Abaloparatide-Zoledronate Combination Protects Against 4- Vinylcyclohexenediepoxide-induced Postmenopausal Osteoporosis in Mice: An Osteoanabolic-Antiresorptive Approach

Abaloparatide-Zoledronate Combination Protects Against 4- Vinylcyclohexenediepoxide-induced Postmenopausal Osteoporosis in Mice: An Osteoanabolic-Antiresorptive Approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Abaloparatide-Zoledronate Combination Protects Against 4- Vinylcyclohexenediepoxide-induced Postmenopausal Osteoporosis in Mice: An Osteoanabolic-Antiresorptive Approach Tabasum Ara, Zeenat Iqbal, Shreshta Jain, Aadil Ahmad Sheikh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7657337/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2026 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 10 You are reading this latest preprint version Abstract Introduction Abaloparatide, a 34-amino acid synthetic peptide analog of parathyroid hormone-related protein (PTHrP), was approved for treating postmenopausal osteoporosis in high-risk individuals or those resistant to existing drugs. This study aimed to investigate the impact of abaloparatide (20µg/kg/d; s.c.), an osteoanabolic drug with an antiresorptive drug zoledronate (125mg/kg i.v.) twice weekly for a month against a mouse model of 4-vinyl cyclohexene diepoxide (VCD)-induced post-menopausal osteoporosis. Materials and Methods Female Swiss albino mice were made ovotoxic by treatment with VCD (160mg/kg/d) for 15 days to mimic a postmenopausal state, confirmed by primordial follicle destruction in histopathological assessment. Microarchitectural analysis of distal femoral epiphysis and cortical mid-diaphysis was carried out using micro-computed tomography. Histopathological evaluation of bone, along with bone markers such as N-terminal propeptide of type 1 procollagen (P1NP) levels, C-terminal cross-linking telopeptide of type 1 collagen (CTX-1), soluble receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) were assessed. Results The VCD-treated mice exhibited bone loss as evidenced through micro CT and histopathology. Treatment with abaloparatide and zoledronate combination for 30 days reversed VCD-induced alterations of BV/TV, BMD,Tb.N and Tb.Sp. while the individual treatments were only partially effective. Serum analysis indicated reduced bone turnover in VCD-treated mice. The abaloparatide individually and in combination reversed the VCD-induced alterations in P1NP, CTX-1, and RANKL. The combination therapy also lowered the RANKL/OPG ratio. Conclusion These findings suggest that the combined approach of osteoanabolic and antiresorptive treatment may offer superior protection compared to individual therapies, holding promise for postmenopausal osteoporosis treatment. Abaloparatide Zoledronate Osteoporosis 4-Vinylcyclohexenediepoxide Osteoanabolic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoporosis poses a significant economic burden for healthcare systems due to its increasing prevalence (Rashki Kemmak et al. 2020 ). The disease is typically treated with anti-resorptive drugs, such as bisphosphonates, denosumab, estrogen, and selective estrogen receptor modulators such as raloxifene, etc. which increases the bone mineral density by reducing osteoclastic activity. However, these drugs cannot repair the compromised bone microarchitecture as they do not promote bone synthesis. A significant boost in bone mass and improvement in cortical and trabecular microarchitecture can be achieved by using osteoanabolic agents such as teriparatide, abaloparatide, and romosozumab (Tu et al. 2018 ). The amino bisphosphonate zoledronate, an intravenous, highly potent antiresorptive drug, is widely used for the treatment of osteoporosis or low bone mass in patients who have primary or secondary osteoporosis. It has a high affinity for mineralized bone, accumulating quickly and more readily localizing to high bone turnover sites. It is known for its potent inhibition of bone resorption. It works by interfering with the activity of osteoclasts and exhibits strong antiresorptive effects beneficial in excessive bone loss (Drake et al. 2008 ; Holstein 2019 ). Abaloparatide, a synthetic peptide analog of parathyroid hormone-related protein is an osteoanabolic agent approved in the USA in 2017 for the treatment of postmenopausal women at high risk of fracture with osteoporosis. It stimulates osteoblastic activity by binding to the parathyroid hormone receptor, resulting in increased bone formation and improved bone mineral density (Shirley 2017 ; Rachner et al. 2019 ). It has been reported that combining osteoanabolic agents with antiresorptive drugs may offer better improvement in bone mineral density and bone strength as compared to either agent alone (Cosman 2014 ; Leder 2018 ; Zhang and Song 2020 ). Accordingly, various preclinical (Li et al. 2012 ; Yang et al. 2013 ; Vegger et al. 2014 ) and clinical studies (Cosman et al. 2011 ; Walker et al. 2013 ) combining teriparatide with bisphosphonates have been found in the literature with beneficial effects. To the best of our knowledge, we could not find a combination study of abaloparatide with any of the bisphosphonates even though teriparatide has been studied with alendronate (Cosman et al. 1998 ; Johnston et al. 2007 ; Muschitz et al. 2013 ) risedronate (Walker et al. 2013 )ibandronate (Yang et al. 2013 ) and zoledronate (Vegger et al. 2014 ) with improvement in bone mass, trabecular bone volume, trabecular thickness, BMD and bone turnover markers. The combinations were superior to monotherapy in preserving bone strength in most cases. The 4-vinyl cyclohexene di epoxide (VCD) model mimics a postmenopausal osteoporosis state through selective action on ovarian follicles (Kappeler and Hoyer 2012 ). The present work investigated the impact of the combination of osteoanabolic abaloparatide with an antiresorptive zoledronate against VCD-induced postmenopausal osteoporosis in Swiss albino mice. Material and methods Experimental animals : Female Swiss albino mice aged between 10 and 12 weeks were acquired from the on-campus central animal house facility, Jamia Hamdard, New Delhi, after ethical approval by the Institutional Animal Ethics Committee under (Protocol no. 1846, year: 2022). The mice were housed in groups of 8 per cage, with the standard chow diet and water ad libitum, and were allowed to acclimatize for 7 days in the laboratory. The temperature and humidity were maintained at 25 ± 2°C and 55–60%, respectively, along with a 12 h light-dark cycle. The guidelines by the Committee for Control and Supervision of Experiments on Animals (CCSEA), India, have been followed (Jain et al. 2021 ). We followed ARRIVE guidelines for reporting the data. Drugs and doses: Drugs and doses : 4-vinyl cyclohexene di epoxide (VCD) (Sigma-Aldrich, India) was used at a dose of 160mg/kg/d (Brooks et al. 2016 ), Abaloparatide at 20µg/kg/d; Lifetein, LLC Hillsborough USA; Zoledronate at 125µg/kg/d (Zhu et al. 2019 ), Sigma-Aldrich, India as per previous literature. The dose of abaloparatide was translated from a human dose, 80 µg/kg, by calculating the animal equivalent dose (AED) based on body surface area, 20 µg/kg for mice (Miller et al. 2016 ). The formula used was AED (mg/kg) = Human dose (mg/kg) x Km, where the Km ratio for mice is 12.3. The sample size was calculated using G*power software (version 3.1.9.4 for Windows 10) and the values were obtained as: Effect size (d) = 1.74, α error probability = 0.05, power(1-β) = 0.95, allocation ratio (N2/N1) = 1, sample size estimate = 8, actual power = 0.9516546 as per the mean ± SD values of parameters like bone volume/trabecular volume (BV/TV) in control and VCD treated groups for VCD induced ovarian failure as reported in previous literature (Wright et al. 2008 ). VCD-induced ovotoxicity Swiss strain albino female mice were made menopausal by inducing ovotoxicity using vinyl cyclohexene di epoxide (VCD, 160mg/kg for 15 days followed by 30 days drug-free period) as confirmed by ovarian histopathology and as previously standardized in our lab (Pottoo et al. 2014 ; Kalam et al. 2016 ). Bone histology For histological evaluation, each femur from the experimental group was initially fixed in 4% formalin and subsequently subjected to decalcification using 10% ethylene diamine tetra acetic acid (EDTA) for 14 days. Following decalcification, the samples were processed through a series of dehydration and clearing steps, then embedded in paraffin to facilitate sectioning. Longitudinal sections approximately 5 µm thick were obtained using a microtome and mounted on glass slides. These sections were stained with hematoxylin and eosin (H&E) to visualize the trabecular bone architecture and assess the presence of various bone cells, including osteoblasts, osteoclasts, and osteocytes, across different treatment groups. Microscopic analysis was performed under a light microscope at magnification of 20x to evaluate histological changes (Nirwan and Vohora 2022 ). Treatment schedule : After induction of ovotoxicity, treatment commenced with abaloparatide at a dose of 20 µg/kg/day, administered subcutaneously (s.c) for 30 days, alongside zoledronate at a dose of 125 µg/kg, administered intravenously (i.v.) twice weekly (Fig. 1 ). Female Swiss albino mice were randomly divided into 7 groups, each containing 8 animals and the treatment was administered as: Group1 (control; Vehicle i.p), Group2 (4- VCD;160mg/kg i.p), Group 3 (VCD + Abaloparatide; 160mg/kg; i.p; 15 days + 20 µg/kg; s.c; 30 days), Group 4 (VCD + Zoledronate;160mg/kg; i.p;15 days + 125µg/kg/twice weekly i.v; 30 days), Group 5 (VCD + Abaloparatide + Zoledronate; 160mg/kg; i.p; 15 days + 20µg/kg s.c; 30 days + 125µg/kg/twice weekly i.v for 30 days;) Group 6 (Abaloparatide per se 20 µg/kg; s.c; 30 days) and Group 7 (Zoledronate per se 125µg/kg/twice weekly i.v; 30 days). Microarchitectural analysis of femur and cortical bone using micro-CT In our study, after euthanasia via CO₂ inhalation, the femurs were carefully excised, ensuring minimal damage to the bone structure. The bones were then cleaned of soft tissue and preserved in 10% neutral buffered formalin at 4°C until micro-CT analysis. Microcomputed tomography of the trabecular region was performed using an ex vivo micro-CT scanner (µCT 40, SCANCO Medical AG, Brüttisellen, Switzerland) at 50 kV of source voltage, 8W power, and current of 145µA with a 200ms integration time. Approximately 50 slices of 0.4 mm thickness were determined with high resolution at 8 µm voxel size with 1000 projections. Reconstruction was performed on the 50 slices, and the region of interest was the secondary spongiosa of the trabecular region (distal femur), eliminating the primary spongiosa. Ellipsoid contours were drawn using CT Analyzer (CTAn, Sky Scan) in the selected trabecular region of bone The bone mineral density (BMDmgHA/ccm), bone volume to tissue volume ratio (BV/TV), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, 1/mm), and trabecular separation (Tb. Sp, mm) were evaluated using the built-in software. A total of 50 slices at the femoral mid-diaphysis were scanned for quantification analysis using Scanco software to evaluate the cortical bone. A threshold of 275 was established by adjusting the threshold lines to avoid noise fluctuations in the central area of the display (vertically). The bone micro-architecture parameters measured included Total cross-sectional tissue area (Tt. Ar, mm²), Cortical Bone area (Ct. Ar, mm²), Cortical Thickness (Ct. Th, mm), and Bone mineral density (BMDmgHA/ccm) (Nirwan and Vohora 2022 ). The person performing the micro-CT data analysis was blinded to the treatment groups. Serum analysis : Blood was drawn from euthanized animals using cardiac puncture as a terminal procedure. Using a microcentrifuge at 4°C for 10 minutes, serum was separated and frosted at -80°C. Serum markers of bone formation and bone resorption were measured: soluble receptor activator of nuclear factor-kappa B ligand (sRANKL); osteoprotegerin (OPG); Mouse N- terminal propeptide of collagen (P1NP) and cross-linked c-telopeptide of type 1 collagen (CTX-1 using mouse-specific ELISA assay kits (Cat numbers: KTE70129; KTE70129; E2686Mo; E0184Mo respectively) from Abbkine and BT LAB (Arlt et al. 2020 ). Statistical analysis The data was analyzed using one-way ANOVA followed by Tukey Kramer’s multiple comparison tests. Data was represented as mean ± standard deviation. All statistical tests were performed using the Prism software package (version 8, GraphPad, San Diego, CA). Results Ovary Histopathology Ovaries of control animals showed a normal number of primordial follicles along with maturing follicles. Graafian follicle was also seen having oocyte, antrum, lined by many layers of granulosa cells and theca externa. Histopathological examination of VCD-treated ovaries depicted a significant reduction in the number of primordial follicles, retardation in oocyte growth, and also a reduction in ovarian size (Fig. 2 ). Bone histopathology H&E staining showed the regular pattern and normal thickness of control bone with properly differentiated cells indicating trabeculae (red arrow), osteocytes (green arrow), bone marrow (black arrow), osteoblasts (blue arrow) and osteoclasts (yellow arrow). However in VCD treated group bone marrow changes, thinner trabeculae, decreased osteoblast number, fewer osteocytes with empty lacunae were observed. Abaloparatide treatment has improved trabecular thickness, osteocyte and osteoblast number. Zoeldronate has shown minimal improvement, similar to the VCD- treated group. The combination has improved trabecular connectivity and osteocyte number. Perse groups resembled the control groups ( Fig. 3 ). Bone Microarchitecture (femur) Bone microarchitectural analysis by micro-CT indicated that the distal femoral epiphysis of the VCD-treated groups (160mg/kg; i.p.) had significantly decreased BV/TV (p < 0.001), Tb. N 1/mm (P < 0.05), Tb. Th mm (p < 0.01), and BMD (mgHA/ccm) (p < 0.001) compared to the control group. Tb.Sp mm was also significantly increased due to VCD-induced ovarian toxicity (p < 0.001). Treatment with abaloparatide (20µg/kg; s.c.) for 30 days in VCD-treated mice resulted in a significant reversal of reduced BV/TV (p < 0.05). Treatment with zoledronate (125mg/kg i.v) twice weekly for 30 days, however, could not reverse the VCD-induced alterations. However, when combined with abaloparatide, there was a significant reversal of VCD-induced reduction in BV/TV (p < 0.001), Tb. N 1/mm and BMD mgHA/ccm (p < 0.01). Tb. Sp (mm) was also decreased by this combination compared to the VCD-treated groups (p < 0.01) (Figs. 4 and 5 ). Evaluation of the cortical bone microarchitecture at the femoral mid-diaphysis was also done. Parameters such as Total cross-sectional tissue area (Tt. Ar), Cortical Bone area (Ct. Ar), Cortical Thickness (Ct.Th), and bone mineral density (BMD mgHA/ccm) were analyzed. However, no statistically significant differences were observed among the groups. These findings, along with representative 3D images, are provided in the Supplementary File as Figure S1 and Figure S2. Bone Turnover Markers The serum levels of bone turnover markers RANKL (pg/ml) and CTX-1 (ng/ml) were increased, (p < 0.01); while the serum levels of OPG (ng/ml) (p < 0.01) and P1NP (pg/ml) (p < 0.001) were decreased in VCD-treated mice (160mg/kg; i.p). Abaloparatide treatment (20µg/kg s.c) for 30 days resulted in a reversal in increased RANKL (p < 0.05) and CTX-1 (P < 0.01) levels and reduced P1NP in serum compared to VCD-treated groups. Interestingly, the combination of abaloparatide and zoledronate produced a better effect in decreasing RANKL (p < 0.01), CTX-1 and RANKL/OPG levels (p < 0.001) and increasing PINP and OPG levels (Fig. 6 ). Discussion Recently, a growing interest in combining anabolic and antiresorptive agents to improve bone density, microarchitecture, and bone strength has been reported (Cosman 2014 ; Zhang and Song 2020 ). Pairing a bone-forming agent with one that inhibits resorption holds significant promise for both maintaining and augmenting bone mass (Garcés and García 2006 ). Simultaneous combination therapy involving an antiresorptive with parathyroid hormone (PTH) is considered appealing due to the potential synergistic benefits derived from their distinct mechanisms of action. By concurrently inhibiting bone resorption (antiresorptive) and stimulating bone formation (osteoanabolic), the combined therapeutic approach may yield superior outcomes compared to using either agent in isolation (Bilezikian 2008 ). The same has been reported for teriparatide in combination with bisphosphonates (Li et al. 2012 ; Yang et al. 2013 ; Vegger et al. 2014 ; Cosman et al. 2011 ; Walker et al. 2013 ). No combination studies using abaloparatide with zoledronate were reported previously and hence in this work, we combined bone anabolic (abaloparatide) and antiresorptive (zoledronate) for treating VCD-induced post-menopausal osteoporosis. The advantage of taking abaloparatide instead of teriparatide is that this drug has potent bone osteoanabolic activity and is reported to reduce both vertebral and nonvertebral fracture rates along with improved stability, safety, and tolerance. Additionally, the calcium mobilizing capacity of this drug is lower than that of teriparatide, thus causing lesser hypercalcemia (Tella et al. 2017 ; Bhattacharyya et al. 2019 ). Our findings showed that the combination afforded better protection than either treatment alone as observed through bone microarchitecture and turnover markers in mice. We observed an improved trabecular bone micro-architecture in VCD-induced ovotoxic mice using the abaloparatide-zoledronate combination. The VCD model used in our study has an advantage over ovariectomy because it induces ovarian failure gradually, and yet retains residual ovarian tissue which may more closely mimic the natural progression of ovarian decline observed in post-menopausal women and allows for a more distinct study of the effects on bone health (Wright et al. 2008 ; Mashouri et al. 2024 ). Abaloparatide treatment resulted in a significant reversal of VCD-induced alterations of bone microarchitecture. Our result is supported by the previous study where the abaloparatide, at the dose of 30ug/kg/day for 28 days has shown similar results in C57BL6/J mice where it enhanced bone volume (Makino et al. 2021 ). We could not, however, observe any significant effects with zoledronate (125ug/kg twice a week) (i.v) given twice weekly for a month. Previous studies have shown variable results with zoledronate depending on the strain of mice, frequency of administration, dosage, and duration of the experiment. Longer duration of treatment has shown more favorable results (Shuai et al. 2015 ; Aref et al. 2016 ; Reid et al. 2020 ) which could be one of the reasons for zoledronate not showing any meaningful effects in the present work. However, when combined with abaloparatide, it demonstrated a significant reversal of VCD-induced alterations. It has been reported that the timing of administering an anabolic agent (pre-, mid, or post-antiresorptive treatment) affects therapeutic outcomes (Leder 2018 ) Hence, teriparatide administered post bisphosphonate resulted in a blunted BMD response (Boonen et al. 2008 ; Eastell et al. 2009 ). However, when teriparatide was administered pre-bisphosphonate, it resulted in an increasing BMD response (Rittmaster et al. 2000 ; Kurland et al. 2004 ; McClung 2017 ). Interestingly, there are also studies where the anabolic agent was co-administered with an antiresorptive agent with beneficial effects(Cosman et al. 2011 ; Muschitz et al. 2013 ). In our study, we co-administered abaloparatide daily with zoledronate twice weekly for a month and observed improvement in BMD with no blunting of response. Our results are in agreement with a previous study where a combination of teriparatide daily administration with zoledronate single infusion increased lumbar spine and hip femoral BMD more than teriparatide (Cosman et al. 2011 ). Other studies have shown improvement using sequential therapy – teriparatide treatment followed by zoledronate (Shimizu et al. 2017 ). CTX-1 is a bone resorption marker released during the breakdown of type 1 collagen. Elevated levels in the blood indicate increased bone resorption. RANKL is an important cytokine produced primarily by osteoblast lineage cells. When RANKL binds to its receptor (RANK) on osteoclast progenitor cells, it stimulates osteoclast formation and activation. This leads to enhanced bone resorption. OPG is the decoy receptor for RANKL. It acts as a "decoy" by binding to RANKL and preventing it from interacting with its receptor (RANK). This binding inhibits osteoclast formation and activation, and it promotes apoptosis of osteoclasts, ultimately reducing bone resorption (Kuo and Chen 2017 ). Abaloparatide individually as well as in combination with zoledronate has shown decreased levels of RANKL and CTX-1 in VCD-treated mice. This is supported by a previous study where abaloparatide at the dose of 40ug/kg for 12 consecutive days in C57Bl6/J mice significantly lowered the serum CTX-1 levels compared to vehicle (Arlt et al. 2020 ). P1NP is a bone formation marker, produced by the synthesis of type 1 collagen. Elevated levels in blood are indicative of increased bone formation. We observed increased P1NP levels in the abaloparatide group as well as in the combination group compared to the VCD-treated groups. This is as per a previous study in C57Bl6/J mice receiving 30ug/kg/day for 28 days where the abaloparatide significantly enhanced PINP serum levels (Makino et al. 2021 ). One more study in wild-type (WT) female mice receiving abaloparatide (20-80ug/kg/d) for 30 days has shown a prominent increase in P1NP (Sahbani et al. 2019 ). The effect on OPG was not significant when abaloparatide or zoledronate were administered individually, however, in combination, an increase in OPG was observed. A previous study in wild-type (WT) female mice receiving abaloparatide (20–80 ug/kg/d) for 30 days indicated a lack of significant impact on OPG concentration (Sahbani et al. 2019 ). We then looked at the RANKL OPG ratio. The balance between RANKL and OPG, expressed as the RANKL/OPG ratio, plays a crucial role in controlling bone resorption. A higher ratio is associated with increased bone resorption, while a lower ratio indicates a shift toward reduced bone resorption and possibly increased bone formation (Sahbani et al. 2019 ). In our study, the combination of abaloparatide and zoledronate lowered the VCD-induced increase in the ratio of RANKL/OPG. Sahbani et al. 2019 reported in their study that abaloparatide (80ug/kg/d s.c.) for 30 days in C57Bl/6J mice, has lowered the RANKL/OPG levels (Sahbani et al. 2019 ) ( Fig. 7 ). To the best of our knowledge, ours is the first study reporting the combined effect of abaloparatide with bisphosphonate in improving osteoporosis. However, our study has limitations. Only one animal model was employed to study the combination. Future investigations with abaloparatide may explore combined therapies involving other animal models such as on glucocorticoids-induced osteoporosis and with other anti-resorptive treatments to unveil potential benefits. However, any possible benefit needs to be weighed in terms of cost and side effects with polytherapy, and any possible long-term effects for which evidence is currently limited for the osteoanabolic-antiresorptive combinations, requiring further investigations. Abbreviations abl Abaloparatide AED Animal equivalent dose BMD Bone Mineral Density BV/TV bone volume to tissue volume CCSEA Committee for control and supervision of experiments on animals CTX-1 C-terminal cross-linking telopeptide of type 1 collagen IAEC Institutional Animal Ethics Committee MICRO-CT Microcomputed tomography OPG Osteoprotegerin PINP N-terminal propeptide of type 1 procollagen PTHrP Parathyroid hormone related protein RANKL Receptor activator of nuclear factor-kappa B ligand Tb. N trabecular number Tb. Sp trabecular separation Tb. Th trabecular thickness VCD 4-vinylcyclohexene diepoxide zol Zoledronate Declarations Funding The work was carried out in the University Grants Commission Special Assistance Program (UGC SAP) and DST-FIST-supported Neurobehavioral Pharmacology laboratory. We sincerely acknowledge the micro-computed tomography facility provided under the UGC SAP DRS-II grant to DV. The financial support by DST-PURSE in providing fellowship to TA is gratefully acknowledged. Conflict of interest All authors declare that they have no conflicts of interest. Author’s contribution Tabasum Ara : Conceptualization, Methodology, Visualization, Formal analysis, Investigation, Writing – original draft. Divya Vohora : Conceptualization, Writing – original draft, Writing - Review and Editing, Supervision, Visualization, Formal Analysis, Funding acquisition, Resources, Project administration. Zeenat Iqbal : Review, Supervision. Shreshta Jain: Investigation, Aadil Ahmad Sheikh: Investigation. The authors declare that all data were generated in-house, that no paper mill was used and that no AI tool has been used for the generation of text or figures. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Supplementary data associated with this article can be found in the online version. References Aref, M. W., E. M. McNerny, D. Brown, K. J. Jepsen, and M. R. Allen. 2016. 'Zoledronate treatment has different effects in mouse strains with contrasting baseline bone mechanical phenotypes', Osteoporos Int , 27: 3637-43. Arlt, H., T. Mullarkey, D. Hu, R. Baron, M. S. Ominsky, B. Mitlak, B. Lanske, and T. Besschetnova. 2020. 'Effects of abaloparatide and teriparatide on bone resorption and bone formation in female mice', Bone Rep , 13: 100291. Bhattacharyya, S., S. Pal, and N. Chattopadhyay. 2019. 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09:21:52","extension":"xml","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111558,"visible":true,"origin":"","legend":"","description":"","filename":"362bccb44c0d4fff87f961a5839b4ecc1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/7b6700336ec1b5f280b0849a.xml"},{"id":94823395,"identity":"b1fe2b54-af34-4a70-a074-aa33db80b123","added_by":"auto","created_at":"2025-10-31 06:47:17","extension":"html","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123521,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/82cd8a7f86e0d06db576f095.html"},{"id":94744112,"identity":"b1611aa4-3846-4969-a1c3-075a1e6b53b9","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105506,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic representation of the experimental design of the study. VCD= 4-vinyl cyclohexene diepoxide; abl=abaloparatide; zol=zoledronate.\u003c/p\u003e","description":"","filename":"FIGURE1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/30e797ec28a761da809ef89e.jpg"},{"id":94744113,"identity":"101bab75-9672-41dd-8981-08c950b1416f","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":689249,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian histopathology of control (normal saline) and 4-vinyl cyclohexene diepoxide (VCD) treated female Swiss albino mice: \u003cstrong\u003e(A)\u003c/strong\u003e Section shows normal primordial (\u003cstrong\u003ePrF\u003c/strong\u003e), primary follicles (\u003cstrong\u003ePF\u003c/strong\u003e), primary oocyte (\u003cstrong\u003eOO\u003c/strong\u003e), many layers of granulosa cells (GC), theca externa (\u003cstrong\u003eTE) \u003c/strong\u003eand antrum \u003cstrong\u003e(AN\u003c/strong\u003e)) \u003cstrong\u003e(B)\u003c/strong\u003e Secondary follicles (\u003cstrong\u003eSF\u003c/strong\u003e). Section \u003cstrong\u003eC\u003c/strong\u003e\u0026amp;\u003cstrong\u003eD\u003c/strong\u003etreated with VCD (160mg/kg/d i.p.) over 15 days and a 30-day drug-free period showed a reduction in the number of primordial follicles, follicle loss, and reduction in ovary size (400x; H\u0026amp;E; scale bar 100µm).\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/a91004dbe694e3105e45ec22.jpg"},{"id":94744116,"identity":"ccf0e8d4-582b-4e43-85dc-5f55ac9afabf","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":492128,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of abaloparatide, zoledronate, and their combination on bone histopathology. Representative images of the H\u0026amp;E staining of the trabecular region (distal femoral epiphysis) of the femur bone (20x; H\u0026amp;E; scale bar 100µm).\u003c/p\u003e","description":"","filename":"FIGURE3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/be05fe5abc216641ff043e19.jpg"},{"id":94744115,"identity":"d45b171e-c0e2-4262-8561-06c4fbfb698c","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":108460,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of abaloparatide, zoledronate, and their combination on VCD-induced alterations of bone microarchitecture using microcomputed tomography analysis. Trabecular bone volume (BV/TV, ), trabecular number (Tb. N, 1/mm), trabecular thickness (Tb. Th, mm), trabecular separation (Tb. Sp, mm), bone mineral density (BMD mgHA/ccm) was measured in the distal femoral epiphysis. One-way ANOVA and Tukey’s multiple comparison test were used for analysis and values are expressed as mean±SD (n=8). The significance is determined as \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001,\u003csup\u003e **\u003c/sup\u003ep\u0026lt;0.01,\u003csup\u003e *\u003c/sup\u003ep\u0026lt;0.05 vs Control,\u003csup\u003e ###\u003c/sup\u003ep\u0026lt;0.001,\u003csup\u003e ##\u003c/sup\u003ep\u0026lt;0.01 vs VCD. vcd=4-vinylcyclohexenediepoxide; abl=abaloparatide; zol=zoledronate.\u003c/p\u003e","description":"","filename":"FIGURE4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/a5d7695fde44e2354e82b308.jpg"},{"id":94744117,"identity":"255ce5bb-072b-4005-beb5-d6db99a7a90f","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201920,"visible":true,"origin":"","legend":"\u003cp\u003eThe three-dimensional images of the mouse distal femoral epiphysis's trabecular region are shown in the figure. (A) control (B) vcd (C) vcd+abl (D) vcd+zol (E) vcd+abl+zol (F) abl per se (G) zol per se.\u003c/p\u003e","description":"","filename":"FIGURE5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/13ddce0cf5190e03ed4bb478.jpg"},{"id":94744122,"identity":"669bb530-ce7a-486f-8daa-7cec59567730","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267627,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of abaloparatide, zoledronate, and their combination on VCD-induced alterations in bone turnover markers in mouse serum. (A) RANKL (pg/ml); (B) OPG (ng/ml); (C) CTX-1 (ng/ml); (D) P1NP (pg/ml) and (E) RANKL/OPG. Analyzing the data by one-way ANOVA followed by Tukey’s multiple comparison test, values are expressed as mean±SD (n=8). The significance is determined as \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001,\u003csup\u003e **\u003c/sup\u003ep\u0026lt;0.01, vs Control,\u003csup\u003e ###\u003c/sup\u003e p\u0026lt;0.001,\u003csup\u003e ##\u003c/sup\u003e p\u0026lt;0.01,\u003csup\u003e #\u003c/sup\u003e p\u0026lt;0.05 vs VCD. vcd=4 vinyl cyclohexene diepoxide; abl=abaloparatide; zol=zoledronate.\u003c/p\u003e","description":"","filename":"FIGURE6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/1f49613ffd31f32a3668fb73.jpg"},{"id":94744123,"identity":"c0521d30-fdb6-4659-a3a4-0aa641d4e946","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":224543,"visible":true,"origin":"","legend":"\u003cp\u003eThe diagrammatic representation of mechanistic pathways of abaloparatide and zoledronate and the effect of their combination on bone turnover markers. ABL= Abaloparatide, PTH1R=Parathyroid Hormone Receptor, GPCR= G-Protein Coupled Receptor; AC= Adenylyl Cyclase, CAMP= Cyclic Adenosine Monophosphate; PKA= Protein Kinase A; FPPS=farnesyl pyrophosphate synthase RANKL=Receptor Activator of NF-kB ligand; P1NP=procollagen Type 1N-terminal propeptide; OPG=Osteoprotegerin; CTX-1= C- terminal telopeptide.\u003c/p\u003e","description":"","filename":"FIGURE7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/07117295735a5f463df3124b.jpg"},{"id":101152748,"identity":"d159aa04-3b64-4301-ac40-79d3a83b7d23","added_by":"auto","created_at":"2026-01-26 16:12:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2821867,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/f2c13673-6617-47aa-9981-2cc6a63fb3a0.pdf"},{"id":94823018,"identity":"e5668a88-1ca9-4e2c-ac54-6c4122036a35","added_by":"auto","created_at":"2025-10-31 06:45:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128767,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/e7f2540772aad13aeb20ac0c.docx"},{"id":94744120,"identity":"b171e935-0488-4195-870f-9ebf9967f918","added_by":"auto","created_at":"2025-10-30 09:21:51","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":467490,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7657337/v1/1d9658fe119b193b5977e2a9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Abaloparatide-Zoledronate Combination Protects Against 4- Vinylcyclohexenediepoxide-induced Postmenopausal Osteoporosis in Mice: An Osteoanabolic-Antiresorptive Approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis poses a significant economic burden for healthcare systems due to its increasing prevalence (Rashki Kemmak et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The disease is typically treated with anti-resorptive drugs, such as bisphosphonates, denosumab, estrogen, and selective estrogen receptor modulators such as raloxifene, etc. which increases the bone mineral density by reducing osteoclastic activity. However, these drugs cannot repair the compromised bone microarchitecture as they do not promote bone synthesis. A significant boost in bone mass and improvement in cortical and trabecular microarchitecture can be achieved by using osteoanabolic agents such as teriparatide, abaloparatide, and romosozumab (Tu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The amino bisphosphonate zoledronate, an intravenous, highly potent antiresorptive drug, is widely used for the treatment of osteoporosis or low bone mass in patients who have primary or secondary osteoporosis. It has a high affinity for mineralized bone, accumulating quickly and more readily localizing to high bone turnover sites. It is known for its potent inhibition of bone resorption. It works by interfering with the activity of osteoclasts and exhibits strong antiresorptive effects beneficial in excessive bone loss (Drake et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Holstein \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e Abaloparatide, a synthetic peptide analog of parathyroid hormone-related protein is an osteoanabolic agent approved in the USA in 2017 for the treatment of postmenopausal women at high risk of fracture with osteoporosis. It stimulates osteoblastic activity by binding to the parathyroid hormone receptor, resulting in increased bone formation and improved bone mineral density (Shirley \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rachner et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that combining osteoanabolic agents with antiresorptive drugs may offer better improvement in bone mineral density and bone strength as compared to either agent alone (Cosman \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Leder \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang and Song \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Accordingly, various preclinical (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vegger et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and clinical studies (Cosman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Walker et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) combining teriparatide with bisphosphonates have been found in the literature with beneficial effects. To the best of our knowledge, we could not find a combination study of abaloparatide with any of the bisphosphonates even though teriparatide has been studied with alendronate (Cosman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Johnston et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Muschitz et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) risedronate (Walker et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)ibandronate (Yang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and zoledronate (Vegger et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with improvement in bone mass, trabecular bone volume, trabecular thickness, BMD and bone turnover markers. The combinations were superior to monotherapy in preserving bone strength in most cases. The 4-vinyl cyclohexene di epoxide (VCD) model mimics a postmenopausal osteoporosis state through selective action on ovarian follicles (Kappeler and Hoyer \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The present work investigated the impact of the combination of osteoanabolic abaloparatide with an antiresorptive zoledronate against VCD-induced postmenopausal osteoporosis in Swiss albino mice.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eExperimental animals\u003c/b\u003e: Female Swiss albino mice aged between 10 and 12 weeks were acquired from the on-campus central animal house facility, Jamia Hamdard, New Delhi, after ethical approval by the Institutional Animal Ethics Committee under (Protocol no. 1846, year: 2022). The mice were housed in groups of 8 per cage, with the standard chow diet and water ad libitum, and were allowed to acclimatize for 7 days in the laboratory. The temperature and humidity were maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 55\u0026ndash;60%, respectively, along with a 12 h light-dark cycle. The guidelines by the Committee for Control and Supervision of Experiments on Animals (CCSEA), India, have been followed (Jain et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We followed ARRIVE guidelines for reporting the data.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDrugs and doses: Drugs and doses\u003c/b\u003e: 4-vinyl cyclohexene di epoxide (VCD) (Sigma-Aldrich, India) was used at a dose of 160mg/kg/d (Brooks et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Abaloparatide at 20\u0026micro;g/kg/d; Lifetein, LLC Hillsborough USA; Zoledronate at 125\u0026micro;g/kg/d (Zhu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Sigma-Aldrich, India as per previous literature. The dose of abaloparatide was translated from a human dose, 80 \u0026micro;g/kg, by calculating the animal equivalent dose (AED) based on body surface area, 20 \u0026micro;g/kg for mice (Miller et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The formula used was AED (mg/kg)\u0026thinsp;=\u0026thinsp;Human dose (mg/kg) x Km, where the Km ratio for mice is 12.3. The sample size was calculated using G*power software (version 3.1.9.4 for Windows 10) and the values were obtained as: Effect size (d)\u0026thinsp;=\u0026thinsp;1.74, α error probability\u0026thinsp;=\u0026thinsp;0.05, power(1-β)\u0026thinsp;=\u0026thinsp;0.95, allocation ratio (N2/N1)\u0026thinsp;=\u0026thinsp;1, sample size estimate\u0026thinsp;=\u0026thinsp;8, actual power\u0026thinsp;=\u0026thinsp;0.9516546 as per the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values of parameters like bone volume/trabecular volume (BV/TV) in control and VCD treated groups for VCD induced ovarian failure as reported in previous literature (Wright et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eVCD-induced ovotoxicity\u003c/strong\u003e\u003cp\u003eSwiss strain albino female mice were made menopausal by inducing ovotoxicity using vinyl cyclohexene di epoxide (VCD, 160mg/kg for 15 days followed by 30 days drug-free period) as confirmed by ovarian histopathology and as previously standardized in our lab (Pottoo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kalam et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBone histology\u003c/strong\u003e\u003cp\u003eFor histological evaluation, each femur from the experimental group was initially fixed in 4% formalin and subsequently subjected to decalcification using 10% ethylene diamine tetra acetic acid (EDTA) for 14 days. Following decalcification, the samples were processed through a series of dehydration and clearing steps, then embedded in paraffin to facilitate sectioning. Longitudinal sections approximately 5 \u0026micro;m thick were obtained using a microtome and mounted on glass slides. These sections were stained with hematoxylin and eosin (H\u0026amp;E) to visualize the trabecular bone architecture and assess the presence of various bone cells, including osteoblasts, osteoclasts, and osteocytes, across different treatment groups. Microscopic analysis was performed under a light microscope at magnification of 20x to evaluate histological changes (Nirwan and Vohora \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment schedule\u003c/b\u003e: After induction of ovotoxicity, treatment commenced with abaloparatide at a dose of 20 \u0026micro;g/kg/day, administered subcutaneously (s.c) for 30 days, alongside zoledronate at a dose of 125 \u0026micro;g/kg, administered intravenously (i.v.) twice weekly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Female Swiss albino mice were randomly divided into 7 groups, each containing 8 animals and the treatment was administered as: Group1 (control; Vehicle i.p), Group2 (4- VCD;160mg/kg i.p), Group 3 (VCD\u0026thinsp;+\u0026thinsp;Abaloparatide; 160mg/kg; i.p; 15 days\u0026thinsp;\u003cb\u003e+\u003c/b\u003e\u0026thinsp;20 \u0026micro;g/kg; s.c; 30 days), Group 4 (VCD\u0026thinsp;+\u0026thinsp;Zoledronate;160mg/kg; i.p;15 days\u0026thinsp;\u003cb\u003e+\u003c/b\u003e\u0026thinsp;125\u0026micro;g/kg/twice weekly i.v; 30 days), Group 5 (VCD\u0026thinsp;+\u0026thinsp;Abaloparatide\u0026thinsp;+\u0026thinsp;Zoledronate; 160mg/kg; i.p; 15 days\u0026thinsp;\u003cb\u003e+\u003c/b\u003e\u0026thinsp;20\u0026micro;g/kg s.c; 30 days\u0026thinsp;+\u0026thinsp;125\u0026micro;g/kg/twice weekly i.v for 30 days;) Group 6 (Abaloparatide per se 20 \u0026micro;g/kg; s.c; 30 days) and Group 7 (Zoledronate per se 125\u0026micro;g/kg/twice weekly i.v; 30 days).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMicroarchitectural analysis of femur and cortical bone using micro-CT\u003c/strong\u003e\u003cp\u003eIn our study, after euthanasia via CO₂ inhalation, the femurs were carefully excised, ensuring minimal damage to the bone structure. The bones were then cleaned of soft tissue and preserved in 10% neutral buffered formalin at 4\u0026deg;C until micro-CT analysis. Microcomputed tomography of the trabecular region was performed using an ex vivo micro-CT scanner (\u0026micro;CT 40, SCANCO Medical AG, Br\u0026uuml;ttisellen, Switzerland) at 50 kV of source voltage, 8W power, and current of 145\u0026micro;A with a 200ms integration time. Approximately 50 slices of 0.4 mm thickness were determined with high resolution at 8 \u0026micro;m voxel size with 1000 projections. Reconstruction was performed on the 50 slices, and the region of interest was the secondary spongiosa of the trabecular region (distal femur), eliminating the primary spongiosa. Ellipsoid contours were drawn using CT Analyzer (CTAn, Sky Scan) in the selected trabecular region of bone The bone mineral density (BMDmgHA/ccm), bone volume to tissue volume ratio (BV/TV), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, 1/mm), and trabecular separation (Tb. Sp, mm) were evaluated using the built-in software. A total of 50 slices at the femoral mid-diaphysis were scanned for quantification analysis using Scanco software to evaluate the cortical bone. A threshold of 275 was established by adjusting the threshold lines to avoid noise fluctuations in the central area of the display (vertically). The bone micro-architecture parameters measured included Total cross-sectional tissue area (Tt. Ar, mm\u0026sup2;), Cortical Bone area (Ct. Ar, mm\u0026sup2;), Cortical Thickness (Ct. Th, mm), and Bone mineral density (BMDmgHA/ccm) (Nirwan and Vohora \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The person performing the micro-CT data analysis was blinded to the treatment groups.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSerum analysis\u003c/b\u003e: Blood was drawn from euthanized animals using cardiac puncture as a terminal procedure. Using a microcentrifuge at 4\u0026deg;C for 10 minutes, serum was separated and frosted at -80\u0026deg;C. Serum markers of bone formation and bone resorption were measured: soluble receptor activator of nuclear factor-kappa B ligand (sRANKL); osteoprotegerin (OPG); Mouse N- terminal propeptide of collagen (P1NP) and cross-linked c-telopeptide of type 1 collagen (CTX-1 using mouse-specific ELISA assay kits (Cat numbers: KTE70129; KTE70129; E2686Mo; E0184Mo respectively) from Abbkine and BT LAB (Arlt et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eThe data was analyzed using one-way ANOVA followed by Tukey Kramer\u0026rsquo;s multiple comparison tests. Data was represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. All statistical tests were performed using the Prism software package (version 8, GraphPad, San Diego, CA).\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOvary Histopathology\u003c/strong\u003e\u003cp\u003eOvaries of control animals showed a normal number of primordial follicles along with maturing follicles. Graafian follicle was also seen having oocyte, antrum, lined by many layers of granulosa cells and theca externa. Histopathological examination of VCD-treated ovaries depicted a significant reduction in the number of primordial follicles, retardation in oocyte growth, and also a reduction in ovarian size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBone histopathology\u003c/strong\u003e\u003cp\u003eH\u0026amp;E staining showed the regular pattern and normal thickness of control bone with properly differentiated cells indicating trabeculae (red arrow), osteocytes (green arrow), bone marrow (black arrow), osteoblasts (blue arrow) and osteoclasts (yellow arrow). However in VCD treated group bone marrow changes, thinner trabeculae, decreased osteoblast number, fewer osteocytes with empty lacunae were observed. Abaloparatide treatment has improved trabecular thickness, osteocyte and osteoblast number. Zoeldronate has shown minimal improvement, similar to the VCD- treated group. The combination has improved trabecular connectivity and osteocyte number. Perse groups resembled the control groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003c/p\u003e\n\u003ch3\u003eBone Microarchitecture (femur)\u003c/h3\u003e\n\u003cp\u003eBone microarchitectural analysis by micro-CT indicated that the distal femoral epiphysis of the VCD-treated groups (160mg/kg; i.p.) had significantly decreased BV/TV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Tb. N 1/mm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tb. Th mm (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and BMD (mgHA/ccm) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the control group. Tb.Sp mm was also significantly increased due to VCD-induced ovarian toxicity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with abaloparatide (20\u0026micro;g/kg; s.c.) for 30 days in VCD-treated mice resulted in a significant reversal of reduced BV/TV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Treatment with zoledronate (125mg/kg i.v) twice weekly for 30 days, however, could not reverse the VCD-induced alterations. However, when combined with abaloparatide, there was a significant reversal of VCD-induced reduction in BV/TV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), Tb. N 1/mm and BMD mgHA/ccm (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Tb. Sp (mm) was also decreased by this combination compared to the VCD-treated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Evaluation of the cortical bone microarchitecture at the femoral mid-diaphysis was also done. Parameters such as Total cross-sectional tissue area (Tt. Ar), Cortical Bone area (Ct. Ar), Cortical Thickness (Ct.Th), and bone mineral density (BMD mgHA/ccm) were analyzed. However, no statistically significant differences were observed among the groups. These findings, along with representative 3D images, are provided in the Supplementary File as Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Figure S2.\u003c/p\u003e\n\u003ch3\u003eBone Turnover Markers\u003c/h3\u003e\n\u003cp\u003eThe serum levels of bone turnover markers RANKL (pg/ml) and CTX-1 (ng/ml) were increased, (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); while the serum levels of OPG (ng/ml) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and P1NP (pg/ml) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were decreased in VCD-treated mice (160mg/kg; i.p). Abaloparatide treatment (20\u0026micro;g/kg s.c) for 30 days resulted in a reversal in increased RANKL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and CTX-1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) levels and reduced P1NP in serum compared to VCD-treated groups. Interestingly, the combination of abaloparatide and zoledronate produced a better effect in decreasing RANKL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), CTX-1 and RANKL/OPG levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and increasing PINP and OPG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRecently, a growing interest in combining anabolic and antiresorptive agents to improve bone density, microarchitecture, and bone strength has been reported (Cosman \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang and Song \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Pairing a bone-forming agent with one that inhibits resorption holds significant promise for both maintaining and augmenting bone mass (Garc\u0026eacute;s and Garc\u0026iacute;a \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Simultaneous combination therapy involving an antiresorptive with parathyroid hormone (PTH) is considered appealing due to the potential synergistic benefits derived from their distinct mechanisms of action. By concurrently inhibiting bone resorption (antiresorptive) and stimulating bone formation (osteoanabolic), the combined therapeutic approach may yield superior outcomes compared to using either agent in isolation (Bilezikian \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The same has been reported for teriparatide in combination with bisphosphonates (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vegger et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cosman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Walker et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). No combination studies using abaloparatide with zoledronate were reported previously and hence in this work, we combined bone anabolic (abaloparatide) and antiresorptive (zoledronate) for treating VCD-induced post-menopausal osteoporosis. The advantage of taking abaloparatide instead of teriparatide is that this drug has potent bone osteoanabolic activity and is reported to reduce both vertebral and nonvertebral fracture rates along with improved stability, safety, and tolerance. Additionally, the calcium mobilizing capacity of this drug is lower than that of teriparatide, thus causing lesser hypercalcemia (Tella et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bhattacharyya et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our findings showed that the combination afforded better protection than either treatment alone as observed through bone microarchitecture and turnover markers in mice.\u003c/p\u003e\u003cp\u003eWe observed an improved trabecular bone micro-architecture in VCD-induced ovotoxic mice using the abaloparatide-zoledronate combination. The VCD model used in our study has an advantage over ovariectomy because it induces ovarian failure gradually, and yet retains residual ovarian tissue which may more closely mimic the natural progression of ovarian decline observed in post-menopausal women and allows for a more distinct study of the effects on bone health (Wright et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mashouri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Abaloparatide treatment resulted in a significant reversal of VCD-induced alterations of bone microarchitecture. Our result is supported by the previous study where the abaloparatide, at the dose of 30ug/kg/day for 28 days has shown similar results in C57BL6/J mice where it enhanced bone volume (Makino et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We could not, however, observe any significant effects with zoledronate (125ug/kg twice a week) (i.v) given twice weekly for a month. Previous studies have shown variable results with zoledronate depending on the strain of mice, frequency of administration, dosage, and duration of the experiment. Longer duration of treatment has shown more favorable results (Shuai et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Aref et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Reid et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) which could be one of the reasons for zoledronate not showing any meaningful effects in the present work. However, when combined with abaloparatide, it demonstrated a significant reversal of VCD-induced alterations.\u003c/p\u003e\u003cp\u003eIt has been reported that the timing of administering an anabolic agent (pre-, mid, or post-antiresorptive treatment) affects therapeutic outcomes (Leder \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Hence, teriparatide administered post bisphosphonate resulted in a blunted BMD response (Boonen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Eastell et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, when teriparatide was administered pre-bisphosphonate, it resulted in an increasing BMD response (Rittmaster et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kurland et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; McClung \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, there are also studies where the anabolic agent was co-administered with an antiresorptive agent with beneficial effects(Cosman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Muschitz et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In our study, we co-administered abaloparatide daily with zoledronate twice weekly for a month and observed improvement in BMD with no blunting of response. Our results are in agreement with a previous study where a combination of teriparatide daily administration with zoledronate single infusion increased lumbar spine and hip femoral BMD more than teriparatide (Cosman et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Other studies have shown improvement using sequential therapy \u0026ndash; teriparatide treatment followed by zoledronate (Shimizu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCTX-1 is a bone resorption marker released during the breakdown of type 1 collagen. Elevated levels in the blood indicate increased bone resorption. RANKL is an important cytokine produced primarily by osteoblast lineage cells. When RANKL binds to its receptor (RANK) on osteoclast progenitor cells, it stimulates osteoclast formation and activation. This leads to enhanced bone resorption. OPG is the decoy receptor for RANKL. It acts as a \"decoy\" by binding to RANKL and preventing it from interacting with its receptor (RANK). This binding inhibits osteoclast formation and activation, and it promotes apoptosis of osteoclasts, ultimately reducing bone resorption (Kuo and Chen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Abaloparatide individually as well as in combination with zoledronate has shown decreased levels of RANKL and CTX-1 in VCD-treated mice. This is supported by a previous study where abaloparatide at the dose of 40ug/kg for 12 consecutive days in C57Bl6/J mice significantly lowered the serum CTX-1 levels compared to vehicle (Arlt et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eP1NP is a bone formation marker, produced by the synthesis of type 1 collagen. Elevated levels in blood are indicative of increased bone formation. We observed increased P1NP levels in the abaloparatide group as well as in the combination group compared to the VCD-treated groups. This is as per a previous study in C57Bl6/J mice receiving 30ug/kg/day for 28 days where the abaloparatide significantly enhanced PINP serum levels (Makino et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One more study in wild-type (WT) female mice receiving abaloparatide (20-80ug/kg/d) for 30 days has shown a prominent increase in P1NP (Sahbani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe effect on OPG was not significant when abaloparatide or zoledronate were administered individually, however, in combination, an increase in OPG was observed. A previous study in wild-type (WT) female mice receiving abaloparatide (20\u0026ndash;80 ug/kg/d) for 30 days indicated a lack of significant impact on OPG concentration (Sahbani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We then looked at the RANKL OPG ratio. The balance between RANKL and OPG, expressed as the RANKL/OPG ratio, plays a crucial role in controlling bone resorption. A higher ratio is associated with increased bone resorption, while a lower ratio indicates a shift toward reduced bone resorption and possibly increased bone formation (Sahbani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, the combination of abaloparatide and zoledronate lowered the VCD-induced increase in the ratio of RANKL/OPG. Sahbani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e reported in their study that abaloparatide (80ug/kg/d s.c.) for 30 days in C57Bl/6J mice, has lowered the RANKL/OPG levels (Sahbani et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, ours is the first study reporting the combined effect of abaloparatide with bisphosphonate in improving osteoporosis. However, our study has limitations. Only one animal model was employed to study the combination. Future investigations with abaloparatide may explore combined therapies involving other animal models such as on glucocorticoids-induced osteoporosis and with other anti-resorptive treatments to unveil potential benefits. However, any possible benefit needs to be weighed in terms of cost and side effects with polytherapy, and any possible long-term effects for which evidence is currently limited for the osteoanabolic-antiresorptive combinations, requiring further investigations.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eabl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbaloparatide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAED\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnimal equivalent dose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBone Mineral Density\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBV/TV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebone volume to tissue volume\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCommittee for control and supervision of experiments on animals\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTX-1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eC-terminal cross-linking telopeptide of type 1 collagen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIAEC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInstitutional Animal Ethics Committee\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMICRO-CT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMicrocomputed tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOPG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOsteoprotegerin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePINP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eN-terminal propeptide of type 1 procollagen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePTHrP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParathyroid hormone related protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRANKL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReceptor activator of nuclear factor-kappa B ligand\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTb. N\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etrabecular number\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTb. Sp\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etrabecular separation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTb. Th\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etrabecular thickness\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVCD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e4-vinylcyclohexene diepoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ezol\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZoledronate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was carried out in the University Grants Commission Special Assistance Program (UGC SAP) and DST-FIST-supported Neurobehavioral Pharmacology laboratory. We sincerely acknowledge the micro-computed tomography facility provided under the UGC SAP DRS-II grant to DV. The financial support by DST-PURSE in providing fellowship to TA is gratefully acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTabasum Ara\u003c/strong\u003e: Conceptualization, Methodology, Visualization, Formal analysis, Investigation, Writing \u0026ndash; original draft. \u003cstrong\u003eDivya Vohora\u003c/strong\u003e: Conceptualization, Writing \u0026ndash; original draft, Writing - Review and Editing, Supervision, Visualization, Formal Analysis, Funding acquisition, Resources, Project administration. \u003cstrong\u003eZeenat Iqbal\u003c/strong\u003e: Review, Supervision. \u003cstrong\u003eShreshta Jain:\u003c/strong\u003e Investigation, \u003cstrong\u003eAadil Ahmad Sheikh:\u003c/strong\u003e Investigation. The authors declare that all data were generated in-house, that no paper mill was used and that no AI tool has been used for the generation of text or figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary data associated with this article can be found in the online version.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAref, M. W., E. M. McNerny, D. Brown, K. J. Jepsen, and M. R. Allen. 2016. \u0026apos;Zoledronate treatment has different effects in mouse strains with contrasting baseline bone mechanical phenotypes\u0026apos;, \u003cem\u003eOsteoporos Int\u003c/em\u003e, 27: 3637-43.\u003c/li\u003e\n\u003cli\u003eArlt, H., T. Mullarkey, D. Hu, R. Baron, M. S. Ominsky, B. 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Song. 2020. \u0026apos;Combination Therapy of PTH and Antiresorptive Drugs on Osteoporosis: A Review of Treatment Alternatives\u0026apos;, \u003cem\u003eFront Pharmacol\u003c/em\u003e, 11: 607017.\u003c/li\u003e\n\u003cli\u003eZhu W, Xu R, Du J, Fu Y, Li S, Zhang P, Liu L, Jiang H. Zoledronic acid promotes TLR-4-mediated M1 macrophage polarization in bisphosphonate-related osteonecrosis of the jaw. FASEB J. 2019 Apr;33(4):5208-5219. doi: 10.1096/fj.201801791RR. Epub 2019 Jan 9. PMID: 30624969.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Abaloparatide, Zoledronate, Osteoporosis, 4-Vinylcyclohexenediepoxide, Osteoanabolic","lastPublishedDoi":"10.21203/rs.3.rs-7657337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7657337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbaloparatide, a 34-amino acid synthetic peptide analog of parathyroid hormone-related protein (PTHrP), was approved for treating postmenopausal osteoporosis in high-risk individuals or those resistant to existing drugs. This study aimed to investigate the impact of abaloparatide (20µg/kg/d; s.c.), an osteoanabolic drug with an antiresorptive drug zoledronate (125mg/kg i.v.) twice weekly for a month against a mouse model of 4-vinyl cyclohexene diepoxide (VCD)-induced post-menopausal osteoporosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale Swiss albino mice were made ovotoxic by treatment with VCD (160mg/kg/d) for 15 days to mimic a postmenopausal state, confirmed by primordial follicle destruction in histopathological assessment. Microarchitectural analysis of distal femoral epiphysis and cortical mid-diaphysis was carried out using micro-computed tomography. Histopathological evaluation of bone, along with bone markers such as N-terminal propeptide of type 1 procollagen (P1NP) levels, C-terminal cross-linking telopeptide of type 1 collagen (CTX-1), soluble receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) were assessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe VCD-treated mice exhibited bone loss as evidenced through micro CT and histopathology. Treatment with abaloparatide and zoledronate combination for 30 days reversed VCD-induced alterations of BV/TV, BMD,Tb.N and Tb.Sp. while the individual treatments were only partially effective. Serum analysis indicated reduced bone turnover in VCD-treated mice. The abaloparatide individually and in combination reversed the VCD-induced alterations in P1NP, CTX-1, and RANKL. The combination therapy also lowered the RANKL/OPG ratio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the combined approach of osteoanabolic and antiresorptive treatment may offer superior protection compared to individual therapies, holding promise for postmenopausal osteoporosis treatment.\u003c/p\u003e","manuscriptTitle":"Abaloparatide-Zoledronate Combination Protects Against 4- Vinylcyclohexenediepoxide-induced Postmenopausal Osteoporosis in Mice: An Osteoanabolic-Antiresorptive Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 09:21:46","doi":"10.21203/rs.3.rs-7657337/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-11T12:56:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T08:24:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118357404430449208752197049948960441444","date":"2025-10-17T10:59:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T02:43:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246943874654372204862460168065444949502","date":"2025-10-16T02:26:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175061266233484333170996007261702222350","date":"2025-10-15T11:53:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T10:04:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-02T13:12:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-02T13:11:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-09-19T10:01:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0bec93c2-c31b-4160-97ce-e24917614a56","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:09:32+00:00","versionOfRecord":{"articleIdentity":"rs-7657337","link":"https://doi.org/10.1007/s00210-026-04984-9","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2026-01-24 15:57:19","publishedOnDateReadable":"January 24th, 2026"},"versionCreatedAt":"2025-10-30 09:21:46","video":"","vorDoi":"10.1007/s00210-026-04984-9","vorDoiUrl":"https://doi.org/10.1007/s00210-026-04984-9","workflowStages":[]},"version":"v1","identity":"rs-7657337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7657337","identity":"rs-7657337","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}