Macrophage function - inhibitor [1]
- Specifically inhibits lipopolysaccharide (LPS)-stimulated production of interleukin-6 (IL-6) and tumor necrosis factor (TNF) in RAW 264 macrophages. [1]

Osteoclasts are induced to function swiftly by clodronate disodium salt, which blocks the absorption, internalization, and translocation of ADP into the mitochondria. Consequently, the inhibition of ATP generation causes the release of cytochrome C into the cytoplasm, which causes cellular fluorescence [2].

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In murine macrophage-like RAW 264 cells, lipopolysaccharide (LPS, 10 μg/ml) stimulation for 24 hours induced substantial production of IL-6 and TNF. Unstimulated cells did not produce detectable amounts of these cytokines. [1]
- Liposome-encapsulated clodronate (1, 10, 100 μM) inhibited LPS-stimulated IL-6 production in a dose-dependent manner. At 100 μM, IL-6 production was completely abolished. Non-loaded liposomes at the highest concentration reduced IL-6 to 43% of control, indicating the effect is mostly due to the drug. [1]
- Free clodronate (10, 100, 1000 μM) also inhibited IL-6 production dose-dependently, but required at least ten times higher concentrations to achieve the same level of inhibition as the liposomal formulation. [1]
- Liposome-encapsulated clodronate also inhibited LPS-stimulated TNF production, although to a lesser extent than IL-6. At 100 μM, TNF production was reduced to about 20% of control. Non-loaded liposomes had only a minor effect, suggesting the drug is primarily responsible for TNF inhibition. [1]
- Free clodronate inhibited TNF production less potently, requiring higher concentrations for an effect comparable to the liposomal form. [1]
- The inhibition of cytokine production was not attributed to non-specific cytotoxicity. MTT staining assays showed that liposome-encapsulated clodronate and non-loaded liposomes caused only a slight decrease in cell viability (approximately 20% at the highest concentration), while free clodronate had no effect on MTT staining. [1]
- This is the first reported observation of the cytokine-inhibitory property of clodronate. [1]

At doses of 12.5 and 25 mg/kg, clodronate disodium salt (6.25–25 mg/kg; lateral; once daily for 28 days) somewhat reduced hindlimb edema [3].

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In a 3-year, prospective, open-label, randomized, controlled study, 30 postmenopausal women with osteoporosis received oral clodronic acid 800 mg daily, plus 500 mg calcium and 400 IU vitamin D. A control group of 49 women received only calcium and vitamin D. [2]
- After 36 months, the clodronic acid group showed a significant increase in bone mineral density (BMD) from baseline at the femoral neck (3.2 ± 2.9%), trochanter (2.2 ± 2.9%), and lumbar spine (3.1 ± 3%). In contrast, the control group experienced significant decreases in BMD at all these sites. The differences between groups were statistically significant (p < 0.05 to p < 0.01). [2]
- Clodronic acid treatment resulted in a significant decrease in urinary hydroxyproline (a bone resorption marker) of 38.3% from baseline at 3 years (p < 0.05). No significant change in hydroxyproline was observed in the control group. [2]
- Markers of bone formation (serum alkaline phosphatase and osteocalcin) were not suppressed compared to baseline in the clodronic acid group, suggesting an uncoupling of bone turnover (decreased resorption without decreased formation). At 3 years, these markers were significantly lower in the clodronic acid group than in the control group. [2]
- No significant changes in serum calcium, phosphate, or parathyroid hormone (PTH) were observed in the clodronic acid group compared to baseline over the 3-year period. [2]
- The incidence of new vertebral fractures was 2/30 in the clodronic acid group versus 1/49 in the control group (p = 0.19). Peripheral fracture rates were similar between groups. [2]

Cell Culture and Drug Treatment: Murine macrophage-like RAW 264 cells were plated in 96-well plates (2 × 10⁵ cells/well) and allowed to adhere. After washing, cells were exposed to various concentrations of liposome-encapsulated clodronate (1, 10, 100 μM), corresponding concentrations of non-loaded liposomes, or free clodronate (10, 100, 1000 μM) for 20 hours. Control wells were treated with buffer. [1]
- Cytokine Induction: After drug exposure, the medium was aspirated, cells were washed, and 100 μl of serum-free medium containing LPS (10 μg/ml) was added to each well. Cells were incubated for an additional 24 hours to induce cytokine production. Non-LPS treated control wells were included. [1]
- Cytokine Analysis (DELFIA): Cell-free supernatants were collected and assayed for IL-6 and TNF using a non-competitive immunoassay based on the dissociation-enhanced lanthanide fluoroimmunoassay principle. Microtiter wells were coated with capture antibodies, samples or standards were added, followed by biotinylated detection antibodies, and finally europium-labeled streptavidin. Fluorescence was measured after enhancement, and cytokine concentrations were quantified against standard curves. This method allowed detection at pg levels with good reproducibility (CV% typically <5). [1]
- Cytotoxicity Assay (MTT): In separate plates, cells were treated with the drug preparations as described above. Instead of LPS induction, the medium was replaced with 100 μl of serum-free medium containing MTT (0.5 mg/ml). After 2 hours incubation, 100 μl of lysis buffer (20% SDS, 50% DMF in water) was added. After overnight incubation, absorbance was measured at 570 nm to assess cell viability. [1]

Animal/Disease Models: Male Lewis rat, 7 years of age [3]
Doses: 6.25, 12.5, and 25 mg/kg
Route of Administration: Oral; one time/day for 28 days
Experimental Results: 25 mg/kg on days 21 and 28 Hind paw swelling was Dramatically less in the adjuvant arthritis (AA) control group (87% and 88% of the AA control group, respectively) and on day 28 in the 12.5 mg/kg dose group.

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The paper provides background information: clodronate is a small, highly water-soluble compound that does not readily enter cells. It binds avidly to hydroxyapatite and accumulates mainly in calcified tissue in vivo, with soft tissues exposed to the drug for only a short period. [1]

In this in vitro study, free clodronate at concentrations up to 1000 μM showed no cytotoxic effect on RAW 264 cells, as measured by the MTT assay. [1]
- Liposome-encapsulated clodronate and non-loaded liposomes caused a slight, dose-dependent decrease in MTT staining (approximately 20% reduction at the highest concentration of 100 μM drug/lipid), suggesting mild cytotoxicity primarily attributable to the liposomal lipids rather than the drug itself. [1]

[1]. Clodronate (dichloromethylene bisphosphonate) inhibits LPS-stimulated IL-6 and TNF production by RAW 264 cells. Life Sci. 1994;54(14):PL229‐PL234.

[2]. Clodronic acid in the treatment of postmenopausal osteoporosis. Clin Drug Investig. 2007;27(6):419‐433.

[3]. Effects of clodronate and alendronate on local and systemic changes in bone metabolism in rats with adjuvant arthritis. Inflammation. 2004;28(1):15‐21.

Clodronic acid disodium salt is the disodium salt of Clodronic acid. It inhibits bone resorption and soft tissue calcification and is usually used in tetrahydrate form as adjunctive therapy for severe hypercalcemia associated with malignant tumors, as well as for osteolytic lesions and bone pain associated with bone metastases. It plays a role in maintaining bone density. It is an organosodium salt and a one-carbon compound. It contains the Clodronic acid ion (2-). Clodronic acid disodium salt is the disodium salt of a nitrogen-free bisphosphonate analog of natural pyrophosphate. Clodronic acid binds to calcium, inhibiting osteoclast-mediated bone resorption and the formation and dissolution of hydroxyapatite crystals, thereby reducing bone turnover. This drug can control hypercalcemia associated with malignant tumors, inhibit osteolytic bone metastases, and relieve pain. A bisphosphonate that affects calcium metabolism. It inhibits bone resorption and soft tissue calcification.

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Clodronate is a bisphosphonate drug, primarily known as a potent inhibitor of osteoclastic bone resorption. It is used clinically for conditions like osteoporosis and hypercalcemia of malignancy. [1]
- This study investigates a novel property of clodronate: its ability to inhibit cytokine production by macrophages. It demonstrates that liposome-encapsulated clodronate potently inhibits LPS-stimulated IL-6 and TNF production in RAW 264 cells, with IL-6 being more sensitive than TNF. Free clodronate is significantly less potent, highlighting the importance of liposomal delivery for targeting macrophages. [1]
- The mechanism of action is not fully elucidated in this study. It suggests that the growth inhibitory action of clodronate in macrophages may involve intracellular iron depletion, and similar mechanisms might underlie the cytokine inhibitory effect. [1]
- The findings support the potential use of liposomal clodronate as a macrophage-suppressive agent in chronic inflammatory diseases like rheumatoid arthritis, where macrophage-derived cytokines play a key pathogenic role. Local administration (e.g., intra-articular injection) could inactivate macrophages in the joint cavity. [1]

CH2CL2NA2O6P2

288.86

287.849

22560-50-5

Clodronate disodium tetrahydrate;88416-50-6;Clodronic acid;10596-23-3

31195

White to off-white solid powder

2.306g/cm3

474.7ºC at 760mmHg

>330ºC

240.9ºC

2.55E-10mmHg at 25°C

1.307

2

6

2

13

206

0

HJKBJIYDJLVSAO-UHFFFAOYSA-L

InChI=1S/CH4Cl2O6P2.2Na/c2-1(3,10(4,5)6)11(7,8)9;;/h(H2,4,5,6)(H2,7,8,9);;/q;2*+1/p-2

disodium;[dichloro-[hydroxy(oxido)phosphoryl]methyl]-hydroxyphosphinate

clodronic acid disodium salt; Dichloromethylenediphosphonic acid disodium salt; Clodronate sodium; disodium dichloromethylene diphosphonate; Foreign brand names: Bonefos; Clasteon; Difosfonal; Loron; Mebonat; Ossiten. Code name: CL2MDP; DMDP.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~346.19 mM)
DMSO :< 1 mg/mL

Solubility in Formulation 1: 100 mg/mL (346.19 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.)