assistance continuously since 1958 by the Muscular Dystrophy Foundation of Indiana Inc., and The Indiana Neuromuscular Research Laboratory Inc.; horn 1959 to 1982 by the United Way of Greater Indianapolis Inc.; from 1979 through 1988 by Community Hospitals Foundation Inc.; in 1989-1990 by the Muscular Dystrophy Association; by an anonymous donor, the Estates of Frank and Anna Landwerlen; and countless other heIpfu1 individuals and corporations. Special thanks are extended to Dr Clifford Wfiliams. Dr Alexander Ross and Mr Kurt Pantzer Jr, who made this long-term study possible. Thanks also to Drs Mark Dyken, Vimal Patel, Jam Muller, and Karen West who gave invaluable assistance along the way. Also to Drs David Henry, Martin Badger and Mike Schmidt and the Eli Lily Company who provided advice and testing during the glucagon aspect of this study.

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