Bisphosphonate (BP), with potent anti-resorptive; RANKL
Zoledronic acid primarily targets the enzyme farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway. By inhibiting FPPS, it prevents the synthesis of isoprenoid lipids (farnesyl pyrophosphate and geranylgeranyl pyrophosphate), which are essential for the prenylation of small GTPases such as Ras and Rho. This inhibition disrupts the function of osteoclasts, leading to their apoptosis and reduced bone resorption. Zoledronic acid also indirectly inhibits osteoclast maturation by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. Additionally, it suppresses RANKL-mediated NF-kB and JNK signaling pathways.

In osteocyte-like MLO-Y4 cells, zoledronic acid monohydrate (0.1–1 µM; 48 hours) enhances the expression of sclerostin and receptor activator of nuclear factor kB ligand (RANKL) mRNA[2]. The expression of osteoclastogenesis supporting factor from MLO-Y4 cells is increased by zoledronic acid monohydrate[2]. In MLO-Y4 cells, zoledronic acid monohydrate increases the production of RANKL through the IL-6/JAK2/STAT3 pathway[2]. Monohydrate zoledronic acid suppresses osteoclast development and function by modulating the JNK and NF-κB signaling pathways[3]. In MC3T3-E1 cells, zoledronic acid monohydrate (10-100 µM; 1-7 days) significantly lowers viability[4]. In MC3T3-E1 cells, zoledronic acid monohydrate (10-100 µM; 1-7 days) causes apoptosis[4]. Because it induces apoptosis, zoledronic acid monohydrate (10–100 µM; 4 days) reduces cell viability[4]. At concentrations less than 1 µM, zoledronic acid monohydrate inhibits the differentiation and maturation of MC3T3-E1 cells[4].
In vitro, zoledronic acid (0.1-1 µM; 48 hours) increases receptor activator of nuclear factor kB ligand (RANKL) and sclerostin mRNA expressions in osteocyte-like MLO-Y4 cells. It enhances RANKL expression via the IL-6/JAK2/STAT3 pathway in MLO-Y4 cells. Zoledronic acid inhibits osteoclast differentiation and function through the regulation of NF-κB and JNK signalling pathways. It also strongly inhibits TNF-α- and RANKL-induced upregulation of RANK in a dose-dependent manner. Zoledronic acid (10-100 μM; 1-7 days) markedly reduces the viability of MC3T3-E1 cells.

For three weeks, zoledronic acid monohydrate (0.05 mg/kg; intraperitoneally; weekly) enhances the density and content of bone mineral[5]. In vivo bone remodeling and osteoclast and osteoblast function are inhibited by zoledronic acid monohydrate (0.5–1 mg/kg; i.p.; weekly; for 3 weeks), which interferes with the mechanical characteristics of bone[5].
In vivo, zoledronic acid is a potent inhibitor of bone resorption. It inhibits osteoclastogenesis and bone resorptive function by suppressing RANKL-mediated NF-kB and JNK pathways. Zoledronic acid reduces bone turnover and has been shown to alleviate osteoporosis by suppressing osteoclastogenesis via regulating RANKL expression. It also inhibits RANKL and reduces bone turnover, which plays an important role in the development of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

Zoledronic acid's activity can be assessed using in vitro enzyme assays measuring FPPS activity. The compound's inhibition of FPPS can be evaluated by measuring the accumulation of its substrate, isopentenyl pyrophosphate (IPP), or the depletion of its products. Its effects on the RANKL/RANK pathway can be studied using osteoclast precursor cells such as RAW264.7, where zoledronic acid inhibits RANKL-induced NF-κB activity. The compound's binding affinity to hydroxyapatite (bone mineral) can be assessed using in vitro binding assays.

Cell Viability Assay[4]
Cell Types: MC3T3-E1 cells
Tested Concentrations: 0.02 µM, 0.1 µM, 1 µM, 10 µM, 100 µM
Incubation Duration: 1 day, 3 days, 5 days, 7 days
Experimental Results: decreased cells viability at 10 µM and 100 µM.

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Apoptosis Analysis[4]
Cell Types: MC3T3-E1 cells
Tested Concentrations: 0.02 µM, 0.1 µM, 1 µM, 10 µM, 100 µM
Incubation Duration: 1 days, 4 days, 7 days
Experimental Results: Increased the number of early apoptotic cells and late apoptotic or necrotic cells at dose-dependent and time-dependent (high concentrations).

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Western Blot Analysis[4]
Cell Types: MC3T3-E1 cells
Tested Concentrations: 0.02 µM, 0.1 µM, 1 µM, 10 µM, 100 µM
Incubation Duration: 4 days
Experimental Results: Down-regulated the protein level of inactive caspase-3 and up-regulated the protein level of active caspase-3 at the concentrations of 10 and 100 µM.
Cellular assays for zoledronic acid involve treating osteoclasts or cancer cells with the compound and measuring cell viability, apoptosis, and function. Osteoclast apoptosis can be assessed using TUNEL staining or caspase activation assays. Osteoclast resorption activity is measured using pit formation assays on bone or dentine slices. The compound's effects on RANKL expression can be evaluated in osteocyte-like MLO-Y4 cells by measuring mRNA and protein levels.

Animal/Disease Models: Fiveweeks old C57BL6 mice[5]
Doses: 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg
Route of Administration: intraperitoneal (ip)injection, weekly, for 3 weeks
Experimental Results: Inhibited both osteoclast and osteoblasts function and bone remodeling at 0.5 mg/kg and 1 mg/kg.
In vivo animal model protocols for zoledronic acid involve its administration (typically intravenous or subcutaneous) to animal models of osteoporosis or cancer. In osteoporosis models (e.g., ovariectomized rats), bone mineral density is measured by DEXA, and bone histomorphometry is performed. In cancer models, tumor growth, bone metastases, and skeletal-related events are monitored. Pharmacokinetic and pharmacodynamic studies are also performed to assess the compound's effects on bone turnover markers.

Zoledronic acid is administered intravenously. It has a unique pharmacokinetic profile characterized by rapid binding to bone mineral and a long residence time in bone. The compound is not metabolized and is excreted unchanged in the urine. The terminal half-life of zoledronic acid in bone is extremely long (estimated to be several months to years), reflecting its slow release from bone tissue. In plasma, the half-life is approximately 7 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Since there is currently no information regarding the use of zoledronic acid during lactation, alternative medications are recommended, especially for breastfed newborns or premature infants. However, breastfed infants are unlikely to absorb zoledronic acid.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.
Zoledronic acid has a well-characterized safety profile. Dose-dependent acute-phase reactions (fever, flu-like symptoms) occur in some patients. Hypocalcemia is a risk, particularly in patients with vitamin D deficiency. Renal impairment can occur with rapid infusion or in patients with pre-existing kidney disease. Osteonecrosis of the jaw (ONJ) is a rare but serious complication associated with long-term bisphosphonate use.

[1]. Various pathways of zoledronic acid against osteoclasts and bone cancer metastasis: a brief review. BMC Cancer. 2020; 20: 1059.

[2]. Zoledronate Enhances Osteocyte-Mediated Osteoclast Differentiation by IL-6/RANKL Axis. Int J Mol Sci. 2019 Mar; 20(6): 1467.

[3]. Zoledronic acid inhibits osteoclast differentiation and function through the regulation of NF-κB and JNK signalling pathways. Int J Mol Med. 2019 Aug;44(2):582-592.

[4]. Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro. Mol Med Rep. 2016 Jan; 13(1): 613-622.

[5]. High-dose zoledronic acid impacts bone remodeling with effects on osteoblastic lineage and bone mechanical properties. Clin Cancer Res. 2009 Sep 15;15(18):5829-39.

[6]. Oral Zoledronic acid bisphosphonate for the treatment of chronic low back pain with associated Modic changes: A pilot randomized controlled trial. J Orthop Res. 2022 Feb 23.

Zoledronic acid is a synthetic imidazole bisphosphonate analogue, a pyrophosphate analogue with anti-bone resorption activity. As a third-generation bisphosphonate, zoledronic acid binds to hydroxyapatite crystals in the bone matrix, slowing their dissolution and inhibiting the formation and aggregation of these crystals. The drug also inhibits farnesyl pyrophosphate synthase, an enzyme involved in the biosynthesis of terpenoids. Inhibition of this enzyme prevents the biosynthesis of isoprene lipids, which are donor substrates for farnesylation and geranylation during the post-translational modification of small GTPase signaling proteins, modifications that play an important role in osteoclast turnover. The reduction in bone turnover and the stabilization of the bone matrix contribute to the analgesic effect of zoledronic acid on painful osteoblastic lesions. The drug also reduces serum calcium concentrations associated with hypercalcemia.
An iminobisphosphonate bone resorption inhibitor used to treat malignant tumor-associated hypercalcemia, osteitis deformans, and osteoporosis.

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Drug Indications
Prevention of skeletal-related events (pathological fracture, spinal cord compression, bone radiation or surgery, or tumor-induced hypercalcemia) in adult patients with advanced bone malignancies. Treatment of hypercalcemia caused by tumors in adults.
Treatment of osteoporosis: postmenopausal women; men; those at increased risk of fracture, including those with recent low-energy hip fractures. Treatment of osteoporosis associated with long-term systemic glucocorticoid therapy in postmenopausal women and men at increased fracture risk. Treatment of Paget's disease.
Prevention of skeletal-related events (pathological fracture, spinal cord compression, bone radiation or surgery, or tumor-induced hypercalcemia) in adult patients with advanced bone malignancies. Treatment of hypercalcemia caused by tumors (TIH) in adults.
4 mg/5 ml and 4 mg/100 ml: Prevention of skeletal-related events (pathological fracture, spinal cord compression, bone radiation or surgery, or tumor-induced hypercalcemia) in adult patients with advanced bone malignancies. Treatment of hypercalcemia caused by tumors (TIH) in adults. 5 mg/100 ml: Treatment of osteoporosis: Suitable for postmenopausal women; men; those at increased risk of fracture, including those with recent low-energy hip fractures. Treatment of osteoporosis associated with long-term systemic glucocorticoid therapy: Suitable for postmenopausal women; men; those at increased risk of fracture. Treatment of Paget's disease in adults.
Prevention of skeletal-related events and treatment of hypercalcemia caused by tumors.
Prevention of skeletal-related events (pathological fractures, spinal cord compression, bone radiation or surgery, or hypercalcemia caused by tumors) in adult patients with advanced bone malignancies. Treatment of hypercalcemia (TIH) caused by tumors in adults.
Treatment of osteoporosis: Postmenopausal women; men; those at increased risk of fracture, including those with recent low-energy hip fractures. Treatment of osteoporosis associated with long-term systemic glucocorticoid therapy: Postmenopausal women; men; those at increased risk of fracture. Treatment of Paget's disease in adults. Treatment of osteoporosis in postmenopausal women increases the risk of fractures in adult men, including those with recent low-energy hip fractures. Treatment of osteoporosis in postmenopausal women associated with long-term systemic glucocorticoid therapy increases the risk of fractures in adult men. Treatment of Paget's disease in adults. Prevention of skeletal-related events in patients with advanced bone malignancies.
Zoledronic acid hydrate (CGP 42446, CAS# 165800-06-6) is a hydrate form of zoledronic acid, a potent, third-generation bisphosphonate. It is a synthetic imidazole bisphosphonate analog with potent anti-bone-resorption activity. Zoledronic acid is a widely used medication for the treatment of osteoporosis, Paget's disease, and bone metastases. It is also known by the brand names Zometa and Reclast. Zoledronic acid primarily targets FPPS in the mevalonate pathway and also modulates the RANKL/RANK pathway to inhibit osteoclast differentiation and function.

C5H12N2O8P2

290.1

290.006

C, 22.07; H, 3.70; N, 10.30; O, 41.16; P, 22.77

165800-06-6

Zoledronic Acid;118072-93-8;Zoledronic acid disodium tetrahydrate;165800-07-7; 165800-06-6 (free acid hydrate); 131654-46-1 (disodium); 165800-08-8 (trisodium hydrate); 827573-11-5 (trisodium); 165800-07-7 (disodium hydrate);

121586

White to off-white solid powder

764ºC at 760 mmHg

245 °C(dec.)

415.8ºC

1.53E-24mmHg at 25°C

-2.3

6

9

4

17

327

0

C(N1C=CN=C1)C(P(=O)(O)O)(P(=O)(O)O)O.O

FUXFIVRTGHOMSO-UHFFFAOYSA-N

InChI=1S/C5H10N2O7P2.H2O/c8-5(15(9,10)11,16(12,13)14)3-7-2-1-6-4-7;/h1-2,4,8H,3H2,(H2,9,10,11)(H2,12,13,14);1H2

(1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl)diphosphonic acid hydrate

CGP42446; CGP42446A; ZOL446; CGP-42446; CGP-42446A; ZOL-446; CGP 42446; CGP 42446A; ZOL 446; Zoledronate, trade names: Zometa; Zoledronic acid monohydrate; Zoledronic acid (monohydrate); Zometa; Zoledronate monohydrate; (1-Hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl)diphosphonic acid hydrate; Zoledronate hydrate; Reclast.

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 : ~14.29 mg/mL (~49.26 mM)
DMSO :< 1 mg/mL

Solubility in Formulation 1: 3.33 mg/mL (11.48 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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