Intermittent Fugu parathyroid hormone 1 (1–34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats

Bone 42 (2008) 1164 – 1174 www.elsevier.com/locate/bone

Intermittent Fugu parathyroid hormone 1 (1–34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats ☆ Julie F. McManus a,⁎, Rachel A. Davey a , Helen E. MacLean a , Elizabeth A. Doust a , W.S. Maria Chiu a , Natalie A. Sims b , Mary L. Bouxsein c , Vaida Glatt c , Jeffrey D. Zajac a , Janine A. Danks a,b,1 a

Department of Medicine, University of Melbourne, Austin Health, Studley Road, Heidelberg, VIC, Australia b St Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, VIC, Australia c Department of Orthopedics, Beth Israel Deaconess Medical Center, Boston, MA, USA Received 19 October 2007; revised 21 December 2007; accepted 25 January 2008 Available online 13 February 2008

Abstract Human parathyroid hormone (hPTH) is currently the only treatment for osteoporosis that forms new bone. Previously we described a fish equivalent, Fugu parathyroid hormone 1 (fPth1) which has hPTH-like biological activity in vitro despite fPth1(1–34) sharing only 53% identity with hPTH(1–34). Here we demonstrate the in vivo actions of fPth1(1–34) on bone. In study 1, young male rats were injected intermittently for 30 days with fPth1 [30 µg–1000 µg/kg body weight (b.w.), (30fPth1–1000fPth1)] or hPTH [30 µg–100 µg/kg b.w. (30hPTH–100hPTH)]. In proximal tibiae at low doses, the fPth1 was positively correlated with trabecular bone volume/total volume (TbBV/TV) while hPTH increased TbBV/TV, trabecular thickness (TbTh) and trabecular number (TbN). 500fPth1 and 1000fPth1 increased TbBV/TV, TbTh, TbN, mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) with a concomitant decrease in osteoclast surface and number. In study 2 ovariectomized (OVX), osteopenic rats and sham operated (SHAM) rats were injected intermittently with 500 µg/kg b.w. of fPth1 (500fPth1) for 11 weeks. 500fPth1 treatment resulted in increased TbBV/TV (151%) and TbTh (96%) in the proximal tibiae due to increased bone formation as assessed by BFR/BS (490%) and MAR (131%). The effect was restoration of TbBV/TV to SHAM levels without any effect on bone resorption. 500fPth1 also increased TbBV/TV and TbTh in the vertebrae (L6) and cortical thickness in the mid-femora increasing bone strength at these sites. fPth1 was similarly effective in SHAM rats. Notwithstanding the low amino acid sequence homology with hPTH (1–34), we have clearly established the efficacy of fPth1 (1–34) as an anabolic bone agent. © 2008 Elsevier Inc. All rights reserved. Keywords: Osteoporosis; Parathyroid hormone; Anabolic; Bone formation; Fish

Introduction Re-modeling of the skeleton relies on the tight coupling of bone formation and bone resorption. Imbalance between these ☆ Disclosure summary: J.M., R.D., H.M., E.D., W.C., N.S., M.B. and V.G. have nothing to declare. J.Z. and J.D. have equity interests in Teeleostin Pty Ltd. J.Z. and J.D. are inventors on (Australia) (951372, AU03/01201, 2003258389), (USA) (10/490.319), (Canada) (TBA), (China) (TBA), (South Africa) (2005/ 02299), (India) (TBA). ⁎ Corresponding author. Department of Medicine, University of Melbourne, Austin Health, Studley Road, Heidelberg, VIC 3084, Australia. Fax: +613 9457 5485. E-mail address: [email protected] (J.F. McManus). 1 Current address: School of Medical Sciences, RMIT University, Bundoora, VIC, Australia.

8756-3282/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.01.015

two processes can result in bone disease. One in 2 postmenopausal women and 1 in 3 men over the age of 60 will have a fracture due to osteoporosis [1]. Agents used to treat osteoporosis include estrogen [2], calcitonin and bisphosphonates [3], synthetic estrogen receptor modulators [4,5] and strontium ranelate [5]. None of these treatments increase bone formation. The major action of parathyroid hormone (PTH) is the regulation of calcium homeostasis by stimulating calcium release from bone and increasing renal and intestinal absorption of calcium [6]. However, PTH increases bone formation if administered intermittently. Several studies have demonstrated that human PTH (hPTH) when administered intermittently can increase bone volume in ovariectomized (OVX) rodents [7–17] and increase bone density in post-menopausal women [18,19]. The studies in OVX rodents showed that hPTH increases

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trabecular thickness (TbTh), bone formation rate (BFR/BS) [13–15] and mineral apposition rate (MAR) [10,11,14,15]. These findings suggest that hPTH acts to increase bone formation by increasing osteoblast number and activity and is thus the only current treatment with anabolic actions on bone. Although human PTH has been administered as the fulllength 84 amino acid peptide or as the active N-terminal portion of the peptide (1–34) with effective results [18,19] some side effects of hPTH (1–34) however have been documented, the most common being hypercalcaemia, nausea, headache, lightheadedness and leg cramps [18,20]. Furthermore, treatment of rats with hPTH (1–34) for two years at doses ranging from 5 to 75 µg/kg b.w. per day, as compared with the 20–40 µg daily human therapeutic dose, demonstrated an increased incidence in osteosarcoma [21]. Despite the adverse findings in rats and the risk of hypercalcaemia in humans, hPTH (1–34) has been approved for use in osteoporosis. Toxicology studies have limited the clinical treatment period to 18 months [19] and consequently there is a need to characterize other potential anabolic agents to increase treatment options for osteoporosis. We have previously described a parathyroid hormone derived from the Japanese puffer fish Fugu rubripes (fPth1) [22]. fPth1 shares 32% homology overall with hPTH and 53% identity with hPTH (1–34) and interestingly, threonine occupies amino acid position one which contrasts with a serine/alanine in that position in hPTH and other mammalian and chicken sequences [22]. A synthetic 34 amino acid peptide of fPth1 (1– 34) was found to stimulate adenylate cyclase in the osteoblast osteosarcoma cell line, UMR106 [22] indicating that fPth1 (1– 34) can act through the PTH1 receptor (PTH1R) despite low homology with mammalian PTH. Although less potent than hPTH at lower doses, the maximal amplitude of fPth1 (1–34) was significantly greater than that achieved with the highest doses of hPTH. The C-terminal portion of mammalian PTH (1–34) has been demonstrated to be involved in binding to the PTH1R, whereas the N-terminal portion is required for receptor activation [23]. The decreased potency of fPth1 in UMR106 cells may be due to decreased affinity for the PTH1R, as a number of residues in the C-terminal portions differ between fPth1 and mammalian PTH (Val17, Gln18, Arg23, Gln25, His29, Gly80 and Pro33) [23]. However, data show that amino acid residue one of mammalian PTH contacts residues in the vicinity of extracellular loop 3 and helix 6 of the PTH1R, and is required for both cAMP and phosphoinositide signaling [23]. The substitution of the threonine for serine/alanine introduces a larger side chain that could potentially impact on receptor activation and signaling, which is consistent with our previous in vitro data [22] showing a higher maximal amplitude of cAMP production in UMR106 cells by fPth1(1–34) versus hPTH(1–34). For these reasons, we are now investigating the biological activity of fPth1 in vivo, by examining the effects on bone turnover in two rat models: 4–5 week old male rats treated for 30 days and 27 week old OVX osteopenic female rats treated for 11 weeks. These studies were designed to establish the anabolic bone activity of fPth1 (1–34) and show despite the difference in homology compared with hPTH, that fPth1 (1–34) is capable of

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forming new bone. The differing amino acid sequence of fPth1 (1–34) may confer different bioavailability or pharmacokinetics or lend itself to modification thus allowing the generation of an anabolic agent based on fPth1 (1–34) with potentially fewer side effects. Materials and methods Rats Three to four week old male and 11 to 12 week old female Sprague Dawley rats were purchased from the Australian Resource Centre, Perth, Australia. Rats were housed in a 12 h light and dark cycle with food and water provided ad libitum except as described in study 2. All procedures were approved by the Austin Health Animal Ethics Committee.

Peptides Human and Fugu PTH peptides were synthesized by either Auspep (Australia) or by GL Biochem (Shanghai, China). Purity of peptides was confirmed by MALDI-TOF mass spectrometrometry, amino acid analysis and their biological activity established by an adenylate cyclase assay in UMR106 cells [22].

Experimental protocol Study 1: Young male rats In the first of two experiments in young male rats, weanling rats (4–5 weeks old) were divided randomly into five groups of 12, and administered by subcutaneous injection either 30 or 100 µg of fPth1(1–34)/kg body weight (b.w.) (30fPth1 or 100fPth1) or 30 or 100 µg hPTH (1–34) /kg b.w. (30hPTH or 100hPTH) or vehicle alone (2% rat serum [derived from male Sprague Dawley rats] in normal saline) in a total volume of 150 µl daily for 30 days. In the second experiment, young male rats were divided randomly into four groups of 8, 8, 12 and 12, and were treated with vehicle, 100hPTH, 500 or 1000 µg fPth (1–34)/kg b.w. (500fPth1 or 1000fPth1) daily for 30 days respectively. For both these experiments, rats were weighed twice weekly and PTH doses adjusted accordingly. Study 2: OVX osteopenic female rats At 12 weeks of age female rats were divided randomly into two groups of 22. Half underwent bilateral ovariectomy under isoflurane anesthesia while the other half was subjected to sham operation where ovaries were examined and then replaced intact. Following surgery, rats were limited to 20 g per rat of standard chow per day to circumvent the hyperphagia and subsequent weight gain associated with ovariectomy [24]. Water was supplied ad libitum. Fifteen weeks post ovariectomy, SHAM and OVX rats were further divided randomly into 2 subgroups to give the following 4 experimental groups: SHAM + vehicle (SHAM-VEH) (n = 10), OVX + vehicle (OVX-VEH) (n = 10), SHAM + 500 µg fPth(1–34)/kg b.w. (SHAM-500fPth1) (n = 12) and OVX + 500 µg fPth(1–34)/kg b.w. (OVX-500fPth1) (n = 12). SHAM and OVX rats received in a total volume of

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150ul, either vehicle (2% rat serum [derived from female Sprague Dawley rats] in normal saline) or 500fPth1, five days per week for 11 weeks. PTH doses were adjusted in line with rat weights which were measured once per week.

Blood samples were taken one hour post administration of PTH or vehicle once per week from selected animals throughout the OVX study via the tail vein for total calcium and total protein. Biochemical analyses were performed by Network Pathology, Austin Health, Victoria, Australia. Ultrafiltrable calcium (UFCa) was calculated based on total calcium and total protein [25].

Sections were then stained with toluidine blue for standard histomorphometry [26]. Histomorphometry was carried out according to standard procedures using the Osteomeasure Image analysis system (Osteometrics, Decatur, GA) in the secondary spongiosa of the proximal tibial metaphysis as previously described [26]. The region measured was a 1 mm long by 1.4 mm wide rectangle commencing 3.5 mm below the growth plate to avoid the primary spongiosa and subcortical bone. Histomorphometric parameters determined included trabecular bone volume (TbBV/TV), trabecular thickness (TbTh), trabecular number (TbN), trabecular separation (TbSp), osteoclast surface (OcS), osteoclast number (OcN), double labeled surface (dLS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS).

Tissue collection

Micro-computed tomography

Ten and three days prior to sacrifice, all rats received 12 mg calcein (Sigma, St Louis, USA) per kg b.w. by intraperitoneal injection. In study 1, on day 31 of injections, young male rats were asphyxiated using CO2 and tibiae harvested and transferred to 70% ethanol. In study 2 on day 74 of injections, rats were asphyxiated using CO2 and tibiae, femora and vertebrae were harvested. Tibiae collected for histomorphometry were placed in 4% paraformaldehyde in PBS overnight at 4 °C and then transferred to 70% ethanol. Vertebrae (L6) were stripped of soft tissue, wrapped in saline soaked gauze and frozen at − 20 °C prior to analysis. Femora for micro-computed tomography (µCT) were stripped of soft tissue and stored in 70% ethanol at 4 °C prior to analysis while femora for mechanical testing were treated as described for the vertebrae.

Micro-computed tomography (µCT) (μCT40, Scanco Medical AG, Basserdorf Switzerland), was used to assess trabecular and cortical bone architecture in study 2 in OVX osteopenic and SHAM rats. Trabecular bone architecture was evaluated by scanning the entire 6th lumbar vertebra, and cortical bone morphology was evaluated at the mid-femoral diaphysis. In addition, thickness of the anterior vertebral cortex and the bone volume fraction of the entire vertebral body (trabecular plus cortical bone, excluding transverse and posterior processes) were evaluated. For all μCT evaluations, a nominal isotropic voxel size of 34 μm was used. Images were subjected to Gaussian filtration and segmented using an adaptive-iterative algorithm applied to each specimen [27]. Morphometric parameters, including bone volume fraction, trabecular number, trabecular thickness, trabecular separation, structure model index, and connectivity density were computed without assumptions regarding the underlying bone architecture [28]. At the mid-femoral diaphysis, 50 transverse CT slices were obtained and used to compute the total cross-sectional area, cortical bone area, medullary area, cortical thickness and bone area fraction.

Biochemical analyses

Histomorphometry Fixed tibiae were bisected transversely, dehydrated in acetone and infiltrated with methylmethacrylate resin. 5 µm longitudinal sections were cut on a Leica 2165 microtome and mounted.

Fig. 1. Effects of treatment with vehicle, 30 or 100hPTH or 30 or 100fPth1 in young male rats on histomorphometric markers in proximal tibiae: (A) Relationship between fPth1 dose and TbBV/TV (B) TbBV/TV (C) TbTh (D) TbN (E) TbSp (F) OcS/BS (osteoclast surface/bone surface). Bars represent the mean ± SEM for n = 5–8 per group. □ vehicle, 30hPTH, 100hPTH, 30fPth1, 100fPth1. ap b 0.01, bp b 0.05 vs vehicle control, cp b 0.05 vs 30fPth1.

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Bone mineral density measurements Bone mineral density (BMD, g/cm2) of intact, excised femurs was determined using peripheral dual-energy X-ray absorptiometry (PIXImus2, GELunar, Madison, WI). Mechanical testing Compression testing of lumbar vertebrae and bending tests of the femoral diaphysis were performed using a materials test system (Synergie 200, MTS Systems, Minneapolis, MN) equipped with a 200 N load cell. Vertebrae were prepared by removing the posterior and transverse processes. To achieve plano-parallel ends, vertebrae were fixed in a custom-made jig. A low-speed diamond saw (Isomet, Beuhler Corp) [29] was configured with two parallel blades, 4.2 mm apart, and the cranial and caudal ends were removed under constant saline irrigation. In addition, a moment relief was incorporated into the compression testing platens. Specimens were then mounted in a custom uniaxial testing fixture, tested to failure in compression at 1.0 mm/min. Femora were tested to failure in a three-point bending configuration with a support span of 14 mm. Loading was applied to the posterior aspect of the mid-diaphysis of each femur at 6.0 mm/min [30]. For both vertebrae and femora, load and displacement data were sampled at 100 Hz and used to determine stiffness, failure load and energy-to-failure. Esti-

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mated material properties (elastic modulus and ultimate stress) were computed by use of standard engineering methods [31] along with relevant cross-sectional geometry measurements. Statistical analyses Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test to identify significant differences. If the Levene's test of homogeneity indicated unequal variance, Tamhane's post-hoc test was applied. Statistics were calculated using the SPSS 13.0 statistics package. A two-variable regression was used to evaluate the effects of fPth1 dose. A value of p b 0.05 was considered significant. Results Study 1 Effect of fPth1 in young growing male rats To investigate the potential anabolic actions of short-term fPth1 administration in vivo, the effect of fPth1 treatment was investigated in young growing male rats. There were no adverse side effects such as behavioral changes, illness or death following fPth1 treatment. There was no significant difference in weight gain between any of the treatment groups in either experiment (data not shown).

Fig. 2. Effects of treatment with vehicle, 100hPTH, 500 or 1000fPth1 in young male rats on histomorphometric markers in proximal tibiae: (A) TbBV/TV (B) TbTh (C) TbN and (D) TbSp, n = 7–9 per group. (E) OcS/BS (osteoclast surface/bone surface), n = 5–8 per group. (F) dLS/BS (double labeled surface/bone surface) (G) MAR (mineral apposition rate) and (H) BFR/BS (bone formation rate/bone surface), n = 4–5 per group. Bars represent the mean ± SEM. □ vehicle, 100hPTH, 500fPth1, 1000fPth1. ap b 0.0001, bp b 0.001, cp b 0.005, dp b 0.01, ep b 0.05 vs vehicle control, fp b 0.05, gp = 0.05 vs hPTH.

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fPth1 increases trabecular bone volume in young growing male rats In the young male rats administered 30fPth1 and 100fPth1 there was a significant dose relationship between fPth1 and trabecular bone volume/total volume (TbBV/TV) ( p b 0.05) in the proximal tibiae (Fig. 1A). However TbBV/TV, TbTh, trabecular number (TbN) and trabecular separation (TbSp) were not significantly altered compared to vehicle controls (Figs. 1B–E). Equivalent doses of hPTH i.e. 30hPTH and 100hPTH resulted in significantly increased TbBV/TV compared with vehicle treatment (146% and p b 0.01 for both doses) (Fig. 1B). There was also an increase in TbTh in response to 30hPTH and 100hPTH (48%, p b 0.05 and 56% p b 0.01 respectively) (Fig. 1C) and increased TbN (72%, p b 0.05 and 64%, p b 0.05 respectively) (Fig. 1D). The findings for 100hPTH were associated with a decrease in osteoclast number ( p b 0.05) (data not shown) while OcS was not altered (Fig. 1F). Given the relatively minor anabolic effect of the lower doses of fPth1 in the young male rats we investigated the effects of higher doses of fPth1, 500 µg/kg b.w. (500fPth1) and 1000 µg/kg b.w. (1000fPth1), as well as a lower, but still supra-pharmacological dose of hPTH, 100 µg/kg b.w. (100hPTH). At these higher doses of fPth1 there was a significant anabolic effect on the bones of the young growing male rats. Both 500fPth1 and 1000fPth1 significantly increased TbBV/TV, by 322% ( p b 0.0001), and 449% ( p b 0.0001) respectively in young male rats compared with vehicle controls (Fig. 2A). The increased TbBV/TV was associated with increases in both TbTh ( p b 0.001 for both doses) and TbN ( p b 0.0001 for both doses) (Figs. 2B,C) and a decrease in TbSp ( p b 0.01) (Fig. 2D). As expected, 100hPTH also significantly increased TbBV/TV ( p b 0.0001), by

286% compared with vehicle control (Fig. 2A). This increase in TbBV/TV elicited by the 100hPTH dose was similar in magnitude to the effect of 500fPth1 (Fig. 2A), whereas 100fPth1 had no significant effect on bone volume in the young male rats (Fig. 1B). fPth1 increases bone formation and decreases markers of bone resorption in young male rats To measure more directly the effects of fPth1 on osteoblast and osteoclast number and function, dynamic histomorphometry was performed on bones from young male rats treated with 500 and 1000fPth1 and 100hPTH. Results of dynamic histomorphometry on the tibiae revealed that 500fPth1 ( p b 0.05) and 1000fPth1 ( p b 0.005) and 100hPTH ( p b 0.05) significantly increased BFR/BS to an equivalent extent compared with vehicle controls (Fig. 2H). 500fPth1 and 1000fPth1 also increased MAR compared with vehicle controls by 33% ( p b 0.05) and 38% ( p b 0.005) respectively whereas 100hPTH had no effect (Fig. 2G). The extent of mineralizing surface (dLS/BS) was equally increased by 100hPTH ( p b 0.005) and 1000fPth1 ( p b 0.05) (Fig. 2F). Osteoid surface was significantly increased by both the fPth1 and hPTH treatments ( p b 0.0001) compared with vehicle controls (data not shown). 500fPth1 was more effective at increasing osteoid surface than 100hPTH ( p b 0.05) (data not shown). All parathyroid hormone treatments equally increased osteoblast number ( p b 0.0001) and surface ( p b 0.0001) (data not shown) compared with vehicle controls. In addition histomorphometric markers of bone resorption, osteoclast surface (Fig. 2E) and number (data not shown) were significantly reduced by all the PTH treatments ( p b 0.005).

Fig. 3. Effects of treatment of SHAM rats with vehicle (SHAM-VEH) or 500fPth1 (SHAM-500fPth1) and OVX rats with vehicle (OVX-VEH) or 500fPth1 (OVX500fPth1) on histomorphometric markers in proximal tibiae: (A) TbBV/TV (B) TbTh (C) TbN and (D) TbSp, n = 9–10 per group. (E) OcS/BS (osteoclast surface/bone surface), n = 8–9 per group. (F) dLS/BS (double labeled surface/bone surface) (G) MAR (mineral apposition rate) and (H) BFR/BS (bone formation rate/bone surface), n = 7–9 per group. Bars represent the mean ± SEM. □ SHAM-VEH, SHAM-500fPth1, OVX-VEH, OVX-500fPth1, ap b 0.0001, bp b 0.001, cp b 0.005, d p b 0.01, ep b 0.05 vs SHAM-VEH, fp b 0.01, gp b 0.05 vs OVX-VEH, hp b 0.005, ip b 0.01, jp b 0.05 vs SHAM-500fPth1.

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Study 2 Effect of fPth1 in OVX osteopenic female rats The strong anabolic effects of fPth1 in young male rats prompted us to investigate fPth1 in a rat model of osteopenia, OVX osteopenic female rats. Based on the study in which young male rats were treated with 500fPth1 and 1000fPth1, we chose the lowest dose of fPth1 necessary to achieve the maximum bone formation, namely 500 µg/kg b.w. (500fPth1). Fifteen weeks post-surgery, SHAM or OVX osteopenic female rats were treated 5 days per week for 11 weeks with either vehicle or 500fPth1. Despite restricting the daily food allowance of SHAM and OVX rats, as expected, the average weight of OVX-VEH began to diverge from SHAM-VEH 3 weeks post-operatively. On completion of the study the OVX-VEH weighed 17% more than SHAM-VEH ( p b 0.001) (data not shown) and the mean weight of OVX-500fPth1 was higher than SHAM-VEH ( p = 0.056).

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There was no difference in weight between SHAM rats receiving fPth1 (1–34) and those in the other treatment groups. Serum calcium and total protein were measured in randomly selected rats one each from the groups administered vehicle and 3 each from the groups administered fPth1, on a weekly basis. The UFCa levels were calculated for the purpose of ensuring that the rats administered fPth1 remained normocalcemic throughout the treatment period. Further studies with larger sample numbers are required to investigate any possible effects of fPth1 on serum calcium. fPth1 increases trabecular bone volume by increasing bone formation in osteopenic OVX female rats Ovariectomy resulted in a significant decrease of 62% in TbBV/TV in the proximal tibiae of OVX-VEH versus SHAMVEH ( p b 0.0001) (Fig. 3A). OVX-VEH rats had a 60% decrease in TbN ( p b 0.0001), while TbTh was unchanged compared with SHAM-VEH (Figs. 3B, C).

Fig. 4. Micro CT of vertebrae (L6) in SHAM rats treated with vehicle (SHAM-VEH) or 500fPth1 (SHAM-500fPth1) and OVX rats treated with vehicle (OVXVEH) or 500fPth1 (OVX-500fPth1). (A) Two-dimensional µCT images (B) TbBV/TV (C) TbTh (D) TbN (E) TbSp (F) Conn D (connectivity density) (G) SMI (structure model index) (H) cortical thickness (I) CSA (cross-sectional area). Bars represent the mean ± SEM of n = 5–11 per group. □ SHAM-VEH, SHAM500fPth1, OVX-VEH, OVX-500fPth1, ap b 0.001, bp b 0.05 vs SHAM-VEH, cp b 0.005, dp b 0.05 vs OVX-VEH, ep b 0.0001, fp b 0.001, gp b 0.005, hp b 0.01, i p b 0.05 vs SHAM-500fPth1.

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Fig. 5. Microarchitecture of mid-femoral diaphysis in SHAM rats treated with vehicle (SHAM-VEH) or 500fPth1 (SHAM-500fPth1) and OVX rats treated with vehicle (OVX-VEH) or 500fPth1 (OVX-500fPth1). (A) total area (B) cortical thickness (C) medullary area. Bars represent the mean ± SEM of n = 9–11 per group. □ SHAM-VEH, SHAM-500fPth1, OVX-VEH, OVX-500fPth1, a p b 0.0001, bp b 0.001, cp b 0.005 vs SHAM-VEH, dp b 0.0001 vs OVX-VEH, (D) Two-dimensional µCT images of mid-femoral diaphysis in SHAM rats treated with vehicle (SHAM-VEH) or 500fPth1 (SHAM-500fPth1) and OVX rats treated with vehicle (OVX-VEH) or 500fPth1 (OVX-500fPth1).

fPth1 treatment also increased TbBV/TV in SHAM rats compared to SHAM-VEH ( p b 0.05) (Fig. 3A), which was also accompanied by an increase in TbTh ( p b 0.05) (Fig. 3B). TbN was not changed following fPth1 treatment in either SHAM or OVX rats compared with their respective controls (Fig. 3C). BFR/BS was not altered in the proximal tibiae of OVX-VEH rats compared with SHAM-VEH controls (Fig. 3H) and as expected, ovariectomy resulted in significant increases in osteoclast surface ( p b 0.05) (Fig. 3E) and number ( p b 0.05) (data not shown) compared with SHAM-VEH controls. fPth1 treatment of OVX rats markedly increased mineralizing surface (dLS/BS), MAR and BFR/BS by 196% ( p b 0.01), 131% ( p b 0.05) and 490% ( p b 0.01) respectively compared with OVX-VEH (Figs. 3F, G, H). We observed no change in osteoclast surface in OVX rats post 500fPth1 treatment (Fig. 3E). Mineralizing surface (dLS/BS) ( p b 0.005), MAR ( p b 0.05) and BFR/BS ( p b 0.01) were also increased in the SHAM500fPth1 compared with SHAM-VEH (Figs. 3F, G, H). Osteoclast number (data not shown) and surface in SHAM rats were not influenced by fPth1 treatment (Fig. 3E). The findings of µCT analyses conducted on the vertebrae of rats in the different treatment groups mirrored those obtained by histomorphometry of the proximal tibiae. TbBV/TV was decreased in OVX-VEH compared with SHAM-VEH and TbBV/TV of OVX-500fPth1 was increased by 15% (p b 0.05) and TbTh by 25% ( p b 0.005) compared with OVX-VEH (Figs. 4A, B, C). TbN in OVX rats was unchanged by fPth1 treatment (Fig. 4D). TbBV/TV ( p b 0.001) and TbTh ( p b 0.001) were also significantly increased in the SHAM-500fPth1 group (Figs. 4A, B, C). As observed in OVX rats, 500fPth1 did not increase TbN in SHAM rats (Fig. 4D) although TbSp was reduced ( p b 0.05) (Fig. 4E), associated with increased TbTh. Connectivity density and structure model index were unchanged by ovariectomy (Figs. 4F, G), nor did connectivity density or structure model index change with fPth1 treatment of OVX rats compared to OVX-VEH (Figs. 4F, G). In SHAM rats the mean value for connectivity density was decreased ( p = 0.051) Table 1 Microarchitecture of the mid-femoral diaphysis of OVX osteopenic and sham operated female rats following treatment with 500fPth1 SHAM-VEH SHAM-500fPth1 OVX-VEH OVX-500fPth1 (n = 10) (n = 11) (n = 9) (n = 10)

In OVX-500fPth1 rats, TbBV/TV was markedly increased by 151% ( p b 0.01) compared with OVX-VEH (Fig. 3A). This was associated with an increase in TbTh of 96% ( p b 0.05) (Fig. 3B) thus restoring the TbBV/TV to the levels observed in SHAM-VEH. TbTh in OVX-500fPth1 was increased above levels observed in SHAM-VEH ( p b 0.05) (Fig. 3B). The mean value for TbSp was lower in the OVX-500fPth1 than in OVXVEH although this did not reach significance ( p = 0.057) (Fig. 3D).

Bone area 6.6 ± 0.2 (mm2) Bone area 66.8 ± 0.8 fraction (%) Femur length 35.9 ± 0.7 (mm)

6.7 ± 0.1

6.6 ± 0.1

7.1 ± 0.1bd

72.9 ± 0.5a

65.0 ± 0.8

73.1 ± 0.6ac

37.3 ± 0.3

36.7 ± 0.4

37.3 ± 0.2

SHAM-VEH and OVX-VEH, sham operated and OVX rats treated with vehicle; SHAM-500fPth1 and OVX-500fPth1, sham operated and OVX rats treated with 500fPth1. Results represent the mean ± SEM. a p b 0.0001 vs SHAM-VEH. b p b 0.05 vs SHAM-VEH. c p b 0.0001 vs OVX-VEH. d p b 0.05 vs OVX-VEH.

J.F. McManus et al. / Bone 42 (2008) 1164–1174 Table 2 Mechanical properties of vertebrae (L6) in OVX osteopenic and sham operated female rats following 500fPth1 treatment SHAM-VEH SHAM-500fPth1 OVX-VEH OVX-500fPth1 (n=6) (n=10) (n=4) (n=10) Structural properties Failure load (N) 266 ± 26 Stiffness (N/nm) 931 ± 118 Energy absorbed 67 ± 11 to failure (N⁎mm) Estimated material properties Ultimate stress 32.4 ± 2.2 (MPa) Modulus (MPa) 583 ± 110

515 ± 45a 1175 ± 111 182 ± 19a

270 ± 8 963 ± 57 62 ± 4

363 ± 25c 1136 ± 73 101 ± 19cd

54.4 ± 4.9b

27.3 ± 1.2

39.9 ± 3.2d

686 ± 68

536 ± 37

688 ± 52

SHAM-VEH and OVX-VEH, sham operated and OVX rats treated with vehicle; SHAM-500fPth1 and OVX-500fPth1, sham operated and OVX rats treated with 500fPth1. Results represent the mean ± SEM. a p b 0.005 vs SHAM-VEH. b p b 0.05 vs SHAM-VEH. c p b 0.05 vs OVX-VEH. d p b 0.05 vs SHAM-500fPth1.

with fPth1 treatment, compared to SHAM-VEH which was accompanied by a decrease in structure model index ( p b 0.05) (Figs. 4F, G). Thickness of the vertebral cortex was not influenced by ovariectomy, or by fPth1 treatment in OVX rats (Fig. 4H). However, cortical thickness of the vertebrae was significantly greater in the SHAM-500fPth1 group versus the SHAM-VEH group ( p b 0.001) (Fig. 4H). Cross-sectional area was not altered in either OVX or SHAM rats by fPth1 (Fig. 4I). As we observed in the vertebrae, cortical thickness at the midfemoral diaphysis was unchanged by ovariectomy (Figs. 5B, D). Treatment of OVX rats with fPth1 resulted in a significant increase in the bone area fraction of the mid-femoral diaphysis ( p b 0.0001) (Table 1). This was associated with an increase in cortical thickness ( p b 0.0001) and a decrease in medullary area ( p b 0.0001) compared with OVX-VEH, without any change in total area (Figs. 5A, B, C, D). Cortical thickness was also significantly increased ( p b 0.005) and medullary area decreased ( p b 0.001) in SHAM rats treated with fPth1 compared with SHAM-VEH (Figs. 5B, C, D). Femoral length was not altered by fPth1 treatment in SHAM or OVX rats (Table 1).

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Whole femoral BMD was significantly increased in response to fPth1 treatment in OVX and SHAM rats by 11% ( p b 0.05) and 17% ( p b 0.0001) respectively compared with controls (data not shown). Distal femoral BMD and content were also increased by fPth1 treatment in OVX rats (23% [ p b 0.0001] and 24% [ p b 0.005] respectively) and SHAM rats (28% [ p b 0.005] and 32% [ p b 0.0001] respectively) (data not shown). Increased bone volume is associated with increased bone strength in OVX osteopenic female rats To determine whether the effects of fPth1 on bone structure had a corresponding effect on bone strength, mechanical testing was performed. Vertebral compressive biomechanical properties were similar in vehicle-treated OVX and SHAM rats. Compared to vehicle, vertebral failure load and energy absorbed to failure were significantly increased in both OVX ( p b 0.05 for both parameters) and SHAM ( p b 0.005 for both parameters) rats treated with fPth1 (Table 2). Neither OVX nor fPth1 treatment influenced structural biomechanical properties at the femoral mid-shaft, as assessed by 3-point bending. However, ultimate stress was increased ( p b 0.005), suggesting that the intrinsic properties of the bone matrix were improved by fPth1 treatment (Table 3). Discussion In these studies we examined the effects of an N-terminal peptide from the Japanese puffer fish Fugu rubripes parathyroid hormone, fPth1 (1–34), on bone in vivo. We showed that intermittent treatment with varying doses of fPth1 (1–34) in two different rat models of elevated bone turnover, young growing male rats and OVX osteopenic female rats, has significant anabolic actions on bone. We demonstrated increased markers of bone formation in the proximal tibiae, vertebrae and femora which translated into increased strength at least in vertebrae and femora. These data suggest that despite fPth1 (1–34) sharing only 53% identity with hPTH (1–34), fPth1 has similar anabolic actions on bone to hPTH in vivo. We initially tested the actions of fPth1 in young growing male rats to demonstrate that fPth1 (1–34) has anabolic activity in bone in vivo, in addition to the biological activity we previously demonstrated in vitro in UMR106 cells [22]. There

Table 3 Mechanical properties of femora in OVX osteopenic and sham operated female rats following 500fPth1 treatment SHAM-VEH (n = 9) Structural properties Maximum load (N) Bending stiffness (N⁎mm/mm) Energy to failure (N⁎mm2)

207 ± 6 1522 ± 93 355 ± 17

Estimated material properties Ultimate stress (MPa) Modulus (MPa)

214 ± 9 17757 ± 1441

SHAM-500fPth1 (n = 10) 205 ± 3 1735 ± 41 299 ± 24

240 ± 4 a 22668 ± 657 (p = 0.058 vs SHAM-VEH)

OVX-VEH (n = 10)

OVX-500fPth1 (n = 11)

203 ± 6 1571 ± 73 373 ± 24

218 ± 8 1698 ± 45 366 ± 24

199 ± 6 17344 ± 833

235 ± 6 b 19867 ± 741

SHAM-VEH and OVX-VEH, sham operated and OVX rats treated with vehicle; SHAM-500fPth1 and OVX-500fPth1, sham operated and OVX rats treated with 500fPth1. Results represent the mean ± SEM. a p b 0.05 vs SHAM-VEH. b p b 0.005 vs OVX-VEH.

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was no significant increase in parameters of bone modeling in the proximal tibiae of rats treated with 30 µg/kg b.w. (30fPth1) and 100 µg/kg b.w. (100fPth1). Despite these findings, we were able to measure a significant dose response relationship for TbBV/TV which may suggest that fPth1 has anabolic activity in bone even at these low doses. Our results are similar to another study undertaken in young male rats [32] where no increase was reported in TbBV/TV or TbTh of lumbar vertebrae in response to treatment with daily doses of 10 and 40 µg/kg b.w. hPTH for a period of 28 days. Our findings of relatively little effect with low doses of fPth1 treatment contrasted with our results using the same doses of hPTH suggesting that hPTH is a more potent anabolic agent that fPth1 (1–34). At higher doses of fPth1 (1–34), 500 µg/kg b.w. (500fPth1) and 1000 µg/kg b.w. (1000fPth1), we observed marked increases in TbBV/TV with an increase in both TbTh and TbN. As we observed an increase in markers of osteoblast activity and a decrease in osteoclast number and surface the increase in bone volume appears likely due to both increased bone formation and decreased resorption. However since hPTH acts to mainly increase bone formation in the early phase of treatment [33– 35], the reduction in osteoclast surface resulting from both hPTH and fPth1 treatment in fast growing rats may indicate a different mechanism in this young rat model. However we cannot rule out the possibility that the increased area occupied by osteoblastic activity reduces the available area for resorption to occur. For most bone histomorphometric parameters there was little difference in the anabolic activity of 500fPth1 versus 1000fPth1, whereas these doses were clearly more potent than 30fPth1 or 100fPth1. Thus we concluded that the lowest dose of fPth1 at which maximal bone formation can be achieved is 500fPth1. In comparing the relative potency of human and Fugu PTH, the extent of the increase in bone formation rate in 100hPTH treated rats was equivalent to 500fPth1 so we can deduce that 500fPth1 is at least as effective a bone anabolic agent as 100hPTH. The lower potency of fPth1 (1–34) compared with hPTH (1–34) is perhaps not surprising given that our in vitro studies in UMR106 cells [22] demonstrated at doses of 10 nmol/L, fPth (1–34) had lower biological activity than hPTH (1–34), and the fact that fPth1 has lower homology with mammalian PTH in the Cterminal region (residues 15–34), involved in receptor binding. These findings suggest the possibility that fPth1 has a lower affinity for the PTH receptor(s), the in vivo kinetics of fPth1 differ from hPTH or fPth1 signals through different messenger pathways, all of which requires further investigation. Following on from the study in young male rats, we tested the efficacy of 500fPth1 in OVX osteopenic female rats, a well established in vivo model for investigating potential bone anabolic agents. The basis for choosing the fPth1 dose was that 500 μg/kg b.w. was the lowest dose demonstrated to achieve maximum bone formation and was equivalent in potency to 100 μg/kg b.w. hPTH. The effect of ovariectomy in the female rats was increased bone turnover, as evidenced by decreased bone volume and increased osteoclast number and surface in proximal tibiae. The result of the increased bone turnover was significantly decreased TbBV/TV attributable to decreased TbN in both proximal tibiae

and vertebrae. There was no decrease in TbTh at either of these sites. Other studies in rats describe similar findings in response to ovariectomy [8,10–13,16]. Ovariectomy results in greater loss of bone from the proximal tibiae than the vertebrae in rodents [16]. Our findings support this observation with 62% loss in TbBV/TV in the proximal tibiae versus 16% in vertebrae. While TbBV/TV was decreased in vertebrae and proximal tibiae by ovariectomy, cortical thickness was unchanged in vertebrae or femora. Similar findings have been reported elsewhere [11]. Treatment of OVX osteopenic female rats with 500fPth1 increased TbBV/TV and in line with the relative bone losses in proximal tibiae and vertebrae following ovariectomy, we observed correspondingly greater increases in TbBV/TV in the proximal tibiae (172%) compared with vertebrae (16%) in response to fPth1. In fPth1 treated SHAM female rats we also noted larger increases in TbBV/TV in the proximal tibiae (87%), compared with vertebrae (21%). The increases in TbBV/TV we observed in the proximal tibiae and vertebrae in OVX osteopenic rats in response to 500fPth1 parallel findings in osteopenic rat models treated with mammalian PTH [8,10–15]. Restoration of TbBV/TV in the OVX rats to SHAM-VEH levels by 500fPth1 is attributed to increased TbTh without any change in TbN [8,10–15] This signifies increased bone modeling as evidenced by increased mineralizing surface, MAR and BFR/BS as has been described in response to hPTH [8,10–15,17,36]. In contrast to the study in young male rats, we did not observe a decrease in resorption markers in response to fPth1 in the OVX osteopenic female rats. The lack of effect on resorption is consistent with findings in humans where hPTH activates remodeling only after treatment exceeds a period of several months [33–35]. The differences we observed in osteoclast parameters between young male rats and OVX osteopenic rats may be explained by the fact that although both are models of high bone turnover, in the OVX model, bone resorption is exceeding formation while in the model of fast growing rats bone is accrued predominantly by bone formation which may impact on response to anabolic agents. No effect on osteoclast number or surface was detected in SHAM rats following 500fPth1 treatment which was as expected given the length of treatment [33–35]. These findings suggest that in OVX osteopenic rats and SHAM rats alike, fPth1 increases TbBV/TV by increasing bone formation without any change in bone resorption. fPth1 significantly increased cortical thickness and decreased the medullary area at the mid-femoral diaphysis in OVX osteopenic rats without a change in total area, indicating increased endocortical bone formation. This is similar to findings in young male rats in response to hPTH [32] but differs from a study in OVX rats [37] where hPTH increased periosteal as well as endocortical apposition. The different observations may be dose dependent. Kneissel [38] demonstrated that cortical bone growth at the periosteum and endosteum in response to hPTH is highly dose dependent with apposition at these surfaces increasing with increasing dose. The hPTH doses at which periosteal bone growth was observed in the studies of Mosekilde (80 µg/kg b.w.) [37] and Kneissel (100 µg/kg b.w.) [38] were at least double that used in the study by Frolik [32]. Given that we consider the 100 µg/kg b.w. dose of hPTH to be equivalent in potency to 500fPth1, it is

J.F. McManus et al. / Bone 42 (2008) 1164–1174

tempting to speculate that fPth1 acts specifically on the endocortical surface of bone. Structural changes in cortical bone suggest that 500fPth1 also promoted bone formation only at the endocortical surface of vertebrae and femora in SHAM rats. Mechanical testing was performed to determine whether the anabolic actions of fPth1 result in increased bone strength, an important end-point for any potential osteoporosis therapeutic. The positive effects of fPth1 on bone volume and architecture lead to increased compressive biomechanical properties at the lumbar vertebrae, and also suggested improved bone material properties at the femoral diaphysis. fPth1, like hPTH [7], improves bone strength of vertebrae and femora. The increase in bone strength in the vertebrae of SHAM rats treated with fPth1 was twice that achieved in OVX osteopenic rats, reflecting the larger increases in trabecular BV/TV we observed in SHAM rats compared with OVX osteopenic rats. Thus, our data demonstrate that fPth1 is capable of anabolic actions on bone, with associated effects on bone strength, in rat models of both normal and high turnover. Here we demonstrated the anabolic actions of fPth1 (1–34) in two rat models. Our data suggest that fPth1 increases TbBV/ TV in young growing male rats and OVX osteopenic female rats by increasing bone formation. In the OVX osteopenic rat model fPth1 restores bone volume to pre-ovariectomy levels. The increased bone formation in both vertebrae and femora after 11 weeks of treatment resulted in an increase in mechanical strength, an outcome which suggests that fPth1 (1–34) could potentially be effective in preventing fractures. fPth1 (1–34) is capable of increasing bone formation in SHAM rats which shows that this peptide also has anabolic activity in normal bone turnover states. We have demonstrated conclusively that although structurally dissimilar to mammalian PTH, fPth1 has similar biological activity in vivo and is an effective bone forming agent. In addition, the unique amino acid sequence of fPth1 may alter its bioavailability or pharmacokinetics or lend itself to amino acid substitution to generate an anabolic agent with fewer side effects and as such merits further investigation. Acknowledgments Supported by TeeleOstin Pty Ltd. HEM is supported by a National Health and Medical Research Council RD Wright Career Development Award and RAD is supported by National Health and Medical Research Council project grant #350356. References [1] Nguyen T, Sambrook P, Kelly P, Jones G, Lord S, Freund J, Eisman J. Prediction of osteoporotic fractures by postural instability and bone density. BMJ 1993;307:1111–5. [2] Hutchinson TA, Polansky SM, Feinstein AR. Post-menopausal oestrogens protect against fractures of hip and distal radius. A case-control study. Lancet 1979;2:705–9. [3] Bruyere O, Edwards J, Reginster JY. Fracture prevention in postmenopausal women. Clin Evid 2003:1304–22. [4] O'Neill S, MacLennan A, Bass S, Diamond T, Ebeling P, Findlay D, et al. Guidelines for the management of postmenopausal osteoporosis for GPs. Aust Fam Physician 2004;33:910–9.

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