Jukka Mönkkönen

Cellular and molecular mechanisms of action of bisphosphonates

Bisphosphonates currently are the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor‐associated osteolysis and hypercalcemia, Pagets disease, and osteoporosis. These compounds have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone‐resorbing osteoclasts and inhibit osteoclast function.

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(β,γ-dichloromethylene) triphosphate, by mammalian cells in vitro

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage‐like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5′‐(β,γ‐dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome‐encapsulated clodronate to the same ATP analog. Liposome‐encapsulated adenosine 5′‐(β,γ‐dichloromethylene) triphosphate was more potent than liposome‐encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome‐encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.

Biochemical and molecular mechanisms of action of bisphosphonates

This review describes the key discoveries over the last 15 years that have led to a clearer understanding of the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Once released from bone mineral surfaces during bone resorption, these agents accumulate intracellularly in osteoclasts. Simple bisphosphonates such as clodronate are incorporated into non-hydrolysable analogues of adenosine triphosphate, which induce osteoclast apoptosis. The considerably more potent nitrogen-containing bisphosphonates are not metabolised but potently inhibit farnesyl pyrophosphate (FPP) synthase, a key enzyme of the mevalonate pathway. This prevents the synthesis of isoprenoid lipids necessary for the post-translational prenylation of small GTPases, thereby disrupting the subcellular localisation and normal function of these essential signalling proteins. Inhibition of FPP synthase also results in the accumulation of the upstream metabolite isopentenyl diphosphate, which is incorporated into the toxic nucleotide metabolite ApppI. Together, these properties explain the ability of bisphosphonate drugs to inhibit bone resorption by disrupting osteoclast function and survival. These discoveries are also giving insights into some of the adverse effects of bisphosphonates, such as the acute phase reaction that is triggered by inhibition of FPP synthase in peripheral blood monocytes.

A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. On the basis of their molecular mechanism of action, bisphosphonates can be divided into two pharmacological classes; nitrogen‐containing (N‐BPs) and non‐nitrogen‐containing bisphosphonates (non‐N‐BP). Both classes induce apoptosis but they evoke it differently; N‐BPs by inhibiting the intracellular mevalonate pathway and protein isoprenylation, and non‐N‐BPs via cytotoxic ATP analog‐type metabolites. N‐BPs are not metabolized to ATP analogs, but we report here that these bisphosphonates can induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of the mevalonate pathway in cells. We also investigated whether ApppI is involved in the apoptosis induced by N‐BPs. Mass spectrometry and NMR were used to identify ApppI in N‐BP treated osteoclasts, macrophages and glioma cells. The potency of different bisphosphonates to promote ApppI production was tested in J774 macrophages. The effects of ApppI on ADP/ATP translocase in isolated mitochondria and its capability to induce apoptosis in osteoclasts were also studied. ApppI production correlated well with the capacity of N‐BPs to inhibit mevalonate pathway. ApppI inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts. In conclusion, these findings provide the basis for a new mechanism of action for N‐BPs. Some of these very potent bisphosphonates, such as zoledronic acid, represent a third class of bisphosphonates that can act both via the inhibition of the mevalonate pathway and by the blockade of mitochondrial ADP/ATP translocase, which is known to be involved in the induction of apoptosis.

Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

Interaction of liposomes with human skin in vitro — The influence of lipid composition and structure

Liposomes have been suggested as a vehicle for dermal and transdermal drug delivery, but the knowledge about the interaction between lipid vesicles and human skin is poor. Therefore, we visualized liposome penetration into the human skin by confocal laser scanning microscopy (CLSM) in vitro. Liposomes were prepared from phospholipids in different compositions and labeled with a fluorescent lipid bilayer marker, N-Rh-PE (L-alpha-phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl). Fluorescently labelled liposomes were not able to penetrate into the granular layers of epidermis. However, the fluorescence from liposome compositions containing DOPE (dioleylphosphatidyl ethanolamine) was able to penetrate deeper into the stratum corneum than that from liposomes without DOPE. Pretreatment of skin with unlabeled liposomes containing DOPE or lyso-phosphatidyl choline (lyso-PC) enhanced the subsequent penetration of the fluorescent markers, N-Rh-PE and sulforhodamine B into the skin, suggesting possible enhancer activity, while most liposomes did not show such enhancement. Resonance energy transfer (RET) and calcein release assay between stratum corneum lipid liposomes (SCLLs) and the phospholipid vesicles suggested that the liposomes containing DOPE may fuse or mix with skin lipids in vitro and loosen the SCLL bilayers, respectively. Among the factors not affecting stratum corneum penetration were: negative charge, cholesterol inclusion and acyl chain length of the phospholipids. In conclusion, fusogenicity of the liposome composition appears to be a prerequisite for the skin penetration.

Macrophage Depletion by Clodronate-Containing Liposomes Reduces Neointimal Formation After Balloon Injury in Rats and Rabbits

Background—Inflammation is critical to vascular repair after mechanical injury. Excessive inflammation enhances neointimal formation and restenosis. We examined whether transient systemic inactivation of macrophages at the time of vascular intervention could attenuate the degree of expected restenosis. Methods and Results—Liposomal clodronate (LC) inhibited the growth of cultured macrophages but had no effect on endothelial or smooth muscle cells and suppressed neointimal hyperplasia in hypercholesterolemic rabbits and rats after intravenous administration of LC, with no adverse effects. LC treatment reduced the number of blood monocytes and decreased macrophage infiltration in the injured arteries as well as smooth muscle cell proliferation, interleukin-1&bgr; transcription, and production and matrix metalloproteinase-2 activity. Conclusions—Macrophages play a pivotal role in vascular repair after mechanical arterial injury. Systemic inactivation and depletion of monocytes and macrophages by LC reduce neointimal hyperplasia and restenosis.

Effect of liposomal and free bisphosphonates on the IL-1β, IL-6 and TNFα secretion from RAW 264 cells in vitro

AbstractPurpose. In order to evaluate the possible antiinflammatory action of bisphosphonates, the effect of the drugs on the secretion of proinflammatory cytokines (IL-lβ, IL-6 and TNFα) from macrophages was studied. Liposomes or high concentration of extracellular calcium was used to enhance the intracellular delivery of bisphosphonates. Methods. RAW 264 cells were used as macrophage model, and they were induced with lipopolysaccharide to produce the cytokines. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Results. As a free drug, clodronate and pamidronate, but not etidronate, inhibited LPS-stimulated secretion of the cytokines from macrophage-like RAW 264 cells. Low concentrations of pamidronate, however, induced the IL-6 secretion, and the cytokine inhibitory action at the higher concentrations of pamidronate was attributed to cytotoxicity of the compound. The cytokine induction or toxic effects were not observed with clodronate or etidronate. When the drugs were encapsulated in negatively charged unilamellar liposomes, the inhibitory potency of both clodronate and etidronate enhanced by a factor of 10-20, while that of pamidronate was not increased. The complex formation of bisphosphonates with extracellular calcium, although enhancing the uptake of the compounds by macrophages, did not considerably increase their cytokine inhibitory potency. Conclusions. Bisphosphonates have inhibitory action on cytokine secretion by macrophages. The non-cytotoxic cytokine inhibition by liposome encapsulated clodronate could be beneficial in local inflammatory diseases, where the inflammation is sustained by the excessive amounts of inflammatory cytokines produced by activated macrophages.

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

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

Nitrogen‐containing bisphosphonates indirectly activate Vγ9Vδ2 T cells through inhibition of farnesyl pyrophosphate synthase and intracellular accumulation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but the cells responsible for Vγ9Vδ2 T cell activation through IPP/DMAPP accumulation are unknown. Treatment of human peripheral blood mononuclear cells (PBMCs) with a pharmacologically relevant concentration of zoledronic acid induced accumulation of IPP/DMAPP selectively in monocytes, which correlated with efficient drug uptake by these cells. Furthermore, zoledronic acid‐pulsed monocytes triggered activation of γδ T cells in a cell contact‐dependent manner. These observations identify monocytes as the cell type directly affected by bisphosphonates responsible for Vγ9Vδ2 T cell activation.