Effect Of Clodronate Research Articles

Effect of clodronate on gene expression in the peripheral blood of horses.

There are two FDA-approved bisphosphonate products, clodronate (Osphos®) and tiludronate (Tildren®), for use in horses. It is hypothesized that bisphosphonates can produce analgesic effects and prevent proper healing of microcracks in bone. Therefore, bisphosphonate use is banned in racehorses. However, bisphosphonates have a short detection window in the blood before sequestration in the skeleton, making the reliability of current drug tests questionable. Seven exercising Thoroughbred horses were administered clodronate (1.8 mg/kg i.m.), and four were administered saline. RNA was isolated from peripheral blood mononuclear cells (PBMCs) collected immediately before a single dose of clodronate or saline and then on Days 1, 6, 28, 56 and 182 post-dose. mRNA was sequenced and analysed for differentially expressed transcripts. While no single transcripts were differentially expressed, pathway analysis revealed that p38 MAPK (p = .04) and Ras (p = .04) pathways were upregulated, and cadherin signalling (p = .02) was downregulated on Day 1. Previously investigated biomarkers, cathepsin K (CTSK) and type 5 acid phosphatase (ACP5), were analysed with RT-qPCR in a targeted gene approach, with no significant difference observed. A significant effect of time on gene expression for ACP5 (p = .03) and CTSK (p < .0001) was observed. Thus, these genes warrant further investigation for detecting clodronate use over time.

In vitro evidence for the involvement of H2S pathway in the effect of clodronate during inflammatory response

Clodronate is a bisphosphonate agent commonly used as anti-osteoporotic drug. Throughout its use, additional anti-inflammatory and analgesic properties have been reported, although the benefits described in the literature could not solely relate to their inhibition of bone resorption. Thus, the purpose of our in vitro study is to investigate whether there are underlying mechanisms explaining the anti-inflammatory effect of clodronate and possibly involving hydrogen sulphide (H2S). Immortalised fibroblast-like synoviocyte cells (K4IM) were cultured and treated with clodronate in presence of TNF-α. Clodronate significantly modulated iNOS expression elicited by TNF-α. Inflammatory markers induced by TNF-α, including IL-1, IL-6, MCP-1 and RANTES, were also suppressed following administration of clodronate. Furthermore, the reduction in enzymatic biosynthesis of CSE-derived H2S, together with the reduction in CSE expression associated with TNF-α treatment, was reverted by clodronate, thus rescuing endogenous H2S pathway activity. Clodronate displays antinflammatory properties through the modulation of H2S pathway and cytokines levels, thus assuring the control of the inflammatory state. Although further investigation is needed to stress out how clodronate exerts its control on H2S pathway, here we showed for the first the involvement of H2S in the additive beneficial effects observed following clodronate therapy.

The Rationale for the Intra-Articular Administration of Clodronate in Osteoarthritis.

Background: Several pharmacological therapeutic approaches have been proposed to manage osteoarthritis (OA), including intra-articular (IA) injections. Although the discovery of clodronate, a bisphosphonate, dates back to the 1960s and the effects of its IA administration have been investigated for decades in animal models, mechanisms of action of this drug are not quite clear, particularly in OA. This scoping review is an overview of the biological as well as the clinical role of clodronic acid in OA. Method: A scoping review based on the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews) model was performed to characterize the mechanisms of action of IA clodronate in OA and to evaluate its efficacy from a clinical point of view. Results: Several effects of clodronate have been observed in animal models of OA, including depletion of synovial lining cells that results in reduced production of chemokines (IL-1, TNF- α), growth factors (TGF-β, BMP 2/4), and metalloproteases (MMP 2/3/9); prevention of cartilage damage, synovial hyperplasia, and proteoglycans loss; reduction in joint inflammation, joint swelling, and osteophyte formation. From a clinical perspective, patients with knee OA treated with IA clodronate experienced improvements in pain and joint mobility. Conclusion: Clodronate appears to have different mechanisms of action interfering with the pathogenic processes contributing to OA development and progression. This intervention demonstrated positive effects for patients affected by knee OA.

Clodronate, an inhibitor of the vesicular nucleotide transporter, ameliorates steatohepatitis and acute liver injury

The vesicular nucleotide transporter (VNUT) is responsible for the vesicular storage and release of ATP from various ATP-secreting cells, and it plays an essential role in purinergic signaling. Although extracellular ATP and its degradation products are known to mediate various inflammatory responses via purinoceptors, whether vesicular ATP release affects steatohepatitis and acute liver injury is far less understood. In the present study, we investigated the effects of clodronate, a potent and selective VNUT inhibitor, on acute and chronic liver inflammation in mice. In a model of methionine/choline-deficient diet-induced non-alcoholic steatohepatitis (NASH), the administration of clodronate reduced hepatic inflammation, fibrosis, and triglyceride accumulation. Clodronate also protected mice against high-fat/high-cholesterol diet-induced steatohepatitis. Moreover, prophylactic administration of clodronate prevented d-galactosamine and lipopolysaccharide-induced acute liver injury by reducing inflammatory cytokines and hepatocellular apoptosis. In vitro, clodronate inhibited glucose-induced vesicular ATP release mediated by VNUT and reduced the intracellular level and secretion of triglycerides in isolated hepatocytes. These results suggest that VNUT-dependent vesicular ATP release plays a crucial role in the recruitment of immune cells, cytokine production, and the aggravation of steatosis in the liver. Pharmacological inhibition of VNUT may provide therapeutic benefits in liver inflammatory disorders, including NASH and acute toxin-induced injury.

The Early Progression of Renal Injury in Obese Dahl Salt‐Sensitive Rats is Associated with Increased M2 Macrophage Infiltration

Previously, we reported that, prior to puberty, the obese Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) strain displays proteinuria and renal injury independent of hyperglycemia and elevations in arterial pressure. Recently, we observed that the early development of renal injury in the obese SSLepRmutant strain was associated with a 5‐fold increase in renal infiltration of macrophages compared to SSWT rats. In this study, we examined whether depletion of macrophages with clodronate would reduce renal injury in prepubescent obese SSLepRmutant rats. Four week‐old SSWT and SSLepRmutant rats were separated into four groups (n=8/group): (1) SSWT and (2) SSLepRmutant rats treated with vehicle and (3) SSWT and (4) SSLepRmutant rats treated with clodronate (50 mg/kg, i.p., twice/week) for 4 weeks. While administration of clodronate did not alter the macrophage population in SSWT rats, clodronate reduced macrophages in the kidneys from SSLepRmutant rats compared to vehicle‐treated rats (8±4 vs. 33±10 % gated, respectively; p&lt;0.05 vs. vehicle) without altering the monocyte population. Interestingly, we did not observe any effects on MAP in animals chronically treated with clodronate. At baseline, protein excretion was significantly higher in SSLepRmutant rats compared to SSWT rats (70±14 vs. 10±3 mg/day, respectively; p&lt;0.05 vs. SSWT) that further increased over the course of the study (335±82 vs. 45±14 mg/day, respectively; p&lt;0.05 vs. SSWT). After 2 weeks of treatment with clodronate, protein excretion was markedly reduced in the SSLepRmutant strain (159±32 vs. 303±52 mg/day; p&lt;0.05 vs. vehicle). However, after 4 weeks of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (346±195 vs. 399±50 mg/day). Chronic treatment with clodronate significantly decreased glomerular injury and renal fibrosis in the obese SSLepRmutant strain. Since clodronate depletes all macrophages: M1 (pro‐inflammatory) and M2 (anti‐inflammatory or pro‐inflammatory), we also examined macrophage polarization in the vehicle treated lean SSWT and obese SSLepRmutant strain prior to puberty at 8 weeks of age. We found that the M1/M2 ratio was significantly higher in the lean SSWT versus obese SSLepRmutant rats (1.60±0.05 vs. 0.54±10, respectively; p&lt;0.05 vs. SSWT), demonstrating more M2 macrophages in the kidneys of the obese SSLepRmutant strain. Overall, these data show that the early development of renal injury in the obese SSLepRmutant strain is associated with increased renal M2 macrophages and suggests that M2 macrophages may be a therapeutic target in obesity‐induced renal injury.Support or Funding InformationThis study was supported by DK109133.

Depletion of macrophages with clodronate partially reduces the progression renal injury in obese Dahl salt‐sensitive rats during prepubertal obesity

Prepubertal obesity (PPO) is now considered a risk factor for the development of proteinuria and renal injury in children. Recently, we reported that, prior to puberty, Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) rats display proteinuria and renal injury as early as 6 weeks of age independent of hyperglycemia and elevations in arterial pressure. Moreover, the early development of renal injury was associated with an increase in the renal infiltration of macrophages in the SSLepRmutant strain compared to their lean SSWT counterparts (39±14 vs. 11±3 % gated, respectively; p&lt;0.05 vs. SSWT). Therefore, the current study examined whether depletion of macrophages with clodronate would reduce renal injury in the SSLepRmutant strain during PPO. Four week‐old SSWT and SSLepRmutant rats were separated into four groups (n=4/group): (1) SSWT and (2) SSLepRmutant rats treated with vehicle‐PBS and (3) SSWT and (4) SSLepRmutant rats treated with clodronate (50 mg/kg, i.p., twice/week) for 4 weeks. While administration of clodronate did not have an effect in SSWT rats, clodronate significantly depleted macrophages in the kidneys of SSLepRmutant rats compared to vehicle‐treated rats without altering the monocyte population (8±4 versus 33±10 % gated, respectively; p&lt;0.05 vs. vehicle). At baseline, protein excretion was significantly higher in SSLepRmutant rats compared to SSWT rats (70±14 vs. 10±3 mg/day, respectively; p&lt;0.05 vs. SSWT) that further increased over the course of the study (335±82 vs. 45±14 mg/day, respectively; p&lt;0.05 vs. SSWT). Clodronate had no effect on the progression of proteinuria or renal histopathology in SSWT rats. After 2 weeks of treatment with clodronate, protein excretion was markedly reduced in the SSLepRmutant strain (86±36 vs. 200±35 mg/day; p&lt;0.05 vs. vehicle). However, after 4 weeks of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (270±90 vs. 335±82 mg/day). The kidneys from SSLepRmutant rats displayed glomerular injury with increased mesangial expansion and renal fibrosis versus SSWT rats. Interestingly, treatment with clodronate had no effect on glomerular injury but significantly decreased renal fibrosis in the SSLepRmutant strain compared to their vehicle‐treated obese counterparts. Overall, these data indicate that the early development of renal injury in the SSLepRmutant strain is associated with macrophage infiltration, which can be blunted by chronic macrophage depletion.Support or Funding InformationThis study was supported by GM104357 and DK109133.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tiludronate and clodronate do not affect bone structure or remodeling kinetics over a 60\xa0day randomized trial

BackgroundTiludronate and clodronate are FDA-approved bisphosphonate drug therapies for navicular disease in horses. Although clinical studies have determined their ability to reduce lameness associated with skeletal disorders in horses, data regarding the effect on bone structure and remodeling is lacking. Additionally, due to off-label use of these drugs in young performance horses, effects on bone in young horses need to be investigated. Therefore, the purpose of this randomized, experimental pilot study was to determine the effect of tiludronate and clodronate on normal bone cells, structure and remodeling after 60 days in clinically normal, young horses. Additionally, the effect of clodronate on bone healing 60 days after an induced defect was investigated.ResultsAll horses tolerated surgery well, with no post-surgery lameness and all acquired biopsies being adequate for analyses. Overall, tiludronate and clodronate did not significantly alter any bone structure or remodeling parameters, as evaluated by microCT and dynamic histomorphometry. Tiludronate did not extensively impact bone formation or resorption parameters as evaluated by static histomorphometry. Similarly, clodronate did not affect bone formation or resorption after 60 days. Sixty days post-defect, healing was minimally affected by clodronate.ConclusionsTiludronate and clodronate do not appear to significantly impact bone tissue on a structural or cellular level using standard dose and administration schedules.

Effect of Clodronate on Antigenic Profile, Growth, and Differentiation of Osteoblast-Like Cells

To evaluate the role of osteoblasts in bisphosphonate-related osteonecrosis of the jaw (BRONJ) by studying the effects of different concentrations of clodronate, a non-nitrogen-containing bisphosphonate, on osteoblast growth, differentiation, and antigenic profile. Osteoblast-like cells (MG63) were incubated in culture medium with different doses of clodronate. Their proliferative capacity was determined with a spectrophotometric technique (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium assay). Flow cytometry was used to study the antigenic profile. Cell differentiation was evaluated by nodule formation and alkaline phosphatase (ALP) activity was measured by spectrophotometric assay. Clodronate had a significant stimulatory effect on osteoblast-like cell (MG63) proliferation (P<.05). A significant decrease in the expression of CD54, CD80, CD86, and HLA-DR membrane antigens versus controls was observed after 24hours of treatment with the different clodronate doses assayed (P<.05). A significant decrease (P= .004) in ALP activity was found after 24hours of treatment with the lowest dose (10(-9)mol/L), and a significant decrease in calcium deposition was found after 15 and 21days of treatment (P < .05). Clodronate increases the proliferation of MG63 osteoblast-like cells and decreases their differentiation capacity, generally at low doses, and modulates the expression of costimulatory molecules associated with immune function. Clodronate exerts its effect on osteoblasts by altering their physiology and impairing their repair capacity, which could be related to the development of BRONJ. However, further research is warranted to elucidate fully the mechanisms by which bisphosphonates can produce this disease.

Effects of clodronate and alendronate on osteoclast and osteoblast co-cultures on silk–hydroxyapatite films

The goal of this study was to explore the effects of osteoporosis-related therapeutics on bone remodeling in vitro. A previously established bone-tissue mimetic system consisting of silk protein biomaterials in combination with hydroxyapatite and human cells was used for the study. Silk–hydroxyapatite films were pre-complexed with the non-nitrogenous bisphosphonate clodronate or the nitrogenous bisphosphonate alendronate and cultured with THP-1 human acute monocytic leukemia cell line-derived osteoclasts, human mesenchymal stem cell derived osteoblasts or a direct co-culture of the two cell types. Metabolic activity, calcium deposition and alkaline phosphatase activity were assessed over 12weeks, and reconstructed remodeled biomaterial surfaces were also evaluated for quantitative morphological changes. Increased metabolic activity and increased roughness were found on the clodronate-complexed biomaterial substrates remodeled by osteoblasts and co-cultures of osteoblasts with osteoclasts, even at doses high enough to cause a 90% decrease in osteoclast metabolic activity. Films complexed with low doses of alendronate resulted in increased metabolic activity and calcium deposition by osteoblasts, while higher doses were similarly toxic among osteoclasts, osteoblasts and co-cultures. These results point to the utility of these well-defined bone-mimetic in vitro cultures as useful screens for therapeutics for bone-related diseases, particularly with the ability to conduct studies for extended duration (here for 12weeks) and with pre-complexed drugs to mimic conditions found in vivo.

Clodronate changes neurobiological effects of pulsed magnetic field on diabetic rats with peripheral neuropathy

Several studies have reported that pulsed magnetic fields (PMFs) can be a choice of therapy for diabetic peripheral neuropathy. However, the exact underlying mechanism of PMF is still not known. The purpose of this study was, therefore, to investigate the effects of clodronate encapsulated with liposome, a specific agent depleting macrophage, on PMF-treated streptozotocin-induced type I diabetic rats with peripheral neuropathy. Effects of PMF, liposome-encapsulated clodronate (LEC) or their combined treatments were investigated in diabetic rats by measuring the thermal latencies, mechanical thresholds, whole blood glucose levels, serum insulin level, and body mass. In diabetic rats, PMF exhibited a decrease in the blood glucose levels but did not change the serum insulin level. Both mechanical thresholds and thermal latencies of diabetic rats enhanced throughout the PMF treatment. During the PMF treatment, the administration of LEC suppressed the PMF-induced decrease in blood glucose level, PMF-induced increase in mechanical threshold and thermal latencies in diabetic animals. In addition, PMF reduced the LEC-induced increase in insulin levels of diabetic rats. Findings demonstrated that although effects of both PMF alone and LEC alone on diabetic animals are mostly positive, LEC may remove the therapeutic efficacies of PMF in combined treatment.

The effect of clodronate on a mevalonate kinase deficiency cellular model

A potential anti-inflammatory effect of clodronate--an aminobisphosphonate--was described to antagonize the pro-inflammatory effects of the block in the mevalonate pathway, the main feature of a rare auto-inflammatory disease called mevalonate kinase deficiency (MKD). In this study we evaluated the potential anti-inflammatory effect of clodronate in MKD--a still orphan drug pediatric disease. We studied some biological parameters, nitric oxide production using Griess reagents and programmed cell death by flow cytometry, as common inflammatory parameters in MKD, in the presence of different doses of clodronate (1, 10 and 100 μM). In our cellular model and in monocytes from patients with MKD, clodronate induced an increase in programed cell death and nitric oxide production in comparison with non-treated cells. Our findings suggest that clodronate does not have an anti-inflammatory effect as previously reported but that it increases the epiphenomena of this pediatric disease.

Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells

Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is a side effect primarily in patients receiving highly potent nitrogen-containing bisphosphonates. The exact etiopathology is unknown. In addition to reduced bone remodeling, there may also be an impact on soft tissues. The impact of nitrogen- (ibandronate, pamidronate, zoledronate) and non-nitrogen-containing bisphosphonates (clodronate) on human umbilicord vein endothelial cells (HUVEC), fibroblasts and osteogenic cells was analyzed employing cell viability testing and a scratch wound assay. The impact on the cell morphology of vital-stained osteogenic cells was investigated by cell visualization (confocal laser scanning microscopy). Pamidronate and zoledronate had the greatest negative impact on all cell lines, whereas the impact of ibandronate and clodronate was less distinct. The effect of clodronate on HUVEC and fibroblasts was particularly marginal. BP-ONJ could be a multifactorial event with multicellular impairments. This might result in altered wound healing. The increased impact of the highly potent bisphosphonates, particularly on non-bone cells, may explain the higher occurrence of BP-ONJ.

344 POSTER Skeletal protective effect of clodronate in primary breast cancer-bone mass, bone turnover, and skeletal-related events (SRE's)

344 POSTER Skeletal protective effect of clodronate in primary breast cancer-bone mass, bone turnover, and skeletal-related events (SRE's)

The Effect of Clodronate on the Integrity and Viability of Rat Small Intestine in Vitro-A Comparison with EDTA

Although substances, which can increase the paracellular permeability of intestinal mucosa, could be very helpful for increasing the bioavailability of hydrophilic drugs, they are not used therapeutically due to the possibilities of acute or long-term toxicity (intestinal inflammations due to penetration of bacterial fragments into subepithelial spaces). In this paper the abilities of a calcium chelator EDTA and clodronate (a first generation bisphosphonate) to increase the paracellular permeability were assessed using rat jejunum in side-by-side diffusion chambers while the viability of the tissue was monitored by transepithelial potential difference. Although clodronate is less potent than EDTA in depleting calcium from the intestinal tissue, it significantly increased the paracellular permeability of viable rat jejunum "in vitro" when tested at 15 mM and higher concentrations (the highest therapeutic dose dissolved in 250 ml gives a 22 mM solution of clodronate). This effect was reversible under "high-calcium" conditions. Since clodronate therapy does not have any long-term consequences it was concluded that a safe, transient increase of small intestinal permeability is possible. However, the acute gastrointestinal undesired effects, which can develop during the therapy with high doses of clodronate, might also occur after oral applications of paracellular permeability enhancers. Namely, 30 mM and higher concentrations of clodronate caused a loss of the tissue viability in all rat jejunal segments tested in "in vitro" conditions. A similar effect was observed with much lower concentrations of EDTA.

Effects of bisphosphonates and insulin-like growth factor-1 (IGF-1) on prostate cancer cells in vitro

4758 Background: The bisphosphonate (BP) zoledronic acid (Zol) reduces skeletal morbidity from metastatic bone disease in patients with prostate cancer, presumably through inhibition of osteolysis. Osteolysis undermines skeletal integrity and releases IGF-1, thus stimulating prostate cancer growth. In addition, BPs have been shown to inhibit cancer cell growth. Therefore, we investigated the effects of BPs on prostate cancer cell growth in vitro. Methods: The effects of clodronate (Clod), pamidronate (Pam), ibandronate (Iban), and Zol on human PC-3 cell growth and survival were investigated after 1–6 days in culture. Cells were grown in media containing 1 to 100 microM BPs in the absence or presence (for Clod and Zol only) of 100 ng/mL IGF-1. Cell survival and growth were assessed by MTT assay, DNA content, and crystal violet staining. Proportion of cells in G0/G1 phase was determined by fluorescent staining, and apoptotic cells were identified by annexin staining. Results: Cell survival was not affected by Clod, but decreased in a time- and dose-dependent manner with Pam, Iban, and Zol. The largest effect was observed after 6 days with 100 microM Zol. Clod and Pam had no significant effect on cell cycle distribution. In contrast, 100 microM Zol or Iban produced cell accumulation in G0/G1 after 1, 2, and 4 days. The percentage of apoptotic cells was increased 2-fold by both Pam and Zol, and 1.3-fold by Iban. Clod had no effect. In the presence of IGF-1, cell growth increased by 28% (standard error of the mean [SEM] = 5%) at day 2 and by 23% (SEM = 7%) at day 6 (P < .001). These mitogenic effects were blocked by 1 microM Zol. Cell proliferation in the presence of IGF-1 was blocked by 10 microM Zol, and 100 microM Zol resulted in 85% cell death at day 6. Mitogenesis by IGF-1 was blocked by 10 microM Clod, but cell growth was not inhibited even at 100 microM Clod. Conclusions: Therefore, BPs, especially Zol, have direct effects on prostate cancer cells, inhibiting growth through cytostatic, apoptotic, and antimitogenic activities. These activities could contribute to the beneficial effects of Zol in the treatment of prostate cancer-induced bone disease. No significant financial relationships to disclose.

A double blind, parallel, placebo controlled, dose ranging study on efficacy and safety of oral clodronate in cancer induced osteolysis measured by biochemical markers of bone resorption

8056 Background: The bisphosphonate clodronate is effective in the treatment of osteolytic bone metastases at an oral dose of 1600 mg per day. However other doses such as 2400 mg and 3200 mg have also been used. Using bone markers as surrogate biomarkers for osteolysis, the purpose of the study was to investigate the efficacy of oral clodronate (BONEFOS®) across a range of doses in patients with tumour induced osteolysis. Methods: 125 patients with metastatic bone disease (87 breast, 33 prostate and 5 other tumour types) were randomized to oral clodronate (800, 1600, 2400 and 3200 mg) or placebo in a phase II double blind, dose ranging, multicentre study with ethical approval. During 6 weeks of treatment, bone markers were assessed weekly. Results: Doses of 1600 mg or above were all effective in producing a mean fall in both urinary N-telopeptide (NTX, see table for breast data) and C-telopeptide of over 40 % from baseline values. Moreover, effects of clodronate were seen by 1 week. The 800mg dose gave a smaller effect and placebo gave no effect. The number of adverse events in the treatment arms was not significantly different from the placebo arm, except for the 3200 mg dose, where a higher number of patients had significant diarrhoea. Conclusions: This is the first such dose-ranging study with clodronate, where bone markers have been used as the primary end-point to assess efficacy. The data are consistent with the known clinical use of 1600 mg daily, but suggest that 2400 mg may also be used without increased toxicity. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Schering OY Schering OY Schering OY Schering OY

A double blind, parallel, placebo controlled, dose ranging study on efficacy and safety of oral clodronate in cancer induced osteolysis measured by biochemical markers of bone resorption

8056 Background: The bisphosphonate clodronate is effective in the treatment of osteolytic bone metastases at an oral dose of 1600 mg per day. However other doses such as 2400 mg and 3200 mg have also been used. Using bone markers as surrogate biomarkers for osteolysis, the purpose of the study was to investigate the efficacy of oral clodronate (BONEFOS®) across a range of doses in patients with tumour induced osteolysis. Methods: 125 patients with metastatic bone disease (87 breast, 33 prostate and 5 other tumour types) were randomized to oral clodronate (800, 1600, 2400 and 3200 mg) or placebo in a phase II double blind, dose ranging, multicentre study with ethical approval. During 6 weeks of treatment, bone markers were assessed weekly. Results: Doses of 1600 mg or above were all effective in producing a mean fall in both urinary N-telopeptide (NTX, see table for breast data) and C-telopeptide of over 40 % from baseline values. Moreover, effects of clodronate were seen by 1 week. The 800mg dose gave a smaller effect and placebo gave no effect. The number of adverse events in the treatment arms was not significantly different from the placebo arm, except for the 3200 mg dose, where a higher number of patients had significant diarrhoea. Conclusions: This is the first such dose-ranging study with clodronate, where bone markers have been used as the primary end-point to assess efficacy. The data are consistent with the known clinical use of 1600 mg daily, but suggest that 2400 mg may also be used without increased toxicity. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Schering OY Schering OY Schering OY Schering OY

Effects of bisphosphonates and insulin-like growth factor-1 (IGF-1) on prostate cancer cells in vitro

4758 Background: The bisphosphonate (BP) zoledronic acid (Zol) reduces skeletal morbidity from metastatic bone disease in patients with prostate cancer, presumably through inhibition of osteolysis. Osteolysis undermines skeletal integrity and releases IGF-1, thus stimulating prostate cancer growth. In addition, BPs have been shown to inhibit cancer cell growth. Therefore, we investigated the effects of BPs on prostate cancer cell growth in vitro. Methods: The effects of clodronate (Clod), pamidronate (Pam), ibandronate (Iban), and Zol on human PC-3 cell growth and survival were investigated after 1–6 days in culture. Cells were grown in media containing 1 to 100 microM BPs in the absence or presence (for Clod and Zol only) of 100 ng/mL IGF-1. Cell survival and growth were assessed by MTT assay, DNA content, and crystal violet staining. Proportion of cells in G0/G1 phase was determined by fluorescent staining, and apoptotic cells were identified by annexin staining. Results: Cell survival was not affected by Clod, but decreased in a time- and dose-dependent manner with Pam, Iban, and Zol. The largest effect was observed after 6 days with 100 microM Zol. Clod and Pam had no significant effect on cell cycle distribution. In contrast, 100 microM Zol or Iban produced cell accumulation in G0/G1 after 1, 2, and 4 days. The percentage of apoptotic cells was increased 2-fold by both Pam and Zol, and 1.3-fold by Iban. Clod had no effect. In the presence of IGF-1, cell growth increased by 28% (standard error of the mean [SEM] = 5%) at day 2 and by 23% (SEM = 7%) at day 6 (P < .001). These mitogenic effects were blocked by 1 microM Zol. Cell proliferation in the presence of IGF-1 was blocked by 10 microM Zol, and 100 microM Zol resulted in 85% cell death at day 6. Mitogenesis by IGF-1 was blocked by 10 microM Clod, but cell growth was not inhibited even at 100 microM Clod. Conclusions: Therefore, BPs, especially Zol, have direct effects on prostate cancer cells, inhibiting growth through cytostatic, apoptotic, and antimitogenic activities. These activities could contribute to the beneficial effects of Zol in the treatment of prostate cancer-induced bone disease. No significant financial relationships to disclose.

Regulation of MMP-9 (gelatinase B) in activated human monocyte/macrophages by two different types of bisphosphonates

Metalloproteinases (MMP), particularly MMP-9 produced by the intratumor monocyte/macrophages, play an important role in tumor invasion and metastases. Recent clinical trials in patients with primary breast cancer suggest that bisphosphonates (BP), above all clodronate, may reduce bone metastases. The aim of the present study was to evaluate whether the effects of BPs on cancer dissemination include inhibition of MMP-9 production in human monocyte/macrophages. The effects of clodronate and pamidronate on the MMP-9 expression in and secretion from stimulated human monocyte/macrophages were measured using quantitative reverse transcriptase — polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsorbent assay (ELISA), respectively. The MMP-9 mRNA levels remained relatively stable in the presence of clodronate. In contrast, pamidronate at 30 μM–300 μM increased the mRNA levels 5- to 10-fold. MMP-9 secretion was dose-dependently down-regulated by clodronate whereas pamidronate at 30 μM induced a 50% increase on MMP-9 secretion (p < 0.05), followed by a down-regulation at higher concentrations. The results suggest that MMP-9 is differentially regulated at mRNA and enzyme protein level by BPs, which affect ATP-dependent intracellular enzymes (clodronate) or post-translational modification of GTPases (pamidronate). These findings may have implications for the therapeutic use of these compounds.

The cellular uptake and metabolism of clodronate in RAW 264 macrophages.

Non-nitrogen-containing bisphosphonates, such as clodronate (dichloromethylene bisphosphonate), appear to act as prodrugs, their active form being the AppCp-type analogues of ATP. To further elucidate this, we examined the cellular uptake of clodronate and intracellular accumulation of the metabolite of clodronate (AppCCl2p) in RAW 264 macrophages, the influence of clodronate metabolism on the intracellular ATP concentration, and the time course of clodronate metabolism and the effects of clodronate on cytokine secretion from macrophages. The cellular uptake of clodronate was measured using 14C-labeled clodronate. AppCCl2p was determined in cell extracts by using an ion-pairing HPLC-ESI-MS. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay. Of the clodronate internalized by macrophages in vitro, 30-55% is metabolized to AppCCl2p, which accumulates to high intracellular concentrations during the first 12 h of exposure. This accumulation does not affect the ATP levels in the cells. The time course of metabolite appearance in the cells and the inhibition of cytokine secretion were very similar. These results strongly support the idea that clodronate acts as a prodrug, the active form being its intracellular AppCCl2p metabolite.