- 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]

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]

- 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]

In vitro toxicity to human bone marrow mesenchymal stem cells (hBMSCs): After treatment with pamidronate sodium at concentrations of 10, 50, and 100 μM for 7 days, the apoptosis rate of hBMSCs increased. 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 treatment with pamidronate sodium at concentrations up to 100 μM for 72 hours, no significant cytotoxicity (e.g., cell membrane damage) was observed in canine osteosarcoma cells (D17 and HMPOS), 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) and promotes apoptosis by inhibiting the Wnt/β-catenin signaling pathway in human bone marrow mesenchymal stem cells (hBMSCs), thereby impairing bone repair and leading to osteonecrosis. This finding provides a new perspective for understanding the side effects of bisphosphonates and may help to develop strategies to reduce the risk of osteonecrosis[2].
3. In a rabbit model of osteoarthritis, pamidronate sodium reduced subchondral bone loss by regulating 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 exerting a protective effect on osteoarthritis joints. This suggests that pamidronate sodium may have the potential to treat osteoarthritis [3].
An aminobisphosphonate that inhibits bone resorption is used to treat osteolytic lesions, bone pain, and severe hypercalcemia associated with malignant tumors.

C3H10NNAO7P2

257.05

257.991

C, 14.02; H, 3.92; N, 5.45; Na, 8.94; O, 43.57; P, 24.10

89131-02-2

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

23678535

Typically exists as solid at room temperature

5

8

4

14

278

0

C(O)(CCN)(P(=O)([O-])O)P(=O)(O)O.[Na+]

AFICAMMULQSNNG-UHFFFAOYSA-M

InChI=1S/C3H11NO7P2.Na/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);/q;+1/p-1

sodium;(3-amino-1-hydroxy-1-phosphonopropyl)-hydroxyphosphinate

J710.127I; Pamidronic acid monosodium salt; Pamidronate monosodium

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)