Treatment of Skeletally Mature Ovariectomized Rhesus Monkeys WithPTH(1-84) for 16 Months Increases Bone Formation and Density and

Improves Trabecular Architecture and Biomechanical Propertiesat the Lumbar Spine

John Fox,1 Michael A Miller,1 Michael K Newman,1 Charles H Turner,2 Robert R Recker,3 and Susan Y Smith4

ABSTRACT: Histomorphometric studies of treatments for osteoporosis in humans are restricted to iliac crestbiopsies. We studied the effects of PTH(1-84) treatment at the lumbar spine of skeletally mature ovariecto-mized rhesus monkeys. PTH increased bone turnover, rapidly normalized BMD, and increased vertebralcompressive strength. PTH increased trabecular bone volume primarily by increasing trabecular number bymarkedly increasing intratrabecular tunneling.

Introduction: Histomorphometric studies of the anabolic properties of PTH(1-84) (PTH) and related peptidesin human bone are restricted to iliac crest biopsies. The ovariectomized (OVX) monkey is an accepted modelof human postmenopausal bone loss and was used to study the effects of PTH treatment at clinically relevantskeletal sites.Materials and Methods: Skeletally mature rhesus monkeys were OVX or sham-operated and, after a bonedepletion period of 9 months, treated daily for 16 months with PTH (5, 10, or 25 g/kg). Markers of boneformation (serum osteocalcin) and resorption (urine N-telopeptide [NTX]) and lumbar spine BMD weremeasured throughout the study. Trabecular architecture and vertebral biomechanical properties were quan-tified at 16 months.Results: PTH treatment induced dose-dependent increases in bone turnover but did not increase serumcalcium. Osteocalcin was significantly increased above OVX controls by 1 month. NTX was significantlyelevated at 1 month with the highest dose, but not until 12 months with the 5 and 10 g/kg doses. Lumbar spineBMD was 5% lower in OVX than in sham animals when treatment was started. All PTH doses increasedBMD rapidly, with sham levels restored by 37 months with 10 and 25 g/kg and by 16 months with 5 g/kg.PTH treatment increased trabecular bone volume (BV/TV), primarily by increasing trabecular number, anddose-dependently increased bone formation rate (BFR) solely by increasing mineralizing surface. The largesteffects on BV/TV and yield load occurred with the 10 g/kg dose. The highest dose reduced trabecularthickness by markedly increasing intratrabecular tunneling.Conclusions: PTH treatment of OVX rhesus monkeys increased bone turnover and increased BV/TV, BMD,and strength at the lumbar spine. All PTH doses were safe, but the 10 g/kg dose was generally optimal,possibly because the highest dose resulted in too marked a stimulation of bone remodeling.J Bone Miner Res 2007;22:260273. Published online on November 6, 2006; doi: 10.1359/JBMR.061101

Key words: ovariectomized primate, PTH, bone turnover markers, bone densitometry, bone histomorphom-etry, bone strength

INTRODUCTION

OSTEOPOROSIS IS CHARACTERIZED by impaired bonequality leading to decreased bone strength and in-creased susceptibility to fracture. PTH(1-84) (PTH) or N-terminal fragments and analogs of PTH or PTH-relatedprotein are powerful anabolic agents in the skeleton when

Dr Fox, Mr Miller, and Mr Newman are or were employees ofand own stock in NPS Pharmaceuticals. Dr Turner serves as aconsultant for Charles River Laboratories Preclinical Services. MsSmith is employed by Charles River Laboratories Preclinical Ser-vices. Dr Recker serves as a consultant for Amgen, Eli Lilly & Co.,GlaxoSmithKline, Merck, Novartis, NPS, Proctor & Gamble,Roche, and Wyeth.

1NPS Pharmaceuticals, Salt Lake City, Utah, USA; 2Department of Orthopaedic Surgery, Indiana University, Indianapolis, Indiana,USA; 3Osteoporosis Research Center, Creighton University, Omaha, Nebraska, USA; 4Charles River Laboratories Preclinical Services,Montral, Quebec, Canada.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 22, Number 2, 2007Published online on November 6, 2006; doi: 10.1359/JBMR.061101 2007 American Society for Bone and Mineral Research

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JO603185 260 273 February

administered by daily injection in normal animals, in animalmodels of osteoporosis, and in humans with postmeno-pausal and glucocorticoid-induced osteoporosis.(110)

Treatment with these agents increases BMC and BMD,particularly at skeletal sites such as the lumbar spine thatare rich in trabecular bone. In animal models, these agentsincrease bone strength and toughness at both trabecularand cortical bone sites.(1,35) Large-scale clinical trials havealso shown that PTH and teriparatide, the N-terminal hu-man PTH(1-34) fragment, markedly decrease the incidenceof vertebral fractures in postmenopausal women with os-teoporosis.(7,8)

In humans, study of the mechanisms responsible for theanabolic activity of PTH or related peptides, as well as thesafety of such treatments in bone, is largely restricted toanalysis of iliac crest biopsies using the techniques of his-tomorphometry and CT.(1113) These analyses are limitedby the number of patients who consent to provide a biopsyand the difficulty in obtaining consistent specimens that aresuitable for rigorous examination. A recent report has de-scribed the use in humans of high-resolution pQCT, whichprovides information on trabecular and cortical bone mi-croarchitecture.(14) However, this technique is restricted tothe distal radius and distal tibia and has not yet been usedto study the effects of therapies for osteoporosis. Thus,studies of the mechanisms underlying the effects of PTHtreatment at skeletal sites that are particularly susceptibleto osteoporotic fracture in humans, for example, the spine,are currently only performed in animals.

The vast majority of preclinical studies that have inves-tigated the effects of PTH or related peptides on the skel-eton have used the ovariectomized (OVX) rat model ofosteoporosis.(14) While offering advantages of small sizeand convenience, the OVX rat is not able to predict all theeffects of PTH therapy in bone in humans. For example,PTH increases trabecular bone volume in the rat exclu-sively by increasing trabecular thickness, whereas in humaniliac crest, bone volume increases primarily through in-creased trabecular number.(2,13) It is well recognized thattrabecular number and connectivity are more important de-terminants of bone strength than trabecular thickness.(15)

Moreover, whereas treatment with PTH peptides canstimulate Haversian remodeling in humans,(12,13) intracor-tical remodeling occurs in response to PTH in the rat onlyunder extreme conditions, such as severe secondary hyper-parathyroidism.(16)

The effects of PTH peptides have been evaluated in rab-bits and dogs in which Haversian remodeling occurs,(17,18)

but nonhuman primates are a far superior model of humanestrogen-deficiency bone loss.(19) Despite this, there is verylittle information available on the effects of treatment withPTH peptides in nonhuman primates, and most of the pub-lished papers are derived from a single study with teripa-ratide in cynomolgus monkeys.(2025) However, it was notpossible to assess reversal of bone loss in that study becauseteriparatide treatment was initiated immediately after OVX.

This paper describes the effects of daily treatment withhuman PTH(1-84) for 16 months on bone turnover, density,architecture, and biomechanical properties at the lumbarspine of skeletally mature OVX rhesus monkeys. Impor-

tantly, in this study, treatment was initiated only after a9-month bone depletion phase that followed OVX.

MATERIALS AND METHODS

This study was conducted at Charles River LaboratoriesPreclinical Services, Montral (CRM), and Creighton Uni-versity in accordance with the Good Laboratory Practiceregulations of the United States Food and Drug Adminis-tration (FDA).

Animals, diets, and surgery

Adult female rhesus monkeys (Macaca mulatta) were ob-tained from the Texas Primate Center (TPC; Alice, TX,USA). All animals were of domestic origin; two animalsoriginated from outside the United States but were housedat TPC for at least 8 years before shipment to CRM. Onarrival, the age range of the animals was stated by TPC tobe 1217 years. Examination of radiographs confirmedepiphyseal closure in the long bones. The monkeys werehoused in cages designed for social housing and were of-fered purified water ad libitum and 200 g/day of a standardpelleted diet (Certified Primate Chow 5048; PMI NutritionInternational, Richmond, IN, USA), containing 1.0% cal-cium, 0.6% phosphorus, and 6.6 IU vitamin D3/g. In addi-tion, a discretionary dietary supplement (PMI Prima-Treat)and/or fresh fruit was provided daily to check appetite. Theanimals were housed under targeted conditions of constanttemperature (24 3C) and humidity (50 20%) and a 12h/12 h light/dark cycle. The monkeys were monitored for 9months before OVX or sham surgery, during which timeperiodic laboratory studies were performed including clini-cal biochemistry, fecal and urine analysis, hematology, tu-berculosis testing, and radiography and bone densitometry.Animals were randomized into groups based on wholebody BMC as assessed by DXA. Groups were also homo-geneous by age and body weight.

Animals were OVX or underwent sham surgery 9months before the start of dosing. The animals were pre-anesthetized with glycopyrrolate, ketamine, and xylazine,followed by isoflurane gas anesthesia that was maintainedthroughout the entire surgical procedure. After ligation ofthe utero-ovarian proper ligament and blood vessels, eachovary was excised with as much surrounding tissue as pos-sible. Sham-operated animals underwent the same surgicalprocedure but the ovaries were left intact. Intramuscularinjections of antibiotic (Penlong XL) were given at least 1 hbefore and 2 days after surgery.

Study protocol

A bone depletion period of 9 months was allowed toelapse before the initiation of treatment. At the end of thisperiod, one group of OVX and one group of sham-operatedanimals were killed as baseline controls. The remainingsham group and one group of OVX animals received dailysubcutaneous injections of vehicle (citric acid, 10 mM; tri-sodium citrate, 10 mM; mannitol, 50 mg/ml; pH 5.5) for 16months. The additional three groups of OVX animals re-ceived recombinant human PTH(1-84) at doses of 5, 10, or

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 261

25 g/kg/day. The 5 and 10 g/kg doses were selected basedon the results of a preliminary 9-month dose-ranging trial(1.5, 5, and 10 g/kg/day) in rhesus monkeys that wereOVX 3 months before the start of treatment in which thelargest effects on bone were provided by the two higherdoses.(26) A 25 g/kg dose was also included in this study toevaluate the effects of a higher dose and to comply with theFDA guidance for osteoporosis studies, which states that anoptimally effective dose and one 5-fold higher should betested in preclinical studies.(27) Although not reported here,the pharmacokinetics of PTH was also determined in thisstudy and showed that the 5, 10, and 25 g/kg doses resultedin plasma PTH exposures that were, respectively, 2.1-, 4.1-,and 13.2-fold higher than with the 100 g dose used inpostmenopausal women (unpublished results). The animalswere killed by intravenous sodium pentobarbital anesthesiaand incision of the axillary or femoral arteries.

Analyses

Chemistry: Radioimmunoassays were used to measureserum levels of 25-hydroxyvitamin D [25(OH)D], 1,25-di-hydroxyvitamin D [1,25(OH)2D] (Incstar, Stillwater, MN,USA), and estradiol (Diagnostics Products, Los Angeles,CA, USA) and an immunoradiometric assay to measureserum intact PTH (Diagnostics Products). Other serum andurine measurements were performed using routine clinicalchemistry procedures.

Bone turnover markers: Blood and urine samples werecollected from each animal after an overnight fast (mini-mum 12 h) before surgery, during weeks 12, 25, and 38 ofthe bone depletion period, and during weeks 4, 12, 26, 51,and 68 of the treatment phase of the study. Blood sampleswere collected from the conscious animal for measurementof serum levels of the bone formation markers osteocalcinby radioimmunoassay (DSL-6900; Diagnostic SystemLaboratories, Webster, TX, USA) and bone-specific alka-line phosphatase (BSALP) using an immunoradiometric as-say (Tandem-R Ostase; Hybritech, San Diego, CA, USA).Urine was usually collected 2 or 3 days after blood samplecollection by bladder catheterization using intramuscularglycopyrrolate, ketamine, and xylazine as anesthetic. Uri-nary concentrations of the bone resorption markers N-telopeptide (NTX) and pyridinoline were measured by en-zyme-linked immunosorbent assay (Osteomark; OstexInternational, Princeton, NJ, USA) and high-performanceliquid chromatography, respectively, and were normalizedto urine creatinine concentrations.

In vivo DXA: The animals were anesthetized by an in-tramuscular injection of glycopyrrolate, ketamine, and xyla-zine before scanning. Areal BMD (aBMD), BMC, andbone area (BMA) of the lumbar spine (anterior-to-pos-terior [A/P] projection of L1L4 and lateral projection ofL2L4) were measured by DXA using QDR 2000 plus bonedensitometers (Hologic, Bedford, MA, USA). For lateralDXA, the region of interest was placed over the center ofeach vertebral body to focus on a site with a greater pro-portion of trabecular bone. Scans were performed every35 months throughout the bone depletion and treatmentphases of the study.

Histomorphometry: In addition to the analysis of bones

harvested at the end of the study, biopsies of iliac crest andrib were collected at baseline and at month 6 of the 16-month treatment period. The results of the iliac crest andrib analyses are not reported in this paper. Fluorochromemarkers were injected at 15 and 5 days before bone collec-tion to label bone-forming surfaces. Calcein (8 mg/kg, IV),oxytetracycline (40 mg/kg, IV) and xylenol orange (90 mg/kg, SC) were administered at baseline, month 6, and month16, respectively. After death, the lumbar spine was re-moved, and the individual vertebrae were separated. L1 andL3 were fixed in neutral buffered 10% formalin before ship-ment to the Creighton University Osteoporosis Center in70% ethanol for histomorphometric analysis. Specimenswere processed through steps of dehydration, defatting,embedding in methyl methacrylate, sectioning, and stainingusing Creightons standard operating procedures. Trans-verse (L1) and longitudinal (L3) sections were preparedfrom the middle of each vertebral body, and trabecularbone was analyzed in 2 4-mm subregions. In L1, thesubregion was located in the middle of the section, whereasin L3, the subregion was located at the one third cranial orcaudal aspects of the section. Histomorphometric measure-ments were made using an interactive image analysis sys-tem (Bioquant R&M Biometrics, Nashville, TN, USA). Allparameters related to fluorochrome labels were based on amineralizing surface calculated as double- plus one half thesingle-labeled surface.

Ex vivo pQCT: L2 and L4 were stored at 20C untildensitometric scanning and biomechanical testing. Beforescanning, the vertebrae were thawed overnight at 4C andimmersed in physiological saline. The end plates and spi-nous processes were removed to obtain a specimen withplano-parallel ends. Each vertebra was placed on a posi-tioning aid, and pQCT scans were acquired (voxel size, 0.20mm; contour mode 1; threshold, 0.500 1/cm; peelmode, 20;60% trabecular area) using an XCT Research SA bonescanner (Stratec Medizintechnik, Pforzheim, Germany)with software version 5.40. Volumetric measures of totaland trabecular BMD and BMC were obtained from a 0.8-mm thick A/P slice taken from the middle of each vertebralbody.

Biomechanical testing: The height of each vertebral bodywas measured using digital calipers before compressiontesting using an MTS 858 Mini Bionix servo hydraulic testsystem with a load cell of 15 kN and a loading rate of 20mm/minute. Load and displacement data were collected us-ing Testworks (version 3.8A) for Teststar II (version 4.0c).Many load-displacement curves did not provide a well-defined peak load because of a continuing increase in mea-sured load during compressive deformation. As a result,peak load and other biomechanical properties of bone, suchas work-to-failure and toughness, are not reported in thispaper. We report yield load and yield stress, together withstiffness and modulus.

Statistics: Many histomorphometric parameters of trabec-ular bone structure and formation were quantified at bothL1 and L3, whereas other measures of bone remodelingwere only determined at L3. When histomorphometric andbiomechanical measurements were obtained at both verte-brae, the values were averaged before statistical analysis.

FOX ET AL.262

Data are reported as mean SE and were analyzed byANOVA followed by Fishers protected least significantprocedure to evaluate differences between groups (Stat-view version 5.0; SAS Institute, Cary, NC, USA). Regres-sion analyses were performed to determine the correlationof densitometric measures derived by pQCT (total and tra-becular BMD) versus each biomechanical property of bone.

RESULTSIn-life phase

Serum estradiol levels decreased to very low or undetect-able levels after surgery in all OVX animals but did notchange significantly in sham-operated monkeys (data notshown). One OVX monkey in the vehicle group had in-creased estradiol levels at month 16, with visible evidenceof resumed menses. After necropsy, this animal was foundto have ectopic ovarian tissue, and therefore, all data fromthis animal was excluded from analysis. One animal in the10 g/kg group was killed during week 58 of the treatmentphase after a rapid decline in health status that was attrib-uted to decreased renal function, a finding considered un-related to PTH treatment. Because data collected from thisanimal at week 51 were consistent with that from earliertime-points and with other animals in that group, all in-lifedata from this animal were included in the analyses.

The characteristics of the monkeys before surgery areshown in Table 1. The groups were well matched with re-spect to body weight, serum clinical chemistry, serumvitamin D metabolites, lumbar spine BMD, BMC, andBMA, and whole body BMC. Serum osteocalcin and urineNTX levels were also similar between groups before sur-gery (Fig. 1).

All groups of animals, apart from those receiving the 25g/kg dose, gained weight during the treatment phase of

the study. The body weight gain (kg) was 1.1 0.2, 0.9 0.1,0.7 0.3, 0.7 0.1, and 0.1 0.3 in the sham-vehicle,OVX-vehicle, OVX-5 g/kg, OVX-10 g/kg, and OVX-25g/kg groups, respectively. The body weight gain was sig-nificantly lower in the 25 g/kg group than in all othergroups, which were not significantly different from eachother.

Biochemical markers of bone turnover

Bone turnover was increased within 3 months of OVX.When all OVX animals were compared with the shamgroup, there was a significant increase in serum osteocalcinlevels throughout the bone-depletion phase. Similarly, uri-nary NTX excretion was significantly higher in OVX thanin sham animals at each time-point, although in contrast toserum osteocalcin, NTX levels tended to increase progres-sively during the 9 months after OVX. At the end of thebone-depletion phase, osteocalcin and NTX levels were,respectively, 87% and 100% higher in OVX than in shamanimals (Fig. 1). Similarly, serum BSALP and urine pyridi-noline levels were, respectively, 174% and 39% higher inOVX than in sham animals at the end of the bone-depletionperiod (data not shown).

The increases in osteocalcin and NTX were largely main-tained throughout the treatment phase of the study in theOVX-vehicle group, although levels were not significantlyhigher than sham-vehicle controls at every time-point (Fig.1). Treatment with PTH was associated with a dose-relatedincrease in osteocalcin to levels that were significantlygreater than in OVX-vehicle controls by month 1 with the10 and 25 g/kg doses. The maximum increase in osteocal-cin levels generally occurred at months 3 or 6, and thiselevated level was maintained to the end of the study. Withthe 5 g/kg dose, osteocalcin was significantly elevatedabove OVX-vehicle controls only at months 12 and 16.

TABLE 1. CHARACTERISTICS OF RHESUS MONKEYS BEFORE SURGERY

Baseline VehicleOVX

(5 g/kg)OVX

(10 g/kg)OVX

(25 g/kg)ANOVA

p

Sham OVX Sham OVX*

n 6 7 9 8 10 9 9

Body weight (kg) 7.9 0.5 8.8 0.8 8.8 0.7 7.5 0.7 7.8 0.4 8.0 0.6 8.2 0.7 0.691Serum

Calcium (mg/dl) 8.9 0.2 8.6 0.2 8.9 0.2 9.3 0.3 9.1 0.2 8.6 0.2 8.6 0.2 0.147Phosphate (mg/dl) 3.8 0.3 4.2 0.6 4.8 0.6 4.2 0.2 4.5 0.3 4.1 0.3 4.4 0.3 0.71525(OH)D (ng/ml) 215 18 181 31 226 13 210 13 224 18 222 17 217 22 0.7591,25(OH)2D (pg/ml) 97 22 77 10 89 12 84 12 76 11 94 15 87 17 0.933Estradiol (pg/ml) 23 9 24 10 49 13 24 2 43 17 29 8 32 8 0.550

DXA densitometryL1L4 (A/P)

BMD (g/cm2) 0.73 0.02 0.75 0.03 0.74 0.02 0.73 0.02 0.75 0.02 0.73 0.01 0.74 0.02 0.922BMC (g) 11.5 0.6 11.9 0.6 12.0 0.6 11.0 0.7 11.9 0.7 11.5 0.5 12.2 0.7 0.854BMA (cm2) 15.8 0.8 15.8 0.3 16.1 0.5 15.0 0.5 15.7 0.5 15.8 0.5 16.5 0.5 0.635

L2L4 (lateral)BMD (g/cm2) 0.51 0.03 0.53 0.03 0.55 0.02 0.53 0.03 0.54 0.03 0.51 0.02 0.52 0.02 0.923

Whole body BMC (g) 266 14 272 17 276 16 258 16 265 12 257 13 270 16 0.963

Values are mean SE.* Animal with ectopic ovarian tissue at necropsy excluded.A/P, anterior-to-posterior.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 263

BSALP levels followed a similar pattern of change withPTH treatment (data not shown).

In contrast to the prompt increase in bone formation withall PTH doses, only the 25 g/kg dose significantly in-creased NTX levels at month 1. In the high-dose group,NTX reached maximum levels at month 3 that were main-tained throughout the remainder of the study (Fig. 1). Itwas not until months 12 or 16 that significant increases inNTX above OVX-vehicle control levels were observed withthe 5 and 10 g/kg doses. Similar but more variable changeswere observed in pyridinoline excretion in PTH-treatedanimals (data not shown). The changes at month 16 frompresurgery and baseline levels for each marker are shown inTable 2 and further document the dose-dependent stimu-lation of bone turnover by PTH treatment.

Serum calcium, phosphate, and PTH

Serum total calcium and phosphate levels were measuredat the same time as bone turnover markers. PTH levelswere measured before dosing during the treatment phase ofthe study (Fig. 1). There was no consistent dose-relatedpattern of change in serum calcium, whereas serum phos-phate levels were significantly lower in the 25 g/kg groupduring most of the treatment phase of the study. Therewere no significant differences in predose PTH levels be-tween groups, confirming complete clearance of injectedPTH and absence of calcium-mediated suppression of PTHsecretion.

DXA densitometry

BMD at L1L4 tended to increase throughout the studyin sham-operated animals but was reduced significantly by6 months after OVX (Fig. 2). At baseline, 9 months aftersurgery, BMD was 5.1% lower in OVX animals than insham controls. The OVX-induced decrease in BMC (8.3%)was larger than BMD, because BMA was a significant 3.2%lower. The low BMD in control OVX animals was main-tained until month 7 of the treatment phase, but thentended to increase. The pattern and significance of OVX-induced changes in BMD of the lateral L2L4 spine weresimilar to those at L1L4 but, at baseline, the magnitude ofthe decrease relative to sham controls was greater (11.3%;Fig. 2).

Treatment with PTH resulted in rapid increases in BMDat L1L4 (Fig. 2). There were no significant differences inthe increases in BMD between the PTH groups at any time-point, although the initial rate of increase in BMD tendedto be greater with the 10 and 25 g/kg doses. BMD wasrestored to the level of the sham-vehicle group by month 7with the 10 and 25 g/kg doses and by month 16 with 5g/kg. However, whereas the 5 and 10 g/kg doses in-creased BMD progressively, the rate of increase in BMDwith 25 g/kg slowed between months 7 and 12, and BMDdecreased slightly between months 12 and 16. Thus, the 10g/kg dose produced the largest increase in BMD at month16. PTH treatment-induced changes in BMD at L2L4, inwhich a region with a greater proportion of trabecular boneis measured, were similar to those at L1L4, although sham-vehicle levels were restored sooner and the magnitude of

FIG. 1. Effects of daily subcutaneous injection of vehicle or PTHfor 16 months in OVX rhesus monkeys on (A) serum osteocalcin,a marker of bone formation, (B) urine NTX, a marker of boneresorption, (C) serum total calcium, (D) serum phosphate, and(E) serum PTH levels. All samples were collected before dosing.OVX or sham surgery occurred at month 9 and treatmentstarted at month 0. Values are mean SE, n 810/group.*Pooled OVX and sham groups significantly different (p < 0.05).a,b,c,dp < 0.05: significance of difference from sham-vehicle, OVX-vehicle, OVX-5 g/kg, and OVX-10 g/kg groups, respectively.

FOX ET AL.264

the increase in BMD was greater (Fig. 2). However, in con-trast to L1L4, the increases in BMD were similar with the10 and 25 g/kg doses.

The percent changes in BMD, BMC, and BMA relativeto both presurgery and baseline levels at L1L4 in eachgroup are shown in Table 3 and document that (1) theincrease in BMC was greater than the increase in BMDbecause BMA also increased, albeit not significantly, byPTH treatment; (2) the 10 g/kg dose of PTH produced thegreatest mean increases in BMD, BMC, and BMA; and (3)there were no significant differences in the increases be-tween PTH doses. Table 3 also documents that the in-creases in BMD at L2L4 were greater than at L1L4.

Histomorphometry

Trabecular architecture: Mean trabecular bone volume(BV/TV), thickness (Tb.Th), and number (Tb.N) were gen-erally lower, and trabecular separation (Tb.Sp) was higherin OVX compared with sham animals at baseline and at theend of the study, but none of the differences were signifi-cant (Fig. 3). Treatment with PTH increased BV/TV, butthe largest effect occurred in the 10 g/kg dose group. Theincrease in BV/TV in PTH-treated animals was caused pri-marily by a dose-dependent increase in Tb.N; there were nosignificant effects of PTH on Tb.Th. However, the lowerBV/TV in the 25 g/kg group compared with the 10 g/kggroup occurred because there was a strong tendency formean Tb.Th to be reduced in the high-dose animals. Thechanges in Tb.N and Tb.Th resulted in a significant, dose-dependent decrease in Tb.Sp. These differences in trabec-ular architecture in animals given the 10 and 25 g/kg dosesof PTH are shown in Fig. 4.

Bone turnover: OVX increased trabecular bone turnover.Mean activation frequency (Ac.f), osteoclast surface (Oc.S/BS), and eroded surface (ES/BS) were higher, and resorp-tion period (Rs.P) was lower in OVX compared with the

sham groups, although most of these differences were notsignificant (Table 4). Mean osteoblast (Ob.S/BS) and oste-oid (OS/BS) surfaces were 7.7- and 1.9-fold higher in theOVX-vehicle than in the sham-vehicle group, but neitherdifference was significant (Fig. 5). Similar nonsignificantincreases in osteoid volume (OV/BV, OV/TV), mineraliz-ing surface (MS/BS), and surface- (BFR/BS) and volume-referent bone formation rate (BFR/BV, BFR/TV) werealso observed in OVX animals, but osteoid thickness(O.Th) was unaffected (Table 4; Fig. 5). Apart from signifi-cantly shorter formation (FP) and remodeling periods(Rm.P) in OVX animals at baseline, there were no signifi-cant effects of OVX on mineral appositional rate (MAR),mineralization lag time (Mlt), wall thickness (W.Th), FP, orRm.P (Table 4).

Although there was a tendency for PTH treatment tostimulate bone resorption, only the 25 g/kg dose causedsignificant changes compared with OVX-vehicle controls.Ac.f and ES/BS were significantly higher and Rs.P was sig-nificantly shorter in the high-dose group, whereas, para-doxically, Oc.S/BS was significantly lower (Table 4). In con-trast, treatment with PTH resulted in significant dose-dependent increases in several parameters related to boneformation, although none of the changes with the 5 g/kgdose group were significantly different from OVX-vehiclecontrols. Ob.S/BS, OS/BS, and OS/BV were significantlyhigher and FP was shorter with the 10 and/or 25 g/kgdoses of PTH. There was no effect of PTH treatment onO.Th (Figs. 4 and 5). Mean W.Th was significantly lower inanimals receiving the 25 g/kg dose. There were dose-dependent increases in surface- and volume-referent BFRthat were solely the result of increased MS/BS; there wasno effect of PTH treatment on MAR (Fig. 5). Finally,Rm.P was shortened significantly in PTH-treated animals(Table 4).

The stimulatory effects of PTH treatment on fluoro-

TABLE 2. PERCENT CHANGES IN SERUM LEVELS OF OSTEOCALCIN AND BSALP AND URINE LEVELS OF NTX AND PYRIDINOLINE ATMONTH 16 COMPARED WITH LEVELS AT PRESURGERY (MONTH 9) AND BASELINE (MONTH 0) IN OVX RHESUS MONKEYS TREATED

DAILY WITH PTH FOR 16 MONTHS

Vehicle

OVX (5 g/kg) OVX (10 g/kg) OVX (25 g/kg)Sham OVX

OsteocalcinVs. presurgery 106 37 214 42 478 99* 793 103* 1098 125*

Vs. baseline 8 17 10 9 63 11 138 37* 184 24*

BSALPVs. presurgery 5 13 43 12 206 39 194 24 661 224*

Vs. baseline 22 15 13 4 50 18 67 20 208 40*

NTXVs. presurgery 76 43 201 64 360 107* 412 85* 675 130*

Vs. baseline 16 19 27 8 1 11 60 24* 88 14*

PyridinolineVs. presurgery 17 15 32 18 51 14 63 22 162 43*

Vs. baseline 11 8 36 5 15 7 1 10 52 16*

Values are mean SE, n 810/group.* p < 0.05 significance of difference from sham-vehicle group. p < 0.05 significance of difference from OVX-vehicle group. p < 0.05 significance of difference from OVX-5 g/kg group. p < 0.05 significance of difference from OVX-10 g/kg group.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 265

chrome label incorporation into newly mineralizing boneare also shown in Fig. 4. These images show the presence ofcalcein and oxytetracycline in trabecular bone at L3 thatwere injected 16 and 10 months earlier as well as xylenolorange that was administered in the 2 weeks before death.An increase in the amount of label, but not in MAR, isreadily apparent in PTH-treated animals. Of note was thevirtual absence of calcein and oxytetracycline labels in themonkey receiving the 25 g/kg dose, a reflection of the highlevel of bone remodeling occurring with this dose.

A potential mechanism responsible for the increase inTb.N in PTH-treated animals is shown in Fig. 6. These lightand UV images show the phenomenon of intratrabeculartunneling in which thickened trabeculae are split into two

thinner trabeculae by tunneling osteoclasts. The resultantthinner trabeculae are thickened by new bone formation.Intratrabecular tunneling was observed in all treatmentgroups but occurred much more frequently in animals re-ceiving the 25 g/kg dose, accounting for the tendency fora lower mean Tb.Th in this group.

Bone safety: As noted above, there was no increased os-teoid accumulation in PTH-treated monkeys. Neither wasthere any evidence of woven bone, marrow fibrosis, or ab-normal bone cells at either L1 or L3 in any animal.

pQCT

The effects of OVX and PTH treatment on volumetrictotal (vBMD) and trabecular (vTbBMD) BMD and BMCare shown in Table 5. As was observed with longitudinalmeasurements of aBMD by DXA, there was a nonsignifi-cant tendency for vBMD and vTbBMD to be higher in thesham-vehicle group relative to sham-baseline controls, butthese measures were relatively stable in control OVX ani-mals. These effects were, in general, mirrored by similarchanges in BMC. PTH treatment resulted in dose-relatedincreases in vBMD and vTbBMD (Table 5). All doses ofPTH significantly increased vBMD and vTbBMD com-pared with the OVX-vehicle group. Moreover, vTbBMDwas significantly higher than in the sham-vehicle group inanimals that received PTH doses 10 g/kg.

Biomechanical testing

There were no significant differences between groups inthe heights of the trimmed vertebral bodies. The averageheights of L2 and L4 were 7.93 0.03 and 7.97 0.02 mm,respectively. The effects of OVX and treatment with PTHon biomechanical properties of bone are shown in Table 5.Consistent with the increase in BMD, there was a nonsig-nificant tendency for all biomechanical properties to behigher in sham-vehicle relative to sham-baseline controls.All mean values from biomechanical testing were alsolower in OVX animals than in the corresponding sham con-trols, but the differences were not significant. All doses ofPTH increased all biomechanical properties to levels thatwere not significantly different from sham-vehicle controls,although there was no consistent evidence of a dose re-sponse. Compared with OVX-vehicle controls, yield loadwas significantly higher in all PTH groups, whereas only the10 g/kg dose resulted in a yield stress that was significantlygreater (Table 5). Stiffness was significantly greater in the 5and 25 g/kg PTH groups than in OVX-vehicle controls.The effects of OVX and PTH treatment on modulus weresimilar to those observed with the other biomechanicalproperties, but the differences were not significant.

Regression analysis

To better understand the relationships between the den-sitometric measures of total and trabecular bone obtainedby pQCT and the biomechanical properties of lumbar ver-tebrae, regression analyses were performed. These analysesshowed that most biomechanical properties were positivelyand highly significantly correlated with the densitometricmeasures. The strongest correlations (all p < 0.001) for yield

FIG. 2. Time-course of changes in areal BMD at the lumbarspine of sham-operated and OVX rhesus monkeys treated dailywith vehicle or PTH (5, 10, or 25 g/kg) for 16 months. (A)Changes in BMD measured by standard anterior-to-posteriorDXA at L1L4. (B) Results from lateral DXA scans of L2L4 withthe region of interest placed over the center of each vertebralbody. OVX or sham surgery occurred at month 9 and treatmentstarted at month 0. Values are mean SE, n 810/group.*Pooled OVX and sham groups significantly different (p < 0.05).a,b,cp < 0.05: significance of difference from Sham-vehicle, OVX-vehicle, and OVX-5 g/kg groups, respectively.

FOX ET AL.266

stress and modulus were with vBMD (r 0.76 and 0.56,respectively) and for yield load and stiffness with totalBMC (r 0.78 and 0.58, respectively). Figure 7 shows thehighly significant correlation of total BMC versus yieldload. The correlations between trabecular densitometricmeasures and biomechanical properties were similar to orlower than for total bone (data not shown).

DISCUSSION

This paper describes the results of the first doseresponsestudy of the effects of any PTH peptide on bone of OVXmonkeys with established bone loss. As noted previously,the OVX rhesus macaque is an excellent animal model ofhuman postmenopausal bone loss that allows detailed studyof the effects of therapies at skeletal sites that are relevantin the treatment of osteoporosis.(19,28) The use of a 9-monthbone depletion period after OVX in this study was particu-larly advantageous because it was sufficiently long to resultin a stabilization of bone after the initial rapid phase ofbone loss. Bone turnover markers, densitometry, and his-tomorphometry together showed that bone turnover in-creased rapidly after OVX, and bone loss occurred at thelumbar spine in a similar fashion as occurs in postmeno-pausal women. However, vitamin D metabolism seems todiffer in rhesus monkeys compared with humans in thatserum 25(OH)D (215 ng/ml) and 1,25(OH)D (86 pg/ml)levels were high. Elevated 25(OH)D levels can be partiallyexplained by high dietary intake of vitamin D3, which con-tributed 175 IU/kg body weight per day. High serum25(OH)D and 1,25(OH)2D levels have been reported pre-viously in rhesus and cynomolgus macaques and may be theresult, in part, of differing binding kinetics of these vitaminD metabolites to vitamin D binding protein.(20,29,30)

Treatment with PTH resulted in dose-dependent in-creases in biochemical markers of bone turnover. The ef-fects of the 25 g/kg dose on bone turnover were markedly

greater than with the two lower doses, reflecting the higherPTH exposure at this dose. Markers of bone formation in-creased within 1 month with all PTH doses, whereas it was12 or 16 months before resorption markers were signifi-cantly elevated with the 5 and 10 g/kg doses. A delayedincrease in bone resorption relative to formation has beenobserved in studies of PTH and teriparatide in women withosteoporosis.(6,31) This study, therefore, provided evidencefor the existence of an anabolic window in monkeystreated with PTH as has been observed humans. However,one difference between humans and rhesus monkeys is thatthe elevated levels of bone formation markers were main-tained to the end of the study in monkeys, whereas in hu-mans treated with teriparatide, elevated formation markersreturned toward baseline levels by 18 months.(31) Boneturnover markers were measured in cynomolgus monkeystreated with teriparatide for 12 months but pooled resultsfrom months 3, 6, 9, and 12 showed only modest nonsignif-icant increases.(20)

When expressed on a per kilogram body weight basis, thedoses of PTH used in this study were higher than those inhumans, but the increases in bone turnover markers rela-tive to OVX-vehicle controls were of a similar magnitude(1.5- to 2.5-fold) to those observed in humans given PTH orteriparatide.(6,31) Moreover, there was no sustained in-crease in serum calcium levels, even in animals given thehighest dose, whereas elevated serum calcium has been re-ported in human trials. The reason for these differencesbetween humans and monkeys is unclear, but it is not aresult of a more-rapid metabolism of PTH in monkeys be-cause the plasma exposure relative to dose was similar be-tween the species. For example, the 25 g/kg dose in mon-keys is 16-fold the 100 g human dose and the plasmaPTH exposure was 13-fold higher.

For reasons that are unclear, lumbar spine BMD tendedto increase progressively throughout the study in sham-operated animals. Increases in BMD were also observed in

TABLE 3. PERCENT CHANGES IN LUMBAR SPINE BMD, BMC, AND BMA AT MONTH 16 COMPARED WITH LEVELS AT PRESURGERY(MONTH 9) AND BASELINE (MONTH 0) IN OVX RHESUS MONKEYS TREATED DAILY WITH PTH FOR 16 MONTHS

Vehicle

OVX (5 g/kg) OVX (10 g/kg) OVX (25 g/kg)Sham OVX

L1L4 (anterior-to-posterior view)Vs. presurgery

BMD 4.3 1.8 2.5 1.3* 3.9 1.8 7.9 3.1 3.0 2.1BMC 8.7 3.5 1.2 1.8 9.3 2.5 12.4 3.7 4.6 3.1BMA 4.0 1.8 3.8 0.8 5.1 1.2 4.1 1.6 1.4 1.4

Vs. baselineBMD 5.0 1.4 3.0 1.1 9.6 1.4 14.9 3.6* 10.5 1.7

BMC 5.7 2.8 4.1 1.6 13.7 2.4 20.3 4.5* 14.7 2.9*

BMA 0.6 1.5 1.1 0.8 3.6 1.0 4.6 1.1 3.6 1.2L2L4 (lateral view)

Vs. presurgeryBMD 8.2 3.8 4.3 3.7* 8.5 3.8 15.2 5.5 16.8 5.5

Vs. baselineBMD 3.3 1.9 0.3 1.9 17.9 3.4* 24.1 6.8* 23.9 3.7*

Values are mean SE, n 810/group.* p < 0.05 significance of difference from sham-vehicle group. p < 0.05 significance of difference from OVX-vehicle group.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 267

sham animals in an 18-month study in cynomolgus mon-keys.(20) The initial rate of increase in aBMD tended to begreater with the two higher doses of PTH, and levels similarto sham animals were achieved within 37 months, reflect-ing the rapid and greater increase in bone formation mark-

ers in those groups. The dose dependency of the rapid ini-tial increase in BMD was most apparent with the lateralDXA scans of L2L4, which quantified trabecular bone pre-dominantly. However, the rate of increase slowed andaBMD tended to plateau or decrease later in the study. Thelate decline in aBMD, particularly with the 25 g/kg dose,may result from the markedly greater increase in bone turn-over in this group and is discussed further below. Treatmentof cynomolgus monkeys with teriparatide also resulted inprogressive, dose-dependent increases in lumbar spineaBMD, although, because treatment was started immedi-ately after surgery, the effects of treatment on OVX-in-duced bone loss were not assessed.(20,24)

The changes in aBMD in response to OVX and PTHtreatment were paralleled by similar, although more vari-able, changes in BMC. However, the magnitude of thechange in BMC was greater than BMD because BMA alsoincreased. An increase in lumbar spine BMA has been ob-served in PTH-treated postmenopausal osteoporoticwomen.(6) It has been noted that the parallel changes thatoccur in BMA when BMD is altered may be an artifact ofhow bone edges are detected by DXA.(32) Nevertheless,these results suggest that changes in aBMD may underes-timate the anabolic response in bone to treatment withPTH.

To complement the results obtained from bone turnovermarkers and densitometry, histomorphometry provided adetailed mechanistic understanding of the response of lum-bar vertebrae to PTH treatment. The increase in BV/TVwith the 10 and 25 g/kg doses was primarily the result ofa dose-dependent increase in Tb.N to levels that were sig-nificantly greater than observed in sham animals. Therewere no significant changes in Tb.Th. Analysis by CT ofT10 from these animals showed similar changes in trabecu-lar architecture and also increased trabecular connectivitywith PTH treatment.(33) However, there were tendenciesfor BV/TV to be lower in the 25 g/kg than in the 10 g/kggroup, a result of a lower mean Tb.Th in the high-dosegroup. Responses similar to those observed in the 5 and 10g/kg groups have been observed at the iliac crest of post-menopausal osteoporotic women treated with PTH for 18months.(13) In contrast, no effect of teriparatide treatmenton Tb.N was observed in iliac crest biopsies from post-menopausal osteoporotic women.(11,12) Increased trabecu-lar number and connectivity would be expected to increasebone strength,(15) and assessment of the biomechanicalproperties of the vertebral bodies supported this notion.There was a strong positive correlation of BMC and vBMDwith yield load. However, mean yield load was highest inthe 10 g/kg group, despite the greater vBMD and Tb.N inthe 25 g/kg group, suggesting that the very high rate ofbone remodeling in the high-dose group may have nega-tively affected bone strength.

The increase in Tb.N assessed by histomorphometry inboth PTH- and teriparatide-treated monkeys seemed to re-sult from intratrabecular tunneling in which a thickenedtrabecula was divided longitudinally by osteoclastic activ-ity.(24) Bone formation followed the resorption, therebymaintaining normal Tb.Th. Qualitatively, this phenomenonwas more apparent in monkeys receiving the 25 g/kg dose

FIG. 3. Effects of daily subcutaneous injection of vehicle or PTHfor 16 months in OVX rhesus monkeys on (A) trabecular bonevolume, (B) trabecular thickness, (C) trabecular number, and (D)trabecular separation at L1 and L3. Values are mean SE, n 610/group. a,b,c,d,ep < 0.05 significance of difference fromsham-baseline, OVX-baseline, sham-vehicle, OVX-vehicle, andOVX-5 g/kg groups, respectively.

FOX ET AL.268

TABLE 4. BONE REMODELING PARAMETERS IN LUMBAR VERTEBRAE OF OVX RHESUS MONKEYS TREATED DAILY WITH PTH FOR16 MONTHS

Baseline VehicleOVX

(5 g/kg)OVX

(10 g/kg)OVX

(25 g/kg)Sham OVX Sham OVX

Ac.f (/year) 0.90 0.20 2.00 0.44 0.64 0.12 1.33 0.36 2.13 0.53 2.34 0.24 6.50 0.80***Oc.S/BS (%) 0.71 0.24 1.16 0.31 0.48 0.11 0.80 0.21 0.53 0.13 0.52 0.07 0.32 0.07

ES/BS (%) 1.16 0.27 1.46 0.29 2.64 0.42* 2.99 0.60* 2.71 0.53* 2.98 0.39* 4.36 0.57***Rs.P (months) 0.20 0.07 0.12 0.04 0.75 0.24* 0.42 0.10 0.24 0.06 0.15 0.02 0.09 0.02

OV/BV (%) 1.00 0.31 0.94 0.21 1.14 0.25 2.22 0.48 1.57 0.36 3.29 0.47* 5.26 0.64***OV/TV (%) 0.31 0.11 0.26 0.06 0.33 0.08 0.64 0.13 0.55 0.14 1.23 0.19* 1.88 0.24***BFR/BV

(mm2/mm2/year) 0.16 0.05 0.32 0.07 0.18 0.03 0.40 0.09 0.56 0.11* 0.62 0.06* 1.43 0.10***BFR/TV

(mm2/mm2/year) 0.07 0.02 0.12 0.03 0.06 0.01 0.11 0.03 0.19 0.04* 0.24 0.03* 0.51 0.03***Mlt (days) 23.5 7.7 9.0 1.2 17.9 6.8 13.8 2.5 9.0 1.8 12.1 1.2 9.1 1.3W.Th (m) 15.2 2.5 16.2 2.2 25.0 1.4* 25.0 1.3* 23.3 1.3* 22.3 0.8* 18.7 1.0

FP (months) 2.08 0.77 0.76 0.19* 2.17 0.54 1.80 0.26 1.33 0.31 1.42 0.09 0.82 0.11*

Rm.P (months) 2.28 0.81 0.90 0.21* 2.91 0.64 2.23 0.36 1.56 0.36 1.58 0.10 0.91 0.11*

Values are mean SE, n 610/group. See text for abbreviations.BFR/BV and BFR/TV values are the means of results from L1 and L3; all other parameters are from L3 only.* p < 0.05 significance of difference from sham-baseline group. p < 0.05 significance of difference from OVX-baseline group. p < 0.05 significance of difference from sham-vehicle group. p < 0.05 significance of difference from OVX-vehicle group. p < 0.05 significance of difference from OVX-5 g/kg group.** p < 0.05 significance of difference from OVX-10 g/kg group.

FIG. 4. Trabecular bone histology and bone formation at L3 of OVX rhesus monkeys treated daily with vehicle or PTH for 16 months.(AC) Trabecular bone histology (trabecular bone, solid arrows; bone marrow, open arrows) in OVX monkeys receiving vehicle orPTH at 10 or 25 g/kg/day, respectively. Note the increased trabecular bone volume with the 10 g/kg dose and the markedly increasednumber of thinner trabeculae with the 25 g/kg dose. (DF) Bone surfaces covered with osteoid (trabecular bone, solid arrows; osteoid,open arrows) in the same groups of animals. Note increased osteoid surface but no increase in osteoid thickness in PTH-treated animals.(GI) Fluorochrome labels depicting new bone mineralization (trabecular bone, solid arrows; bone marrow, open arrows; xylenolorange labels, white arrows) in the same groups of animals. Calcein (green), oxytetracycline (yellow), and xylenol orange (red) weregiven 16 months, 10 months, and 2 weeks, respectively, before death. Note the increased bone formation in PTH-treated animals and,in particular, the virtual absence of calcein and oxytetracycline labels in animals given the 25 g/kg dose.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 269

Fig 4 live 4/C

FIG. 5. Effects of daily subcutaneous injection of vehicle or PTH for 16 months in OVX rhesus monkeys on (A) osteoblast surface,(B) osteoid surface, (C) osteoid thickness, (D) mineralizing surface, (E) mineral appositional rate, and (F) surface-referent BFR intrabecular bone at L1 and L3. Values are mean SE, n 610/group.

a,b,c,d,e,fp < 0.05 significance of difference from sham-baseline,OVX-baseline, sham-vehicle, OVX-vehicle, OVX-5 g/kg, and OVX-10 g/kg groups, respectively.

FIG. 6. Intratrabecular tunneling as a mechanism responsible for increased trabecular number at L3 of OVX rhesus monkeys treateddaily with vehicle or PTH for 16 months. The images show (AC) Goldners-stained sections and (DF) unstained sections viewedunder UV light. Shown is (a) active trabecular remodeling and (b) intratrabecular tunneling both created by osteoclasts (solid arrows)and followed by bone-forming osteoblasts. Osteoid (open arrows) and xylenol orange labels (white arrows) show actively forming andmineralizing surfaces, respectively. Intratrabecular tunneling was observed in OVX monkeys receiving vehicle or PTH at 10 g/kg, butoccurred at a much greater frequency with the 25 g/kg dose of PTH.

FOX ET AL.270

Fig 6 live 4/C

of PTH and best explains the higher Tb.N and tendency toa lower Tb.Th in this group. Evidence of active intratra-becular tunneling and increased Tb.N has also been ob-served in iliac crest biopsies from women treated with PTHfor 18 months.(13)

Treatment with PTH resulted in a dose-dependent in-crease in BFR that was solely the result of increased MS/

BS; there was no effect on MAR. As was observed in boneformation markers, the increase in BFR was markedlygreater at 25 g/kg than with the two lower PTH doses. Theincrease in bone formation was associated with increasedOV/BV that was solely the result of increased OS/BS be-cause O.Th was unaffected in PTH-treated animals. Thisindicates that osteoid mineralization was normal and, in-deed, there was no effect of PTH treatment on Mlt.

There were significant dose-dependent increases in Ac.fand ES/BS and a decrease in resorption period but, forreasons that are unclear, Oc.S/BS was significantly lower inthe high-dose PTH group. The modest effect of PTH treat-ment on bone resorption at the two lower doses is entirelyconsistent with the relatively small changes observed inbone resorption markers. Of interest was the significantdecrease in mean wall thickness in the 25 g/kg dose group.Whereas the mechanism responsible for this is unknown,the shorter resorption period, possibly resulting in de-creased erosion depth, may play a role. In vitro studies inbone cells lacking the PTH-1 receptor have suggested thatthe C-terminal region of PTH may increase apoptosis.(34)

Thus, it is possible that a greater generation of C-terminalPTH fragments by peripheral PTH metabolism in the high-dose group may limit osteoblast longevity and, becauseMAR was unaffected, results in a shorter formation periodand reduced wall thickness. No effects of teriparatide treat-ment on wall thickness were observed in the study ofJerome et al.(5) Wall thickness was not reported in the otherteriparatide study in cynomolgus monkeys.(22,22,24)

There were no safety issues in lumbar vertebrae thatwere related to daily treatment with PTH for 16 months inany animal. Specifically, there was no woven bone forma-tion, even in the high-dose group in which bone turnoverwas markedly elevated, and no evidence of osteoid accu-mulation that would indicate a mineralization defect ormarrow fibrosis typical of hyperparathyroid bone disease.

FIG. 7. Regression analysis of BMC quantified by pQCT vs.yield load in L2 and L4 of sham-operated or OVX rhesus monkeysbefore treatment and after daily administration of vehicle or PTH(5, 10, or 25 g/kg) for 16 months. n 57/regression.

TABLE 5. pQCT ANALYSIS AND BIOMECHANICAL TESTING OF LUMBAR VERTEBRAE OF OVX RHESUS MONKEYS TREATED DAILYWITH PTH FOR 16 MONTHS

Baseline VehicleOVX

(5 g/kg)OVX

(10 g/kg)OVX

(25 g/kg)Sham OVX Sham OVX

vBMD (mg/cm3) 483 16 449 13 506 10 460 10 520 17 539 25* 550 14*

BMC (mg/mm) 78.4 3.3 74.6 2.9 81.2 3.3 69.5 2.8 86.5 2.9* 83.0 2.9 95.9 6.0***vTbBMD (mg/cm3) 320 21 295 10 346 9 301 10 386 20* 409 26* 450 18*

TbBMC (mg/mm) 31.0 1.8 29.4 1.4 33.4 1.5 27.3 1.3 38.2 1.4* 37.6 1.8* 47.0 3.2***Yield load (N) 3743 171 3473 202 4054 175 3523 198 4150 153 4406 269* 4341 267

Yield stress (MPa) 23.1 0.7 20.9 1.1 25.4 1.1 23.3 1.1 25.1 1.2 28.7 2.1* 25.0 0.9**Stiffness (N/mm) 16,867 745 15,908 1050 19,488 1411 16,512 828 20,001 732 19,448 1343 21,436 1471*

Modulus (MPa) 829 31 767 41 960 60 884 61 960 61 1006 60 999 75

In preparation for biomechanical testing and before pQCT scanning of the vertebral midsection, the spinous processes and end-plates were removed toobtain a specimen with plano-parallel ends.

Values are mean SE of results averaged from L2 and L4, n 610/group.* p < 0.05 significance of difference from sham-baseline group. p < 0.05 significance of difference from OVX-baseline group. p < 0.05 significance of difference from sham-vehicle group. p < 0.05 significance of difference from OVX-vehicle group. p < 0.05 significance of difference from OVX-5 g/kg group.** p < 0.05 significance of difference from OVX-10 g/kg group.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 271

Finally, there were no other abnormal cells detected, in-cluding osteosarcoma, either on the bone surfaces or withinthe marrow.

In conclusion, in OVX rhesus monkeys with establishedbone loss, daily treatment with PTH safely restored BMDat the lumbar spine to levels in sham-operated animals byincreasing trabecular bone formation and volume. The in-crease in BV/TV in PTH-treated animals occurred primar-ily by an increase in Tb.N, which seemed to result fromincreased intratrabecular tunneling. The 10 g/kg dose ofPTH provided the greatest effect on vertebral strength, pos-sibly because the highest dose resulted in too marked astimulation of bone remodeling. However, when increasedremodeling is associated with substantial gains in bonemass, the latter more than offsets the former.

ACKNOWLEDGMENTS

This study was funded by NPS Pharmaceuticals.

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Address reprint requests to:John Fox, PhD

NPS Pharmaceuticals, Inc.383 Colorow Drive

Salt Lake City, UT 84108, USAE-mail: jfox@npsp.com

Received in original form March 8, 2006; revised form August 30,2006; accepted November 1, 2006.

PTH INCREASES BONE FORMATION IN OVX RHESUS MONKEYS 273