Nitrogen containing Bisphosphonates

Author: Alessandro Mussa
Date: 31/07/2007



Nitrogen-containing bisphosphonates act intracellularly by inhibiting farnesyl diphosphate synthase (FPP), an enzyme of the mevalonate pathway, thereby preventing prenylation of small GTPase signaling proteins required for normal cellular function. Inhibition of farnesyl diphosphate synthase also seems to account for their antitumor effects observed in vitro and for the activation of g,yTcells, a feature of the acute phase response to bisphosphonate treatment in humans. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that seems to involve primarily the formation of cytotoxic metabolites in osteoclasts.


MolecularMechanisms of Action of Bisphosphonates: Current Status

The Use of Molecular Markers of Bone Turnover in the Management of Patients with Metastatic Bone Disease

Effect of NBPs on Mevalonate Pathway

The inhibition of farnesyl diphosphate synthase (FPP) leads to

  • an increase of uphill substrates (IPP)
  • a decrease of downhill substrates (cholesterol, Coenzyme Q, farnesylPP and related metabolism)

Increase of IPP

Zoledronic acid-induced IPP/ApppI production in vivo 2007

ApppI is a toxic metabolite carrying a negative info (AMP= low ATP; IPP=lack of NADPH)

Mevalonate pathway intermediates downregulate zoledronic acid-induced isopentenyl pyrophosphate and ATP analog formation in human breast cancer cells 2010

Decrease of

Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by

targeting the mevalonate pathway, 2010

Clinically compatible doses of ZA inhibits the activity of the Mev pathway, but not the proliferative rate of TUBO cells in vitro. (A) ZA induces a dose-dependent inhibition of FPP synthase in TUBO cells, as shown by the significant intracellular reduction of FPP, ubiquinone and cholesterol, and the significant increase of IPP (P-values from <0.02 to <0.001 for all ZA concentrations, Student’s t-test). Results are expressed as the mean ± S.E.M. of three independent experiments. (B) Western blot analysis of prenylated (P) and unprenylated (U) Ras and pull-down assay for Ras-GTP in TUBO cells cultured in the absence (CTRL), or in the presence of 1, 10 and 100 μM ZA. The specific farnesyl transferase inhibitor (FTI) 277 (10 μM) was used as a positive control of Ras inhibition. The expression of GAPDH was used as a control of equal protein loading. Results are from one representative out of three experiments. © ZA inhibition of TUBO cells proliferation was assessed by measuring BrdU incorporation. TUBO cells were incubated in quadruplicates without ZA (♦) or with ZA at 1 μM (▪), 10 μM (▴) or 100 μM (▾). BrdU incorporation was measured after 8, 48, 96 and 102 hrs as the mean optical density measured for each quadruplicate. As shown, ZA 1 μM never affected tumour cell proliferation, whereas 10 μM ZA induced a time-dependent inhibition and 100 μM ZA a complete abrogation of BrdU incorporation. Results are expressed as mean ± S.E.M. of three experiments.

Modulation of macrophages functions by low-dose ZA. Cells were isolated from control BALB-neuT mice and left untreated (CTRL) or treated ex vivo for 24 hrs with 1 μM ZA (ZA 24 hrs), or isolated from ZA-treated mice (ZA) as reported in the ‘Materials and methods’ section. (A) Inhibition of FPP synthase. A dose-dependent reduction of intracellular FPP, ubiquinone and cholesterol (always *P < 0.01, Student’s t-test), together with a significant increase of IPP (*P < 0.05, Student’s t-test) were displayed by ZA 24 hrs and ZA macrophages as compared to CTRL macrophages. Results are shown as mean ± S.E.M. of three experiments. (B) Inhibition of Ras prenylation and Ras activity. Ras pull-down assay and band shift assay in CTRL, ZA 24 hrs and ZA macrophages. (C, D) Activation of NF-κB. © Western blot analysis of IκB and GAPDH in CTRL, ZA 24 hrs and ZA macrophages. N11 glial cells cultured with (LPS N11) or without (CTRL N11) LPS 20 μg/ml were used as controls. (D) NF-κB translocation detected by EMSA on nuclear extracts from cell preparations run in lanes labelled as in ©. (E, F) Induction of iNOS expression and nitrite production. (E) Western blot analysis of iNOS and GAPDH in protein lysates from cell preparations run in lanes labelled as in (C and D). Results shown in (B–E) are from one representative out of three experiments. (F) Nitrite secretion by CTRL, ZA 24 hrs and ZA macrophages (MAC). Untreated (N11 CTRL) or LPS-treated (N11 LPS) N11 glial cells were used as controls. Results are expressed as mean ± S.E.M. of three experiments. The difference between ZA 24 hrs and CTRL macrophages is statistically significant (*P < 0.01, Student’s t-test). (G) Nitrotyrosine expression (arrowheads) in the tumour stroma of ZA-treated (right panel) versus control (left panel) BALB-neuT mice. Scale bars, 40 μm.

IPP effect on γδ T cell

Peripheral blood monocytes are responsible for γδ T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP 2008

in cultures of PBMCs, only CD14+ monocytes internalized large amounts of fluorescently labelled N-BP, while little or no uptake by other cell types, including CD3+ lymphocytes, could be detected

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between V{gamma}9V{delta}2 T Cells, {alpha}{beta} CD8+ T Cells, Regulatory T Cells, and Dendritic Cells. 2011

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer.

Papers Monocytes gammadelta

Side effects

Denosumab versus zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: a randomised, double-blind study. 2011

Comment in: Lancet. 2011 Mar 5;377(9768):785-6.

BACKGROUND: Bone metastases are a major burden in men with advanced prostate cancer. We compared denosumab, a human monoclonal antibody against RANKL, with zoledronic acid for prevention of skeletal-related events in men with bone metastases from castration-resistant prostate cancer.

METHODS: In this phase 3 study, men with castration-resistant prostate cancer and no previous exposure to intravenous bisphosphonate were enrolled from 342 centres in 39 countries. An interactive voice response system was used to assign patients (1:1 ratio), according to a computer-generated randomisation sequence, to receive 120 mg subcutaneous denosumab plus intravenous placebo, or 4 mg intravenous zoledronic acid plus subcutaneous placebo, every 4 weeks until the primary analysis cutoff date. Randomisation was stratified by previous skeletal-related event, prostate-specific antigen concentration, and chemotherapy for prostate cancer within 6 weeks before randomisation. Supplemental calcium and vitamin D were strongly recommended. Patients, study staff, and investigators were masked to treatment assignment. The primary endpoint was time to first on-study skeletal-related event (pathological fracture, radiation therapy, surgery to bone, or spinal cord compression), and was assessed for non-inferiority. The same outcome was further assessed for superiority as a secondary endpoint. Efficacy analysis was by intention to treat. This study is registered with, number NCT00321620, and has been completed.

FINDINGS: 1904 patients were randomised, of whom 950 assigned to denosumab and 951 assigned to receive zoledronic acid were eligible for the efficacy analysis. Median duration on study at primary analysis cutoff date was 12·2 months (IQR 5·9-18·5) for patients on denosumab and 11·2 months (IQR 5·6-17·4) for those on zoledronic acid. Median time to first on-study skeletal-related event was 20·7 months (95% CI 18·8-24·9) with denosumab compared with 17·1 months (15·0-19·4) with zoledronic acid (hazard ratio 0·82, 95% CI 0·71-0·95; p = 0·0002 for non-inferiority; p = 0·008 for superiority). Adverse events were recorded in 916 patients (97%) on denosumab and 918 patients (97%) on zoledronic acid, and serious adverse events were recorded in 594 patients (63%) on denosumab and 568 patients (60%) on zoledronic acid. More events of hypocalcaemia occurred in the denosumab group (121 [13%]) than in the zoledronic acid group (55 [6%]; p<0·0001). Osteonecrosis of the jaw occurred infrequently (22 [2%] vs 12 [1%]; p = 0·09).

INTERPRETATION: Denosumab was better than zoledronic acid for prevention of skeletal-related events, and potentially represents a novel treatment option in men with bone metastases from castration-resistant prostate cancer

inhibition of FarnesylPP synthesis

  • decreased CoQ10 synthesis
  • decreased dolichol synthesis
  • decreased cellular cholesterol synthesis

alendronate cholesterol

creatine kinase alendronate

zoledronate cholesterol

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