ORIGINAL ARTICLE

Positive skeletal effects of cladrin, a naturally occurringdimethoxydaidzein, in osteopenic rats that were maintainedafter treatment discontinuation

K. Khan & K. Sharan & G. Swarnkar & B. Chakravarti &M. Mittal & T. K. Barbhuyan & S. P. China & M. P. Khan &

G. K. Nagar & D. Yadav & P. Dixit & R. Maurya &

N. Chattopadhyay

Received: 11 February 2012 /Accepted: 6 August 2012# International Osteoporosis Foundation and National Osteoporosis Foundation 2012

AbstractSummary Effects of cladrin treatment and withdrawal inosteopenic rats were studied. Cladrin improved trabecularmicroarchitecture, increased lumbar vertebral compressivestrength, augmented coupled remodeling, and increasedbone osteogenic genes. A significant skeletal gain wasmaintained 4 weeks after cladrin withdrawal. Findings sug-gest that cladrin has significant positive skeletal effects.Introduction We showed that a standardized extract ofButea monosperma preserved trabecular bone mass in ovari-ectomized (OVx) rats. Cladrin, the most abundant bioactivecompound of the extract, promoted peak bone massachievement in growing rats by stimulating osteoblast func-tion. Here, we studied the effects of cladrin treatment andwithdrawal on the osteopenic bones.Methods Adult female Sprague–Dawley rats were OVx andleft untreated for 12 weeks to allow for significant estrogendeficiency-induced bone loss, at which point cladrin (1 and10 mg/kg/day) was administered orally for another 12 weeks.Half of the rats were killed at the end of the treatments and theother half at 4 weeks after treatment withdrawal. Sham-operated

rats and OVx rats treated with PTH or 17β-estradiol (E2) servedas various controls. Efficacy was evaluated by bone microarch-itecture using microcomputed tomographic analysis and fluo-rescent labeling of bone. qPCR and western blotting measuredmRNA and protein levels in bone and uterus. Specific ELISAwas used for measuring levels of serum PINP and urinary CTx.Results In osteopenic rats, cladrin treatment dose dependentlyimproved trabecular microarchitecture, increased lumbar ver-tebral compression strength, bone formation rate (BFR), cor-tical thickness (Cs.Th), serum PINP levels, and expression ofosteogenic genes in bones; and reduced expression of boneosteoclastogenic genes and urinary CTx levels. Cladrin had nouterine estrogenicity. Cladrin at 10mg/kgmaintained acquiredskeletal gains 4 weeks after withdrawal.Conclusion Cladrin had positive skeletal effects in osteo-penic rats that were maintained after treatment withdrawal.

Keywords Bone formation . Bone strength .

Microarchitecture . Phytoestrogen . Treatment withdrawal

Introduction

In recent years, naturally occurring dietary compounds havereceived greater attention in the field of osteoporosis preven-tion and treatment research [1–3]. A reduced prevalence ofosteoporotic fractures in East Asian women has been attribut-ed to their soy-rich diet and, in view of which, trials haveaddressed skeletal effects of soy isoflavones in postmenopaus-al women [4–9]. However, results of the clinical and epide-miological studies on the effects of consumption of soy foodsenriched in isoflavones on bone health have failed to providebone-conserving effects in postmenopausal osteoporosis[10–12], thereby leaving the scope for identifying more potentforms of isoflavones with respect to skeletal effect.

Electronic supplementary material The online version of this article(doi:10.1007/s00198-012-2121-8) contains supplementary material,which is available to authorized users.

K. Khan :K. Sharan :G. Swarnkar : B. Chakravarti :M. Mittal :T. K. Barbhuyan : S. P. China :M. P. Khan :G. K. Nagar :D. Yadav :N. Chattopadhyay (*)Division of Endocrinology,CSIR—Central Drug Research Institute,Chattar Manzil, P.O. Box 173, Lucknow, Indiae-mail: n_chattopadhyay@cdri.res.in

P. Dixit :R. MauryaDivision of Medicinal & Process Chemistry,CSIR—Central Drug Research Institute,Chattar Manzil, P.O. Box 173, Lucknow, India

Osteoporos IntDOI 10.1007/s00198-012-2121-8

Daidzein is a major constituent of soy isoflavone. Al-though no human studies have been conducted to assessskeletal effects of daidzein, ipriflavone, a synthetic deriva-tive of daidzein effectively reduced bone loss in postmeno-pausal women in several small intervention studies [13–15].On the other hand, in a large RCT, ipriflavone failed to showany skeletal advantage in postmenopausal women comparedto the placebo group [16]. Notwithstanding variable efficacyoutcomes of the clinical trials with ipriflavone, it appearsthat this daidzein analog may have some mitigating effect ondecreasing bone turnover and maintaining BMD [16–20].

Daidzein showed favorable effects on bone cells by stimu-lation of osteoblast [21–23] and inhibition of osteoclast func-tions [24] acting via the estrogen receptors (ER). Often theconcentrations of daidzein used in bone cell cultures are inthe micromolar ranges that are unlikely to be achieved in vivodue to extensive metabolic alterations in the intestine and liver(glucoronidation and sufonylation), the major causes for itsreduced bioavailability. In ovariectomized (OVx) rats, daidzeinreduced bone turnover but failed to reverse a previously estab-lished bone loss [25]. Reports suggest that the O-methoxysubstitutions of free phenolic hydroxyl groups enhance lip-ophilicity and lead to the production of derivatives that arenot susceptible to glucuronic acid or sulfate conjugation [26].Thus O-methoxy substitution of daidzein could improve itspharmacokinetic/metabolic stability profiles and consequently,enhance the pharmacodynamic effect (in vivo potency) [27].Daidzein also exhibits varying degrees of estrogenic and anti-estrogenic effects in a species-specific manner [28]. The estro-genic effect of daidzein, which appears to underlie its beneficialskeletal outcomes, is mediated by equol, a major metabolite ofdaidzein formed by the action of intestinal microflora [29–31].Studies suggest that almost one third of humans are unable tobiotransform daidzein to equol in the intestine, which preventsthem from having beneficial effects of daidzein [32]. Methoxysubstitution could also overcome the unfavorable estrogeniceffect of isoflavonoids such as daidzein [33]. Together, itappears that if daidzein is considered as a ‘lead’ pharmacophorefor postmenopausal osteoporosis, then overcoming the existingdisadvantages, including poor bioavailability, estrogenicity, andbiopotency towards promoting skeletal function, could beachieved via its methoxylation. So far, no systematic study isavailable on the skeletal effect of O-methoxy derivative ofdaidzein in osteopenic condition.

In our phytopharmacological evaluation program aimed atfinding effective alternative therapy against postmenopausalosteoporosis, we showed that a total extract and a standardizedfraction from the stem-bark of Butea monosperma attenuatedOVx-induced bone loss in rats. To this effect, the standardizedfraction was tenfold potent than the total extract of B. mono-sperma [34]. There were five methoxyisoflavones present in theextract and fraction, including cajanin (7-methoxy genistein),medicarpin (a methoxypterocarpan with cyclized genistein ring

structure), isoformononetin (7-methoxy dadizein), formonone-tin (4′-methoxy daidzein), and cladrin (3′4,-dimethoxy daid-zein). Each of them stimulated osteoblast differentiation invitro and all but formononetin promoted modeling-directedbone growth in growing female rats. Out of these compounds,medicarpin, isoformononetin, and cladrin were enriched in thestandardized fraction that likely contributed to its enhancedbiopotency over the total extract. Cladrin was the highestenriched of the three methoxydaidzein analogs in the fraction.Cladrin was 104-fold more potent than daidzein in inducingosteoblast proliferation and differentiation in vitro by signalingthrough extracellular signal-regulated kinase (Erk). Pharmaco-kinetic studies revealed superior oral bioavailability of cladrinover daidzein, and unlike in the case of the later, the formationof estrogenic metabolite, equol was not observed with cladrin[35, 36]. Together, from these reports, it appears that in cladrin,the aforementioned limitations of daidzein have been overcome.

The present studywas designed to assess the effects of cladrintreatment in osteoporotic bones as well as the skeletal responseafter withdrawal of treatment by using static and dynamic his-tomorphometries, evaluation of biomechanical competence, andexpression levels of osteogenic and osteoclastogenic genes inthe long bones and biochemical parameters relevant to bonemetabolism. As one of the prerequisites for any postmenopausaltherapy is the absence of estrogenicity of a given agent, wemadea comprehensive investigation of the uterine effects of cladrin.Because cladrin is classified under phytoestrogen, its skeletaleffects were compared with 17β-estradiol (E2). Towards assess-ing the bone gain effect of cladrin, the clinically used anabolictherapy, intermittent injection of human parathyroid hormone(PTH), was included within the study.

Material and methods

Reagents and chemicals

All fine chemicals including E2, calcein, and tetracyclinewere purchased from Sigma-Aldrich (St. Louis, MO, USA).Human PTH (1–34) was purchased from Calbiochem, USA.Cladrin, initially isolated from B. monosperma [37], wassubsequently synthesized in gram scale for in vivo studiesby previously published protocol [38]. Synthesized com-pounds were matched with the data of the authentic samplesand the purities of the compounds were confirmed by HPLCand nuclear magnetic resonance analytical methods [37].

Animals and experimental procedures

All animal care and experimental procedures were approvedby the Institutional Animal Ethics Committee (IAEC). Fe-male Sprague–Dawley rats were obtained from the NationalLaboratory Animal Centre, CSIR-CDRI. Animals were kept

Osteoporos Int

in a 12 h light–dark cycle, with controlled temperature (22–24 °C) and humidity (50–60 %). Standard laboratory rodentchow diet (devoid of soy proteins) and water were providedad libitum [39, 40].

Figure 1 summarizes the study timeline and experimentalgroups. One hundred and twenty adult Sprague–Dawley rats(10–12 weeks old; 200±20 g each) were randomly divided intosix equal groups (n020 rats/group) as follows: sham operated(ovary intact) + vehicle (gum acacia in distilled water p.o.),OVx + vehicle, OVx+40.0μg/kg PTH (5 days/weeki.p.), OVx+100 μg/kg E2 (5 days/weeks.c.), OVx+1 mg/kg/day cladrin,and OVx+10 mg/kg/day p.o. cladrin in gum acacia. Sincecladrin has been reported to enhance modeling-directed bonegrowth at 10 mg/kg/day dose [36], we used this dose and alower dose for the current study. For cladrin treatment, 1% gumacacia was used as vehicle based on our previous studies as wellas the report that it possesses characteristics to act as suspendingagent, stabilizer, and thickener in pharmaceutical formulations[41]. Because gum acacia is reported to alter calcium absorption[42], we used it at only 1 % and the same was given to thecontrol group as vehicle to cancel out any modulatory effect oncalcium absorption. The dosing and regimen of PTH and E2were based on previous reports [43–46]. The dose of E2(100 μg/kg) was higher than normally used in rodents becauseit is reported that at higher doses, E2 could stimulate boneformation along with suppression of resorption [47].

Rats were bilaterally OVx and left untreated for 12 weeksfor osteopenia to develop [48]. Various treatments describedabove started 12 weeks after the surgery and continued for12 weeks, after which ten rats from each group were killed(baseline), and the remaining animals were continued foradditional weeks without any active treatments. At 2 and4 weeks postwithdrawal, μCT scans of proximal tibia of live

rats of all groups were used to monitor changes in bonevolume fraction (BV/TV). At the end of withdrawal span(4 weeks), the rest of the animals (n010 rats/group) from eachgroup were killed (Fig. 1). On the day of sacrifice, femur, tibia,and L5 vertebrae were collected, cleaned off the soft tissues,and stored in 70 % isopropanol at 4 °C until further analysis.

For dynamic histomorphometry measurements, each ani-mal received calcein (20 mg/kgi.p.) and tetracycline (20 mg/kgi.p.), respectively, 15 and 3 days before sacrifice (baseline);bone mechanical strength was examined by compression testof the L5 vertebrae. Urine and serum samples were collectedafter 24-h starvation to assess changes in biochemical markers(urinary CTx/Cr and serum PINP) of bone metabolism. Theuterine histomorphometric measurement was done amongvarious treatment groups to determine estrogenicity.

μCT

Ex vivo μCT scanning of femur, tibia, and L5 vertebrae wascarried out using the SkyScan 1076 μCT scanner (Aartselaar,Belgium) as described before [48, 49]. In vivo μCT scans ofproximal tibia metaphysis were obtained after 12 weeks oftreatment and at 2- and 4-week withdrawal after anesthetizingrats with ketamin (90 mg/kg) and xylazine (10 mg/kg) duringthe scan, which lasted about 20 min. From the in vivo scan,hundred projections were acquired at 360° angular range withspecial modewidth adjusted to full. Reconstruction was carriedout using a modified Feldkamp algorithm using the SkyScanNRecon software. To analyze, ROI was drawn at a total of 100slices in the region of secondary spongiosa situated 1.5 mmaway from the distal border of growth plate excluding allprimary spongiosa and cortical bone, and analyzed with theCT analyzer (CTAn, SkyScan) software. Trabecular bone

Fig. 1 Study design. Treatmentstarted 12 weeks after OVxsurgery in Sprague–Dawleyrats. Treatments were given for12 weeks. Ten animals pergroup were killed at baseline,after treatment and 4 weeksafter discontinuation oftreatment (end point). Blood,urine (24 h), uterus, tibia, fe-mur, and vertebrae were har-vested at both baseline and endpoint

Osteoporos Int

volume, trabecular number, and trabecular separation of femurepiphysis, proximal tibia metaphysis, and L5 vertebrae werecalculated by the mean intercept length method. Trabecularthickness was calculated according to themethod of Hildebrandand Ruegsegger [50]. 3-D parameters were based on analysis ofa Marching cubes-type model with a rendered surface. Corticalthickness (Cs.Th), cortical area, and periosteal area (T.Ar) werecalculated by 2-D analysis of cortical bones of femur (mid-diaphysis). To ensure consistent measurement of corticalparameters, the beginning of the growth plate served as thereference point from where 350 serial image slides were dis-carded to exclude the trabecular region, and the following 200consecutive image slides (representing only cortical bone) wereselected for analysis and quantification using CTAn software.

Fluorochrome labeling and bone histomorphometry

Methods followed our previously published protocol withsome modifications. Cross sections (50 μm) of distal regionsof undecalcified femur diaphysis of each rat were obtainedusing an IsoMet Low Speed Bone Cutter (Buehler, LakeBluff, IL, USA). Images were captured using Leica Qwinsoftware, and periosteal (p) bone-forming rate/bone surface(pBFR/BS) and mineral appositional rate (pMAR) were cal-culated [51]. Histomorphometric measurements included peri-osteal perimeters, single-labeled surface (sLS), double-labeledsurface (dLS), and interlabeled thickness (IrLTh). These datawere used to calculate the mineralizing surface/bone surface(MS/BS), mineral apposition rate (MAR), and bone formationrate (BFR) as follows: pMS/BS0(1/2 sLS + dLS) / BS (%);pMAR 0 IrLTh/12 days (μm/day); pBFR/BS 0 MAR × MS/BS (μm3/μm2/day). To correct for label escape error, the levelof bone surface actively mineralizing was calculated as thesum of the dLS plus half of the sLS [52]. When double labelswere missing in femur sections of the same surface, but singlelabels were present, MARwas set to the biological lower limitof 0.3 μm/day [53], and if both single and double labels weremissing, MAR was treated as a missing value [54].

Vertebral compression test

Following endplate removal, the fifth lumbar vertebrae (L5)from each rat was isolated for compression testing. Spinousand transverse processes were also removed. In addition, thearticular surfaces were removed to provide two flat andparallel faces for compression testing. The vertebrae weremounted between the faces of a compression jig and aconstant force was applied in the craniocaudal direction ata nominal deformation rate of 2 mm/min [55] using a BoneStrength Tester TK-252C (Muromachi Kikai Co., Ltd.,Tokyo, Japan). The load–displacement curves generatedwere used to calculate the ultimate load (Newton), stiffness(Newton per millimeter), and energy to failure (millijoule).

Estrogenecity assessment

Bodyweight of each animal was taken before the start and endof the experiment. The uterus of each rat was weighed andthen divided into three parts for histomorphometry, qPCRdetermination of mRNA levels, and western blot analysis.

A 6-mm segment from the middle part of each uterus fixedin 2 % paraformaldehyde was dehydrated and embedded inparaffin wax using standard procedures. Transverse sections(5 μm) were stained with hematoxylin and eosin and repre-sentative images were captured. Total uterine area, luminalarea, and luminal epithelial height were measured using LeicaQwin-Semiautomatic Image Analysis software [49, 56].

Total RNA was isolated from total uterus using TRIzol(Gibco BRL, Gaithersburg, MD, USA). From aliquots of2 μg total RNA/tissue sample, cDNA was synthesized withthe Revertaid cDNA Synthesis Kit (Fermentas, Austin, TX,USA). For qPCR, the cDNAwas amplified using SYBR greenchemistry to perform quantitative determination of progesteronereceptor (PR) and the housekeeping gene GAPDH. The designof sense and antisense oligonucleotide primers was based onpublished cDNA sequences using the Universal ProbeLibrary(Roche Applied Science, Indianapolis, IN, USA). The sequenceof the primer pairs for each genes were as follows: PR(NM_022847.1) 5′-GGCAGCTGCTTTCAGTAGTCA-3′ and5 ′-CCTTGAATGTGTAGCTACTGGT-3 ′ ; GAPDH(NM_017008) 5′-TTTGATGTTAGTGGGGTCTCG-3′ and5′-GAAGGGCAACTACTGTTCGA-3′.

Western blotting of uteri was performed using previouslypublished protocol [57]. Tissue was homogenized in an ice-coldPBS with protease inhibitor cocktail (50 μg/g tissue) (Sigma-Aldrich, St Louis, MO, USA). After quantification by Bradfordassay, aliquots of 40 μg protein were separated on 10 % acryl-amide gel. After transfer to Immuno-Blot PVDF membrane,nonspecific sites were blocked with chemiluminescent blocker(Millipore) for 30 min at room temperature and then incubatedovernight at 4 °C with PR antibody at 1:1,000 dilution (SantaCruz Biotechnology, Santa Cruz, CA, USA) in 2 % BSA inTBS. After washing, blots were incubated with secondaryantibody (1:5,000 dilution, conjugated with horseradish perox-idase) for 1 h at room temperature. After repeated washing with0.1 % Tween 20 in TBS, membranes were developed withImmobilon Western Chemiluminescent HRP substrate.

Bone biochemical marker analysis

On the basis of our previously published protocols [58],urinary C-terminal teleopeptide of type I collagen (CTx) lev-els and serum type 1 procollagen N-terminal propeptide(P1NP) were determined by enzyme-linked immunosorbentassay kits purchased from Immunodiagnostic Systems, Inc.(Tyne & Wear, UK) by following the manufacturer's proto-cols. Serum and urine samples from individual rats were

Osteoporos Int

assayed for PINP and CTx, respectively, in duplicate. Urinarycreatinine was measured by an automated biochemistry ana-lyzer (Merk Selectra Junior) for normalizing the CTx data.

Studies on the expression of osteoblast- and osteoclast-specific genes in long bones

Pooled femur and tibia were pulverized in liquid N2. Thefrozen powder was transferred into a tube containing TRIzoland total RNA was isolated and qPCR were performed asdescribed before [40]. qPCR analysis of runt-related tran-scription factor 2 (RUNX2), type I collagen (COL1), osteo-calcin (OCN), osteoprotegrin (OPG), receptor activator ofNF-κB ligand (RANKL), RANK and tartrate resistance acidphosphatase (TRAP) were performed as described before[49]. The housekeeping gene GAPDH was used as theinternal control in this study. The sequence of the primerpairs for each gene was as follows: COL1 (NM_053304) 5′-CATGTTCAGCTTTGTGGACCT-3′ and 5′-TGTAGGGACTTCAGTCGACG-3′; OCN (NM_013414) 5′-ATAGACTCCGGCGCTACCTC-3′ and 5′-GGATGGGTCTAGGGGACC-3′; OPG (U94330.1) 5′-TGAGGTTTCCAGAGGACCAC-3′ and 5′-TGTTGGGTCCTTTGGAAAGG-3′;RANKL (NM_012870.2) 5′-TGAGGTTTCCAGAGGACCAC-3 ′ and 5 ′-TGTTGGGTCCTTTGGAAAGG-3 ′;RUNX-2 (NM_053470), 5′-CCACAGAGCTATTAAAGTGACAGTG-3′ and 5′-CGAACTACTGAAGTTTGGATCAAACAA-3′; RANK (NM_012870.2), 5′-TGAGGTTTCCAGAGGACCAC-3′ and 5′-TGTTGGGTCCTTTGGAAAGG-3′; TRAP (NM_019144.1) 5′-GCCTCTTGCGTCCTCTATGA-3′ and 5′-ACCTATGCACCTACCACGA-3′.

Statistics

Data are expressed as mean ± SEM. The data obtained inexperiments with multiple treatments were subjected to one-way ANOVA followed by post hoc Newman–Keuls multi-ple comparison test of significance using GraphPad Prism 5software. An unpaired form of Student's t test was used tocompare endpoint data with baseline.

Results

Effect on uterine estrogenecity and changes in body weight

Cladrin was generally well tolerated for the duration(12 weeks) of administration. OVx resulted in increasedbody weight compared to the ovary intact sham group. Bodyweight was not different between the E2 and sham groups.PTH or cladrin treatment to OVx rats had no effect on theOVx-induced weight gain (Supplemental Table 1).

Efficacy of OVxwas confirmed by studying uterine param-eters. OVx resulted in reduced uterine weight-to-body weightratio compared to sham group. This ratio was not differentbetween the sham and OVx + E2 groups, and between theOVx and OVx rats treated with cladrin or PTH (Fig. 2a).Analysis of uterine histomorphometry (representative photo-micrograph of the uterine cross section in variousgroups in Fig. 2b) showed that OVx resulted in markedreduction in total uterine area, luminal area, and luminalepithelial cell height compared to the sham group (Fig. 2c–e).E2 supplementation to OVx rats exhibited comparable uterineand luminal areas to the sham but increased luminal epithelialcell height. These parameters were not different between theOVx + veh and OVx rats treated with cladrin or PTH.

To exclude that cladrin may have very low estrogenic po-tency, expression of PR mRNA and protein levels in the uteruswas compared between the various groups. Relative to thesham rats, OVx resulted in the reduction of PR mRNA levelsin the uterus by half, and E2 supplementation to OVx ratsrobustly increased the transcript (Fig. 2f). PR mRNA levelswere not different between the OVx and cladrin groups. WhenPR was assessed at the protein level between the groups, theresults were in agreement with the transcript data (Fig. 2g).

Effect on trabecular microarchitecture in osteopenic rats

In vivo μCT scans of tibia metaphysis exhibited dramaticdecrease in bone volume fraction (BV/TV) 12 weeks afterOVx, suggesting significant induction of osteopenia. Cladrin,PTH, and E2 treatments to osteopenic rats for the next12 weeks increased BV/TV compared to its pretreatmentlevels (Fig. 5).

μCT analysis of the excised femur epiphysis, tibia proximalmetaphysis, and L5 vertebra at the end of treatments confirmedthe expected trabecular bone loss caused by OVx (shamversus OVx after 24 weeks). Various treatments to OVxrats presented improved μCT patterns at different tra-becular sites when compared to OVx rats (representativeμCT images shown in Fig. 6). Trabecular response tocladrin treatment of OVx rats was quantified, and the valueswere compared with that of sham, PTH, and E2 (Table 1).

Femoral data showed that compared with the shamgroup, the OVx + veh group had reduced BV/TV, Tb.N,and Conn.D, and increased Tb.sp, Tb.pf, and SMI (Table 1).Cladrin treatment to OVx rats dose dependently increasedBV/TV and Tb.N, and reduced Tb.pf. Comparison of thecladrin (10 mg/kg) or PTH treatment group with the shamgroup showed no significant differences in BV/TV, Tb.N,Tb.pf, and SMI. Conn.D was lower in the cladrin (10 mg/kg) group compared with the sham but higher than the OVx+ veh group. Tb.Th was not different among the variousgroups except PTH where it was higher than the shamgroup. Tb.sp values were lower in the cladrin (10 mg/kg),

Osteoporos Int

PTH, and E2 groups compared to OVx but higher than thesham group, and they were not different between the cladrin(1 mg/kg) and OVx groups. In the E2 group, BV/TV andTb.N were higher than the OVx but lower than the shamgroup. SMI and Conn.D in the E2 group were not differentfrom the OVx group. Tb.pf was not different between the E2and the sham groups.

Tibial data showed substantial deterioration in all thetrabecular parameters in the OVx + veh group comparedto the sham (Table 1). Cladrin treatment of OVx rats dosedependently reversed OVx-induced changes in BV/TV,Conn.D, Tb.sp, Tb.pf, and SMI. All the parameters betweenthe cladrin (10 mg/kg) and sham groups were comparable.PTH group had higher Tb.Th than the sham group; however,

Fig. 2 Cladrin is devoid of uterine estrogenicity. Uteri were harvestedfrom rats after various treatments. a Compared to sham, uterine-to-body weight ratio in all OVx groups with different treatments exceptOVx + E2. b Representative photomicrographs of the stained (H&E,4×) cross sections of the uteri. Histomorphometric calculations per-formed on these sections showed that compared to the sham c uterinearea, d luminal area, and e luminal epithelial cell heights were reducedin all OVx groups with different treatments except OVx + E2. Uteriwere harvested from the sham, OVx, and OVx treated with cladrin

(10 mg/kg) or E2; f PR mRNA and g PR protein levels were quantifiedafter appropriate loading corrections. OVx and OVx + cladrin hadcomparable mRNA and protein levels of PR, which were less thanE2-replete rats. Data represent the mean ± SE from three independentexperiments. All values are expressed as mean ± SEM (n010 rats/group); ***P<0.001 compared with OVx or as indicated. V vehicle;cladrin 1 and 10 mg/kg/day p.o., PTH 40 μg/kgi.p. 5 times/week; E217β-estradiol 100 μg/kg/days.c. 5 times/week

Osteoporos Int

Tab

le1

Effectof

variou

streatm

entsfor12

weeks

ondifferentbo

neparametersof

osteop

enic

rats(baseline)

Param

eters

Sham

+vehicle

(gum

-acacia)

OVx+vehicle

(gum

-acacia)

OVx+cladrin

(1mg/kg

)OVx+cladrin

(10mg/kg

)OVx+PTH

(40μg

/kg)

OVx+E2

(100

μg/kg)

μCTmeasurementsat

femur

epiphy

sis

BV/TV

(%)

21.43±0.25

2*10

.19±0.10

416

.04±0.50

4***,££,+++

20.86±0.85

**

22.39±0.19

6*16

.86±0.12

3***,££

Tb.N

(1/m

m)

1.88

±0.16

*0.83

3±0.09

51.29

±0.02

8***,£££,+++

1.60

6±0.09

7**

1.67

±0.113*

*1.34

±0.12

5***,£££

Tb.Th(m

m)

0.12

1±0.00

20.12

3±0.00

10.12

9±0.00

30.13

3±0.02

0.15

7±0.00

4*0.12

1±0.00

1

Con

n.D

(1/m

m3)

100.33

±11.15*

46.42±7.48

368

.494

±7.31

90.54±1.75

3***,£££

102.1±7.61

*45

.63±7.56

1

Tb.Sp(m

m)

0.60

6±0.10

2**

1.12

5±0.08

70.95

7±0.07

60.76

7±0.06

***,£££

0.71

8±0.05

4***,£££

0.76

9±0.02

8***,£££

Tb.Pf(1/m

m)

1.61

8±0.31

*5.95

5±0.74

92.16

±0.55

7*,++

1.79

2±0.08

9*1.84

7±0.12

2*2.23

±0.01

2*

SMI

1.54

9±0.08

6*2.12

±0.118

1.97

3±0.07

11.48

7±0.21

9*1.59

2±0.10

4*2.01

±0.17

8

μCTmeasurementsat

prox

imal

tibia

metaphy

sis

BV/TV

(%)

21.885

±0.80

1*5.98

8±0.46

011.57±0.66

**,£££,+++

19.164

±0.52

9*21

.027

±0.28

3*15

.031

±0.14

9*

Tb.N

(1/m

m)

1.48

5±0.22

3*0.69

9±0.10

20.75

5±0.03

71.22

8±0.06

2**

1.27

3±0.05

9**

0.96

1±0.08

9***,££

Tb.Th(m

m)

0.119±0.00

1*,$$$

0.10

3±0.00

080.09

8±0.00

20.12

1±0.00

2*0.14

8±0.00

3*0.117±0.00

04*

Con

n.D

(1/m

m3)

63.784

±7.07

8*22

.448

±2.61

538

.854

±3.29

2***,£££,+++

61.604

±2.48

6*62

.415

±3.89

*61

.915

±2.12

1*

Tb.Sp(m

m)

0.34

8±0.03

1*1.12

8±0.02

20.70

9±0.05

9*,££,+++

0.41

4±0.02

6*0.46

8±0.02

5*0.49

1±0.02

3*

Tb.Pf(1/m

m)

6.33

6±0.75

1*13

.406

±0.67

58.64

5±0.57

1*,£££,+++

6.33

6±0.75

1*6.49

4±0.77

4*12

.186

±0.59

5

SMI

1.61

6±0.14

2*2.91

5±0.05

72.04

2±0.08

8***,££,+++

1.73

7±0.09

4*1.70

7±0.14

3*2.69

±0.29

9

μCTmeasurementsat

L5

BV/TV

(%)

24.62±1.35

8*12

.216

±0.67

919

.733

±0.71

2***,£££,+++

23.82±1.118*

23.98±2.46

4*20

.796

±1.13

2***,£££

Tb.N

(1/m

m)

2.26

3±0.18

6*0.92

1±0.08

21.55

1±0.08

5**,£££,+++

2.09

3±0.06

4*2.08

6±0.07

4*1.95

5±0.07

7*

Tb.Th(m

m)

0.13

7±0.00

4*0.11

±0.00

20.09

7±0.00

10.13

±0.00

1*0.13

7±0.00

2*0.118±0.00

1

Con

n.D

(1/m

m3)

94.85±12

.573

*40

.433

±2.32

257

.766

±6.52

71.683

±4.97

6***,£££

68.65±8.04

3***,£££

72.033

±3.53

4***,£££

Tb.Sp(m

m)

0.36

1±0.02

4*0.64

8±0.02

90.48

±0.04

7***,£££,++

0.38

4±0.01

6*0.33

9±0.01

3*0.45

7±0.01

7***,£££

Tb.Pf(1/m

m)

4.37

5±0.91

7*11.39±0.97

210

.591

±0.91

24.41

2±0.48

2*4.10

7±0.76

8*10

.79±0.27

8

SMI

1.82

8±0.13

0***

2.45

1±0.09

12.32

±0.07

62.41

±0.111

2.05

6±0.07

1***

2.53

±0.90

4

L5compression

test

Ultimateload

(N)

202.07

±24

.107

***

107.32

±5.30

717

9.5±20

.922

$$$

208.71

±9.83

5***

281.55

±42

.105

*17

5.27

±12

.12$

$$

Energy(m

J)28

2.85

±2.84

9*15

0.4±4.09

923

3.3±9.5*

**,££,+++

286.3±4.3*

290.25

±6.84

9*26

3.95

±8.35

**,£££

Stiffness(N

/mm)

161.55

±2.25

**

71.2±2.4

108.65

±5.75

***,+++

159.9±1.1*

*16

3.1±1.09

9*16

0.6±5.5*

μCTmeasurementsat

femur

diaphy

sis

T.Ar(m

m2)

11.16±0.36

3*,++

8.98

±0.33

510

.79±0.33

5**,++

12.81±0.32

3*11.65±0.43

4*,+++

9.76

±0.35

7

B.Ar(m

m2)

5.62

±0.15

9***,$$$

5.12

±0.118

5.77

±0.17

8***

6.02

±0.16

5**

6.31

±0.18

5*5.42

±0.20

1

Cs.Th(μm)

0.49

±0.00

6**

0.43

±0.01

0.49

±0.00

7**

0.51

±0.011*

*0.54

±0.01

8*0.45

±0.01

3

Dyn

amic

histom

orph

ometricmeasurementsat

femur

diaphy

sis

sLS/dLS

0.81

0±0.01

7*0.87

2±0.00

50.85

0±0.00

5££,$$,++

0.80

2±0.00

7*0.79

4±0.00

7*0.86

3±0.00

3£,$,+

Osteoporos Int

all the remaining parameters were not different betweenthese two groups. E2 was not different from the sham whenBV/TV, Tb.Th, Conn.D, and Tb.sp were considered, butlower in the case of Tb.N. SMI and Tb.pf were not differentbetween the E2 and the OVx groups.

Similar to tibia trabeculae, L5 had severe microarchitec-tural erosion due to OVx (Table 1). Cladrin treatment to OVxrats dose dependently reversed BV/TV, Tb.N, and Tb.sp. SMIwas not different between the OVx, cladrin (either doses), andE2 groups. Cladrin (10 mg/kg) and PTH had BV/TV, Tb.th,Tb.sp, and Tb.pf comparable to the sham.

Effect on lumbar vertebral compression in osteopenic rats

Because trabecular microarchitecture is an independent deter-minant of vertebral fractures in humans with osteopenia [59],we studied whether better microarchitectural quality in L5 bycladrin over the OVx group translated to improved compressiveparameters. Table 1 showed that cladrin dose dependentlyincreased ultimate load, energy to failure, and maximum stiff-ness in the OVx rats. There was no difference in these param-eters between sham, cladrin (10 mg/kg), and PTH groups. Incomparison to the sham group, energy to failure was lower inthe E2 group but maximum stiffness was not different betweenthe two groups, while ultimate load was comparable betweenthe E2 and 1 mg/kg dosed cladrin groups.

Effect on bone accrual in osteopenic rats

Dynamic histology in the periosteal region of the femur diaph-ysis was compared in the various treatment groups (Table 1).sLS/dLS was increased in the OVx rats compared to the sham.Cladrin dose dependently reduced it and at the 10 mg/kg dose,it was not different from the sham and PTH groups. Nosignificant difference was observed in pMS/BS betweengroups. pMAR and pBFR/BS were reduced in OVx ratscompared with the sham group. Cladrin dose dependentlyincreased pMAR and pBFR/BS, and these two parameterswere not different between the sham, PTH, and cladrin(10 mg/kg) groups. sLS/dLS, pMAR, and pBFR/BS werenot different between the E2 and OVx groups.

Static histomorphometric (2D-μCT) measurements at thesite of femur mid-diaphysis (Table 1) show that relative to thesham group, the OVx group had reduced cortical thickness(Cs.Th), periosteal area (T.Ar), and cortical mean cross-sectional area (B.Ar). These three parameters were not differentbetween the sham, PTH, and cladrin groups and, between theOVx and E2 groups.

Effect on biochemical markers in osteopenic rats

Serum PINP levels, an accepted osteogenic marker, weresignificantly reduced in OVx rats compared to the shamT

able

1(con

tinued)

Param

eters

Sham

+vehicle

(gum

-acacia)

OVx+vehicle

(gum

-acacia)

OVx+cladrin

(1mg/kg

)OVx+cladrin

(10m

g/kg

)OVx+PTH

(40μ

g/kg

)OVx+E2

(100μg

/kg)

pMS/BS(%

)97

.997

±1.31

93.34±5.53

994

.27±2.46

95.871

±0.89

297

.249

±0.87

93.916

±1.21

4

pMAR(μm/day)

0.79

7±0.02

7**

0.56

6±0.011

0.63

7±0.05

££££,$$$

0.74

3±0.01

2***

0.77

8±0.01

4**

0.59

9±0.02

9££,$$$,+++

pBFR/BS(μm

3/μm

2/year)

4.07

9±0.34

1*2.91

3±0.04

33.28

9±0.18

7£,$$,+

3.89

6±0.54

9*3.95

1±0.20

6*3.28

3±0.02

8£,$$,++

Valuesrepresentmean±SEM;n010

rats/group

BV/TVbo

nevo

lume/trabecular

volume,

Tb.Sp

trabecular

spacing,

Tb.N

trabecular

number,Tb.Thtrabecular

thickn

ess,Tb.pf

trabecular

pattern

factor,Con

n.D

conn

ectio

ndensity,SM

Istructure

mod

elindex,

T.Arperiostealarea,B

.Arcorticalmeancross-sectionalarea,C

s.Thcorticalthickn

ess,sLS/dL

Ssing

le-labeled

surface/do

uble-labeled

surface,pM

S/BSperiostealmineralizingsurface/

bone

surface,pM

ARperiosteal

mineral

appo

sitio

nrate,pB

FR/BSperiosteal

bone

form

ationrate/bon

esurface

*P<0.00

1,**P<0.01

,***P<0.05

comparedtoOVx+veh;

£P<0.00

1,££P<0.01

,£££P<0.05

comparedtosham

;$P<0.00

1,$$P<0.01

,$$$P<0.05

comparedtoOVx+PTH;+

P<0.00

1,++P<0.01

,+++P<0.05

comparedto

OVx+cladrin,

10mg/kg

Osteoporos Int

(Fig. 3a). Cladrin dose dependently increased PINP levels,and these levels were not different between the sham, PTH,and cladrin (10 mg/kg) groups. PINP levels were comparablebetween the E2 and OVx groups.

Urinary CTx levels, an accepted antiresorptive marker,were twofold higher in the OVx rats compared with thesham (Fig. 3b). Cladrin dose dependently reduced CTxlevels, and these levels were not different between the sham,E2, and cladrin (10 mg/kg) groups. CTx levels were com-parable between the E2 and OVx groups.

Effect on the expression of osteoblast-and osteoclast-specific genes in bones

mRNA levels of osteogenic genes, including RUNX2,COL1, OCN, and OPG, were lower in the OVx femurcompared with the sham (Fig. 4a). Cladrin dose depen-dently increased mRNA levels of these genes. At the10 mg/kg dose of cladrin, the levels of RUNX2, col1,OCN, and OPG were higher than the sham. The levelsof RUNX2, col1, and OCN were higher in the PTHgroup compared to the sham. In the E2 group, RUNX2and OPG were higher than the sham but OCN was compara-ble. COL1 levels were not different between the E2 and OVxgroups. RANKL was higher in the OVx rats compared withthe sham, and the cladrin- and PTH-treated groups were notdifferent from the OVx. E2 group had decreased RANKLlevels compared to the sham. The OPG-to-RANKL ratiowas lower in the OVx group compared with the sham. Thisratio was comparable between the cladrin (10 mg/kg) andsham groups, and the cladrin (1 mg/kg), OVx, and PTH

groups. In comparison to the sham group, the OPG-to-RANKL ratio was robustly elevated in the E2 group.

mRNA levels of the osteoclast marker genes, includingRANK and TRAP,were robustly upregulated in the OVx groupcompared to the sham (Fig. 4b). Cladrin dose dependentlyreduced mRNA levels of both genes. Expression levels of thesetwo genes were not different between the cladrin (10 mg/kg)and sham groups, and PTH and OVx groups. RANK andTRAP levels in the E2 group were lower than the sham.

Effect of treatment withdrawal on trabecular bone

We studied the preservation of acquired bone gain after dis-continuation of cladrin treatment. In vivo μCT scans of prox-imal tibia metaphysis for all rats were obtained on the day lasttreatment was dispensed, which served as baseline (Fig. 7).BV/TV was used to monitor the changes at 2 and 4 weeksfollowing cessation of treatments. Four weeks after treatmentwithdrawal, OVx rats treated with cladrin (1 mg/kg) or E2showed significant decrease in BV/TV compared to theirrespective baseline values but it was maintained in cladrin(10 mg/kg) and PTH groups. BV/TV in the sham groupexhibited no differences at these two time points comparedto baseline values (data not shown). All groups were killed4 weeks post withdrawal (end point).

μCT analysis of isolated bones at the end point showedthat among all the trabecular parameters studied at femurepiphysis, cladrin (1 mg/kg) resulted in 53.9 % and 38.25 %reductions in BV/TV and Tb.N, respectively, compared tothe baseline (Table 2). Cladrin (10 mg/kg), PTH, and E2 hadno difference between the end point and baseline values in any

Fig. 3 Cladrin corrects OVx-induced alterations in biochemicalmarkers of bone at the end of the treatments (baseline). a Serum PINPlevels were dose dependently increased by cladrin and at the higherdose, the levels were comparable to the sham. b Urinary CTx levelsadjusted for urinary creatinine (Cr) were dose dependently diminished

by cladrin and at the higher dose, the levels were comparable to thesham. PTH had no effect on OVx-induced increase in urinary CTxlevels. All values are expressed as mean ± SEM (n010 rats/group); *P<0.05, **P<0.01, ***P<0.001 compared with OVx + veh or asindicated

Osteoporos Int

of the parameters. However, in contrast to cladrin (10 mg/kg)or PTH, E2 group at the end point had lower BV/TV andTb.N, and higher Tb.sp and SMI.

In the proximal tibia metaphysis, BV/TV in cladrin(1 mg/kg) at the end point was reduced by 56.78 %, whereas

Tb.pf and SMI were increased, respectively, by 36.28 % and32.37 % when compared to baseline (Table 2). Cladrin(10 mg/kg) and PTH had no difference between the endpoint and baseline in any of the parameters. In the E2 group,BV/TV in the end point was reduced by 26.75 % compared

Fig. 4 Cladrin favorably regulates expression of osteoblast- andosteoclast-specific genes in long bones at the end of the treatments(baseline). Total RNA was isolated from long bones and qPCR wasperformed to examine the mRNA levels of a osteoblast specific genes;RUNX2 (runt-related transcription factor 2), COL1 (collagen type 1),OCN (osteocalcin), OPG (osteoprotegrin), RANKL (receptor activatorof NF-κB ligand). Cladrin treatment significantly increased mRNAexpression of RUNX2 and OCN in a dose-dependent manner and atthe higher dose, RUNX2, COL1, and OCN levels were more than the

sham group. Cladrin at the higher dose restored the OVx-inducedreduction in OPG-to-RANKL ratio to the sham level. b OVx-inducedincrease in expression of osteoclast-specific genes; RANK and TRAP(tartrate-resistant acid phosphatase) were reduced in a dose-dependentmanner with cladrin treatment and are equivalent to sham in 10 mg/kgcladrin treatment group. All values are expressed as mean ± SEM (n010 rats/group); *P<0.05, **P<0.01, ***P<0.001 compared with OVx +vehicle group or as indicated

Osteoporos Int

to the baseline, while other parameters were not different.However, in contrast to cladrin (10 mg/kg) or PTH, E2group at the end point had lower BV/TV and Conn.D, andhigher Tb.sp, Tb.pf, and SMI.

In L5, BV/TV and Tb.N in cladrin (1 mg/kg) at the endpoint was reduced by 13.3 % and 19.08 %, and Tb.sp wasincreased by 28.12 %, in comparison to respective baselinevalues (Table 2). Cladrin (10 mg/kg), PTH, and E2 had nodifference between the end point and baseline values in anyof the parameters. However, in comparison to cladrin(10 mg/kg) or PTH, E2 group at the end point had lowerTb.Th and higher Tb.sp.

Effect of treatment withdrawal on lumbar vertebralcompression

In the cladrin (1 mg/kg) group, end point ultimate load,energy to failure and maximum stiffness were reducedby 23.81 %, 36.17 %, and 38.5 %, respectively, com-pared to the baseline (Table 2). Between baseline andend point, no significant differences in these valueswere observed in cladrin (10 mg/kg), PTH, and E2groups. However, when end point values were consid-ered, E2 had lower maximum stiffness compared tocladrin (10 mg/kg) and PTH groups.

Table 2 Effect of 4-week treatment withdrawal (end point) after the end of 12 weeks of treatment (baseline) on different bone parameters

Parameters OVx + cladrin (1 mg/kg) OVx + cladrin (10 mg/kg) OVx + PTH (40 μg/kg) OVx + E2 (100 μg/kg)

μCT measurements at femur epiphysis

BV/TV −53.9±2.7*** −1.08±0.114 −0.92±0.12 −15.2±1.8£

Tb.N −38.25±3.9*** −1.48±0.067 −1.93±0.103 −16.4±0.35£

Tb.Th −7.5±0.289 −5.569±0.297 −4.946±0.294 −8.346±0.27

Conn.D −1.74±0.021 −2.6±0.056 −2.29±0.181 −3.85±0.079

Tb.Sp 17.18±04.05 2.86±0.226 3.85±0.064 9.9±0.044£££

Tb.Pf 12.65±0.256 6.5±0.158 7.8±0.16 5.44±0.17

SMI 7.132±0.04 1.372±0.035 1.17±0.033 7.95±0.037£££

μCT measurements at proximal tibia metaphysis

BV/TV −56.78±2.27*** −5.95±0.114 −8.036±1.120 −26.75±0.042*,£

Tb.N −4.637±0.039 −1.435±0.067 −1.946±0.103 −2.735±0.062

Tb.Th −1.246±2.89 −1.456±2.97 −3.257±2.94 −1.148±2.74

Conn.D −14.527±0.021 −6.641±0.056 −2.732±0.181 −18.52±0.079££

Tb.Sp 16.83±0.06 4.81±0.05 3.85±0.064 19.90±0.044£

Tb.Pf 36.285±2.56*** 6.685±0.158 7.842±0.16 14.45±0.17£££

SMI 32.375±0.69** 1.567±0.04 1.157±0.035 14.09±0.037£

μCT measurements at L5

BV/TV −13.3±0.113*** −9.86±041 −4.51±0.034 −7.375±0.432

Tb.N −19.08±0.002*** −3.96±0.02 −1.91±0.064 −2.68±0.023

Tb.Th −4.2±0.059 −1.2±0.019 −1.32±0.019 −5.61±0.063£££

Conn.D −4.75±0.025 −3.07±0.119 −4.20±0.029 −2.69±0.03

Tb.Sp 28.12±0.047*** 1.613±0.049 3.1±0.186 7.29±0.028c

Tb.Pf 1.21±0.103 2.78±0.855 1.84±0.124 6.51±1.162

SMI 4.8±0.054 1.42±0.022 3.79±0.075 1.47±0.08

L5 compression test

Ultimate load −23.81±1.031* −5.62±0.582 −3.37±0.813 −14.83±2.47

Energy −36.17±0.137*** −10.73±0.028 −9.87±0.043 −11.72±0.036

Stiffness −38.5±0.183** −5.73±0.0147 −4.57±0.38 −7.39±0.011£££

Biochemical markers of bone metabolism

Serum PINP −21.56±0.033* −1.53±0.062 −3.87±0.054 −10.38±0.122££

Urinary CTx/Cr 58.35±0.214** 4.13±0.0987 6.92±0.214 71.74±0.987***,£

All parameters expressed as mean percentage change from baseline. Values are mean ± SEM from 10 rats/group. Student's t test (unpaired form)was used to compare endpoint data with baseline***P<0.001, **P<0.01, and *P<0.05 compared to baseline; £P<0.001, ££P<0.01, and £££P<0.05 compared to 10 mg/kg cladrin group

Osteoporos Int

Effect of treatment withdrawal on biochemical markers

In the cladrin (1 mg/kg) group, end point serum PINP wasreduced by 21.5 % and urinary CTx elevated by 58 %, com-pared to the baseline (Table 2). Urinary CTx in the E2 groupwas elevated by 71.7 % at the end point over the baseline.Between baseline and end point, no significant differences inPINP and CTx were observed in the cladrin (10 mg/kg) andPTH groups. When end point values were considered, E2 hadlower PINP than the cladrin (10 mg/kg) and PTH groups.

Discussion

Following leads from our previous studies on B. mono-sperma towards potential osteogenic effect of its constitu-ents [34], we took the most abundant osteogenic compound,cladrin, and evaluated its skeletal effects in osteoporotic rats.At a dose of 10 mg/kg, cladrin had the following effects: (1)improved the parameters of trabecular microarchitecture atthe appendicular and axial sites that were mostly compara-ble with the sham group and was at par with PTH at a doseand regimen that have been widely used to demonstraterestoration of osteopenic bones [39]; (2) improved vertebralcompression parameters to the sham levels; (3) exhibitedmineral accrual and BFRs, Cs.Th, expression of the osteo-genic genes in the long bones and serum PINP (osteogenicmarker) levels comparable to the sham; (4) reduced OVx-induced urinary CTx (antiresorptive marker) to the shamlevels; and (5) maintained the positive skeletal effects aftertreatment discontinuation and in this regard, cladrin (10 mg/kg) was equivalent to PTH, whereas E2 effect waned. De-spite being a phytoestrogen, cladrin had no estrogenic effecton the uterus. Thus, cladrin appears to be a novel phytoes-trogen having positive effects on various skeletal parametersthat persists following its discontinuation.

Cladrin promoted modeling-directed bone growth in fe-male rats, which was attributed to its effect of increasing thedifferentiation of bone marrow progenitors to osteogeniclineage [36]. Here, we assessed whether the ability of cla-drin to promote modeling-directed bone growth is translatedto osteogenic action under osteopenic condition. For thispurpose, we used osteopenic rats that had been OVx12 weeks prior to the start of the cladrin treatment and testedif it reversed osteopenia. The preservation of trabecularmicroarchitecture significantly contributes to bone strengthand may reduce fracture risk beyond BMD [60]. Restorationof microarchitecture parameters is necessary to evaluate thetrue impact of anabolic treatment on the quality of trabecularbone given that trabecular bone is more readily lost becauseof OVx in rats [61]. At 1 mg/kg dose, cladrin had alreadypresented improved trabecular response compared to OVxrats, and at 10 mg/kg dose, most of the parameters at all three

sites were comparable to the sham group, suggesting signifi-cant bone gain. Increase in bone volume fraction by cladrinwas caused by trabecular number and thickness, withcorresponding reduction in space. Remarkably, the indicesthat served as surrogate of strength including SMI, Tb.pf,and conn.D were comparable to the sham level at all threecancellous sites by cladrin treatment, while E2 was by andlarge ineffective suggesting that cladrin (10 mg/kg) was supe-rior to E2 in mitigating the OVx-induced microarchitecturaldeterioration of cancellous bones. E2 acts primarily as ananticatabolic agent and thus protects bone from further lossin OVx condition [62–65]. Because the improvement of tra-becular bone by cladrin exceeds the suppressive effect onbone loss by E2, it suggests a likely bone anabolic action ofcladrin. Furthermore, marked improvements in the architec-tural indices in the trabecular rich vertebra by cladrin (10 mg/kg) treated OVx rats translated to greater resistance to com-pressive failure in lumbar vertebra. Since osteoporotic com-pression fracture is closely associated with the mechanicalcharacteristics of trabecular bone [66–68], our data suggestthat cladrin could effectively reduce the risk of this type offracture by improving trabecular microarchitecture in post-menopausal women.

E2 deficiency is characterized by high turnover bone lossthat exhibits increased osteoblastic parameters early on butdiminishes over time. In our case, estrogen deficiency wasmaintained long enough to induce a reduction in osteoblastfunction and the associated parameters. Increases in MARand BFR/BS in OVx rats treated with cladrin suggested anincrease in coupled remodeling likely due to augmented oste-oblast activity in contrast to uncoupled remodeling favoring netbone loss in OVx rats. The effect of cladrin was dose dependentand at the higher dose, the effect was comparable to sham orPTH, while E2 had no effect. At femur diaphysis, cladrininduced greater periosteal apposition, larger cross-sectionalarea, and a thicker cortex compared to OVx group, while E2failed to improve these parameters. Overall, cortical depositionby cladrin was comparable to the sham or PTH group. A majoranabolic aspect of PTH is to increase appositional bone forma-tion that leads to enlarged cortical bone thickness, and cladrinappears to possess this effect. PINP is presently accepted as aclinical serum biomarker for assessing osteoblastic activity andthus, bone formation, in the treatment of bone loss [69, 70].Cladrin had a dose-dependent effect in increasing serum PINPlevels, attaining sham levels at the higher dose, but E2 had noeffect. Together, these data demonstrated that the dose of E2that was effective in restoring complete uterine estrogenicitywas found to be mostly ineffective in augmenting the osteo-blastic parameters in vivo. By contrast, the ‘phytoestrogen-like’cladrin significantly increased the osteoblastic parameters inosteopenic rats without uterine estrogenic effect.

E2 deficiency increases osteoclast production resulting inincreased osteoclastic marker genes, including TRAP and

Osteoporos Int

RANK in bone, as well as osteoclast action leading toaugmented urinary levels of collagen degradation product,CTx. E2 supplementation to OVx rats suppresses excessiveosteoclast function and brings down the levels of osteoclastmarkers to E2 replete state found in animals with normalovarian function. Cladrin treatment dose dependently re-duced urinary CTx as well as TRAP mRNA levels in theOVx bones, suggesting antiresorptive effect. Indeed, thesuppressive effect of cladrin on urinary CTx at the higherdose was comparable to E2, suggesting considerable anti-catabolic action of cladrin. Phytoestrogens are generallyreckoned with an anticatabolic effect on skeleton [71, 72].Cladrin had no effect on the differentiation of the bonemarrow cells to osteoclasts induced by RANKL and M-CSF (data not shown); however, cladrin treatment of OVxrats increased OPG-to-RANKL ratio in the bones. Previous-ly, we had shown that cladrin promoted osteoblast differen-tiation in cultures by activating the MEK–Erk pathway [36].Increased OPG production is a function of osteoblast matu-ration and has an anticatabolic effect due to the inhibitoryeffect of OPG on osteoclast differentiation. Based on ourprevious report and the present data, it appears that twotypes of signals are produced by cladrin simultaneously:anabolic pathways are activated in osteoblasts and an anti-catabolic message is sent from the differentiating osteoblaststo preosteoclasts and osteoclasts through an increase in theOPG-to-RANKL ratio. Concomitant stimulation of osteo-blast and suppression of osteoclast function is akin to the so-called dual (bone anabolic and anticatabolic) mode of actionthat is exhibited by strontium ranelate (SrR), a drug ap-proved in many countries (but not in the US) for reducingthe fracture risk in postmenopausal women [73, 74]. How-ever, unlike SrR, which has a direct inhibitory action on theproduction and viability of osteoclasts, cladrin appears toinhibit osteoclast function only indirectly through increasedosteoblastic production of OPG. Furthermore, inhibition ofTNFα-producing T cells as one of the antiosteoclastogenicmechanisms by which daidzein protects bone loss under E2deficiency may also be true for cladrin [75].

Despite several preclinical reports of positive outcomesof phytoestrogens on the skeleton, their poor bioavailabilityimpedes the application of these promising and relativelynontoxic agents to develop as pharmacological agents forpostmenopausal osteoporosis [76, 77]. Methoxylation ofisoflavones have been reported to augment bioavailabilitywith the potential to enhance pharmacodynamic effects [78,79]. Comparison of the pharmacokinetic profiles of cladrin,daidzein, and formononetin (monomethoxydaidzein)revealed that cladrin possessed the best oral bioavailabilityof the three. The favorable pharmacokinetic feature of cla-drin coupled with its osteoblast stimulatory function [36]appeared to contribute to the sustenance of the acquiredskeletal gains by cladrin at 10 mg/kg but not at 1 mg/kg

dose treatment of OVx rats, indicating that the higher doseof cladrin used in this study would be effective for thepurpose of maintaining bone gain after treatment withdraw-al. With respect to the preservation of the bone gain aftertreatment withdrawal, E2 showed signs of waning. To thebest of our knowledge, this is the first demonstration ofsustenance of skeletal gains by any phytoestrogen followingits discontinuation.

There are several limitations to the study: (1) baselinevalue prior to treatment initiation was limited only to BV/TV and not the entire gamut of the μCT parameters thatwere presented at the end of the treatments (baseline) and,which was necessary to definitely conclude that the treat-ment was anabolic; (2) interlabel period might havestretched beyond the time required for accurate determina-tion of dynamic bone formation in rats and could have led tolabel escape; (3) assessments of bone mass parameters in-cluding bone mineral content and areal bone mineral densitywere not performed; and (4) withdrawal study was kept atonly 4 weeks on account of significant loss of the trabecularbone fraction in the 1 mg/kg cladrin group compared tocorresponding baseline values, thus preventing us from de-termining the exact time of protection against bone loss bycladrin at 10 mg/kg dose following its discontinuation.Future studies are required to determine the time whenskeletal gains achieved by cladrin treatment starts to ebband ultimately lost by discontinuing the treatment beyond4 weeks. Such studies may be useful to determine the extentof the drug-free intervals with cladrin treatment in the futureclinical setting of osteoporosis that is needed for optimumtreatment adherence.

In conclusion, the results of the present study demon-strate that cladrin has positive outcomes on osteopenic bonedue to stimulatory effect on osteoblast differentiation. Dataalso suggest that cladrin has strong anticatabolic effect.Skeletal gains by cladrin in the osteopenic rats were durablealbeit in short-term following its withdrawal. Given its lackof uterine estrogenicity, good tolerability, and prolongedsystemic bioavailability in rats [36], the present findingsthat cladrin improves bone properties at a favorable oraldose of 10 mg/kg makes it an attractive alternative strategyfor the development of new treatment against postmeno-pausal osteoporosis.

Acknowledgments This study was supported by grant-in-aid fromthe Ministry of Health and Family Welfare, Government of India.Funding from the Indian Council of Medical Research, Governmentof India (N.C.) is acknowledged. Research fellowship grants from theDepartment of Biotechnology (K.K., K.S.), University Grants Com-mission (M.M.), Council of Scientific and Industrial Research (G.S.,V.C., T.K.B., S.P.C., D.Y., P.D.), and Indian Council of Medical Re-search (M.P.K.), Government of India, are also acknowledged.

Conflicts of interest None

Osteoporos Int

Appendix

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