Stanozolol in postmenopausal osteoporosis: Therapeutic efficacy and possible mechanisms of action

Stanozolol

in Postmenopausal Osteoporosis: Therapeutic and Possible Mechanisms of Action

Charles H.

Chesnut III,Joel L. Ivey, Helen E. Gruber, Karen Sisom, and David J.

Meredith

Matthews,

Efficacy

Wil B. Nelp,

Baylink

To assess the efficacy of the anabolic steroid stanozolol in the treatment of osteoporosis, a 29-month double-blind study was performed with 23 treated and 23 control postmenopausal osteoporotic women. Drug efficacy was assessed by serial determinations of total body calcium (TX-total bone mass) by neutron activation analysis, regional bone mass (RBM) by single-photon absorptiometry, and by spinal roentgenograms. Total body calcium increased 4.4% from baseline values (P < 0.01) in the treated group and remained unchanged in the control group; the difference in the change in TBC between the treated and control groups was significant ( P = 0.03). The effect of the drug on TBC persisted throughout the 29-month period. In contrast to TBC, measurements of RBM indicated no signficant difference between the treated and placebo groups, suggesting a possible differential response to therapy at various skeletal sites. No new spinal compression fractures were noted in the treated group (compared with three new fractures in the control group). Assessment of serum and urine values indiceted a decrease in the level of urinary calcium and an increase in the level of total urinary cyclic AMP in the treated group. These changes were observed even though the level of serum iPTH was significantly decreased during the study. An analysis of changes in bone biopsy specimens revealed no significant differences between the treated and control groups. Seventy-six percent of the treated subjects developed SGOT elevations or other side effects from the stanozolol therapy; at no time were these effects sufficiently severe to cause termination of medication. The data suggest that long-term use of stanozolol increases the net total bone mass above pretreatment levels.

T

HE ROLE of synthetic anabolic steroids in the treatment of postmenopausal osteoporosis has been uncertain. Previous studies’.‘,’ utilizing these agents, while suggesting potential therapeutic benefits, were uncontrolled, included relatively few patients, and demonstrated only short-term (less than 12 months) potential benefits. Recent double-blind controlled studies of the anabolic steroid methandrostenolone4,’ indicated a positive and persistent effect of this medication in postmenopausal osteoporosis over the long term (24-26 months), but the trial utilized essentially one technique only for establishing drug efficacy (total body calcium by neutron activation: TBCNAA). Also, in these and other previous studies, little information was available regarding the mechanism of action of the drug. To further investigate the actions of the synthetic anabolic steroids in postmenopausal osteoporosis, a long-term double-blind controlled clinical study of the anabolic steroid stanozolol (Winstrol, Winthrop Laboratories) was performed using a number of the currently available techniques for determining the therapeutic effect on bone mass, as well as multiple serum, urine, and histologic variables in an attempt to delineate the mode of action of the drug. MATERIALS

AND

METHODS

Patients Studied

Forty-sixpostmenopausalosteqorotic white women aged 50 to 80 yearswereselectedfrom patients self-referred or referred by their physicians to the study. Informed consent was obtained from each patient according to procedures established by the Human Subjects Review Committee of the University of Washington. Selection criteria were vertebral osteopenia (as evidenced by cortical thinning, loss of horizontal trabeculation, and anterior wedging) or one or

more atraumatic spinal compression fractures, normal serum calcium and phosphorus determinations on two occasions, and the absence of recognized causes of secondary osteoporosis or other metabolic bone disease; the latter were excluded by history. physical examination, and appropriate laboratory evaluations. Patients selected for the study were ambulatory and had received no therapy for osteoporosis within six months of the beginning of the study.

Initial Features of the Treated and Placebo Groups Patients selected for the study were randomized according to age and TBC to treated (stanozolol) or control (placebo) groups of 23 patients each. As noted in Table 1, the baseline values for the treated and control groups were comparable. Treated patients received 2 mg of stanozolol three times a day (total daily dosage of 6 mg) for three of every four weeks; control patients received identically packaged placebo medication on the same dosage schedule. Both groups were instructed by professional dietitians on the maintenance of a 1000-mg elemental calcium intake (dietary calcium or calcium supplements or both) throughout the period of participation in the study. Patients were followed in the study for approximately 29 months.

From the Division of Nuclear Medicine, Departments of Medicine and Radiology, University of Washington, Seattle, WA. and the Mineral Metabolism Laboratory, VA Medical Center, American Luke, Tacoma, WA. Acceptedfor publication November 30,1982. This work was supported by the Sterling-Winthrop Research Institute, Rensselaer, NY and Winthrop Laboratories, New York, NY. A portion of this work was conducted through the Clinical Research Centerfacility of the University of Washington, supported by the National Institutes of Health, grant RR37. Portions of this work were presented at the American Society for Bone and Mineral Research, Anaheim. California, 1979. Address reprint requests to Charles H. Chesnut. III, MD, Division of Nuclear Medicine NN203, RC-70. University Hospital, University of Washington, Seattle, WA 98195. 0 1983 by Grune & Stratton, Inc. 0026%0495/83/3206-0007$02.00/0

CHESNUT III ET AL

572

Table 1. Initial Features

in Treated

and Control Patients at Baseline Normal Range

Variable Age fyr)

-

Height (cm) Racial extraction

Treated Patients 68.3

? 1.3

69.0

? 1.5

156.7

* 1.6

156.8

+ 1.4

Northern European

Years since menopause

-

22.5

Dietary calcium intake (mg/24 hr)

497.6

Compression fractures

? 2.0 + 54.4

2.6 ? 0.5

TBC-NAA (gm Cal

678.7-865.9

Control Patients

Northern European 23.2 458.6

+ 3.0 * 88.5

2.5 f 0.5

624.8

+ 22.1

640.8

* 23.1

BMC Sl site (gm/cm)

0.74-0.94

0.66

+ 0.03

0.69

* 0.03

S2 site fgm/cm)

0.70-0.90

0.68

* 0.03

0.70

* 0.03

Serum calcium (mg/ 100 ml)

8.9- 10.2

Serum phosphorus (mg/ 100 ml)

3.0-4.5

Serum alkaline phosphatase (U/liter)

20.0-

Serum iPTH (pg/ml)

86.0-480.0

Fractional calcium absorption f%)*

24.2-40.6

Exchangeable calcium pool size (gm)

3.3-5.6

Urinary calcium (mg/24 hr) Urinary calcium/creatinine (mg/mg/24

105.0

50.0-

0.06-0.23

hr)

Urinary hydroxyproline/creatinine (rg/mg/24

150.0

3.8 f 0.1 76.6 308.1

t 4.4 +_ 19.5

19.6 + 2.9 4.7 * 0.2 124.8

9.4 f 0.1 3.7 * 0.1 68.7 320.7

+ 4.4 + 22.6

19.3 + 1.7 4.4 * 0.2

* 15.4

108.6

* 11 .o

0.18 ? 0.02

0.15

* 0.01

28.4

24.0

+ 1.6

hr)

Total

8.9-33.4

Nondialyzable

1.7-3.2

Urinary cyclic AMP (total) fpmole/24

9.3 f 0.1

hr)

+ 2.0

2.2 t 0.1

2.0 + 0.1

2.6 + 0.4

2.7 f 0.3

Values are mean ? SEM. n = 23 for all variables except dietary calcium intake (17 treated and 15 control), fractional calcium absorption and exchangeable calcium pool (20 treated and 22 control), and urinary cyclic AMP (total) (14 treated and 8 control). No significant differences (as determined by Student f-test) were noted between treated and control groups for any variable. *Determined by dual isotope technique of DeGrazia et al.”

Total Body Calcium Measurements Total body calcium was measured by neutron activation with a precision of r 2%6 and accuracy of k 5.2%’ at intervals of six to nine months throughout the study. Since in the majority of normal and osteoporotic subjects 98% to 99% of TBC is within the skeleton4 TBC is in effect a measure of total bone mass.

Regional Bone Mass Measurements Measurements of regional bone mass (RBM) by photon absorptiometry were also performed at six-to-nine-month intervals: a Norland-Cameron Bone Mineral Analyzer, Model 178, was used. Bone mineral content (BMC) measurements were performed at two sites, Sl and S2 (respectively one-tenth and one-fifth of the forearm length proximal to the styloid process), of the nondominant arm (distal radius): the Sl site contains both cortical and trabecular (12% to 20%) bone, whereas the S2 site consists primarily of cortical bone.’

Serum, Plasma, and Urine Measurements All blood measurements were performed on specimens obtained in the morning after fasting at the baseline and six-to-nine month intervals unless otherwise noted. Variables assessed by standard laboratory techniques included a complete blood count, levels of serum electrolytes, calcium, phosphorus, glucose, creatinine, BUN, and cholesterol (at the baseline and the conclusion of the study); levels for serum SCOT, total alkaline phosphatase, bilirubin, and total protein with albumin/globulin ratio were obtained to assess possible hepatotoxic effects of stanozolol. Serum protein electrophoresis, latex fixation, and thyroxine (T4) were obtained at the baseline (and were within normal limits in all patients). The level of serum iPTH (immunoreactive Parathyroid Hormone) was determined utilizing a radioimmunoassay detecting primarily the amino terminal

portion of native bovine PTH (AS-21 l/32 antiserum, BurroughsWellcome Laboratories, London, England)?~” Twenty-four-hour urinary calcium and creatinine levels were determined by standard laboratory methods at the baseline and six-to-nine-month intervals; also patients were placed on a low hydroxyproline diet for 24 hours preceding urine collection for hydroxyproline determinations. Urine samples for hydroxyproline were stored at - 20 “C; total hydroxyproline was assessed using the method of Prockop and Udenfriend,” and nondialyzahle hydroxyproline was assessed by the method of Krane et al.” The level of urinary cyclic AMP (total) was determined according to the method of Gilman” in randomly selected study participants (14 treated and 8 control subjects at the baseline, 21 treated and 17 control at the conclusion of the study) in an effort to confirm iPTH changes.

Bone Biopsy Measurements Twenty treated and 19 control patients were biopsied at the beginning of the study; 20 treated and 16 control patients were biopsied at the conclusion of the study. With the patient under local anesthesia, biopsy specimens (0.4 x 2-cm cylinders) were obtained with an electric drill from the right iliac crest at the beginning of the study and from the left iliac crest at the conclusion of the study, 2 cm posterior to the anterior superior spine. Before each biopsy, each subject received two courses of tetracycline (generally tetracycline hydrochloride, 250 mg q.i.d. for three days, and 14 to 15 days later, demeclocycline, 150 mg q.i.d. for two days) so that kinetic bone values could be determined. The biopsy specimens were fixed in cold neutral buffered formalin, dehydrated, embedded in methacrylate, and sectioned on a Zeiss Universal Cut-All microtome at 6g for Goldner stain sections,” and at 7.5~ for tetracycline analysis. For quantitation of trabecular bone values,‘5 a Leitz Orthoplan microscope, optical bench, camera lucida, and digitizing tablet

573

STANOZOLOL IN OSTEOPOROSIS

interfaced with a PDP-8/E computer were used. The following variables were obtained from Goldner stained sections: percentageof total bone area (as a percentage of the total tissue area of the biopsy specimen), percentage of osteoid area (as a percentage of the total bone area), percentage of forming surface, percentage of resorbing (crenated) surface, and percentage of neutral surface (as a percentage of the total surface length of the trabeculae); and mean width of osteoid seams (in microns). Unstained 7.5~ sections were examined under a fluorescence microscope to determine the average distance (in microns) between double labels; the bone apposition rate (microns/day) calculated by dividing the mean width between double labels by the number of days between the administration of the first and second tetracycline labels; the mineralization lag time (days) calculated as the mean width of osteoid seams in microns divided by the bone apposition rate; the rate of osteoid maturation (percent/ day) as obtained by dividing 100% by the mineralization lag time; and the bone formation rate (mm’/mm*/day) calculated as the product of the bone apposition rate and the length of the trabecular surface involved in formation (as indicated by osteoid) divided by the total tissue area. All histologic variables were measured with a precision ranging from 1% to 8%.15

Roentgenographic

Measurements

Thoracic and lumbar spine films in anterior and lateral projections were obtained at the baseline, after approximately one year of participation in the study, and at the conclusion of the study. The roentgenograms were evaluated according to the following criteria: the anterior and posterior lateral heights of each vertebral body from T-3 and L-5 were measured with a standard centimeter ruler. A grade reflecting the degree of vertebral collapse resulting from wedging or compression fracture was determined as follows:

(1) Anterior vertebral height/posterior

vertebral

Statistical Analysis Standard statistical methods were used;16 all variables studied were analyzed with respect to all subjects entering the study. Comparison of group means (between-groups analysis), both absolute means and means of the changes between groups, were performed with the Student t-test for independent groups using pooled variance estimates based on the individual variance estimates. Significances of changes within a group (within groups analysis) were assessed using the Student r-test for paired data. Changes in the various parameters within and between groups were computed from the baseline; in addition, interval changes between successive parameter determinations were analyzed. To determine the rate of change, linear regression analysis was used to obtain the best slope estimate for each subject’s data using the actual times between measurements. The mean slope for each group was determined from the individual slope estimates. This approach takes into account variability in measurement times for the subjects. and equally weighs all of a patient’s data in determining the slope estimate. Significance of the slopes (compared with a slope of zero) was evaluated using the Student t-test for paired data, taking the individual slopes as a measure of change per month. When appropriate, nonparametric methods were used to evaluate differences between groups (two-sided Fisher exact test). Paired differences from baseline were evaluated both as actual changes and on a percentage change basis. The statistical significance of changes evaluated in both fashions were in good agreement. Urinary calcium, hydroxyproline, and total urinary cyclic AMP could not be normalized for urine volume or lean body mass by urinary creatinine, as urinary and serum creatinine increased in the treated group during participation in the study.

height x 100 =

RESULTS

A(%).

(2) If A = 100%. a grade of 0 was assigned; if A < 100% and >80%, a grade of I was assigned; if A < 80% and >60%, a grade of 2 was assigned: if A < 60%. a grade of 3 was assigned. (3) If A = 100% but biconcavity grade of 1 was assigned.

of the vertebral

body existed, a

(4) If both the anterior and posterior heights were reduced greater than 30% of the height of the immediately adjacent vertebrae, a grade of 3 was assigned. (5) A grade of I or 2 was defined as vertebral wedging; a grade of 3 was defined as a vertebral compression fracture. The Occurrence of a new wedged vertebral body was classified as a progression from grade 0 to I or 2; worsening of a previous vertebral abnormality was classified as evolution from grade I to 2 or 2 to 3. The occurrence of a new compression fracture was defined as a progression from grade 0 or I to 3. All x-ray films were graded by one investigator (CHC).

Dietary Calcium Measurement Dietary calcium intake was assessed at the baseline in 32 patients (I 7 treated and I5 control); subsequent intake was monitored by a dietitian via a daily calcium intake diary.

Clinical Evaluation Patients were clinically evaluated at approximate six-week intervals with particular attention to potential adverse effects of the study medication (edema, acne, hirsutism, etc.).

The data reported are from all 46 patients entering the study. Thirty-eight patients (21 treated and 17 controls) completed the study; eight subjects (two treated and six control) withdrew before the conclusion of the study, including two control patients who withdrew before their second TBC determinations. No factor (such as subjective awareness of drug effect) could be identified to account for the greater withdrawal in the placebo group. Total Body Calcium A significant 4.4% increase in TBC from the baseline determinations through 29 months in the treated group is noted in Figure 1; such a change was significantly different from that occurring in the control group. An analysis of the mean differences between the treated and control groups in the percentage of change of TBC from the baseline values at each TBC-NAA determination revealed that the magnitude of the mean differences increased throughout the period of study participation (Fig. l), although the mean difference between the two groups was significant only at the final (29-month) TBC-NAA measurement. The absolute changes were statistically comparable

574

CHESNUT III ET AL

significantly (P < 0.01) different from the mean slope value (+0.02 + 0.3) of the control group. I211 9 l I

+3%-

l --4

Regional Bone Mass

/

STANOZOLOC

/

-PLACEBO

i

,I ,I

16 Months

I 32

24

Percentage of change in TBC (grams) as a function of Fig. 1. treatment time in treated and control patients, mean k SEM. (n): l = significantly different from baseline, P < 0.01; a = change from baseline between treated and control groups significantly different, P = 0.03.

with the percentage changes both within and between groups (Table 2); in absolute terms stanozolol-treated subjects gained an average 25.6 t 9.2 (mean * SEM) gm of calcium ( + 30.2 mg/day/patient) while placebo patients lost 0.7 * 6.8 gm (-0.8 mg/day/patient). In the treated group 17 patients gained TBC and 6 patients lost TBC and in the control group 9 patients gained TBC and 12 patients lost TBC. This difference in the distribution of gains and losses between groups was statistically significant (P < 0.03, two-sided Fisher exact test). Significant differences were also noted when the means of individual slope values (TBC versus months from baseline) were evaluated: the mean slope value for treated patients, +0.84 k 0.2 gm calcium/month, differed significantly from zero (P < O.Ol), and was Table 2.

Slope changes in BMC in the treated and control groups were not significant at either the Sl or S2 sites; while a significant (P < 0.01) reduction in BMC at the S2 site was noted within the treated subject group between the baseline values and the 29-month determinations, no significant differences in BMC changes were observed at either site between groups. As noted in Figure 2, a significant correlation (r = 0.55, P -c 0.01) existed between slope changes in BMC (S2 site) and TBC in the treated patients; no other significant correlations between BMC and TBC were noted. Serum Values

The level of serum calcium increased significantly from the baseline values through 29 months in both the treated group (9.32 * 0.06 to 9.65 + 0.06 mg/lOO ml, P < 0.01) and the control group (9.40 + 0.05 to 9.66 * 0.13, P = 0.03), with no significant differences noted between groups in these increases. Significant decreases from the baseline value (P < 0.01) in serum phosphorus were noted at 9, 17, and 23 months in both the treated and control groups; changes between the groups were not significant. The level of serum total alkaline phosphatase decreased significantly (P < 0.01) from the baseline determination at both 9 and 23 (but not at 29) months in the treated group with the largest decrease occurring at 23 months (- 14.2 f 5.83 U/liter). These changes from the baseline value were significantly different from those in the placebo group (P < 0.02). Slope changes were not significant, however, either within or between the treated and control groups.

Total Body Calcium Determinations

in Treated

and Control Patients Interval

Gr0llp

Baseline

Treated Number of patients Actual value (gm; mean + SEM)

23 624.8

+ 22.1

Mean change from baseline (mean k SEMI Controls Number of patients Actual value (gm; mean r SEMI

9 Months

23

k 23.1

Mean change from baseline (mean + SEM)

23 Months

21

t 20.9

629.4

+ 20.9

630.3

c 20.0

-5.1

+ 6.4

+8.5

k 6.3

+9.50

-r 7.4

21

29 Months

21

619.7

23 640.8

17 Months

19

21 646.4 +25.6

18

k 23.1 r 9.2’ 17

632.2

t 26.4

650.1

+ 21.4

637.3

t 24.7

630.7

+ 23.6

-4.6

+ 7.8

+3.9

k 4.4

-0.9

+ 6.1

-0.7

t 6.8

Significance of difference between treated and control Actual Mean interval change *Significantly different from zero, P < 0.01.

NS -

NS

NS

NS

NS

NS

NS

NS

P = 0.03

STANOZOLOL IN OSTEOPOROSIS

575

min/globulin ratio, or cholesterol were noted in the treated or control groups. Urine Values

+

,

-6.0(x10-~)-/

-2

0

-1

+1

+2

f4

+3

+5

A TBC: gramslmcmth Fig. 2. Correlation of slope changes in BMC and TBC determinations at the S2 site of distal radius in treated patients (n = 21) completing the study.

As noted in Figure 3, the level of serum iPTH decreased significantly from the baseline value in the treated group; such a decrease was significantly different from changes in the controls. The treated group displayed a significantly negative slope (- 2.24 k 0.98 pg/ml/month, P < 0.05), which was significantly (P < 0.01) different from the slope of the controls. The level of serum creatinine increased significantly (P < 0.01) from the baseline value through 29 months (0.81 + 0.03 to 0.98 k 0.03 mg/lOO ml) in the treated group, and when compared with the control group (P < 0.01). No significant changes in the levels of serum electrolytes, glucose, BUN, bilirubin, total protein with albu-

The level of urinary calcium decreased significantly from the baseline determination in the treated group (Fig. 4); such changes were significantly different from changes in the control group at 9 months and approached significance at 29 months (P < 0.07). Based upon group means, total urinary cyclic AMP was significantly greater in the treated patients at 29 months (Fig. 5). As the number of samples obtained was different at the baseline determination and at 29 months, data were also evaluated in terms of paired differences from the baseline values; significant increases (+ 1.1 + 0.36 ymole/24 hr) were observed in the treated group (n = 14), with such increases being significantly different from changes (-0.06 + 0.35) seen in the control group (n = 8). No significant correlations were noted between the levels of total urinary cyclic AMP, and urinary calcium, creatinine, or nondialyzable hydroxyproline, serum calcium, creatinine, or iPTH, or bone biopsy values. The level of urinary creatinine increased significantly (P < 0.001) in the treated patients from the baseline values through 29 months (600.06 2 26.62 to 836.76 IL 37.92 mg/24 hr). No significant changes in creatinine clearance were noted in either the treated or the control groups. The level of total urinary hydroxyproline increased slightly in both groups, with a significant increase from the baseline values in the treated groups at 17 months (+5.23 * 2.43 mg/24 hr, P < 0.05), and in the placebo group at 23 months (+5.36 f 1.49 mg/24 hr. P -c +60

l

1

M

8+80-

F

--+

STANOZOLOL PLACEBO

.$ $+40B F P Og, 8 % -4o$ 2 - 80?I k

G-120-1

0

.---e

STANOZOLOL

v

PLACEBO

8

16 Months

I

24

#

32

Fig. 3. Mean change from baseline value in serum iPTH (pg/ml) es a function of treatment time in treated and control patients, mean + SEM. In); l = significantly different from baseline value, P < 0.01; l * = different from baseline value, P < 0.05; a,b = changes from baseline value between treated and control groups significantly different, P < 0.01 and P c 0.05, respectively.

1

I

- 604 0

8

A x+ Mo%s

24

32

Fig. 4. Mean change from baseline valus in urinary calcium (mg/24 hr) as a function of treatment time in treated and control patients, mean k SEM. (n); l, l * = significantly different from baseline value, P < 0.01, P = 0.02, respectively: a = changes from baseline values between treated and control groups significantly different, P < 0.01.

576

CHESNUT III ET AL

,a,c (21)

groups. Such changes were also noted when nondialyzable hydroxyproline was quantitated as a percentage of total hydroxyproline excreted.

l

T

Bone Biopsy

29 months

Baseline

Fig. 5. Urinary cyclic AMP (pmole/24 hr) in treated and control patients at the baseline and at 29 months, mean + SEM. (n); l = significantly different from baseline value, P = 0.008, (n = 14): a = change from baseline value between treated (II = 141 and control (n = 8) groups significantly different, P < 0.05; c = significantly different from control, P -c 0.001.

in changes between groups were noted. The level of nondialyzable hydroxyproline increased significantly from the baseline value through 17 months in the treated group (Fig. 6): such a change was significantly different from that seen in the control group at 17 months. Slope changes in nondialyzable hydroxyproline through 29 months were not significant, either within or between 0.01).

No significant

differences

No significant differences in biopsy values were found at the baseline determinations between the treated and control patients (Table 3). Likewise, no significant changes, either within or between groups, were noted when all follow-up biopsy specimens were compared with all baseline samples. As with the urinary cyclic AMP samples, the number of biopsy specimens was different at the baseline determinations and at 29 months (and sometimes even included different patients); bone biopsy data was consequently evaluated from the standpoint of paired differences from the baseline values (Table 3). When analyzed in this fashion osteoid width decreased significantly in the control group; in the treated group the rate of bone formation increased: + 1.32 * 0.61 mm2/mm2/day x 10e4, P < 0.06. Roentgenograms No new compression fractures were noted in the treated group, although one treated patient experienced a worsening of the abnormality of the T-l 1 vertebral body (grade 1 progressing to 2). Three new compression fractures were noted in the control group (grade 1 progressing to 3). In addition, three control patients exhibited worsening of previous vertebral abnormalities. This observed distribution of roentgenographic findings between the treated and control groups was not statistically significant (P = 0.071, two-sided Fisher exact test), but it should be recognized that both the number of subjects and the observation periods were too small to adequately assess fracture frequency. Dietary Calcium

-.60

-.90

Dietary calcium increased significantly (P < 0.01) from the baseline values in both the treated and control groups, to respective means of 971.8 ? 56.6 and 1003.8 + 48.7 (maintained throughout the study). Changes from the baseline values between the groups, however, were not significant (P = 0.08).

O---O STANOZOLOL -PLACEBO

I

0

8

16 Months

24

I

32

Fig. 6. Mean change from baseline value in urinary nondialyzable hydroxyproline (mg/24 hr) as a function of treatment time in treated and control patients, mean + SEM. (n): l, l * = significantly different from baseline value, P < 0.01 and P = 0.03, respectively; a = change from baseline value between treated and control groups significantly different. P = 0.02.

Adverse Eflects

As noted in Table 4, the level of SGOT increased significantly (P < 0.001) from the baseline determination to the conclusion of the study in the treated group, although this increase was not significant until the 23-29-month interval; changes between treated and control groups were also significantly (P < 0.001) dif-

STANOZOLOL IN OSTEOPOROSIS

577

Table 3. Bone Bioosv Results in Treated

and Control Patients Controls

Treated Patients Paired Change V&e

Baseline

Paired Change

from Baseline

EaselIne

from Baseline

15.56

+ 0.84 120)

+3.47

t

2.02 (16)

13.95

+ 1.95 (19)

+1.56

-+ 1.83 (13)

Osteoid area (as % of bone)

3.06

+ 0.51 (20)

-0.30

t- 0.85 (16)

2.22

+ 0.40 (19)

f0.06

+ 0.60 (13)

Osteoid width (p)

9.98

+ 0.96 (20)

-2.58

8.26

k 0.82 (19)

-2.68

+ 0.92 (13)*

24.97

k 2.77 (20)

+0.46

? 1.36 (16) + 4.36 (16)

18.27

+ 3.30(19)

+9.17

t 4.81 (13)

Resorbing surface (%)

3.28

f 0.27 (20)

-0.36

+ 0.39 (19)

- 1.14 t 0.72 (13)

0.56

k 0.06 (17)

+0.11

+ 0.58 116) + 0.10(13)

3.96

Rate of bone apposition (p/day)

0.62

k 0.04 (14)

-0.02

Rate of osteoid maturation (%/dav)

7.41

f 0.94 (16)

+2.14

I 2.31 (12)

9.29

* 1.44 (13)

+ 1.33 i 0.94 171

3.71

+ 0.69 (15)

+I.32

3 0.61 (1 11”

2.73

+ 0.53 (131

+ 1.02 i

Total bone area (%I

Forming surface (%)

Rate of bone formation (mm*/mm2/day)

* 0.07 (9)

x

1om4

1.39 (7)

Values are mean -t SEM (n). lP = 0.013. l*P = 0.06.

ferent at study conclusion. Ten (43%) of the 23 treated patients demonstrated an elevated SGOT leve! (greater than the normal range of 8-25 IU) on at least one occasion, with 59 IU as the highest value recorded. As noted in Table 4, however, the mean 29-month SGOT value was 25.8 IU, only slightly above the normal range. In addition, all elevated SGOT values returned to normal following discontinuation of the drug at completion of the study. Of the 23 subjects receiving stanozolol during the study. 13 (57%) developed at least one clinically adverse reaction during the course of participation, including an increase in facial hair (seven patients, 30%), ankle edema (five patients, 22%), hoarseness (five patients, 22%), or acne (two patients, 9%). At no time were these reactions sufficiently severe to cause total termination of the stanozolol therapy. Seven patients (30%) in the placebo group developed similar clinical findings during the study period, including increased facial hair (two patients), and ankle edema/ fluid retention (five patients). Seventy-six percent (16/23) of the treated patients experienced one or more adverse effects (SGOT elevation, clinical effect) during study participation. Table 4.

Groupand Value

DISCUSSION

The current study was designed to evaluate the long-term (29 months) therapeutic efficacy of stanozo101in a large number of relatively homogeneous osteoporotic subjects utilizing a double-blind controlled study format. In this regard TBC (total bone mass) increased significantly in stanozolol-treated patients, with drug effect on bone mass apparently maintained throughout the entire 29-month study. Also, although the number of study participants was not sufficient to determine a true incidence of spinal compression fractures, the occurrence of three new compression fractures in the control group and no new compression fractures in the treated group suggests that the increase in TBC in the treated group represents a stabilization of the structural integrity of the spine. A reasonable assumption is that the observed change in TBC represents a change in total bone mineral rather than a change in extraskeletal calcium (an assumption also inherent in metabolic balance studies); in support of this assumption is the observation that the magnitude of the intergroup TBC difference would require only a 4%-5% difference in bone

SGOT Levels in Treated

Baseline

9 Months

and Control Patients

17 Months

23 Months

29 Months

Treated 22

23

Number of Patients Actual value (IU) Mean change from baseline

17.70

+ 1.45

22.59

k 2.42

f4.45

+ 2.49

+2.24

21

21

21 20.00

_f 1.52 k 1.91

19.67 +1.90

+ 1.56 ? 1.32*

25.76 +8.00

k 2.54 -t 2.38*t

Control 21

23

Number of Patients Actual value (IU)

17.78

k 0.96

17.00 -0.16

Mean change from baseline

f 1.45 + 1.46

-1.63

f

*Change from baseline significantly different from change from baseline in control, P i 0.001 +Significantly different from baseline, P -c 0.00

1.

1.18

f 1.39

Values are mean + SEM.

17

18

19 16.84

14.00 -4.28

* 0.88 + l.llt

15.88 -2.35

+ 1.20 k 1.37

578

calcium but about a six-fold difference in the extraskeletal calcium balance.4 Essentially no significant changes were noted in regional bone mass measurements at the distal radius (BMC decreased at the S2 site in the treated subjects between the baseline determination and 29 months, but slope changes and changes between groups were not significant). However, the failure of the RBM measurement to confirm an apparent beneficial effect of stanozolol in postmenopausal osteoporosis (as indicated by its effect on TBC and spine roentgenograms) is not totally unexpected; a number of previous studies8,‘7have noted differential changes in bone mass between varying skeletal sites, ie, axial (spine), appendicular (radius, metacarpal), and axial and appendicular combined (total bone mass), possibly because of differing amounts of trabecular and cortical bone at each site. Response to therapy, therefore, may not be uniform throughout the osteoporotic skeleton, as suggested by previous studies with estrogen’8,‘9 human parathyroid hormone (PTH l-34),*’ and fluoride” in humans, and with low calcium diets in rats.22 While in this study a significant correlation was noted in the treated group between regional (S2 sites) and total bone mass slope changes (see Fig. 2), in the majority (52%) of these subjects TBC was increasing as RBM was decreasing, supporting the hypothesis of a differential response of various skeletal sites to stanozolol therapy. It should be noted, however, that in these latter subjects the net increase in total bone mass suggests that the overall increase in trabecular bone (or perhaps in cortical bone at sites other than S2) was obviously much greater than the possible loss at the S2 site on the radius. It thus appears that stanozolol does not produce a stereotyped response but that its net effect on skeletal mass is positive. In addition to these findings regarding the effect of stanozolol on bone mass, the data obtained in this study provide insight, unfortunately not definitive, into the effect of the drug on bone metabolism and on associated serum and urine variables. For instance, a decrease in urinary calcium was observed, (an effect noted in previous studies’,2ss) but was somewhat unexpected as it was accompanied by a decrease rather than an increase in serum iPTH. It therefore seems reasonable to assume an action of this drug of decreasing urinary calcium at the kidney level, followed by an observed increase in serum calcium and a counter regulatory iPTH decrease. In addition, stanozolol would appear to increase levels of total urinary cyclic AMP, again a somewhat unanticipated finding as urinary cyclic AMP levels were obtained to confirm the decrease in iPTH levels. This increase in urinary cyclic AMP was unrelated to

CHESNUT Ill ET AL

iPTH levels; whether the observed increase was due to stimulation of production, or to diminished breakdown secondary to phosphodiesterase inhibition, is unclear. Also, the specific physiologic manifestations of the increase in urinary cyclic AMP are not immediately evident from the current data: no significant correlations were noted between total urinary cyclic AMP changes and changes in the other values measured in the study. Because of large interpatient variation and methodologic uncertainties among the various techniques, it is possible that such correlations existed but could not be detected. It is noteworthy, however, that a relationship between the increase in total urinary cyclic AMP and the decrease in urinary calcium was not defined, perhaps because of the fact that such a response has not been consistently observed.23s24 Analysis of the bone biopsy data did not define specific stanozolol effects on the histomorphometric values measured. The observed decrease in iPTH would be expected to result in decreased bone resorption, and previous studies after short-term (2-4 months) anabolic steroid therapy’,* have noted such a decrease. In the current study, however, biopsy data did not reveal a significant change in this value 29 months after the beginning of therapy; a transient decrease in bone resorption within 3 to 6 months of the beginning of treatment cannot be ruled out. Also, while there was an increase of borderline statistical significance in the rate of bone formation in the treated patients, such an increase cannot be attributed to stanozolol since the placebo group also increased slightly and no statistical difference was noted in the amount of change between the groups. Nevertheless, an increase in bone formation would not be unexpected since stanozolol is a known anabolic agent: the drug is thought to increase muscle mass,*j a change documented in this study by definite increases in serum and urine creatinine values. Also, an initial increase of urinary nondialyzable hydroxyproline was noted in the treated subjects through 17 months (a change thought to reflect an enhancement of bone formation).‘* Such a stimulatory effect on bone formation was postulated by Albright and Reifenstein26 and suggested by subsequent investigators following testosterone therapy.27 In addition, a significant (P < 0.001) increase in skeletal alkaline phosphatase activity in serum is noted following stanozolol therapy;*’ experimental evidence29 suggests that the level of serum skeletal alkaline phosphatase reflects the rate of bone formation, Similar increases in this value are noted in response to sodium fluoride therapy,28 an agent known to increase bone formation.30 Other studies utilizing microradiographic’ and radiocalcium’ techniques were

STANOZOLOL IN OSTEOPOROSIS

579

unable to demonstrate a definite effect of anabolic steroids on bone formation; these studies were, however, uncontrolled and involved small numbers of patients. Significant but transient increases in SGOT and other adverse reactions were associated with stanozolol therapy, even when such therapy was administered on an intermittent drug schedule (three out of every four weeks). In no cases, however, were these adverse reactions of sufficient severity to require cessation of drug usage. Also, while SGOT elevation did occur, no other (laboratory or clinical) evidence of possible liver dysfunction was observed, and the deleterious implications of a transient SGOT elevation are unclear. Nevertheless, hepatoma3’ and peliosis hepatis3* have been noted in patients treated with long-term androgenicanabolic steroids; these entities are, however, rare and in general have followed continuous and prolonged therapy with dosages two to three times those used in the present study and have occurred in patients receiving these medications for conditions other than osteoporosis. In conclusion, the present study suggests the potential benefit of stanozolol in postmenopausal osteoporosis. Whether the observed positive effect could be maintained beyond 29 months is not defined by the

current study; even if the observed TBC increase was sustained indefinitely, full repletion of skeletal mass would not be achieved within the remaining life span of many patients. In this regard, any drug arresting the loss of bone mass will obviously be more effective if instituted before severe skeletal depletion has occurred. It should be noted as well that osteoporosis is a severely disabling, morbid, and progressive disease for which there currently is no available cure, and therefore any agent that will arrest bone loss represents a major therapeutic advance. It is also quite possible that this drug, in combination with other drugs, could produce additive effects.

ACKNOWLEDGEMENT We would like to express thanks to Dr. Ronald Kerschner for assistance with statistical analysis, Dr. Norma Maloney for embedment of baseline biopsy specimens, Emily Thompson and Janis Langager for assistance with bone biopsy work, Mario Forte for hydroxyproline determinations, Mike Su for PTH determinations, Harold Fellows for Ca absorption data, Jean Bailey for patient care and data recording, Robert Murano for assistance with total body calcium determinations, and Suzanne Reynolds for assistance with preparation of the manuscript. We would also like to thank Winthrop Laboratories. New York, New York, for kindly supplying the stanozolol (Winstrol) used in this study.

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caland

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