Parathyroid hormone receptor 1 (PTHR1)[1]

Abaloparatide TFA (0-100 nM; 40 min) improves β-arrestin recruitment and Gs/cAMP signaling in MC3T3-E1 cells [1]. With an EC50 value of 0.8 nM, betaloparasite TFA (0-100 nM) efficiently causes PTHR1 internalization in U2OS cells in a dose-dependent manner [1].

In mice, abelaparatide (20–80 µg/kg; s.c.; daily for 30 days) improves cortical structure and bone formation[1].
Teriparatide and abaloparatide are parathyroid hormone receptor 1 (PTHR1) analogs with unexplained differential efficacy for the treatment of osteoporosis. Therefore, we compared the effects of abaloparatide and teriparatide on bone structure, turnover, and levels of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). Wild-type (WT) female mice were injected daily with vehicle or 20-80 µg/kg/day of teriparatide or abaloparatide for 30 days. Femurs and spines were examined by microcomputed tomography scanning and serum levels of bone turnover markers, RANKL, and OPG, were measured by ELISA. Both analogs similarly increased the distal femoral fractional trabecular bone volume, connectivity, and number, and reduced the structure model index (SMI) at 20-80 µg/kg/day doses. However, only abaloparatide exhibited a significant increase (13%) in trabecular thickness at 20 µg/kg/day dose. Femoral cortical evaluation showed that abaloparatide caused a greater dose-dependent increase in cortical thickness than teriparatide. Both teriparatide and abaloparatide increased lumbar 5 vertebral trabecular connectivity but had no or modest effect on other indices. Biochemical analysis demonstrated that abaloparatide promoted greater elevation of procollagen type 1 intact N-terminal propeptide, a bone formation marker, and tartrate-resistant acid phosphatase 5b levels, a bone resorption marker, and lowered the RANKL/OPG ratio. Furthermore, PTHR1 signaling was compared in cells treated with 0-100 nmol/L analog. Interestingly, abaloparatide had a markedly lower EC50 for cAMP formation (2.3-fold) and β-arrestin recruitment (1.6-fold) than teriparatide. Therefore, abaloparatide-improved efficacy can be attributed to enhanced bone formation and cortical structure, reduced RANKL/OPG ratio, and amplified Gs-cAMP and β-arrestin signaling.[1]

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Abaloparatide is a novel 34-amino acid peptide selected to be a potent and selective activator of the parathyroid hormone receptor (PTH1R) signaling pathway with 41% homology to PTH(1-34) and 76% homology to PTHrP(1-34). A 12-month treatment study was conducted in osteopenic ovariectomized (OVX) rats to characterize the mechanisms by which abaloparatide increases bone mass. Sprague-Dawley (SD) rats were subjected to OVX or sham surgery at age 6 months and left untreated for 3 months to allow OVX-induced bone loss. Ten OVX rats were euthanized after this bone depletion period, and the remaining OVX rats received daily subcutaneous injections of vehicle (n = 18) or abaloparatide at 1, 5, or 25 μg/kg/d (n = 18/dose level) for 12 months. Sham controls (n = 18) received vehicle daily. Bone densitometry and biochemical markers of bone formation and resorption were assessed longitudinally, and L3 vertebra and tibia were collected at necropsy for histomorphometry. Abaloparatide increased biochemical bone formation markers without increasing bone resorption markers or causing hypercalcemia. Abaloparatide increased histomorphometric indices of bone formation on trabecular, endocortical, and periosteal surfaces without increasing osteoclasts or eroded surfaces. Abaloparatide induced substantial increases in trabecular bone volume and density and improvements in trabecular microarchitecture. Abaloparatide stimulated periosteal expansion and endocortical bone apposition at the tibial diaphysis, leading to marked increases in cortical bone volume and density. Whole-body bone mineral density (BMD) remained stable in OVX-Vehicle controls while increasing 25% after 12 months of abaloparatide (25 μg/kg). Histomorphometry and biomarker data suggest that gains in cortical and trabecular bone mass were attributable to selective anabolic effects of abaloparatide, without evidence for stimulated bone resorption. © 2016 American Society for Bone and Mineral Research.[2]

PathHunter® eXpress PTHR1 CHO‐K1 β‐arrestin GPCR assay[1]
To assess the effects of Abaloparatide and teriparatide stimulation of PTHR1 on β‐arrestin recruitment to the cell membrane, a PathHunter eXpress PTHR1 Chinese Hamster Ovary‐K1 (CHO‐K1) β‐arrestin GPCR Assay was used. The assay takes advantage of Enzyme Fragment Complementation technology. The PTHR1 is fused in frame with a small enzyme donor fragment ProLink™ (PK) and co‐expressed in CHO‐K1 cells stably expressing a fusion protein of β‐arrestin and the larger, N‐terminal deletion mutant of β‐galactosidase (called enzyme acceptor or EA). Activation of the PTHR1 stimulates binding of β‐arrestin to the PK‐tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active β‐galactosidase enzyme. An increase in enzyme activity is then measured using chemiluminescent PathHunter Detection Reagents. Cell seeding, incubation, and detection were performed as instructed by the manufacturer. Briefly, cells were seeded in a clear bottom white 96‐well plate and incubated for 48 h at 37°C CO2 incubator. Cells were treated with vehicle, teriparatide, or Abaloparatide for 60 min at 37°C in a CO2 incubator. At the end of the incubation, β‐gal enzyme substrate was added for 60 min at room temperature in the dark. Light generation (Relative Light Units, RLU), an indication of β‐gal enzyme fragment complementation and β‐Arrestin/ PTHR1 interaction, was measured using BMG Labtech PHERAstar FS luminescence plate reader.[1]

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PathHunter® eXpress PTHR1 activated GPCR internalization assay[1]
To determine PTHR1 internalization, we used PathHunter eXpress PTHR1 U2OS Activated GPCR Internalization Assay. PathHunter® PTHR1 Activated GPCR Internalization U2OS cell lines are engineered to co‐express an untagged PTHR1, an EA‐tagged β‐arrestin, and a PK tag localized to the endosomes. Activation of the untagged PTHR1 induces β‐arrestin recruitment, followed by internalization of the GPCR‐β‐arrestin‐EA complex in PK‐tagged endosomes. Similar to the β‐arrestin assay format, this internalization forces complementation of the two β‐gal enzyme fragments, forming functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal. U2OS osteoblastic cell line seeding, incubation, and detection were performed as instructed by the manufacturer. Cells were treated with vehicle, teriparatide, or Abaloparatide for 60 min at 37°C in a CO2 incubator. At the end of the incubation, β‐gal enzyme substrate was added for 60 min at room temperature in the dark. Light generation (RLU), an indication of β‐gal enzyme fragment complementation and β‐arrestin/endosome/PTHR1 formation, was measured using BMG Labtech PHERAstar FS luminescence plate reader.

Measurement of intracellular cAMP generation[1]
MC3T3‐E1 cells were seeded at 40,000 cells/well of a 24‐well plate containing 500‐µL alpha‐MEM supplemented with 10% FBS and 1% PS. After culture for 1 week, the medium was removed and replaced with 250 µL of stimulation medium (alpha‐MEM containing 0.05% FBS, 0.1% BSA, 5 mmol/L hepes buffer, and 0.5 mmol/L IBMX) for 15 min. IBMX is a phosphodiesterase inhibitor that prevents degradation of the generated cAMP. Vehicle, Abaloparatide, and teriparatide were then added in 250 µL stimulation medium to achieve final concentrations of 0, 0.01, 0.1, 1, 10, and 100 nmol/L/well. Incubation continued for 40 min at 37°C before the medium was removed and the plates were snap frozen in liquid N3 and stored at −80°C. For extraction of intracellular cAMP, 100 mmol/L Hcl was added and cells were incubated at room temperature for 1 h. Intracellular cAMP was assayed using a cAMP competitive ELISA kit and following the manufacturer protocol and instructions.

Animal/Disease Models: Female SD (Sprague-Dawley) rats (age 22 weeks)[2]
Doses: 1 µg/kg, 5 µg/kg, 25 µg/kg
Route of Administration: subcutaneous (sc) injection; daily; for 12 months
Experimental Results: Increased biochemical bone formation markers, histomorphometric indices of bone formation on trabecular, endocortical, and periosteal surfaces. Induced substantial increases in trabecular bone volume and density and improvements in trabecular microarchitecture. Stimulated periosteal expansion and endocortical bone apposition at the tibial diaphysis, leading to marked increases in cortical bone volume and density. Whole-body bone mineral density (BMD) was increasing 25%.

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Animal/Disease Models: 16weeks old wild-type (WT) female C57BL/6J mice[1]
Doses: 20-80 µg/kg
Route of Administration: Sc; daily for 30 days
Experimental Results: Efficiently expanded cortical thickness (Ct. Th) at both doses of 20 and 80 µg/kg/day by 17% and 18%, respectively, increased P1NP levels to 227% and 407% at 20 and 80 µg /kg/day, respectively.

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16-week-old wild-type (WT) female C57BL/6J mice[1]
20-80 µg/kg
S.c.; daily for 30 days

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All experiments were conducted on 16‐week‐old wild‐type (WT) female C57BL/6J mice (Stock number 664). Vehicle (0.9% NaCl/10 mmol/L acetic acid) or 20–80 µg/kg/day teriparatide or abaloparatide was injected subcutaneously (SC) daily (except Sunday) and continued for 30 days. No peptide injection was performed on the day of animal sacrifice.[1]

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A total of 13 rats were euthanized or found dead between study days 117 to 358 before study termination: 6 in the sham control group, 2 in the OVX-Veh group, 3 in the OVX + abaloparatide 1 μg/kg/d group, 1 in the OVX + abaloparatide 5 μg/kg/d group, and 1 in the OVX + abaloparatide 25 μg/kg/d group. For these animals’ data, absolute values were reported if collected, and data based on % change from baseline were censored as required. Five deaths were likely secondary to complications from blood collection, whereas the remaining deaths were attributed to incidental age-related pathologies.[1]
Study design and dose selection[1]
After a 13-week postsurgical bone depletion period, one group of untreated OVX rats was euthanized as a pretreatment baseline group for histomorphometry data. The remaining groups were given daily s.c. injections of vehicle (Vehicle; 0.9% sodium chloride) or one of three dose levels of abaloparatide in a 0.1 mL/kg volume. Abaloparatide dose levels were 1 μg/kg/d (OVX-ABL1), 5 μg/kg/d (OVX-ABL5), and 25 μg/kg/d (OVX-ABL25), with dosing guided by weekly body weight measurements. Preliminary results from another rat study indicated that 6 weeks of abaloparatide at 1.25 μg/kg/d completely reversed OVX-induced bone loss (Radius Health, Inc., Waltham, MA, USA). This led to selection of 1 μg/kg as the low dose, and also 5- and 25-fold multiples of this dose to provide safety margins.[1]

Absorption
The absolute bioavailability of abaloparatide in healthy women after subcutaneous administration of an 80 mcg dose was 36%. Following subcutaneous administration of 80 mcg abaloparatide in postmenopausal women with osteoporosis for seven days, the mean (SD) Cmax was 812 (118) pg/mL and the AUC0-24 was 1622 (641) pgxhr/mL. The median Tmax was 0.51 hours, with a range from 0.25 to 0.52 hours.

Route of Elimination
The peptide fragments of abaloparatide are primarily eliminated through renal excretion.

Volume of Distribution
The volume of distribution was approximately 50 L.

Clearance
The mean apparent total plasma clearance for subcutaneous administration is 168 L/h in healthy subjects.

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Metabolism / Metabolites
Abaloparatide is metabolized into smaller peptide fragments via non-specific proteolytic degradation.

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Biological Half-Life
The mean half-life of abaloparatide is approximately one hour.

Protein Binding
In vitro, abaloparatide was approximately 70% bound to plasma proteins.

[1]. Sahbani K, et al. Abaloparatide exhibits greater osteoanabolic response and higher cAMP stimulation and β-arrestin recruitment than teriparatide. Physiol Rep. 2019 Oct;7(19):e14225.
[2]. Varela A, et al. One Year of Abaloparatide, a Selective Activator of the PTH1 Receptor, Increased Bone Formation and Bone Mass in Osteopenic Ovariectomized Rats Without Increasing Bone Resorption. J Bone Miner Res. 2017 Jan;32(1):24-33.

Abaloparatide is an N-terminal analog of parathyroid hormone-related protein (PTHrP) and an agonist at the parathyroid hormone type 1 (PTH1) receptor. It is a synthetic 34 amino acid peptide with 41% homology to human parathyroid hormone 1-34 and human PTHrP 1-34. Abaloparatide and PTHrP share the first 21 amino acids and the receptor-activating domain. Abaloparatide is an osteoanabolic agent that stimulates bone formation. It was first approved by the FDA on April 28, 2017, for the treatment of osteoporosis in postmenopausal women and is also used to increase bone density in men with osteoporosis. In October 2022, the EMA's Committee for Medicinal Products for Human Use (CHMP) recommended abaloparatide be granted marketing authorization in Europe and the drug was fully authorized by the European Commission on December 19, 2022.

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Abaloparatide is a 34 amino acid synthetic analog of human parathyroid hormone-related protein (PTHrP) (PTHrP(1-34) analog), with bone-growing and bone density conserving activities. Upon subcutaneous administration, abaloparatide acts similar to PTHrP and targets, binds to and activates parathyroid hormone 1 (PTH1) receptor (PTH1R), a G protein-coupled receptor (GPCR) expressed in osteoblasts and bone stromal cells. PTH1R activates the cyclic AMP (cAMP) signaling pathway and the bone anabolic signaling pathway, leading to bone growth, increased bone mineral density (BMD) and volume. This correlates with increased bone mass and strength and prevents or treats osteoporosis and decreases fractures.

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Drug Indication
Abaloparatide is indicated for the treatment of postmenopausal women with osteoporosis at high risk for fracture (defined as a history of osteoporotic fracture or multiple risk factors for fracture) or patients who have failed or are intolerant to other available osteoporosis therapy. In postmenopausal women with osteoporosis, abaloparatide reduces the risk of vertebral and nonvertebral fractures. Abaloparatide is also indicated to increase bone density in men with osteoporosis at high risk for fracture (defined as a history of osteoporotic fracture or multiple risk factors for fracture) or patients who have failed or are intolerant to other available osteoporosis therapy.

Treatment of osteoporosis in postmenopausal women at increased risk of fracture.
Treatment of osteoporosis.

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Pharmacodynamics
Abaloparatide stimulates bone formation on periosteal, trabecular, and cortical bone surfaces. It increases bone mineral density and bone formation markers in a dose-dependent manner. Abaloparatide causes transient and limited increases in osteoclast bone resorption and increases bone density. In rats and monkeys, abaloparatide exerted anabolic effects, increasing bone mineral density and mineral content correlating with increases in bone strength at vertebral and nonvertebral sites.

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Mechanism of Action
Abaloparatide is an agonist at the PTH1 receptor (PTH1R), a G-protein-coupled receptor (GPCR) that regulates bone formation and bone turnover, as well as mineral ion homeostasis. The PTH1R couples to Gs and Gq, which stimulates adenylyl cyclase (AC), which activates the cAMP/PKA signalling cascade, and phospholipase C (PLC), which activates the IP/PKC signalling cascade. Abaloparatide binds to the PTH1R in target cells to activate the Gs-protein-mediated cAMP signalling pathway, thereby stimulating osteoblastic activity. Abaloparatide also activates Gq and β-arrestin-1 pathway downstream of PTH1R as off-targets in target cells such as the testis and epididymis, which have been associated with anti-inflammatory effects and alleviation of epididymitis and orchitis symptoms. The PTH1R has two conformations with distinct ligand binding profiles. The R0 conformation is a G protein–independent high-affinity conformation, and upon binding, the ligand induces a longer-lasting signalling response that gradually increases cAMP. Due to the prolonged signalling response, ligands selectively binding to the R0 conformation are associated with a risk for increased calcium mobilization and hypercalcemia. Conversely, the RG conformation is G-protein–dependent (GTPγS-sensitive) with a shorter signalling response. Abaloparatide binds to the RG conformation with greater selectivity: it induces more transient signalling responses and favours net bone formation over bone resorption. The drug's relatively low risk for hypercalcemia and osteoclast resorption compared to [teriparatide] is attributed to the preferential binding of abaloparatide to the RG conformation.

C176H301N56F3O51

4074.61

Abaloparatide;247062-33-5

Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-{Aib}-Lys-Leu-His-Thr-Ala-NH2

AVSEHQLLHDKGKSIQDLRRRELLEKLL-{Aib}-KLHTA-NH2

Typically exists as White to off-white solid at room temperature

BIM-44058; Abaloparatide TFA; BA-058; ITM-058; BIM 44058; BA 058;ITM 058; BIM44058; BA058;ITM058; trade name: Tymlos

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~24.54 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)