Treatment of calcinosis universalis with low-dose warfarin

Treatment of Calcii-rosis Universalis with Low-Dose Warfarin

ROBERT G. BERGER, M.D. GERALD L. FEATHERSTONE, B.S. RALPH H. RAASCH, Pharm.D. WILLIAM H. MCCARTNEY, M.D. NORTIN M. HADLER, M.D.

Chapel Hill, North Carolina

From the Division of Rheumatology/lmmunolo- gy, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina. Requests for reprints should be addressed to Dr. Robert G. Berger, Division of Rheumatology, Department of Medicine, University of North Carolina, 932 Faculty Laboratory Office Building, Chapel Hill, North Carolina 27514. Manuscript submitted July 24, 1986, and accepted February 25, 1967.

Patients with calcinosis universalis secondary to dermatomyositis or systemic sclerosis have increased levels of the calcium-binding amino acid, gamma-carboxyglutamic acid. The enzyme that effects gamma carboxylation of glutamic acid is warfarin-sensitive. Four patients with calcinosis universalis were treated with 1 mg per day of warfarin for 18 months in a non-blind initial study. Two patients had both decreased gamma-carboxyglutamic acid urinary concentration and decreased extra-skeletal uptake on technetium 99m-diphosphonate whole-body nuclear scanning. In a subsequent double-blind placebo study, two thirds of the patients receiving 1 mg per day of warfrin had decreases in extra-skeletal nuclear tracer uptake after 18 months, compared with none of the four patients receiving placebo. No patient had a change In clinical assessment, bleeding complication, or baseline normal prothrombin time. This low-dose warfarin regimen appears to have no demonstrable adverse effects, and these results suggest a beneficial effect on the progression of calcinosis in these rheumatic diseases.

In some patients with dermatomyositis or systemic sclerosis, disabling soft tissue calcifications develop. When severe and widespread, this calcinosis universalis may produce more morbidity than the underlying rheumatic disease [I]. Patients with calcinosis universalis have high levels of the calcium-binding amino acid, gamma-carboxyglutamic acid, in involved tissue [2]. Additionally, elevated urinary levels of gamma- carboxyglutamic acid have been found in both patients with calcinosis universalis and patients with systemic sclerosis or dermatomyositis without calcinosis [3,4]. The carboxylation of glutamine is coupled to the vitamin K cycle and is sensitive to the inhibitory effects of warfarin [5,6]. We postulated that the peripheral carboxylation of glutamine might be particularly sensitive to inhibition by a low dose of warfarin. This study reports an initial l&month non-blind study involving four patients with calcinosis universalis who received low-dose warfarin (phase I) and a subsequent 18-month randomized double-blind drug/placebo study involving eight patients (phase II).

PATlENTS AND METHODS

Phase I. Four patients with dermatomyositis or systemic sclerosis and evidencing multiple areas of subcutaneous calcinosis were evaluated over a period of 18 months. Clinical, radiologic, and laboratory assessments were performed at entry and at six-month intervals during the study. All patients received 1 mg per day of warfarin orally. Phase II. Eight patients (including the four patients in the phase I study) with dermatomyositis or systemic sclerosis and evidence of substantial subcutaneous calcification in multiple areas were randomly assigned to

72 July 1987 The American Journal of Medicine Volume 83

LOW-DOSE WARFARIN FOR CALCINOSIS UNIVERSALIS-BERGER ET AL

receive i mg warfarin per day orally or an identical placebo capsule for 18 months. Patients were evaluated at entry to the study and at six-month intervals by similar, although not identical, clinical, radiologic, and laboratory assessments as in the phase I study. In both phases, patients continued to receive any medications they were given at entry. Clinical Assessment. All patients were evaluated clinical- ly by the same examiner who was not aware of the therapy administered to patients in phase II. The presence or ab- sence of rash, muscle weakness, acrosclerosis, or proxi- mal skin thickening was used to assess activity of the underlying rheumatic disease. The clinical extent of the calcinosis was graded as follows: l+, radiologic evidence of calcinosis only, without palpable deposits: 2-j=, multiple small areas of palpable deposits without tumoral calcifications; 3-l-, widespread extensive deposition without tumoral areas; and 4+, widespread extensive deposition with large tumoral areas and/or skin breakdown. The largest diameter of a single “signal lesion” was measured at each visit. Laboratory Assessment. Complete blood count, platelet count, prothrombin time, data from urinalysis, and calcium, phosphorus, creatine phosphokinase, blood urea nitrogen, and creatinine levels were obtained at each visit. During phase I only, at entry, six months, and 12 months, 24-hour urine collections were frozen at -2O’C and analyzed later for creatinine and gamma-carboxyglutamic acid content. Gamma-carboxyglutamic acid concentration was ex- pressed as pmole of gamma-carboxyglutamic acid per g of creatinine. Urinary gamma-carboxyglutamic acid was as- sayed as follows:

Frozen 24-hour urine specimens with sodium azide as preservatives were clarified by centrifugation. Sulfa- salicylic acid, 10 percent, was used to deproteinize and adjust the pH to 2.2. A 5 ml aliquot was put over an AG50= 2X (sodium) column. The water wash was collected as a second sample, not pooled as Kuttan et al [7] had done. The water-eluted peak was split into two equal portions and spun to dryness in a Savant Speed-Vat two times, using water, pH IO, to remove ammonia traces. A half-portion was hydrolyzed for 22 hours in 6 N hydrogen chloride, and the other half was hydrolyzed with sodium hydroxide according to Madar et al [8] in polyallomer (Beckman) tubes inside Glenco vacuum pyrolysis tubes.

All samples were dried in a Speed-Vat, taken up in 0.2 M sodium citrate buffer, and pHadjusted to 2.2 before quanti- tation on a Glenco MM-70 amino acid analyzer. The car- bamazepine-gamma-carboxyglutamic acid standard was kindly supplied by Dr. Richard G. Hiskey of the University of North Carolina-Chapel Hill Chemistry Department. The glu- tamate standard was Pierce Chemical Company’s amino acid standard “H” with an interassay coefficient of variation of 0.23. The nmol of gamma-carboxyglutamic acid per ml of 24=hour collection of urine was determined by the differ- ence of the acid and base hydrolysis for each sample. Radiologic Assessment. In phase I, plane radiographs of a single selected area of calcinosis were obtained from patients at entry and then during each follow-up visit. These were interpreted by the same radiologist. Each patient underwent whole-body bone scintigraphy two hours after injection of 0.21 mCi/kg of technetium 99m=high-density

TABLE i Urinary Gamma-Carboxygiutamic Acid Concentration (pmollg creatinine) in Phase I Patients

Patient Initial Six Months of Warfarin 12 Months of Warfarin

1 133 276 148 2 228 197 *

3 120 172 47 4 199 207 51

l Lost to follow-up.

polyethylene oxidronate. Identical positioning was performed for the serial studies. All scans were interpreted by the same radiologist and were graded on a qualitative scale from no extra-skeletal uptake to mild, moderate, or wide= spread extra-skeletal uptake. Specific areas were not graded. In the phase II study, radiologic assessment was identical except for the grading technique on the bone scanning. On each bone scan, all areas with extra-skeletal uptake were graded according to the following scale: 0, no extra- skeletal uptake; 1, barely perceptible extra-skeletal uptake; 2, extra-skeletal uptake less than in adjacent bone; 3, extra- skeletal uptake equal to that in adjacent bone; 4, extra- skeletal uptake greater than in adjacent bone. A sum of abnormal area scores was obtained for each scan.

RESULTS

Phase I. On entry to the phase I study, none of the patients had clinical or laboratory evidence of activity of their underlying rheumatic disease. Bone scanning and plane radiology demonstrated evidence of extra-skeletal calcification in multiple areas in all four patients.

None of the patients receiving warfarin during the initial 18 months had a change in clinical severity of calcinosis as assessed by the examiner, or change in baseline laboratory assessment. No patient had clinical evidence of activity of the underlying rheumatic disease. Patients 3 and 4 had a marked decrease in urinary concentration of gamma-carboxyglutamic acid at 12 months, as shown in Table I. Patients 1 and 2 had no change in radiologic assessment during the study. Patient 3 had improvement on qualitative interpretation of bone scans over the study course, but no change on plane films. Patient 4 had complete resolution of extra-skeletal uptake on bone scanning, as well as resolution of calcific areas noted on plane films during the 18 months of the study. No patient had bleeding complications or change in baseline normal prothrombin time during the study. Phase II. Eight patients initially entered the double-blind study (including the four patients in the phase I study). One patient in the warfarin-treated group was excluded because of noncompliance, leaving seven patients who completed 18 months of the study. Patient characteristics at entry are shown in Table II. None of the patients had clinical or laboratory evidence of activity of underlying

July 1987 The American Journal of Medicine Volume 83 73


TABLE II Patient Characteristics at Entry to Randomlred Study*

Patient Age Rheumatic

and Sex Disease

Duration of Rheumatic

Dlsease (years)

Duration of Clinical Caicinosis Extent of

(years) Caicinosis Concurrent Concurrent

Drug Therapy Chronic illness

Placebo 6 18F Dermatomyositis 7 20F Sclerodermal

dermatomyositis

1 15F Dermatomyositis

2 66F Scleroderma

Warfarin 5 8OF Scleroderma

3 12M Dermatomyositis/ scleroderma

4 17M Dermatomyositis

l Patients 1 to 4 were involved in Phase I.

9 7 4+ 12 10 4+

6 3 3+

12 6 2+

19 10 2+

5 9 4+

6 4 it

None None Prednisone, 10 mglday; None

amitriptyline, 20 mg/day

Prednisone, 8 mg every Hypertrigiyceridemia other day

insulin, methyidopa, Diabetes meliitus, con- digoxin, hydrochioro- gestive heart failure thiazide

Prednisone, 15 mg/day; None aspirin, 1,800 mg/day; methyldopa, 500 mg/ day; hydrochiorothia- zide, 50 mg/day

Prednisone, 10 mg None every other day

None None

rheumatic disease at entry. Patients 1 and 4 had asymp- tomatic increases in creatine phosphokinase at six-month follow-up that continued throughout the rest of the study. No other patient had either laboratory or clinical evidence of activity of the underlying rheumatic disease during the course of the trU. No patient had bleeding complications or change in baseline normal prothrombin time.

There waSi no change in clinical assessment of calcinosis or plane films in either group during ttie study. Changes in bone scan scores are illustrated in Table III. Two of three patients in the warfarin-treated group had improvement at 18 months in bone scan scores compared with none of four patients in the group receiving placebo. The two patients in the treatment group with improvement in total scores (Patients 4 and 5) had decreases in uptake in all existing areas and no appearance

TABLE III Extra-Skeletal Uptake of Technetium-99m High-Derislty Polyethylene In Phase II Patients

Patient

Placebo 6 7 1 2

Warfarin 5 3 4

inlliai Total Final Total

23 25 11 15 10 10

9 9

21 15 15 17 10 1

of new areas. In both groups, patients with no improvement in total sdore had no change in individual areas of extra-skeletal uptake. Two patients in the placebo-treated group (Patients 6 and 7) had appearance of new areas of uptake during the study.

The serial bone scan results over both phases of the study in Patient 4 are shown in Figure 1 and correlated with the use of warfarin. Because the bone scan grading techniques changed between phase I and II of the study, the phase II bone scans were re-evaluated using the qualitative scale applied in the phase I study. During the phase I study, at 18 months, the patient had no abnormal activity observed on bone scanning and had resolution of calcification on plane radiography as well. At that time, warfarin treatment was stopped between phase I and II. In the ensuing six months, bone scanning again revealed abnormal activity at the previous level, although results of plane radiography remained normal; when warfarin therapy was begun again, he had a similar response with almost no extra-skeletal activity seen on bone scanning at 12 months (Figure 2).

COMMENTS

Although high levels of gamma-carboxyglutamic acid are found in involved tissue and urine of patients with calcinosis universalis, its role in ectopic calcification is not clear. We postulated that gamma-carboxyglutamic acid was indeed of central importance in ectopic calcification, as it is present in high concentration in normal mineralized tissue such as bone and tooth dentin. Likewise, gamma-



Figure 1. Serial bone scan results in Patient 4.

Figure 2. Left, image of anterior tibias from bone scan obtained with technetium-99m high-density polyethylene: image shows markedly increased uptake in the soft tissues of the right medial shin area. Right, image of anterior lower legs from bone scan obtained with techne- Cum-99m high-density polyethylene; image shows marked reduction in degree of soft tissue accumulation of the radi& pharmaceutical compared with the prt- vious study.

Warfarin Warfarin begun stopped begun

0) 5 Moderate

t + c

h 3 5 f 5 $5 0 Mild

g I2 9 ‘;: 0 c 5 6 None

0 6 12 18 24 30 36 42

L Months

carboxyglutamic acid is essential to the coagulation cas- cade because of its calcium-binding properties within the vitamin K-dependent coagulation factors [s]. Gamma- carboxyglutamic acid has also been demonstrated in high concentration in porcine bioprosthetic valves that have undergone calcific degeneration after aortic valve re- placement. There are conflicting reports in the literature on in vivo inhibition of this process by long-term antico- agulant doses of warfarin [9, IO]. lt was our hope that the post-translational carboxylation of glutamic acid residues

to form gamma-carboxyglutamic acid in the involved soft tissues of calcinosis universalis would be inhibitable by smaller doses of warfarin than those used to inhibit the carboxylation of the vitamin K-dependent coagulation factors in the liver. The two patients in the phase I study who had improvement in their bone scan activity both had substantial decreases in the urinary concentration of gamma-carboxyglutamic acid after 12 months of low-dose warfarin therapy, but not at six months. One patient who had no improvement in bone scan activity during the

July 1987 The American Journal of Medicine Volume 83 75


phase I study and who had urine available for serial study had no change in urinary gamma-carboxyglutamic acid concentration. This suggests that the presumed aberrant carboxylation of glutamine is inhibitable by low doses of warfarin given on a long-term basis, but this inhibition is variable, and appears to require prolonged administration of watfarin when used at such a low dose.

Previous attempts at treatment of calcinosis universalis have included use of oral phosphate binders, probenecid, corticosteroids, and most recently diphosphonates [ 11,121. None of these agents has met with success in their use in a limited number of patients. Furthermore, previous studies of these therapeutic agents involved few patients and have used primarily plane radiology to assess the progression of soft tissue calcification. This technique is limited by the lack of sensitivity and technical difficulties in standardizing measurement of plane radiographs. Technetium bone scanning has been used to assess and follow the degree of ectopic calcification in myositis ossificans, and we adapted its use to our patients with calcinosis universalis [ 13,141. It was apparent during the phase I study that we were seeing improvement in the findings on bone scanning interpreted on a qualitative basis, and we instituted a more detailed numerical scoring system for the second phase of the study.

By the use of bone scan scoring in phase II, it is evident that the clinical progression or regression of ectopic calcific lesions is a very slow or intermittent process. No patient in either group had a change in the results of their clinical examination. Furthermore, no patient in the placebo-treated group had worsening of bone scan total

scores in the phase II study, although two had appearance of new areas of extra-skeletal uptake. The two patients receiving warfarin who had improvement in bone scan scores in the phase II study had less extensive calcinosis than did the patient without a response. The patient with the most striking response in the therapeutic group had no clinical calcinosis on physical examination and the lowest initial bone scan score. Perhaps earlier disease without large existing areas of calcium deposition might be more responsive to inhibition of the production of gamma- carboxyglutamic acid in the involved tissue, and accretion of mineral front in existing large lesions may not be inhibited by a decrease in gamma-carboxyglutamic acid concentrations.

The use of 1 mg of warfarin per day orally appears to be unassociated with any side effect, since no patient had increased bleeding tendency or change in baseline normal prothrombin time. Because of the small number of patients studied, we cannot statistically support our argu- ment for the efficacy of low-dose warfarin in calcinosis. Nonetheless, it would appear that it has benefit in those patients with mild clinical disease and perhaps would be beneficial to those patients with more severe disease if used for an extended period.

ACKNOWLEDGMENT

We would like to thank Richard L. Clark, M.D., Depart- ment Of Radiology, University of North Carolina, for his assistance in reviewing the plane radiographs for this study.

REFERENCES

1. Rodnan G: Progressive systemic sclerosis. In: McCarty D, ed. Arthritis and allied conditions. Philadelphia: Lea and Febiger, 1979; 797-798.

2. Lian J, Skinner M, Glimcher M, Gallop P: Presence of gamma carboxyglutamic acid in the proteins associated with ectopic calcification. Biochem Blophys Res Commun 1976; 73: 349-355.

3. Lian J, Pachman L, Gunberg C, Partridge N, Gallop P: Gam- ma carboxyglutamate excretion: a marker in patients with ectopic calcification disorders. Arthritis Rheum 1979; 22: 634-635.

4. Lian J, Pachman L, Gunberg C, Partridge R, Manjowski M: Gamma carboxyglutamate excretion and calcinosis in juvenile dermatomyositis. Arthritis Rheum 1982; 25: 1094-l 100.

5. Gallop P, Lian J, Anuschka P: Carboxylated calcium binding proteins and vitamin K. N Engl J Med 1980; 302: 1460- 1466.

6. Levy R, Lian J: Gamma carboxyglutamate excretion and warfarin therapy. Clin Pharmacol Ther 1979; 25: 562- 570.

7. Kuttan R, Wilson N, Tenni R: Determination of y-carboxyglutamic acid excretion in urine. J Chromatogr 1981; 223:

182-187. 8. Madar DA, Willis RA, Koehler KA, Hiskey RG: Analysis of y-

carboxyglutamic acid via amino acid analysis. Anal Bio- them 1979; 92: 466-472.

9. Stein P, Riddle J, Kemp S, et al: Effect of warfarin on calcification of spontaneously degenerated porcine bioprosthetic valves. J Thorac Cardiovasc Surg 1985; 90: 119-125.

10. Milan0 A, Burtolotti V, Talenti E, et al: Calcific degeneration as the main cause of porcine bioprosthetic valve failure. Am J Cardiol 1984; 53: 1066-1070.

11. Metzger A, Singer F, Bluestone R, Pearson C: Failure of disodium etidronate in calcinosis due to dermatomyositis and scleroderma. N Engl J Med 1974; 291: 1294-1298.

12. Fleisch H: Experimental basis for clinical use of diphosphonates in Paget’s disease of bone. Arthritis Rheum 1980; 23: 1162-1167.

13. Suzuki Y, Hisada .K, Takeda M: Demonstration of myositis ossificans by Tc 99 pyrophosphate bone scanning. Radi- ology 1974; 111: 663-664.

14. Powers TA, Touya JJ: Tc-99m-pyrophosphate bone scan in calcinosis universalis. Clin Nucl Med 1980; 5: 302- 304.


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Treatment of calcinosis universalis with low-dose warfarin