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

Rosario M.Rosario M. PerezPerez11, Flordeliza P. de, Flordeliza P. de VillaVilla11, Russell C., Russell C. TianzonTianzon11, Takashi, Takashi HayashiHayashi11, , IoriIori ItagakiItagaki22, , Tina S.Tina S. BaileyBailey33, Victor, Victor ShenShen33 and Christopherand Christopher B.B. JeromeJerome33

1 1 INA RESEARCH PHILI PPI NES, I NC. PINA RESEARCH PHILI PPI NES, I NC. P--2, B2, B--7, L17, L1--A, Technology Avenue Laguna Technopark, BiA, Technology Avenue Laguna Technopark, Biññan, Laguna,4024, Phili ppinesan, Laguna,4024, Phili ppines2 2 Ina Research Inc., 8047 Nishiminowa, InaIna Research Inc., 8047 Nishiminowa, Ina--shi, Naganoshi, Nagano--ken, 399ken, 399--4501, Japan4501, Japan

3 3 SkeleTech, Inc. SkeleTech, Inc. 22002 26th Avenue SE Suite 104, Bothell , WA 98021, USA22002 26th Avenue SE Suite 104, Bothell , WA 98021, USA

Glucocorticoid (GC) use is the most common form of drugGlucocorticoid (GC) use is the most common form of drug--related osteoporosis, and its longrelated osteoporosis, and its long--term administration for medical term administration for medical disorders such as rheumatoid arthritis and chronic obstructive disorders such as rheumatoid arthritis and chronic obstructive pulmonary disease is associated with a high rate of fracture. pulmonary disease is associated with a high rate of fracture. There has been consistent evidence that exposure to elevated There has been consistent evidence that exposure to elevated levels of glucocorticoids results in decreased osteoblast levels of glucocorticoids results in decreased osteoblast proliferation and protein synthesis. The actions of glucocorticoproliferation and protein synthesis. The actions of glucocorticoids ids on bone cells are mediated, in part, directly via specific on bone cells are mediated, in part, directly via specific receptors. However, several issues regarding glucocorticoid receptors. However, several issues regarding glucocorticoid effects on the bone still remain uncertaineffects on the bone still remain uncertain..

The development of an animal model is considered to be useful The development of an animal model is considered to be useful for the understanding of the pathogenesis of glucocorticoidfor the understanding of the pathogenesis of glucocorticoid--induced bone disease, as well as, in the investigation of induced bone disease, as well as, in the investigation of pharmacological interventions. pharmacological interventions.

ANIMALSANIMALS

Nine (9) male and nine (9) female adult cynomolgus monkeys Nine (9) male and nine (9) female adult cynomolgus monkeys aged 6 years and above were obtained from the Primate Quality aged 6 years and above were obtained from the Primate Quality Control Center (PQCC)Control Center (PQCC), the animal resource facility of INA , the animal resource facility of INA RESEARCH PHILIPPINES, INC. All animals were quarantined RESEARCH PHILIPPINES, INC. All animals were quarantined and conditioned at the main testing facility for at least 4 weekand conditioned at the main testing facility for at least 4 weeks s from their date of arrival. The animals were confirmed to be infrom their date of arrival. The animals were confirmed to be ingood health condition, certified tuberculin negative and free ofgood health condition, certified tuberculin negative and free ofactive active SalmonellaSalmonella and and ShigellaShigella infection. As part of the infection. As part of the acclimation, the animals were preconditioned to restraining and acclimation, the animals were preconditioned to restraining and dosing for at least one month prior to the commencement of the dosing for at least one month prior to the commencement of the study. The animals were housed individually in stainless steel study. The animals were housed individually in stainless steel cages measuring 60 W x 70 D x 77 H cm in an animal room cages measuring 60 W x 70 D x 77 H cm in an animal room which was maintained under the following controlled which was maintained under the following controlled environmental conditions: room temperature and relative environmental conditions: room temperature and relative humidity within the range of 22 to 28humidity within the range of 22 to 28°°C and 30 to 80%, C and 30 to 80%, respectively; 12respectively; 12--hour light/dark cycle (7:00 to 19:00); and, a hour light/dark cycle (7:00 to 19:00); and, a minimum ventilation rate of 10 clean, fresh air changes per hourminimum ventilation rate of 10 clean, fresh air changes per hour. . Each animal was provided daily with approximately 100 g of Each animal was provided daily with approximately 100 g of Certified Primate Diet PS biscuit type (Oriental Yeast Co., Ltd.Certified Primate Diet PS biscuit type (Oriental Yeast Co., Ltd., , Japan). The animals were fasted for at least 16 hours prior to Japan). The animals were fasted for at least 16 hours prior to blood collection for biochemical marker measurements. blood collection for biochemical marker measurements. Chlorinated deepChlorinated deep--well and filtered drinking water was provided well and filtered drinking water was provided ad libitum ad libitum via an automated watering system. via an automated watering system.

All the animals in this study were used in accordance with the All the animals in this study were used in accordance with the ethical procedures approved for the care and use of laboratory ethical procedures approved for the care and use of laboratory animals by the Animal Care and Use Committee of INA animals by the Animal Care and Use Committee of INA RESEARCH PHILIPPINES, INC.RESEARCH PHILIPPINES, INC.

SELECTION AND PRESELECTION AND PRE--STUDY EVALUATIONSTUDY EVALUATION

Animals were initially selected based on the absence of visible Animals were initially selected based on the absence of visible growth plate remnants and abnormalities on xgrowth plate remnants and abnormalities on x--rays of the arms, rays of the arms, legs and lumbar spine. The hypothalamiclegs and lumbar spine. The hypothalamic--pituitarypituitary--adrenal axis adrenal axis was assessed using the dexamethasone suppression test (DST) was assessed using the dexamethasone suppression test (DST) that was done at least one week prior to the start of the twothat was done at least one week prior to the start of the two--week week alternatealternate--day parenteral steroid administration. The DST was day parenteral steroid administration. The DST was done by injecting the monkeys with dexamethasone sodium done by injecting the monkeys with dexamethasone sodium phosphate (Oradexon Organon 5 mg/mL amp) intravenously at a phosphate (Oradexon Organon 5 mg/mL amp) intravenously at a single dose of 0.75 mg/kg immediately after collecting the serumsingle dose of 0.75 mg/kg immediately after collecting the serumsample for cortisol assay at 0 h (21:00). Subsequent serum sample for cortisol assay at 0 h (21:00). Subsequent serum samples for cortisol determination were obtained according to thsamples for cortisol determination were obtained according to the e following schedule: 10 h (7:00), 15 h (12:00), and 19 h (16:00).following schedule: 10 h (7:00), 15 h (12:00), and 19 h (16:00).Serum cortisol concentrations were determined using Serum cortisol concentrations were determined using commercially available reagents (MEGA Diagnostics, Inc., Los commercially available reagents (MEGA Diagnostics, Inc., Los Angeles, CA, USA). Angeles, CA, USA).

EXPERIMENTAL DESIGNEXPERIMENTAL DESIGN

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

Table 1. Outline of experimental designDose Level(mg/kg/day)

I Placebo Distilled Water i.v. 0

II Long-acting Steroid Dexamethasone sodium i.v. 1.0phosphate (DXM)

III Intermediate-acting Methylprednisolone i.v. 4.0Steroid sodium succinate (MP)

Group Treatment Substance Route

Cumulated 24Cumulated 24--hour urine specimen was collected from each hour urine specimen was collected from each monkey on days monkey on days --3 to 3 to --2, 2 to 3, 4 to 5, 8 to 9 and 13 to 14. The 2, 2 to 3, 4 to 5, 8 to 9 and 13 to 14. The total urine volume was measured and recordedtotal urine volume was measured and recorded. An appropriate . An appropriate volume taken from the 24volume taken from the 24--hour sample was centrifuged at 1500 hour sample was centrifuged at 1500 rpm for 5 min at about 4 rpm for 5 min at about 4 °°C and the supernatant was analyzed C and the supernatant was analyzed for Type I collagen crossfor Type I collagen cross--linked Nlinked N--telopeptide (NTx) and telopeptide (NTx) and Creatinine (CREA).Creatinine (CREA).

IN VIVOIN VIVO DEXA BMD AND BMC MEASUREMENTS:DEXA BMD AND BMC MEASUREMENTS:

Measurements of Bone Mineral Density (BMD) and contentMeasurements of Bone Mineral Density (BMD) and content(BMC) of the lumbar vertebrae, (BMC) of the lumbar vertebrae, femur, tibia and radiusfemur, tibia and radius were done were done once on Weeks once on Weeks --1 and 3 using a Lunar DPX1 and 3 using a Lunar DPX-- αα xx--ray bone ray bone densitometer (Lunar Corporation, U.S.A.) at pediatric AP spine densitometer (Lunar Corporation, U.S.A.) at pediatric AP spine mode set at 8 cm scan width and 6 cm/second scan speed.mode set at 8 cm scan width and 6 cm/second scan speed. The The BMD and BMC of the regions of interest, demarcated at a scaling BMD and BMC of the regions of interest, demarcated at a scaling factor of 100 percent, were generated, as desired, using the factor of 100 percent, were generated, as desired, using the Lunar scan software version 1.3h. Lunar scan software version 1.3h.

STATISTICAL ANALYSES:STATISTICAL ANALYSES:

FF--test was used at 10% level of significance to determine test was used at 10% level of significance to determine homogeneity of variances. If variances were homogeneous, homogeneity of variances. If variances were homogeneous, StudentStudent’’s ts t--test was used to compare the means but if the test was used to compare the means but if the variances were heterogeneous, approximate tvariances were heterogeneous, approximate t--test was used for test was used for subsequent comparison. Statistical differences were evaluated subsequent comparison. Statistical differences were evaluated at 5% or less level of significance and presented as p<0.05 or at 5% or less level of significance and presented as p<0.05 or p<0.01.p<0.01.

The equality of variance was tested using FThe equality of variance was tested using F--test before using test before using StudentStudent’’s ts t--test to determine statistical difference on BMD and test to determine statistical difference on BMD and BMC values of the femur, radius, tibia and lumbar. BMC values of the femur, radius, tibia and lumbar.

At the beginning of the study the mean serum cortisol was At the beginning of the study the mean serum cortisol was 34.8 34.8 + 19.9 + 19.9 mmg/dLg/dL in the whole study group. in the whole study group. Maximal suppression of Maximal suppression of serum cortisol levels was noted 15 hours after DXM serum cortisol levels was noted 15 hours after DXM administration confirming that the HPA system of the administration confirming that the HPA system of the cynomolgus monkey was responsive to exogenous cynomolgus monkey was responsive to exogenous glucocorticoids.glucocorticoids. There were no significant differences between There were no significant differences between groups in the serum cortisol values on day groups in the serum cortisol values on day --1. Day 1. Day --1 values 1 values also did not differ significantly from the concentrations at tialso did not differ significantly from the concentrations at time 0 me 0 (0h). After the 2(0h). After the 2--week alternateweek alternate--day steroid treatment, cortisol day steroid treatment, cortisol levels were significantly lower in the DXMlevels were significantly lower in the DXM-- treated group treated group compared to the control. Cortisol levels in the MPcompared to the control. Cortisol levels in the MP--treated group treated group had a tendency to be lower compared to the control but the had a tendency to be lower compared to the control but the difference was not statistically significant. difference was not statistically significant.

The cortisol levels of the cynomolgus monkeys before and after The cortisol levels of the cynomolgus monkeys before and after the twothe two--week alternate day glucocorticoid treatment are shown week alternate day glucocorticoid treatment are shown in the figures below: in the figures below:

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At baseline, there were no differences in the biochemical At baseline, there were no differences in the biochemical markers of bone turnover among the three groups. On days 2, markers of bone turnover among the three groups. On days 2, 4, 8 and 14, the mean percentage serum OC values of the DXM4, 8 and 14, the mean percentage serum OC values of the DXM--treated group were observed to be significantly lower compared treated group were observed to be significantly lower compared to the corresponding values of the placebo group (p<0.01). to the corresponding values of the placebo group (p<0.01).

Figure 2. Mean percent changes of serum biochemical markerss : p < 0.05, § : p < 0.01, significantly different from Group I (t-test)† : p < 0.05, significantly different f rom Group II (t-test)

NTx

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Figure 3. Mean p ercent changes of urine NTx/CREAs : p < 0.05, sign if icantly different from Group I (t-test)† : p < 0.05, sign if icantly different from Group II (t-test)

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Our data corroborated the findings of previous studies which Our data corroborated the findings of previous studies which showed that exposure of cynomolgus monkeys to elevated levels showed that exposure of cynomolgus monkeys to elevated levels of glucocorticoids induced bone loss that was caused by of glucocorticoids induced bone loss that was caused by increased bone resorption and reduced bone formation. The increased bone resorption and reduced bone formation. The significant and early reduction in the bone formation marker, significant and early reduction in the bone formation marker, osteocalcin, a bone osteocalcin, a bone glagla protein produced primarily by protein produced primarily by osteoblasts, was noted as an effect of dexamethasone treatment. osteoblasts, was noted as an effect of dexamethasone treatment. Our results clearly showed the effects of dexamethasone Our results clearly showed the effects of dexamethasone treatment on bone resorption as exemplified by significant treatment on bone resorption as exemplified by significant increases in the specific degradation products of type I collageincreases in the specific degradation products of type I collagen, n, crosscross--linked Clinked C--telopeptides (CTx) and Ntelopeptides (CTx) and N--telopeptides (NTx) in telopeptides (NTx) in the serum and urinary crossthe serum and urinary cross--linked Nlinked N--telopeptides (NTx) telopeptides (NTx) expressed as a ratio to urinary creatinine. expressed as a ratio to urinary creatinine.

Generally, only slight reductions of serum OC and minimal Generally, only slight reductions of serum OC and minimal increases in CTx were associated with MP treatment. No effects increases in CTx were associated with MP treatment. No effects on serum and urinary NTx were seen. It could be possible that on serum and urinary NTx were seen. It could be possible that the alternatethe alternate--day MP administration at 4 mg/kg/day resulted in an day MP administration at 4 mg/kg/day resulted in an insufficient pharmacodynamic effect because of the relatively insufficient pharmacodynamic effect because of the relatively shorter halfshorter half--life of MP compared to dexamethasone.life of MP compared to dexamethasone. The use of The use of higher doses of MP might likewise be necessary to clearly higher doses of MP might likewise be necessary to clearly demonstrate the effects of MP on bone loss.demonstrate the effects of MP on bone loss.

Corticosteroids had been consistently associated with early Corticosteroids had been consistently associated with early changes in the trabecular bone mineral density. However, we changes in the trabecular bone mineral density. However, we were not able to observe such BMD changes within 2 weeks were not able to observe such BMD changes within 2 weeks after initiation of highafter initiation of high--dose steroid administration using dual dose steroid administration using dual energy xenergy x--ray absorptiometry.ray absorptiometry.

IntroductionIntroduction

In this study, normal adult male and female cynomolgus In this study, normal adult male and female cynomolgus macaques (macaques (Macaca fascicularisMacaca fascicularis) were utilized:) were utilized:

•• To characterize the osseous effects of twoTo characterize the osseous effects of two--week alternateweek alternate--day day intravenous glucocorticoid administration by longitudinal intravenous glucocorticoid administration by longitudinal investigations of bone turnover changes.investigations of bone turnover changes.

•• To examine and compare the effects of two commonly used To examine and compare the effects of two commonly used steroids, dexamethasone and methylprednisolone, on the steroids, dexamethasone and methylprednisolone, on the biochemical markers of bone turnover.biochemical markers of bone turnover.

ObjectivesObjectives

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

Discuss ionDiscuss ion

A similar tendency was observed in the MPA similar tendency was observed in the MP--treated group but treated group but the differences were not significant. the differences were not significant. After 2 weeksAfter 2 weeks, OC values , OC values were significantly lower in the DXMwere significantly lower in the DXM--treated group compared to treated group compared to those of the MPthose of the MP--treated group. treated group.

Significantly higher serum CTx values were noted with DXM Significantly higher serum CTx values were noted with DXM treatment compared to the control on day 14. A similar slight treatment compared to the control on day 14. A similar slight tendency was noted in the MPtendency was noted in the MP--treated group compared to the treated group compared to the placebo group but the difference was not statistically significaplacebo group but the difference was not statistically significant. nt. In contrast, DXM caused significant increases in serum NTx on In contrast, DXM caused significant increases in serum NTx on days 4, 8 and 14. Similar effects were not associated with MP. days 4, 8 and 14. Similar effects were not associated with MP. On day 14On day 14, urine NTx values were significantly higher in the , urine NTx values were significantly higher in the DXMDXM--treated group compared to those of the MPtreated group compared to those of the MP--treated group. treated group. No effects on serum BNo effects on serum B--ALP and BMD were associated with ALP and BMD were associated with corticosteroid treament for two weeks. corticosteroid treament for two weeks.

The serum and urinary biochemical markers mean percent The serum and urinary biochemical markers mean percent changes of the three groups are shown below.changes of the three groups are shown below.

ResultsResults

SPECIAL EXAMINATIONS FOR THE DETERMINATION SPECIAL EXAMINATIONS FOR THE DETERMINATION OF CHANGES IN BONE METABOLISM:OF CHANGES IN BONE METABOLISM:

BIOCHEMICAL MARKER ASSAY:BIOCHEMICAL MARKER ASSAY:

Serum samples were collected from all the animals between 8:00 Serum samples were collected from all the animals between 8:00 and 9:00 in the morning on days and 9:00 in the morning on days --2, 2, --1, 2, 4, 8 and 14. The 1, 2, 4, 8 and 14. The following parameters were analyzed: Osteocalcin (OC), bone following parameters were analyzed: Osteocalcin (OC), bone alkaline phosphatase (Balkaline phosphatase (B--ALP), Type I collagen crossALP), Type I collagen cross--linked Clinked C--telopeptide (CTx) and Type I collagen crosstelopeptide (CTx) and Type I collagen cross--linked Nlinked N--telopeptide telopeptide (NTx). (NTx).

ININ--LIFE OBSERVATIONS AND EXAMINATIONS:LIFE OBSERVATIONS AND EXAMINATIONS:

The animals were observed at least once daily during the preThe animals were observed at least once daily during the pre--treatment period and at least twice daily (pretreatment period and at least twice daily (pre-- and postand post--GC GC administration) during the treatment period. Animals that becomeadministration) during the treatment period. Animals that becomesick were treated appropriately. sick were treated appropriately.

Daily food consumption measurements and weekly body weight Daily food consumption measurements and weekly body weight determinations were done during the predeterminations were done during the pre--treatment and treatment treatment and treatment periods. periods. Complete Blood Count, routine blood chemistry Complete Blood Count, routine blood chemistry examinations and urinalysis were conducted on all animals every examinations and urinalysis were conducted on all animals every two weeks during the pretwo weeks during the pre--treatment and treatment periods.treatment and treatment periods.

GLUCOCORTICOID ADMINISTRATION:GLUCOCORTICOID ADMINISTRATION:

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

Materials and MethodsMaterials and Methods