Effects of a Combination Therapy of Sclerostin Antibody III and Raloxifene on Bone Formation Markers in Ovariectomized Rats

Transcription

1 ORIGINAL ARTICLE Effects of a Combination Therapy of Sclerostin Antibody III and Raloxifene on Bone Formation Markers in Ovariectomized Rats Hayam Ibrahim Gad Allam ABSTRACT Objective: To determine the systemic effect of sclerostin monoclonal antibody (Scl-AbIII) administration on markers of bone formation and compare it with a combination of sclerostin antibody and raloxifene. Study Design: Experimental study. Place and Duration of Study: Medical College Animal House at King Khaled University Hospital, Riyadh, Saudi Arabia, from January to November Methodology: Forty-five female rats were divided into 5 groups equally; 1 control group and 4 groups of ovariectomized (OVX) rats: control OVX rats and OVX rats treated by Scl-AbIII, raloxifene or Scl-AbIII+raloxifene one month after ovariectomy, continued for 4 weeks. At the end of treatment, serum levels of Bone Specific Alkaline Phosphatase (BSAP), alkaline phosphatase, osteocalcin, Insulin-like Growth Factor-1 (IGF-1), Parathyroid Hormone (PTH), Ca 2+ and phosphorus were measured. Uterus was weighed and body weight change was calculated. Results: Scl-AbIII or raloxifene treatment produced significant increase of serum BSAP, osteocalcin, IGF-1, PTH and Ca 2+ levels. Raloxifene, either alone or combined with Scl-AbIII attenuated the decrease in uterus wet weight, and the increase in body weight seen in OVX rats. Combination therapy of Scl-AbIII, and raloxifene produced significant increase of serum alkaline phosphatase, osteocalcin and IGF-1 levels than treatment with either Scl-AbIII or raloxifene alone. Conclusion: Combination therapy of Scl-AbIII and raloxifene is an attractive strategy to enhance bone formation and can offer better gain over treatment with either one of them alone. Confirmation of these preliminary observations must await careful long-term studies. Key Words: Osteoporosis. Sclerostin antibody. Raloxifene. Osteogenesis. INTRODUCTION Osteoporosis is accepted as a major public health problem characterized by compromised bone strength, micro-architectural deterioration of bone tissue and increased fracture risk with an important socio-economic burden. 1 It is a multifactorial disease, but the most common form is involutional osteoporosis, which is associated with advancing age and menopause. Along with efforts to develop improved antiresorptive agents, there has been a long-standing goal to develop therapeutics that can stimulate bone formation, to increase bone mass, and strength for low bone mass conditions (e.g. osteoporosis). 2 Wnt proteins are signaling molecules that regulate osteoblasts differentiation into mature osteoblasts. The binding of Wnt proteins to their specific receptors leads to stabilization of β-catenin, which translocates to the nucleus and regulates gene expression. Sclerostin is a natural Wnt antagonists secreted by osteoblasts/ Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia. Correspondence: Dr. Hayam Ibrahim Gad, Physiology Department, College of Medicine, King Saud University, P.O. 2925(29), Riyadh 11461, Saudi Arabia. hayam_gad@hotmail.com Received: January 23, 2015; Accepted: October 12, osteocytes. Sclerostin antibodies (Scl-Abs) have been shown to neutralize the inhibitory effects of sclerostin on Wnt/b-catenin signaling and might be an attractive approach for the development of a novel bone anabolic agent. 3 Estrogen (E2) is involved in the regulation of a number of molecules that have an effect on osteoclasts. E2 limits the size of preosteoclast population by apoptosis and stimulating osteoblastic stromal cells to synthesize more osteoprotegerine (OPG) that inhibits osteoclast differentiation and bone resorption. E2 also stimulates osteoblastic cells to make Transforming Growth Factorbeta (TGF β) which restrains the expression of cathepsin K, which is the molecular shovel that osteoclasts used to dig holes in bone. 4 The Selective Estrogen Receptor Modulators (SERM), such as raloxifene, are nonsteroidal and synthetic class of compounds that reproduce the positive effects of E2 in bone. 5 Ovariectomized (OVX) rat models, mimic changes in bone metabolism observed in postmenopausal osteoporosis and have been most commonly used for the efficacious studies of potential therapeutic agents for the treatment of osteoporosis. 6 The aim of this study was to determine the systemic effect of sclerostin antibody administration on markers of bone formation in rats made estrogen deficient (OVX) and to compare this effect with that of raloxifene and a combination of sclerostin antibody and raloxifene. 46 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50

2 Sclerostin antibody and bone formation markers METHODOLOGY Forty-five female Wistar rats were housed at room temperature (25± C) and were allowed water ad libitum. Their initial weight varied from 225 to 250 grams and age from 19 to 20 weeks. The experiments were conducted in accordance with Institutional Review Board (IRB) at King Khaled University Hospital, Riyadh. The rats were divided randomly into five groups equally. Group I included sham operated (intact) control rats. Group II, III, IV and V were OVX rats received saline, sclerostin monoclonal antibody III (Scl-AbIII), raloxifene or a combination therapy of both Scl-AbIII and raloxifene, respectively. Rats were anesthetized using ketamine hydrochloride (120 mg/kg) and xylazine hydrochloride (24 mg/kg) intramuscularly. 7 The experiment for bilateral ovariectomy was done through abdominal incision to expose the uterus, oviducts and ovaries. The oviduct and blood vessels supplying the ovaries were tied up, and the ovaries were removed through an incision. The uterus and oviducts were put back with the fat tissues around them, and the incision was closed with suture. In sham operated rats, an abdominal incision was performed affecting skin and muscle and peritoneum. No organ was extirpated or handled. The wound was subsequently sealed. The animals were randomized into groups according to the above protocol. Treatments of OVX rats were started one month postsurgery. Twenty-five mg/kg of Scl-AbIII was injected subcutaneous (S.C.) 2 times per week for 4 weeks. 8 Scl- AbIII stock solution was stored at -80 C. The dosing solutions were prepared weekly by completely thawing in a 4 C refrigerator overnight then diluting with saline to a concentration of 5 mg/ml. Raloxifene was purchased from Sigma Aldrich Co. and administered orally at a daily dose of 5mg/kg, for 4 weeks. 9 Body weight was measured at the start and at the end of the experiment. Body weight change was calculated for each rat. At the end of the treatment, blood samples (by cardiac puncture) were collected at 7:00 and 9:00 A.M., centrifuged and frozen within one hour, and stored under identical conditions. The serum samples were assayed for the levels of estradiol by competitive enzyme immunoassay. 10 Bone specific alkaline phosphatase (BSAP) using immunoradiometric assay, 11 alkaline phosphates by colorimetric method, 12 osteocalcin by immunoassay, 11 insulin like growth factor-1 (IGF-1) by enzyme linked immunosorbent assay (IGF-1-ELISA kit), 13 parathyroid hormone (PTH) by enzyme amplifier sensitivity immunoassay (EASIA), 14 and calcium and phosphorous by colorimetric method. 15 Uteri were excised from sacrificed animals and weighed after trimming associated fat and expressing any luminal fluid. Statistical analyses were carried out using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) program for windows version 21. Data were expressed as mean ±SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) with 95% confidence intervals followed by Tukey's multiple comparison tests. Differences were considered to be statistically significant at p < RESULTS OVX-control rats had statistically significant decrease of estradiol level in comparison with the sham-operated controls. Neither Scl-AbIII nor raloxifene administration affected the decrease in estradiol levels (Table I). Both serum BSAP and alkaline phosphatase levels were significantly decreased in control OVX rats as compared to sham-operated controls and OVX rats treated with Scl-AbIII, raloxifene or Scl-AbIII+Raloxifene (p < 0.001). Scl-AbIII administration for four weeks increased bone formation markers, as demonstrated by a 19.8% increase in serum BSAP and a 24.8% increase in serum alkaline phosphatase as compared to control OVX rats (p < 0.001). Similar results were found after 4 weeks of raloxifene treatment. Combined Scl-AbIII+raloxifene treatment resulted in greater increase in serum BSAP and alkaline phosphatase (42.2%, 44.5% respectively Table I: Mean Values ± SD of the measured parameters in controls (group I), ovariectomized rats (OVX) rats (group II), sclerostin antibody III (Scl-AbIII)-treated OVX rats (group III), Raloxifene-treated OVX rats (group IV), and OVX rats treated with both Scl-AbIII and raloxifene (group V). Group I Group II Group III Group IV Group V p-value Serum estradiol (pmol/l) ± ±0.98* ±0.94* ±0.59* ±0.71* Serum alkaline phosphates (U/l) ± ± ± ± ± Serum osteocalcin (ng/ml) 3.86 ± ± ± ± ± Serum insulin like growth factor-1 (ng/ml) ± ± ± ± ± Serum parathyroid hormone (pg/ml) ± ± ± ± ± Serum calcium (mmol/l) 3.53 ± ± ± ± ± Serum phosphorous (mmol/l) 3.65 ± ± ± ± ± Body weight changes (g) ± ±2.50** ±2.15** ± ± Uterus wet weight (g) ± ±4.21^ ±4.25^ ±4.36^ ±4.96^ Significance was considered at p<0.05, 95% confidence interval. (*) p<0.001 versus group I; ( ) p<0.001 versus groups I, III, IV and V; ( ) p<0.01 versus group III, p< 0.05 versus group IV; ( ) versus groups I (p<0.05), III (p<0.01), IV (p<0.05); ( ) p<0.05 versus groups I, III, IV and V; ( ) versus groups I (p<0.05) and III (p<0.01); (**) p<0.001 versus groups I, IV and V; (^) p<0.001 versus group I ( ) p<0.001 versus groups II, III. Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1):

3 Hayam Ibrahim Gad Allam Figure 1: Effect of sclerostin antibody III (Scl-AbIII) and raloxifene on serum bone specific alkaline phosphatase in controls (group I), ovariectomized rats (OVX) rats (group II), sclerostin antibody III (Scl-AbIII)-treated OVX rats (group III), Raloxifene-treated OVX rats (group IV) and OVX rats treated with both Scl-AbIII and raloxifene (group V). All values are represented as mean±s.d. * = p<0.001 versus all other groups, = p<0/001 versus groups III and IV. higher as compared to control OVX rats, p < 0.001). Moreover, Scl-AbIII+raloxifene-treated rats had higher BSAP and alkaline phosphatase levels as compared to rats treated by either Scl-AbIII or raloxifene alone (Table I, Figure 1). Control OVX rats had significantly lower serum osteocalcin levels as compared to sham-operated controls. Combined Scl-AbIII+raloxifene treatment significantly increased osteocalcin level than treatment with Scl-AbIII (p < 0.01) or raloxifene (p < 0.05) each separately. The increases in osteocalcin levels were 67.8%, 47.9%, 40.6% higher in Scl-AbIII+raloxifene (gp V), raloxifene (gp IV) and Scl-AbIII-treated rats (gp III) respectively as compared to control OVX rats (p < 0.001). No significant change could be detected between Scl-AbIII-treated rats and raloxifene-treated rats (Table I). The decrease in serum level of IGF-1 was evident in control OVX rats as compared to sham-operated controls and OVX-treated rats by either Scl-AbIII or raloxifene alone or combined. Combined treatment of OVX rats with Scl-AbIII and raloxifene significantly increased IGF-1 level as compared to sham-operated controls (p < 0.05), OVX treated rats with Scl-AbIII (p < 0.01) or raloxifene (p < 0.05) each separately (Table I). Control OVX rats showed significant decrease of serum PTH and Ca 2+ levels and significant increase of serum phosphorous levels as compared to sham-operated controls and OVX rats treated with either Scl-AbIII, raloxifene or Scl-AbIII+raloxifene. PTH levels increased significantly with Scl-AbIII (26.34%), raloxifene (32.38%) or Scl-AbIII+raloxifene (34.72%) as compared to control OVX rats (p < 0.001). Combined treatment of OVX rats with Scl-AbIII+raloxifene did not affect the increase of Ca 2+ or the decrease of phosphorous levels achieved by treatment of either of Scl-AbIII or raloxifene alone (Table I). After 4 weeks of observation, body weight increased in all groups. However, when comparing the different study groups, the weight gain of control OVX rats and those rats treated with Scl-AbIII rats at the end of the study period was more pronounced than that of shamoperated controls (99.61 ±2.50, ±2.15 vs ±2.52 g, respectively). Raloxifene therapy, either alone or combined with Scl-AbIII provided to OVX rats, kept weight changes comparable with those seen in shamoperated controls (74.58 ±3.45, ±3.36 vs ±2.52 g, respectively, Table I). Bilateral ovariectomy induced a substantial decrease of uterine wet weight in relation to the sham-operated controls, expected results of a lowered E2 level, and was 23.92% of the uterine weight of the intact control. Although administration of Scl-AbIII to OVX rats did not affect uterine wet weight; administration of raloxifene, either alone or combined with Scl-AbIII, attenuated the decrease of uterus mass in the OVX rats (36.97%, 37.12% respectively of the uterine weight of the shamoperated controls (Table I). DISCUSSION The present study showed an increase in serum BSAP and alkaline phosphatase after 4 weeks administration of either Scl-AbIII or raloxifene. While, combined treatment with Scl-AbIII and raloxifene resulted in more increase in BSAP and serum alkaline phosphatase as compared to non-treated OVX rats (p < 0.001). The marked improvements in bone formation markers in Scl-AbIII-treated OVX rats provide the evidence that inhibition of sclerostin can enhance bone formation, which may be directly related to the effects of reversing sclerostin's inhibitory effect on osteoblast activity. The mechanism by which sclerostin negatively regulates bone formation is an area of continuing investigation. One body of research supports the hypothesis that sclerostin inhibits Wnt-β-catenin signaling by interacting with Wnt coreceptors and thus impairing osteoblast differentiation and function. 16 Consistent with the present findings is the human clinical study of Padhi et al. 17 who found that a single dose of a humanized sclerostin monoclonal antibody in healthy men and postmenopausal women resulted in a dose-dependent increase in the concentrations of the bone-formation markers (BSAP and osteocalcin) and decreased the bone-resorption marker (serum C-telopeptide). In addition, Tasci et al. found high serum alkaline phosphatase in raloxifene treated osteoporotic rats. 18 Serum levels of osteocalcin are regarded as sensitive and specific marker of osteoblastic activity and the rate of bone formation. Hence, the finding of the present 48 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50

4 Sclerostin antibody and bone formation markers work of reduced serum osteocalcin in OVX rats suggests that reduced osteoblast activity may be responsible for the osteoporosis. Evidence of a stimulatory effect of Scl-AbIII or raloxifene on serum osteocalcin level was apparent following their administration for 4 weeks. Combined Scl-AbIII+raloxifene treatment was more effective than Scl-AbIII treatment alone in improving serum osteocalcin levels. This is in accordance with the study of Li et al. who reported significant increase of serum osteocalcin, recruitment and functional longevity of osteoblasts after Scl-Ab treatment. 6 Humans with inherited sclerostin deficiency have increased bone mass and are resistant to fracture. 19 However, the extent to which sclerostin might regulate bone formation and bone mass in a normal aged skeleton is not clear. The data of the current study describes the powerful anabolic response to sclerostin inhibition in OVX rats and clearly shows that sclerostin functions as a pivotal negative regulator of bone formation. The evidence for an antiresorptive effect with Scl-Ab administration was observed in ovariectomy-induced bone loss rats 6 and gonad-intact female monkey models. 8 Alaee et al. showed enhanced bone repair following treatment of the rat femoral defect model with Scl-Ab. 20 Furthermore, their results showed significantly 44% higher osteocalcin levels at 2 weeks of Scl-Ab treatment compared to the vehicle group. Tian demonstrated significant increase of bone mass by Scl- Ab treatment of immobilization/disuse rat model compared with their controls. 21 Moreover, Sinder et al. reported that treatment of mouse model of osteogenesis imperfecta for 5 weeks with Scl-Ab had increased bone mass, cortical bone strength and induced bone formation on surfaces that are normally quiescent or resorptive in rapidly growing mice. 22 Taken together, these findings suggested that antibody-mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization-induced osteopenia and a therapy during fracture healing to improve bone formation. 23 Several reports suggest modulating effect of raloxifene on production of various factors involved in osteoclastogenesis and osteoclast survival, resulting in suppression of bone resorption. 24 In this study, the treatment combined with Scl-AbIII and raloxifene significantly increased BSAP, osteocalcin and IGF-1 levels in comparison with Scl-AbIII treatment alone. Raloxifene is a unique agent that apparently possesses sufficient intrinsic activity to act like an agonist in bone and liver, but is a relatively pure antagonist in uterine tissue. In this study, raloxifene caused statistically significant increase in uterine weight in relation to the OVX controls. This effect may be related to the slight hypertrophy of the myometrium and endometrial stroma, which, in previous work, has been attributed to water retention. 25 CONCLUSION Combination therapy of Scl-AbIII and raloxifene can offer better gain over treatment with either one of them alone. These results support the potential of Scl-AbIII and raloxifene as attractive strategy to enhance bone formation. Confirmation of these preliminary observations must await careful long-term studies. Acknowledgment: This research project was supported by a grant from the Research Center of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University. REFERENCES 1. Ishtiaq S, Fogelman I, Hampson G. Treatment of postmenopausal osteoporosis: beyond bisphosphonates. J Endocr Invest 2015; 38: Cairoli E, Zhukouskaya V, Eller-Vainicher C, Chiodini I. Perspectives on osteoporosis therapies. J Endocr Invest 2015; 38: Leucht P, Helms JA. Wnt signaling: An emerging target for bone regeneration. J Am Acad Orthop Surg 2015; 23: Zhao R. Immune regulation of osteoclast function in postmenopausal osteoporosis: a critical interdisciplinary perspective. Int J Med Sci 2012; 9: Gennari L, Merlotti D, Nuti R. Selective estrogen receptor modulator (SERM) for the treatment of osteoporosis in postmenopausal women: focus on lasofoxifene. Clin Interv Aging 2010; 5: Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009; 24: Black LJ, Sato M, Rowley E, Magee D, Bekele A, Williams D, et al. Raloxifene (LY HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J Clin Invest 1994; 93: Ominsky MS, Vlasseros F, Jolette J, Smith SY, Stouch B, Doellgast G, et al. Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength. J Bone Miner Res 2010; 25: Folwarczna J, Kaczmarczyk-Sedlak I, Pytlik M, Nowinska B, Cegiela U, Sliwinski L, et al. Effect of low-dose tacrolimus coadministered with raloxifene on the skeletal system in male rats. Acta Poloniae Pharmaceutica 2009; 66: Cao ZT, Swift TA, West CA, Rosano TG, Rej R. Immunoassay of estradiol: unanticipated suppression by unconjugated estriol. Clin Chem 2004; 50: Azinge EC, Bolarin DM. Osteocalcin and bone-specific alkaline phosphatase in sickle cell haemoglobinopathies. Niger J Physiol Sci 2006; 21: Scariano JK, Garry PJ, Montoya GD, Chandani AK, Wilson JM, Baumgartner RN. Serum leptin levels, bone mineral density Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1):

5 Hayam Ibrahim Gad Allam and osteoblast alkaline phosphatase activity in elderly men and women. Mech Ageing Dev 2003; 124: Tschuor F, Zini E, Schellenberg S, Wenger M, Boretti FS, Reusch CE. Evaluation of four methods used to measure plasma insulin-like growth factor 1 concentrations in healthy cats and cats with diabetes mellitus or other diseases. Am J Vet Res 2012; 73: Findlay JW, Dillard RF. Appropriate calibration curve fitting in ligand binding assays. AAPS J 2007; 9:E Burtis CA, Ashwood ER, Bruns DE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 5th ed. St Louis: Elsevier Saunders; pp Lin C, Jiang X, Dai Z, Guo X, Weng T, Wang J, et al. Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/β-catenin signaling. J Bone Miner Res 2009; 24: Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J Bone Miner Res 2011; 26: Tasci A, Bilgili H, Altunay H, Gecit M, Keskin D. Prospective evaluation of Vitamin K2, Raloxifene and their coadministration in osteoporotic rats. Eur J Pharm Sci 2011; 43: Li C, Ominsky MS, Tan HL, Barrero M, Niu QT, Asuncion FJ, et al. Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene. Bone 2011; 49: Alaee F, Virk MS, Tang H, Sugiyama O, Adams DJ, Stolina M, et al. Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model. J Orthop Res 2014; 32: Tian X, Jee WS, Li X, Paszty C, Ke HZ. Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model. Bone 2011; 48: Sinder BP, Salemi JD, Ominsky MS, Caird MS, Marini JC, Kozloff KM. Rapidly growing Brtl/+ mouse model of osteogenesis imperfecta improves bone mass and strength with sclerostin antibody treatment. Bone 2015; 71: Bukata SV. Systemic administration of pharmacological agents and bone repair: what can we expect. Injury 2011; 42: Takeda S, Sakai S, Shiraishi A, Koike N, Mihara M, Endo K. Combination treatment with eldecalcitol (ED-71) and raloxifene improves bone mechanical strength by suppressing bone turnover and increasing bone mineral density in ovariectomized rats. Bone 2013; 53: Peano BJ, Crabtree JS, Komm BS, Winneker RC, Harris HA. Effects of various selective estrogen receptor modulators with or without conjugated estrogens on mouse mammary gland. Endocrinology 2009; 150: Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (1): 46-50