effect of d-003, a mixture of very high molecular weight aliphatic acids, on prednisolone-induced...

Drugs R D 2004; 5 (5): 281-290ORIGINAL RESEARCH ARTICLE 1174-5886/04/0005-0281/$31.00/0

© 2004 Adis Data Information BV. All rights reserved.

Effect of D-003, a Mixture of VeryHigh Molecular Weight AliphaticAcids, on Prednisolone-InducedOsteoporosis in Sprague-Dawley RatsMiriam Noa, Sarahi Mendoza, Rosa Mas, Nilda Mendoza and Felipe Leon

Center of Natural Products from the National Center for Scientific Research, Havana City, Cuba

Background: Drugs inhibiting cholesterol biosynthesis may affect bone metabo-Abstractlism through inhibition of the mevalonate pathway resulting in the inhibition ofprotein prenylation required for osteoclast activity. D-003 is a mixture of highmolecular weight aliphatic primary acids purified from sugar-cane (Saccharumofficinarum) wax, with cholesterol-lowering effects demonstrated in experimentaland clinical studies. D-003 inhibits cholesterol biosynthesis through indirectregulation of HMG-CoA reductase activity. A previous study demonstrated thatD-003 prevented bone loss and bone resorption on ovariectomy-induced osteo-porosis in rats. Corticosteroid-induced osteoporosis is the result of changesaffecting calcium homeostasis, but the hallmark of corticosteroid-induced boneloss is the direct effects on bone cells, such as inhibition of osteoblastogenesis,promotion of apoptosis of osteoblasts and osteocytes, and decrease in boneformation.Objective: To determine whether D-003 could prevent the bone loss induced withprednisolone in Sprague-Dawley rats.Methods: Rats were randomly distributed in five groups (ten rats per group): asham-operated control and four groups orally treated with prednisolone 6 mg/kgfor 80 days; a positive control orally treated with vehicle; and three groups orallytreated with D-003 at 5, 25 and 200 mg/kg, respectively. Rats were killed, bonesremoved and histological variables of bone resorption and formation studied forhistomorphometry.Results: Compared with the sham group, prednisolone significantly (p < 0.01)reduced trabecular bone volume (TBV), while D-003 significantly (p < 0.001) anddose-dependently prevented the prednisolone-induced reduction of TBV. Treat-ment with prednisolone lowered (p < 0.001) trabecular thickness (TbTh) andnumber (TbN), while increasing (p < 0.001) the gap between trabeculae. D-003(5, 25 and 200 mg/kg/day) significantly (p < 0.001) and dose-dependently pre-vented the reduction of TbTh and TbN and the increase of trabecular gap inducedwith prednisolone. Treatment with prednisolone increased both the surface andnumber of osteoclasts compared with sham (p < 0.001). D-003 (5–200 mg/day),however, prevented this effect (p < 0.001 for all comparisons). D-003 alsoprevented (p < 0.001) the reduction of osteoblast surface (ObS/BS) induced by

282 Noa et al.

prednisolone. Osteonecrotic areas were observed in all positive controls, but innone of the sham animals. Positive controls showed hypertrophy of bone marrowadipocytes and lipid-laden pluripotential stromal cells in bones. A significant anddose-dependent reduction of the frequency of animals showing prednisolone-induced osteo-necrosis was observed across the doses of D-003 (5, 25 and200 mg/kg) investigated here.Conclusions: D-003 (5, 25 and 200 mg/kg) prevented trabecular bone loss andfemoral neck osteonecrosis induced with prednisolone in Sprague Dawley rats,also increasing osteoblast surface and reducing bone resorption parameters. Theseresults suggest that D-003 could be useful for managing corticosteroid-inducedosteoporosis.

Osteoporosis is characterised by low bone mass pression, receptor binding or binding proteinlevels.[10,11]and abnormal bone architecture, leading to aug-

Prednisolone is a corticosteroid used for inducingmented bone fragility and fracture risk. The frequen-osteoporosis experimentally[12,13] and for evaluatingcy of osteoporosis increases with age, and it is adrug-dependent inhibition of the osteoporosis pro-major cause of morbidity in the elderly.[1]

cess.[14]The adverse effects of corticosteroids on bone

Nutritional and pharmacological interventionshave been documented,[2-5] but the cellular and mo-are indicated for osteoporosis management.[15] Tolecular basis of such action remains elusive. Cur-date, bisphosphonates are among the most effectiverently, corticosteroid-induced osteoporosis rankstherapies for preserving and building bone mass.[16]

third in frequency after postmenopausal and senileBisphosphonates affect osteoclast activity by inhib-osteoporosis.[3]

iting osteoclastic bone resorption throughout theCorticosteroid-induced osteoporosis is the result inhibition of farnesyl diphosphate synthase, a key

of changes affecting calcium homeostasis caused by enzyme in the formation of isoprenoids via thedecreased intestinal calcium absorption and in- mevalonate pathway. The inability to producecreased renal excretion.[4,5] Nevertheless, increasing prenylated proteins in the osteoclast caused by de-evidence of the contribution of direct effects of pletion of isoprenoids impairs both osteoclast for-corticosteroids on bone cells is available. Thus, bone mation and function.[17,18]

loss induced by corticosteroids is due to the inhibi- On the other hand, inhibitors of HMG-CoAtion of osteoblastogenesis, an increase in osteoblast reductase (statins) inhibit cholesterol synthesisapoptosis and decreased bone formation.[6-9] Never- through direct competitive inhibition of the conver-theless, early bone loss induced by corticosteroids is sion of HMG-CoA to mevalonate.[19] Inhibition ofcaused by extension of the lifespan of pre-existing the mevalonate pathway ultimately results in theosteoclasts, an effect not preventable by inhibition of protein prenylation because of the de-bisphosphonates.[9] Corticosteroids activate osteo- pletion of farnesyl diphosphate or geranylgeranylclasts probably via osteoblast activation, thus pro- diphosphate involved in osteoclastic activity.[17,20]

moting bone resorption by stimulating osteoclas- Hence, the rationale that drugs acting on mevalonatetogenesis via the increased expression of RANK synthesis can modify bone metabolism is supported.ligand and decreased expression of its decoy recep- Thus, experimental evidence supports the conten-tor, osteoprotegerin, osteoblastic signals acting as tion that statins increase new bone formation infinal mediators of osteoclastogenesis. Corticoste- rodents and human cells in vitro,[19] and that atorvas-roids decrease osteoblastic function directly and in- tatin 5 mg/kg/day for 6 weeks increased femoral anddirectly through modulation of growth factor ex- vertebral bone strength in Sprague-Dawley rats with

© 2004 Adis Data Information BV. All rights reserved. Drugs R D 2004; 5 (5)

D-003 in Prednisolone-Induced Osteoporosis 283

osteoporosis induced by ovariectomy or corticoste- 50 and 200 mg/kg on ovariectomy-induced osteo-roids.[21] porosis demonstrated that D-003 prevented bone

loss and inhibited parameters of bone resorption.[31]Other processes contribute to osteoporosis devel-

Considering this background, this study was con-opment. Thus, oxidised lipids can exert oppositeducted to determine whether D-003 could preventeffects in vascular calcification and bone cellularthe bone loss induced by prednisolone in Sprague-differentiation, since they can inhibit the differentia-Dawley rats.tion and activity of osteoblasts and induce monocyte

migration, leading to increased osteoclasts in boneMaterials and Methodstissue.[22] Some antioxidants, such as tocopherol (vi-

tamin E), stimulate bone formation in chicks fed ahigh-fat diet, suggesting that inhibition of lipid oxi- Animalsdation can induce normal bone growth.[23]

Female Sprague-Dawley rats (weighingD-003 is a mixture of higher aliphatic primary225 ± 20g and aged 3 months) from the Nationalacids purified from sugar-cane (Saccharum of-Center for Laboratory Animals Productionficinarum) wax, in which octacosanoic acid (C28) is(CENPALAB, Havana, Cuba) were adapted to labo-the main component, followed by triacontanoicratory conditions for 2 weeks with free access to(C30), dotriacontanoic (C32) and tetratriacontanoicfood and water.(C34) acids, as well as hexacosanoic (C26),

Animal handling was conducted in accordanceheptacosanoic (C27), nonacosanoic (C29), hen-with the Cuban Regulations for the Use of Laborato-triacontanoic (C31) and tritriacontanoic (C33) acids.ry Animals. Study protocol was consistent with ourTetracosanoic (C24), pentacosanoic (C25) andapproved standard operational procedures.pentatriacontanoic (C35) acids are also present as

minor components.[24]

Administration and DosageD-003 lowers cholesterol through inhibition ofcholesterol biosynthesis between acetate consump- D-003 was obtained from the Center for Naturaltion and mevalonate production, throughout the in- Products (National Center for Scientific Research,direct regulation of the enzyme activity in a lipid- Havana City, Cuba) and its purity checked by gasdepleted medium, by depression of de novo synthe- chromatography. D-003 was suspended in Tween/sis of HMG-CoA reductase and/or stimulation of its H20 vehicle and administered orally by gastric ga-degradation.[25] Hence, effects associated with the vage (5–10 mL/kg). After checking the stability ofinhibition of cholesterol biosynthesis are expected the suspensions, they were prepared weekly andfor D-003. concentrations were adjusted according to the

D-003 has shown cholesterol-lowering effects in bodyweight gain of the animals.rabbits[26,27] and healthy human volunteers,[28,29]

Rats were randomly distributed in five groupscharacterised by a significant reduction of total and (ten rats per group). Groups comprised a negativelow-density lipoprotein (LDL) cholesterol. On the control and four groups treated with prednisolone: aother hand, D-003 inhibits the susceptibility of rat positive control orally receiving the vehicle only andlipoprotein and human LDL to undergo lipid perox- three groups treated with oral D-003 5, 25 andidation induced by different agents.[30]

200 mg/kg, respectively. Control groups were givenConsidering that D-003 inhibits cholesterol bio- similar volumes of the vehicle by gastric gavage. All

synthesis via HMG-CoA reductase activity regula- treatments were administered once daily (5–6 daystion and inhibits susceptibility of lipoproteins to weekly) from 9 to 11am for 80 days. Treatmentlipid peroxidation, effects of D-003 in preventing started the same day as prednisolone administration,development of osteoporosis were expected. A pre- but was administered 2 hours before prednisolone.vious study investigating the effects of D-003 Doses were adjusted according to bodyweight gain.


284 Noa et al.

The rationale for the minimum dose of D-003 Evaluation of Osteonecrosisinvestigated (5 mg/kg) was based on the observation

Bone samples (eight regions per animal) werethat D-003 orally administered at this dose wasexamined under a light microscope for histopatho-effective for lowering cholesterol in rabbits[26,27] andlogical changes, such as haematopoietic cell necro-for inhibiting rat lipoprotein peroxidation.[30] Also,sis with cytolysis, karyorrhexis and/or karyolysis,considering that D-003 50 and 200 mg/kg preventedfat cell necrosis with the loss of nuclei and distinctbone loss and resorption in ovariectomised rats,[31]

cell borders, and osteonecrosis (ON).we assessed whether doses of D-003 from 5 toAnalysis of ON occurrence was performed as by200 mg/kg could prevent corticosteroid-induced os-

Irisa et al.[34] ON occurrence was considered asteoporosis.being present if bone necrosis associated with emptylacunae or pycnotic nuclei of osteocytes and sur-

Bodyweight Assessment rounding bone marrow necrosis (necrosis of adipo-cytes and haematopoietic cells) were present. All

Bodyweight was monitored every 7 days during rats that had at least one osteonecrotic lesion of eightthe first 30 days, and at 45, 60 and 80 days on areas observed were considered to have ON, where-treatment. as those with no osteonecrotic lesions were consid-

ered as being ON-free.Induction ofCorticosteroid-Induced Osteoporosis Histomorphometric Study

Morphometry was performed as described.[35]For inducing osteoporosis, prednisolone (6 mg/

The primary efficacy variables were the changes inkg) was orally administered for 80 days to Sprague-the measurements related to trabecular bone volumeDawley rats, according to Oxtaf and Oxlund.[12]

and structure, such as trabecular thickness (TbTh, inmicrometres), number (TbN, number per millime-

Microscopic Studies tre) and separation (TbSp, in micrometres), osteo-clast number and surface (OcS/BS). The percentage

At study completion, rats were fasted for 12 of osteoblast surface (ObS/BS) was also calculated.hours and killed under ether anaesthesia. Then the All values of histomorphometric parameters wereright femur and fifth lumbar vertebra from each rat derived from primary measurements of areas andwere removed for the morphological study. perimeters.

Treatment effects were assessed through micro- The calculation related to trabecular bone volumescopic and morphometric studies. The following (TBV) for estimation of bone mass was performedspecimens were obtained from each animal: fifth according to Ke et al.,[36] considering BV/TV × 100,lumbar vertebral body, femoral neck and distal fe- where BV is the trabecular bone area and TV themur. The right femur was cut through the inter- total area. Histomorphometric analysis was carriedtrochanteric line to create a wide and flat base for out using the image analysis system Digipatadequate positioning of the femoral neck before (ECISOFT, Havana City, Cuba).[37]

embedding, as reported by Bagi et al.;[32] while thedistal femur was always taken at the second 0.5cm Statistical Analysisfrom the distal end of the femur.

The specimens were processed as described pre- Comparisons between groups were done usingviously.[33] In brief, bones were decalcified in 0.5 the nonparametric Mann-Whitney U-test, while anmol/L disodium edetic acid (pH 7.4) at 4°C for 4 ANOVA test was used for bodyweight analysis.weeks, embedded in paraffin, sectioned and stained Comparisons between groups of animals showingwith haematoxylin and eosin. ON were performed with Fisher’s Exact Probability



Table I. Effects of D-003, after different periods of treatment, on bodyweight (mean ± SD) of rats treated with prednisolone

Group Dose Bodyweight (g)

(mg/kg) baseline 7 days 14 days 30 days 45 days 60 days 80 days

Sham 0 270.3 ± 23.0 281.8 ± 28.2 286.0 ± 29.4 302.4 ± 33.3 307.4 ± 33.0 313.2 ± 31.0 320.0 ± 32.9

Positive 0 267.2 ± 20.7 261.1 ± 17.8* 262.0 ± 20.3* 271.7 ± 21.9* 279.4 ± 24.9* 282.4 ± 24.7* 288.0 ± 27.7*control

D-003 5 267.5 ± 24.5 264.2 ± 25.8* 263.5 ± 26.6* 272.0 ± 25.0* 278.3 ± 32.3* 282.1 ± 32.0* 289.1 ± 29.2*

D-003 25 265.5 ± 20.9 264.0 ± 20.3* 262.8 ± 24.9* 273.0 ± 28.5* 278.7 ± 31.2* 282.4 ± 31.5* 288.3 ± 34.6*

D-003 200 269.9 ± 12.7 272.3 ± 22.7 275.5 ± 22.3 277.5 ± 32.0 280.7 ± 22.2 285.7 ± 27.4 292.1 ± 32.1

test. An α = 0.05 was selected a priori for statistical between trabeculae (table II). D-003 (5, 25 andsignificance. All analyses were performed using the 200 mg/kg/day) significantly (p < 0.001) and dose-statistical package Windows program. dependently prevented the reduction of TbTh and

TbN and the increase of trabecular separation in-duced by prednisolone, as revealed by the compari-Resultssons with positive controls.

Bodyweight data are shown in table I. After The effects of D-003 on both the surface and7 days on treatment, prednisolone had significantly number of osteoclasts are summarised in table III.(p < 0.05) reduced bodyweight compared with While prednisolone increased both variables withsham, values being slightly lower than at baseline. respect to the sham group (p < 0.001), D-003Bodyweights of prednisolone-positive control rats (5–200 mg/day) significantly prevented this effectwere lower than baseline values during the first 14 (p < 0.001 for all comparisons). D-003 significantlydays, but were lower than sham values throghout

(p < 0.001) prevented the reduction of ObS/BSthe study, recovery starting after 30 days on

induced by prednisolone (figure 2).prednisolone.Figure 3 illustrates the effects of D-003 on theCompared with sham, prednisolone administered

proportion of animals showing osteonecrotic areasfor 80 days significantly (p < 0.01) reduced TBVin the femoral neck. No animal from the sham groupvalues (figure 1). D-003 significantly (p < 0.001)showed ON. Osteonecrotic areas were observed inand dose-dependently prevented the reduction ofall positive controls (8/8, 100%), which showedTBV induced by prednisolone.hypertrophy of bone marrow adipocytes and lipid-Prednisolone significantly (p < 0.001) lowered

TbTh and TbN, while increasing (p < 0.001) the gap laden pluripotential stromal cells in bones.

10

15

20

25

30

Sham+ Control5 mg/kg25 mg/kg200 mg/kg

5th lumbar vertebra Femoral neck Distal femur

Trab

ecul

ar v

olum

e

††

†

†

*

††

†† †

*

Fig. 1. Effects of D-003 on trabecular bone volume in bones of prednisolone-treated rats. * p < 0.001 vs sham group (Mann-Whitney U-test);† p < 0.001 vs positive control (+ Control) group (Mann-Whitney U-test).


286 Noa et al.

The occurrence of ON in positive controls was Corticosteroids induce complex effects on thethe specific change induced by prednisolone. Osteo- body, since they control energy balance, appetite,petrosis, a lesion characterised by the lack of the cell proliferation and apoptosis, stress response,normal clear zone and ruffled border of active osteo- metabolic rate, inflammation and repair systems.[38]

clasts and marrow spaces filled with necrotic calci- Decrease in rat bodyweight after treatment withfied cartilage was observed in positive controls, prednisolone has been reported by other investiga-whose primary trabeculae containing remnants of tors; it is a complex effect including a multiplecartilage tissue persisted within the epi-meta and rather than a single mechanism. Nevertheless, suchdiaphysis of long bones.

an effect has been related to the effects of corticoste-A significant and dose-dependent reduction of

roids on muscle mass and function.[39-41] Our resultsthe frequency of animals showing prednisolone-

showed that D-003 200 mg/kg prevented the impair-induced ON was observed across the doses of D-003ment of bodyweight growth induced by(5, 25 and 200 mg/kg) investigated. D-003 practical-prednisolone. This preventive effect of D-003, how-ly abolished the presence of hypertrophy of boneever, cannot be explained merely by its effects onmarrow adipocytes and lipid-laden pluripotentialbone indicators, since at 5 and 100 mg/kg, dosesstromal cells in bones.effective for preventive bone loss induced byprednisolone, it did not protect againstDiscussionprednisolone-induced bodyweight growth impair-ment. Thus, probably another systemic effect ofThe results of the present study showed thatD-003, not yet understood, could contribute to theD-003 5, 25 and 200 mg/kg/day prevented theprotection exerted by the high dose of D-003, or itchanges in rat bones induced by prednisolone, and iscould be a random result. This aspect needs furtherthe first report of the effects of D-003 on corticoste-

roid-induced osteoporosis. investigation.

Table II. Effects of D-003 on bones from prednisolone-treated rats; morphometric study

Group Dose (mg/kg) TbTh (μm) TbN (no./mm) TbSp (μm)

Femoral neck

Sham 0 83.83 ± 2.63 7.33 ± 0.36 191.88 ± 1.98

Positive control 0 58.67 ± 3.15* 4.38 ± 0.21* 357.38 ± 5.54*

D-003 5 71.75 ± 1.15†† 6.38 ± 0.21† 211.75 ± 6.06†††

D-003 25 78.71 ± 2.72††† 6.88 ± 0.35††† 209.46 ± 2.68†††

D-003 200 80.88 ± 2.0††† 7.63 ± 0.12††† 195.17 ± 3.90†††

Distal femur

Sham 0 93.00 ± 1.41 1.50 ± 0.18 203.54 ± 1.75


D-003 5 87.42 ± 2.79†† 1.13 ± 0.31† 224.46 ± 6.40†††

D-003 25 89.79 ± 1.14†† 1.21 ± 0.31†† 208.33 ± 2.47†††

D-003 200 93.04 ± 2.03††† 1.38 ± 0.28†† 203.00 ± 2.59†††

Fifth vertebra

Sham 0 85.21 ± 0.96 3.50 ± 0.18 216.67 ± 1.30


D-003 5 80.50 ± 1.71†† 3.33 ± 0.18††† 215.50 ± 10.41†††

D-003 25 80.54 ± 2.68†† 3.50 ± 0.25††† 205.38 ± 2.26†††

D-003 200 87.00 ± 2.06††† 3.71 ± 0.21††† 204.67 ± 2.16†††

TbN = trabecular number; TbSp = trabecular separation; TbTh = trabecular thickness; * p < 0.0001 vs sham group (Mann-Whitney U-test);† p < 0.05, †† p < 0.01, ††† p < 0.001 vs positive control group (Mann-Whitney U-test).



Table III. Effects of D-003 on bones from prednisolone-treated rats; morphometric study of bone resorption parameters

Group Dose (mg/kg) Osteoclasts (no./mm) OcS/BS (%)

Femoral neck

Sham 0 0.44 ± 0.02 5.41 ± 0.18

Positive control 0 0.68 ± 0.04* 8.19 ± 0.16*

D-003 5 0.46 ± 0.02† 5.48 ± 0.32†

D-003 25 0.43 ± 0.02† 5.35 ± 0.24†

D-003 200 0.42 ± 0.02† 5.25 ± 0.19†

Distal femur

Sham 0 0.91 ± 0.03 3.99 ± 0.29


D-003 5 1.03 ± 0.04† 3.99 ± 0.19†

D-003 25 0.93 ± 0.06† 3.92 ± 0.03†

D-003 200 0.87 ± 0.03† 3.88 ± 0.04†

Fifth vertebra

Sham 0 0.27 ± 0.02 1.18 ± 0.15


D-003 5 0.29 ± 0.02† 0.79 ± 0.06†

D-003 25 0.23 ± 0.02† 0.60 ± 0.03†

D-003 200 0.21 ± 0.03† 0.58 ± 0.03†

OcS/BS = osteoclast surface/bone surface; * p < 0.01 vs sham group (Mann-Whitney U-test); † p < 0.001 vs positive control group (Mann-Whitney U-test).

The positive control group showed the changes id-laden pluripotential stromal cells in bones, a re-described for the model of corticosteroid-induced sult consistent with previously reported data show-osteoporosis, which includes occurrence of ON, a ing that mice treated with dexamethasone had bonesignificant reduction of TBV, TbN, TbTh and ObS/ marrow infiltrated by numerous lipid-laden, pluri-BS, and increased number and perimeter of osteo- potential stromal cells.[42] In turn, pluripotentialclasts.[6] Such data reinforce the validity of our marrow stromal cells cultured in dexamethasone-results and confirm that the effects reported here are enriched medium differentiate into adipocytes andactually D-003 dependent. partly lose the expression of osteocalcin messenger

RNA and type I collagen, paralleling the suppressedPositive controls showed ON, with the presenceof hypertrophy of bone marrow adipocytes and lip- maturation of mesenchymal cells into osteoblasts.[43]

10

15

20

25

30

5th lumbar vertebra Femoral neck Distal femur

Sham+ Control5 mg/kg25 mg/kg200 mg/kg

ObS

/BS *

†† ††††

††

*

†† ††

*

† † †

Fig. 2. Effect of D-003 on osteoblast surface (ObS/BS) of prednisolone-treated rats. * p < 0.001 vs sham group (Mann-Whitney U-test);† p < 0.01, †† p < 0.001 vs positive control (+ Control) group (Mann-Whitney U-test).


288 Noa et al.

cannot discard the possibility that a similar effectcould explain, at least partially, the effects of D-003.

A previous study demonstrating that D-003 sig-nificantly prevented bone resorption inovariectomised rats showed that D-003 preventedthe decrease of TbN and TbTh as well as the in-crease of trabecular separation and osteoclast num-ber and perimeter, but failed to change ObS/BS, ahistomorphometric indicator of bone formation.[31]

0

25

50

75

100 Sham+ Control5 mg/kg25 mg/kg200 mg/kg

0/8

8/8

3/8

2/8

1/8

*

†††

††

Ani

mal

s w

ith O

N (

%)

Fig. 3. Effect of D-003 on femoral neck osteonecrosis (ON) in ratswith prednisolone-induced osteoporosis. * p < 0.001 vs sham group(Fisher’s Exact Probability test); † p < 0.05, †† p < 0.01 vs positivecontrol (+ Control) group (Fisher’s Exact Probability test).

The possible explanation for the different effects ofD-003 on osteoblasts in the two osteoporosis modelsCorticosteroid-induced osteoporosis shows simi-must consider the absence of lipid-laden mesenchy-larities to that induced by bilateral ovariectomy, inmal cells differentiating into adipocytes, but not intothat both models reveal bone loss and changes inosteoblasts in the ovariectomised rat model.

bone microarchitecture.[44] Nevertheless, there areFurthermore, ON or avascular necrosis, a com-some differences; the hallmark of corticosteroid-

mon complication present in approximately 25% ofinduced osteoporosis is the decrease in bone forma-patients receiving corticosteroid therapy,[42,43] main-tion with reduction of osteoblasts and concomitantly affecting the femoral head, was prevented with D-presence of ON. The role of bone resorption in the003. Boss and Misselevich[42] reported that lipid-pathogenesis of osteoporosis induced by long-terminduced hypertrophy of the fat cells of rabbits treat-treatment with corticosteroids is uncertain, and sev-ed with corticosteroids cannot expand the marroweral authors have reported an early increase of bonecavity within the inflexible osseous cage. Conse-resorption in animals and humans.[8-11] Our resultsquently, the pressure within the bone rises, leadingwere consistent with these findings, since bone re-to sinusoidal compression, venous stasis and, even-sorption parameters were increased compared withtually, arterial obstruction, accounting for ischaemicthose in negative controls. This finding could be theON. The great reduction of hypertrophic bone mar-main reason for the rapid bone loss observed inrow adipocytes in D-003-treated animals could ex-humans after the initiation of corticosteroid therapy,plain the reduction of ON observed in our study.and also explains the effectiveness of antiresorptive

agents in the management of corticosteroid-induced The preventive effects of D-003 on the osteo-osteoporosis. porotic changes induced with prednisolone in rats

demonstrated here confirmed the hypothesis sup-D-003 significantly and dose-dependently pre-porting the objective of this study. These effectsvented such changes, also increasing ObS/BS, acould be related, at least partially, to the inhibition ofmarker of bone formation, in comparison to thecholesterol biosynthesis prior to mevalonate forma-positive control group. The results suggest that D-tion induced by D-003.[25] Thus, such an effect must003 prevented the reduction of bone formation in-reach the extravascular system, preventing osteo-duced by corticosteroids. The mechanism wherebyporosis development, as occurs with other inhibitorsD-003 prevented the reduction of osteoblasts in cor-of cholesterol biosynthesis, such as statins.[19,20]

ticosteroid-induced osteoporosis in rats is beyondthe objective of this study. Nevertheless, consider- Also, since inhibition of lipid oxidation has beening that lovastatin, an HMG-CoA reductase inhibi- linked to bone functioning, the ability of D-003 totor, in vitro inhibited the corticosteroid-induced ex- prevent plasma lipoprotein peroxidation[29,30] couldpression of adipocytic genes and counteracted the also contribute to preventing development of osteo-

porosis.inhibition of expression of osteoblastic genes,[45] we



13. King C, Wein E, Gundberg C, et al. Effects of continuousConclusionsglucorticoid infusion on bone metabolism in the rat. CalcifTissue Int 1996; 59: 184-91D-003 5, 25 and 200 mg/kg/day prevented tra-

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Clin Invest 1999; 104: 1363-74mental studies must evaluate the direct effect of18. Fisher JE, Rogers MJ, Halasy JM, et al. Alendronate mechanismD-003 on bone formation and apoptosis of bone

of action: geranylgeraniol, an intermediate of the mevalonatecells, and clinical data using bone resorption mark- pathway, prevents inhibition of osteoclasts formation, bone

resorption and kinase activation in vitro. Proc Natl Acad Sci Uers and densitometry are required to confirm theS A 1999; 96: 133-8clinical relevance of these potential effects.

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1946-9received by any of the authors for conducting the study.21. Bayraktar F, Oge A, Uyulgan B, et al. Atorvastatin increases

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