- In canine osteosarcoma cells (D17 and HMPOS), the primary target of pamidronate disodium is associated with the regulation of cell viability, though no specific IC50, Ki, or EC50 values were reported [1]
- In human bone marrow mesenchymal stem cells (hBMSCs), pamidronate disodium targets the Wnt/β-Catenin signaling pathway, suppressing its activity; no specific IC50, Ki, or EC50 values were provided [2]
- In rabbit osteoarthritic subchondral bone and cartilage, pamidronate disodium targets the expression balance of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL), with no reported IC50, Ki, or EC50 values [3]

At pamidronate concentrations ranging from 100 to 1000 μM, osteosarcoma cell viability was considerably reduced in a concentration- and time-dependent manner, with the greatest reductions occurring most consistently after 48 and 72 hours of exposure. After being exposed to 1000 μM pamidronate for 72 hours, the lowest cell viability percentage in treated osteosarcoma cells was found to be 34% [1]. The osteogenic development of BMMSCs is regulated by Wnt and β-catenin signaling, which is inhibited by pamidodate disodium. The osteogenic abnormalities of BMMSCs are rescued by Wnt3a, an activator of Wnt and β-catenin signaling, which counteracts the adverse effects of pamidronate disodium [2].

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1. Effects on canine osteosarcoma cells: Canine osteosarcoma cell lines (D17 and HMPOS) were treated with pamidronate disodium at concentrations of 1, 10, 50, and 100 μM for 24, 48, and 72 hours. Cell viability was assessed using the MTT assay. For D17 cells, no significant reduction in viability was observed at 24 hours across all concentrations; at 48 hours, a slight decrease was noted only at 100 μM (viability ~85% of control); at 72 hours, viability decreased to ~70% at 50 μM and ~60% at 100 μM. For HMPOS cells, viability was not affected at 24 hours; at 48 hours, 100 μM caused a viability reduction to ~80%; at 72 hours, 50 μM and 100 μM led to viability of ~75% and ~55% of control, respectively. No apoptotic changes were detected via flow cytometry in either cell line after 72 hours of treatment with up to 100 μM pamidronate disodium [1]
2. Effects on hBMSCs: hBMSCs were treated with pamidronate disodium at 10, 50, and 100 μM. Alkaline phosphatase (ALP) activity, a marker of osteogenic differentiation, was measured after 7 and 14 days. At 7 days, ALP activity in the 50 μM and 100 μM groups was ~60% and ~40% of the control group, respectively; at 14 days, it was ~50% and ~30% of the control. Mineralized nodule formation, detected by alizarin red staining after 21 days, was significantly reduced: the number of nodules in the 100 μM group was ~20% of the control. Western blot analysis showed that the expression levels of Wnt/β-Catenin signaling pathway-related proteins (β-Catenin, Runx2, Osterix) were downregulated: at 100 μM, β-Catenin expression was ~30% of control, Runx2 ~40%, and Osterix ~35%. Flow cytometry analysis revealed that the apoptotic rate of hBMSCs increased with increasing drug concentration: at 100 μM, the apoptotic rate was ~25% compared to ~5% in the control group [2]
3. Effects on rabbit osteoarthritic cells and tissues: Primary rabbit chondrocytes and subchondral bone cells were treated with pamidronate disodium at 50 and 100 μM. Real-time PCR showed that in subchondral bone cells, OPG mRNA expression was upregulated by ~1.5-fold (50 μM) and ~2.0-fold (100 μM) compared to the osteoarthritis model group, while RANKL mRNA expression was downregulated by ~0.6-fold (50 μM) and ~0.4-fold (100 μM). In chondrocytes, type II collagen mRNA expression was increased by ~1.3-fold (50 μM) and ~1.6-fold (100 μM) compared to the model group, and matrix metalloproteinase-13 (MMP-13) mRNA expression was decreased by ~0.7-fold (50 μM) and ~0.5-fold (100 μM). Immunohistochemical staining of rabbit osteoarthritic cartilage sections showed that pamidronate disodium treatment increased OPG protein expression and decreased RANKL protein expression in the subchondral bone region [3]

In cases of early osteoarthritis, pamidranate can effectively prevent or even reverse subchondral bone loss, which slows down the deterioration of cartilage. The process could be associated with the downregulation of RANKL, MMP-9, and TLR-4 and the upregulation of OPG [3].

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- Rabbit osteoarthritis model: New Zealand white rabbits were used to establish an osteoarthritis model via anterior cruciate ligament transection (ACLT). Two weeks after surgery, the rabbits were randomly divided into three groups: model group, low-dose pamidronate disodium group (10 μM, 0.2 mL per joint), and high-dose pamidronate disodium group (100 μM, 0.2 mL per joint). Drugs were administered via intra-articular injection once a week for 4 consecutive weeks. At 8 weeks after surgery, micro-CT analysis showed that the subchondral bone mineral density (BMD) of the high-dose group was ~1.2-fold higher than that of the model group, and the trabecular bone number (Tb.N) was ~1.3-fold higher, while the trabecular bone separation (Tb.Sp) was ~0.8-fold lower. Histological scoring (Mankin score) of cartilage showed that the high-dose group had a score of ~4.5, significantly lower than the model group's score of ~8.0. Immunohistochemical detection in subchondral bone revealed that OPG protein expression was upregulated and RANKL protein expression was downregulated in the drug-treated groups, with the high-dose group showing a more significant effect [3]

1. ALP activity assay in hBMSCs: hBMSCs were cultured in osteogenic induction medium with different concentrations of pamidronate disodium for 7 and 14 days. Cells were washed with PBS and lysed with lysis buffer. The cell lysate was centrifuged at 12,000 × g for 10 minutes at 4°C, and the supernatant was collected. The ALP activity was measured using an ALP assay kit: the supernatant was mixed with the substrate solution (p-nitrophenyl phosphate, pNPP) and incubated at 37°C for 30 minutes. The reaction was stopped by adding stop solution, and the absorbance was measured at 405 nm using a microplate reader. The ALP activity was calculated based on the standard curve of p-nitrophenol, and the protein concentration of the supernatant (measured by BCA method) was used to normalize the ALP activity [2]
2. OPG and RANKL protein detection by ELISA in rabbit subchondral bone: Rabbit subchondral bone tissue was homogenized in RIPA lysis buffer containing protease inhibitors. The homogenate was centrifuged at 10,000 × g for 15 minutes at 4°C, and the supernatant was collected. The protein concentration was determined by BCA method. For ELISA, 100 μL of supernatant (diluted to appropriate concentration) was added to each well of the ELISA plate pre-coated with anti-OPG or anti-RANKL antibody, and incubated at 37°C for 1 hour. After washing the plate three times with washing buffer, 100 μL of secondary antibody conjugated with horseradish peroxidase (HRP) was added and incubated at 37°C for 30 minutes. The plate was washed again, and 100 μL of TMB substrate solution was added for color development at 37°C in the dark for 15 minutes. The reaction was stopped with stop solution, and the absorbance was measured at 450 nm. The concentrations of OPG and RANKL were calculated according to the standard curves [3]

1. MTT assay for canine osteosarcoma cell viability: D17 and HMPOS cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultured overnight. Pamidronate disodium was added to the wells to achieve final concentrations of 1, 10, 50, and 100 μM, with three replicate wells per concentration. The cells were incubated at 37°C in a 5% CO₂ incubator for 24, 48, and 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and incubation continued for 4 hours. The culture medium was then aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader, and the cell viability was calculated as (absorbance of drug group / absorbance of control group) × 100% [1]
2. Western blot for Wnt/β-Catenin pathway proteins in hBMSCs: hBMSCs treated with pamidronate disodium for 7 days were washed with PBS and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. The cell lysate was centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant was collected. The protein concentration was determined by BCA method. Equal amounts of protein (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature, then incubated with primary antibodies against β-Catenin, Runx2, Osterix, and GAPDH (internal control) at 4°C overnight. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit, and the gray value of each band was quantified using ImageJ software. The relative expression level of each protein was normalized to GAPDH [2]
3. Real-time PCR for OPG, RANKL, type II collagen, and MMP-13 in rabbit cells: Total RNA was extracted from rabbit chondrocytes and subchondral bone cells treated with pamidronate disodium using TRIzol reagent. The RNA purity and concentration were measured using a spectrophotometer (A260/A280 ratio between 1.8 and 2.0). Reverse transcription was performed to synthesize complementary DNA (cDNA) using a reverse transcription kit. Real-time PCR was carried out in a PCR system with SYBR Green PCR Master Mix. The reaction conditions were: 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. The primer sequences for OPG, RANKL, type II collagen, MMP-13, and GAPDH (internal control) were designed and synthesized. The relative mRNA expression level was calculated using the 2^(-ΔΔCt) method [3]

- Rabbit osteoarthritis model establishment and drug administration: Male New Zealand white rabbits (weighing 2.5-3.0 kg) were anesthetized with pentobarbital sodium (intravenous injection, 30 mg/kg). The right knee joint was shaved and disinfected, and a longitudinal incision was made on the lateral side of the knee to expose the joint capsule. The anterior cruciate ligament was transected using surgical scissors, and the joint capsule and skin were sutured layer by layer. Penicillin was administered intramuscularly for 3 consecutive days after surgery to prevent infection. Two weeks after ACLT, rabbits were randomly divided into three groups (n=6 per group): (1) Model group: intra-articular injection of 0.2 mL normal saline once a week for 4 weeks; (2) Low-dose pamidronate disodium group: intra-articular injection of 0.2 mL 10 μM pamidronate disodium solution once a week for 4 weeks; (3) High-dose pamidronate disodium group: intra-articular injection of 0.2 mL 100 μM pamidronate disodium solution once a week for 4 weeks. The pamidronate disodium solution was prepared by dissolving the drug in normal saline. At 8 weeks after surgery (4 weeks after the end of drug administration), the rabbits were euthanized by intravenous injection of excessive pentobarbital sodium. The right knee joint was dissected, and the femoral condyle and tibial plateau were collected for micro-CT, histological, and immunohistochemical analyses [3]

Absorption, Distribution and Excretion
For patients with creatinine clearance >90 mL/min, the 90 mg intravenous dose achieves a Cmax of 1.92 ± 1.08 µg/mL, a Tmax of 4 h, and an AUC of 10.2 ± 6.95 µgh/mL. In patients with creatinine clearance 61–90 mL/min, the 90 mg intravenous dose achieves a Cmax of 1.86 ± 0.50 µg/mL, a Tmax of 4 h, and an AUC of 10.7–3.91 µgh/mL. [A203264] In patients with creatinine clearance 30–60 mL/min, the 90 mg intravenous dose achieves a Cmax of 1.84 ± 0.58 µg/mL, a Tmax of 4 h, and an AUC of 10.1 ± 3.38 µgh/mL. For patients with creatinine clearance <30 mL/min, a 90 mg intravenous dose achieves a Cmax of 1.93 ± 0.53 µg/mL, a Tmax of 4 h, and an AUC of 34.0 ± 8.37 µgh/mL. Pamidronate sodium is completely excreted in the urine. 120 hours after administration, 46 ± 16% of the dose has been excreted in the urine. The mean total clearance of pamidronate sodium is 107 ± 50 mL/min, and the mean renal clearance is 49 ± 28 mL/min. Metabolites/Metabolites: Pamidronate sodium is not metabolized in the body. Pamidronate sodium is not metabolized and is completely excreted by the kidneys. Excretion route: Pamidronate sodium is not metabolized and is completely excreted by the kidneys. Half-life: The mean ± standard deviation elimination half-life is 28 ± 7 hours.

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Biological Half-Life
The mean elimination half-life of pamidronate sodium is 28 ± 7 hours.

Toxicity Summary
Pamidronate sodium's mechanism of action is the inhibition of bone resorption. Pamidronate adsorbs calcium phosphate (hydroxyapatite) crystals in bone and may directly block the dissolution of this mineral component in bone. In vitro studies have also shown that inhibiting osteoclast activity helps suppress bone resorption. Pamidronate sodium also targets farnesyl pyrophosphate (FPP) synthase. Nitrogenous bisphosphonates (such as pamidronate sodium, alendronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid sodium) appear to act as lipid analogs of isoprene diphosphate, thereby inhibiting an enzyme in the mevalonate pathway—FPP synthase. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprene lipids (FPP and GGPP), which are crucial for the post-translational farnesylation and geranylation of small GTPase signaling proteins. This activity inhibits osteoclast activity and reduces bone resorption and bone turnover. In postmenopausal women, it reduces elevated bone turnover, resulting in a net increase in bone mass on average.

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Effects during pregnancy and lactation
◉ Overview of use during lactation
Limited information suggests that the concentration of the drug in breast milk is very low after a mother receives an intravenous injection of 30 mg pamidronate sodium. Because pamidronate sodium has a serum half-life of approximately 3 hours, is highly bound to calcium, and has a low oral absorption rate (0.3% to 3% in adults), it is unlikely that breastfed infants will absorb pamidronate sodium. [1] Until more data are available, stopping breastfeeding 12 to 24 hours after administration should ensure that breastfed infants are minimally exposed to pamidronate sodium. Other evidence suggests that breastfeeding after long-term pamidronate sodium treatment appears to have no adverse effects on infants. Some experts recommend that if the mother has received pamidronate sodium treatment during pregnancy or lactation, the infant's serum calcium levels should be monitored during the first two months postpartum. [2]
◉ Effects on breastfed infants
A mother began receiving monthly 30 mg intravenous pamidronate sodium 6 months postpartum. After each administration, she expressed and discarded the milk for 48 hours. During the mother's pamidronate sodium treatment, approximately 80% of the infant's milk was breastfed, and the infant remained healthy and grew normally during this period. [1]
Because pamidronate sodium can persist in the body for several years after long-term administration, the following case may be of reference value. Three women with osteogenesis imperfecta or McCune-Albright syndrome received intravenous pamidronate sodium at cumulative doses of 6, 7.5, and 9 mg/kg per year for 2, 4, and 2.2 years, respectively. Their last administrations were 3 months, 3 months, 48 months (two infants), and 21 months before conception, respectively. None of the women resumed pamidronate during lactation, but all breastfed postpartum, one for 18 months, two for an unknown duration, and one for 6 weeks. None of the infants experienced adverse reactions to pamidronate. [3] Two other mothers received intravenous pamidronate before and during pregnancy. One mother received a total of 240 mg of pamidronate, with the last dose given in early pregnancy. She exclusively breastfed her infant for 6 months and continued breastfeeding until the infant was 12 months old. Her infant grew and developed normally without adverse reactions. [4] Another woman received alendronate for 6 months one year before pregnancy, followed by pamidronate every 4 months thereafter. Her infant was breastfed for 3 months (the extent of breastfeeding was not specified). The infant developed mild hypocalcemia at 2 months of age, but calcium levels were normal at 5 months of age, and long bone development was normal. [5]
A woman experienced transient osteoporosis during pregnancy, accompanied by foot pain. She received 30 mg of pamidronate sodium intravenously on days 3 and 8 postpartum, and 2 months later. She was instructed by her physician to discard breast milk within 24 hours after each administration. Her breastfed infant (feeding extent not specified) was growing normally at 15 months of age. [6]
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
The protein binding rate of pamidronate sodium in serum is approximately 54%.

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- In vitro toxicity to human bone marrow mesenchymal stem cells (hBMSCs): Treatment with pamidronate sodium at concentrations of 10, 50, and 100 μM for 7 days increased the apoptosis rate of hBMSCs. Flow cytometry analysis showed that the apoptosis rate in the control group was approximately 5%, while the apoptosis rates in the 10 μM, 50 μM and 100 μM drug groups were approximately 10%, 18% and 25%, respectively. After treating canine osteosarcoma cells (D17 and HMPOS) with sodium pamidronate at concentrations up to 100 μM for 72 hours, no significant cytotoxicity (e.g., cell membrane damage) was observed, as evidenced by no significant increase in lactate dehydrogenase (LDH) release compared to the control group [1][2].

[1]. Investigation of the effect of pamidronate disodium on the in vitro viability of osteosarcoma cellsfrom dogs. Am J Vet Res. 2005 May;66(5):885-91.

[2]. Pamidronate Disodium Leads to Bone Necrosis via Suppression of Wnt/β-Catenin Signaling in Human Bone Marrow Mesenchymal Stem Cells In Vitro. J Oral Maxillofac Surg. 2017 Jul;75(7):1476.e1-1476.e15.

[3]. Effects of pamidronate disodium on the loss of osteoarthritic subchondral bone and the expression of cartilaginous and subchondral osteoprotegerin and RANKL in rabbits. BMC Musculoskelet Disord. 2014 Nov 6;15:370.

1. Pamidronate sodium is a bisphosphonate drug commonly used to treat osteoporosis and bone metastases and other bone-related diseases. This study aims to explore its potential inhibitory effect on canine osteosarcoma cells and provide a theoretical basis for its application in the treatment of canine osteosarcoma [1]. 2. The study revealed a new mechanism by which pamidronate sodium induces osteonecrosis: the drug inhibits osteogenic differentiation (reduces alkaline phosphatase activity and mineralization) by inhibiting the Wnt/β-catenin signaling pathway in human bone marrow mesenchymal stem cells (hBMSCs) and promotes apoptosis, thereby impairing bone repair and leading to osteonecrosis. This finding provides new insights into the side effects of bisphosphonates and may help develop strategies to reduce the risk of osteonecrosis [2]
3. In a rabbit model of osteoarthritis, pamidronate sodium reduced subchondral bone loss by modulating the OPG/RANKL balance in subchondral bone (upregulating OPG and downregulating RANKL) and protected cartilage by increasing the expression of type II collagen in chondrocytes and decreasing the expression of MMP-13, thereby playing a protective role in osteoarthritis joints. This suggests that pamidronate sodium may have the potential to treat osteoarthritis [3]

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Pharmacodynamics>
Pamidronate is a second-generation nitrogenous bisphosphonate that inhibits osteoclast-mediated bone loss. It has a wide therapeutic index and a long duration of action, and for some indications, it can be administered once every 3-4 weeks. Patients should be informed of the risks of elevated blood urea nitrogen, renal tubular necrosis, and nephrotoxicity.

C3H11NO7P2

235.0695

235.001

C, 15.33; H, 4.72; N, 5.96; O, 47.64; P, 26.35

40391-99-9

Pamidronate Disodium;57248-88-1; 89131-02-2 (monosodium); 109552-15-0 (disodium pentahydrate);40391-99-9 (free acid)

4674

White to off-white solid powder

1.998 g/cm3

658.7ºC at 760 mmHg

226-228ºC

352.2ºC

1.611

-6.9

6

8

4

13

243

0

OC(P(O)(O)=O)(P(O)(O)=O)CCN

WRUUGTRCQOWXEG-UHFFFAOYSA-N

InChI=1S/C3H11NO7P2/c4-2-1-3(5,12(6,7)8)13(9,10)11/h5H,1-2,4H2,(H2,6,7,8)(H2,9,10,11)

(3-amino-1-hydroxy-1-phosphonopropyl)phosphonic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

H2O : ~5 mg/mL (~21.27 mM)
DMSO : ~1 mg/mL (~4.25 mM)

Solubility in Formulation 1: 2 mg/mL (8.51 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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