Glucocorticoid-induced Osteoporosis in Adult Cynomolgus Monkeys (Macaca fascicularis): Glucocorticoidfascicularis): Effects on Biochemical Markers of Bone Turnover

Rosario M. Perez1, Flordeliza P. de Villa1, Russell C. Tianzon1, Takashi Hayashi1, Iori Itagaki2, Tina S. Bailey3, Victor Shen3 and Christopher B. Jerome3
1

INA RESEARCH PHILIPPINES, INC. P-2, B-7, L1-A, Technology Avenue Laguna Technopark, Bian, Laguna,4024, Philippines P- B- L1Bi 2 Ina Research Inc., 8047 Nishiminowa, Ina-shi, Nagano-ken, 399-4501, Japan InaNagano3993 SkeleTech, Inc. 22002 26th Avenue SE Suite 104, Bothell, WA 98021, USA

Introduction
Glucocorticoid (GC) use is the most common form of drug drugrelated osteoporosis, and its long -term administration for medical longdisorders such as rheumatoid arthritis and chronic obstructive pulmonary disease is associated with a high rate of fracture. There has been consistent evidence that exposure to elevated levels of glucocorticoids results in decreased osteoblast proliferation and protein synthesis. The actions of glucocortico ids glucocorticoids on bone cells are mediated, in part, directly via specific receptors. However, several issues regarding glucocorticoid effects on the bone still remain uncertain. uncertain. The development of an animal model is considered to be useful for the understanding of the pathogenesis of glucocorticoidglucocorticoidinduced bone disease, as well as, in the investigation of pharmacological interventions.

GLUCOCORTICOID ADMINISTRATION:
Dexamethasone sodium phosphate (Oradexon Organon 5 mg/mL amp, Organon Phils., Inc.) or methylprednisolone sodium Phils., succinate (Solu-Medrol 125 mg/ml, Pharmacia & Upjohn, Inc.) (Soluwere injected intravenously into the saphenous vein based on a twotwo - week alternate- day schedule. Control animals were treated alternatewith distilled water. Injections were given between 8:00 and 9:0 0 9:00 in the morning.

A similar tendency was observed in the MP-treated group but MPthe differences were not significant. After 2 weeks, OC values weeks, were significantly lower in the DXM -treated group compared to DXMthose of the MP-treated group. MPSignificantly higher serum CTx values were noted with DXM treatment compared to the control on day 14. A similar slight tendency was noted in the MP-treated group compared to the MPplacebo group but the difference was not statistically significant. significant. In contrast, DXM caused significant increases in serum NTx on days 4, 8 and 14. Similar effects were not associated with MP. On day 14 , urine NTx values were significantly higher in the 14, DXMDXM -treated group compared to those of the MP-treated group. MPNo effects on serum B-ALP and BMD were associated with Bcorticosteroid treament for two weeks. The serum and urinary biochemical markers mean percent changes of the three groups are shown below.
NTx
1 5 0 .0 0

IN-LIFE OBSERVATIONS AND EXAMINATIONS: INThe animals were observed at least once daily during the pre pretreatment period and at least twice daily (pre - and post-GC (prepostadministration) during the treatment period. Animals that become sick were treated appropriately. Daily food consumption measurements and weekly body weight determinations were done during the pre -treatment and treatment preperiods. Complete Blood Count, routine blood chemistry examinations and urinalysis were conducted on all animals every two weeks during the pre -treatment and treatment periods. pre-

OC
s
100.00 75.00

Objectives
In this study, normal adult male and female cynomolgus macaques ( Macaca fascicularis) were utilized: (Macaca fascicularis) To characterize the osseous effects of two -week alternate -day twoalternateintravenous glucocorticoid administration by longitudinal investigations of bone turnover changes. To examine and compare the effects of two commonly used steroids, dexamethasone and methylprednisolone, on the biochemical markers of bone turnover.

1 0 0 .0 0

Gro up I Gro up II Gro up III

5 0 .0 0

50.00 25.00

(% )

SPECIAL EXAMINATIONS FOR THE DETERMINATION OF CHANGES IN BONE METABOLISM: BIOCHEMICAL MARKER ASSAY:
Serum samples were collected from all the animals between 8:00 and 9:00 in the morning on days -2, - 1, 2, 4, 8 and 14. The following parameters were analyzed: Osteocalcin (OC), bone alkaline phosphatase (B-ALP), Type I collagen cross-linked C (BcrossCtelopeptide (CTx) and Type I collagen cross-linked N -telopeptide crossN(NTx). Cumulated 24- hour urine specimen was collected from each 24monkey on days -3 to -2, 2 to 3, 4 to 5, 8 to 9 and 13 to 14. The total urine volume was measured and recorded. An appropriate recorded. volume taken from the 24- hour sample was centrifuged at 1500 24rpm for 5 min at about 4 C and the supernatant was analyzed for Type I collagen cross-linked N-telopeptide (NTx) and crossNCreatinine (CREA).

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(%)

0 .0 0 -2 5 .0 0

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- 100.00

Gro up I Gro up II Gro up III

2 4 8 14

-5 0 .0 0 -7 5 .0 0 -1 0 0 .0 0 2

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14

Day of m easur ement

Day of measurem ent

1 0 0 .0 0 7 5 .0 0 5 0 .0 0 2 5 .0 0

B-ALP

100.00 75.00 50.00 25.00

CTx

Gro up I Gro up II Gro up III

(%)

0.00 - 25.00 - 50.00 - 75.00 - 100.00 2 4 8 14

(%)
Gro up I Gro up II Gro up III

0 .0 0 -2 5 .0 0 -5 0 .0 0 -7 5 .0 0 -1 0 0 .0 0 2 4 8 14

Materials and Methods

ANIMALS Nine (9) male and nine (9) female adult cynomolgus monkeys aged 6 years and above were obtained from the Primate Quality Control Center (PQCC) , the animal resource facility of INA (PQCC), RESEARCH PHILIPPINES, INC. All animals were quarantined and conditioned at the main testing facility for at least 4 weeks weeks from their date of arrival. The animals were confirmed to be in good health condition, certified tuberculin negative and free of active Salmonella and Shigella infection. As part of the acclimation, the animals were preconditioned to restraining and dosing for at least one month prior to the commencement of the study. The animals were housed individually in stainless steel cages measuring 60 W x 70 D x 77 H cm in an animal room which was maintained under the following controlled environmental conditions: room temperature and relative humidity within the range of 22 to 28C and 30 to 80%, 28 respectively; 12- hour light/dark cycle (7:00 to 19:00); and, a 12minimum ventilation rate of 10 clean, fresh air changes per hour . hour. Each animal was provided daily with approximately 100 g of Certified Primate Diet PS biscuit type (Oriental Yeast Co., Ltd. , Ltd., Japan). The animals were fasted for at least 16 hours prior to blood collection for biochemical marker measurements. Chlorinated deep - well and filtered drinking water was provided deepad libitum via an automated watering system. All the animals in this study were used in accordance with the ethical procedures approved for the care and use of laboratory animals by the Animal Care and Use Committee of INA RESEARCH PHILIPPINES, INC. SELECTION AND PRE-STUDY EVALUATION PREAnimals were initially selected based on the absence of visible growth plate remnants and abnormalities on x- rays of the arms, xlegs and lumbar spine. The hypothalamic- pituitary -adrenal axis hypothalamic- pituitarywas assessed using the dexamethasone suppression test (DST) that was done at least one week prior to the start of the two -week twoalternatealternate-day parenteral steroid administration. The DST was done by injecting the monkeys with dexamethasone sodium phosphate (Oradexon Organon 5 mg/mL amp) intravenously at a single dose of 0.75 mg/kg immediately after collecting the serum sample for cortisol assay at 0 h (21:00). Subsequent serum samples for cortisol determination were obtained according to the the following schedule: 10 h (7:00), 15 h (12:00), and 19 h (16:00). Serum cortisol concentrations were determined using commercially available reagents (MEGA Diagnostics, Inc., Los Angeles, CA, USA).

Day of m easur ement

Day of measurem ent

Figure 2. Mean percent changes of serum biochemical markers

s : p < 0.05, : p < 0.01, significantly different from Group I (t-test) : p < 0.05, significantly different from Group II (t-test)
25 0.00

NTx/CREA
Gr oup I Gr oup II Gr oup III

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15 0.00

IN VIVO DEXA BMD AND BMC MEASUREMENTS:

Measurements of Bone Mineral Density (BMD) and content (BMC) of the lumbar vertebrae, femur, tibia and radius were done once on Weeks - 1 and 3 using a Lunar DPX- x-ray bone DPXdensitometer (Lunar Corporation, U.S.A.) at pediatric AP spine mode set at 8 cm scan width and 6 cm/second scan speed. The BMD and BMC of the regions of interest, demarcated at a scaling factor of 100 percent, were generated, as desired, using the Lunar scan software version 1.3h.
(%)

10 0.00

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- 50.00

14

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Day of measurement

Figure 3. Mean percent changes of urine NTx/CREA

s : p < 0.05, significantly different from Group I (t-test) : p < 0.05, significantly different from Group II (t-test)

STATISTICAL ANALYSES:
F-test was used at 10% level of significance to determine homogeneity of variances. If variances were homogeneous, Student tStudents t-test was used to compare the means but if the variances were heterogeneous, approximate t- test was used for tsubsequent comparison. Statistical differences were evaluated at 5% or less level of significance and presented as p<0.05 or p<0.01. The equality of variance was tested using F-test before using FStudent tStudents t-test to determine statistical difference on BMD and BMC values of the femur, radius, tibia and lumbar.

Discussion
Our data corroborated the findings of previous studies which showed that exposure of cynomolgus monkeys to elevated levels of glucocorticoids induced bone loss that was caused by increased bone resorption and reduced bone formation. The significant and early reduction in the bone formation marker, osteocalcin, a bone gla protein produced primarily by osteoblasts, was noted as an effect of dexamethasone treatment. Our results clearly showed the effects of dexamethasone treatment on bone resorption as exemplified by significant increases in the specific degradation products of type I collage n, collagen, crosscross-linked C-telopeptides (CTx) and N-telopeptides (NTx) in CNthe serum and urinary cross-linked N-telopeptides (NTx) crossNexpressed as a ratio to urinary creatinine. Generally, only slight reductions of serum OC and minimal increases in CTx were associated with MP treatment. No effects on serum and urinary NTx were seen. It could be possible that the alternate-day MP administration at 4 mg/kg/day resulted in an alternateinsufficient pharmacodynamic effect because of the relatively shorter half -life of MP compared to dexamethasone. The use of halfhigher doses of MP might likewise be necessary to clearly demonstrate the effects of MP on bone loss. Corticosteroids had been consistently associated with early changes in the trabecular bone mineral density. However, we were not able to observe such BMD changes within 2 weeks after initiation of high-dose steroid administration using dual highenergy x-ray absorptiometry. x-

Results
At the beginning of the study the mean serum cortisol was 34.8 + 19.9 m g/dL in the whole study group. Maximal suppression of serum cortisol levels was noted 15 hours after DXM administration confirming that the HPA system of the cynomolgus monkey was responsive to exogenous glucocorticoids. There were no significant differences between groups in the serum cortisol values on day -1. Day -1 values also did not differ significantly from the concentrations at ti me 0 time (0h). After the 2- week alternate-day steroid treatment, cortisol 2alternatelevels were significantly lower in the DXM - treated group DXMcompared to the control. Cortisol levels in the MP-treated group MPhad a tendency to be lower compared to the control but the difference was not statistically significant. The cortisol levels of the cynomolgus monkeys before and after the two -week alternate day glucocorticoid treatment are shown twoin the figures below:
1 4 0 .0 0 1 2 0 .0 0 1 0 0 .0 0 3 0 .0 2 5 .0 7 5 .0 0 5 0 .0 0 2 5 .0 0
 # %   m  f

3 5 .0

Table 1. Outline of experimental design

Group
I II

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D ay - 1

Placebo Long-acting Steroid

Distilled Water Dexamethasone sodium phosphate (DXM) Methylprednisolone sodium succinate (MP)

i.v. i.v.

0 1.0

III

Intermediate-acting Steroid

i.v.

4.0

At baseline, there were no differences in the biochemical markers of bone turnover among the three groups. On days 2, 4, 8 and 14, the mean percentage serum OC values of the DXM DXMtreated group were observed to be significantly lower compared to the corresponding values of the placebo group (p<0.01).

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Treatment

Substance

Route

Dose Level (mg/kg/day)

5. 0

sy

1 0 .0

4 0. 00

The monkeys were randomly assigned to 3 dose groups (3 males and 3 females per group) as shown below:

( g/dL)

(%)

8 0. 00 6 0. 00

(%)

2 0 .0 1 5 .0

D ay 1 4

EXPERIMENTAL DESIGN

1 0 0 .0 0

0 .0 0 - 2 5 .0 0 - 5 0 .0 0 - 7 5 .0 0

Conclusion
sy

sy

- 1 0 0 .0 0 - 1 2 5 .0 0

Despite the relatively small number of animals used, the following conclusions can be drawn from the results of this study. study. Bone formation, as evidenced by serum osteocalcin levels, was reduced while bone resorption, as shown by urinary collagen crosscross-link excretion and serum levels of specific degradation products of type I collagen (CTx and NTx), increased with the administration of high-dose dexamethasone for two weeks to highadult cynomolgus monkeys. The alternate- day administration of alternateMP at 4 mg/kg/day did not cause significant changes in the biochemical markers of bone turnover.