Myeloma

Targeting an MMP-9-activated prodrug to multiple myeloma-diseased bone marrow: a proof of principle in the 5T33MM mouse model

Abstract

Multiple myeloma (MM) is an incurable B-cell cancer characterised by the monoclonal proliferation of tumour cells in the bone marrow (BM). It has been described that matrix metalloproteinases (MMPs) and especially MMP-9 is secreted by MM cells. In this study, we investigated the possibility to exploit MMP-9 activity to activate prodrugs and to target MM cells as a new tumour-specific therapy. Cleavage of the prodrug EV1-FITC by MMP-9 resulted in release of fluorescence which can be used as a measure of prodrug activation. The 5T33MM mouse model was used in this proof-of-principle study. The prodrug was activated in a higher amount by addition to MMP-9-producing 5T33MMvv cells, homogenates from tumour-bearing organs (BM, spleen) and isolated 5T33MM-diseased BM and spleen cells compared to non-MMP-9-producing 5T33MMvt cells and homogenates/cells from nontumour-bearing organs/mice, as measured by fluorescence release. This fluorescence release could be inhibited by the MMP-2/MMP-9-specific inhibitor, CTT. Activation of the prodrug in the 5T33MM spleen and BM homogenates was confirmed by chromatography. EV1-fluorescein isothiocyanate injection into 5T33MM-diseased animals resulted in a higher fluorescence release by the isolated BM and spleen cells compared to injection into healthy animals. In conclusion, MMP-9 activity can be used to activate prodrugs that target MM.

Introduction

Multiple myeloma (MM) is a B-cell cancer, located in the bone marrow (BM). The presence of a monoclonal serum immunoglobulin (M-component), enhanced angiogenesis, development of osteolytic lesions, anaemia and kidney failure are characteristics of the disease. Conventional therapies and high-dose chemotherapy followed by stem cell transplantation have resulted in prolonged survival, but are not curative. New or improved therapies are necessary. While systemic treatments have as a major disadvantage the toxicity to noninvaded, normal tissues, a new strategy to overcome this problem is the development of nontoxic prodrug forms that can be activated by the tumour cells. Activation of prodrugs by tumour-specific enzymes is one possible approach.1 The use of prostate-specific antigen, legumain, plasmin or undefined tumour-associated proteases to activate anticancer prodrugs has already been reported.2, 3, 4, 5 A family of enzymes described to be overexpressed in many tumours and also in MM are the matrix metalloproteinases (MMPs). MMPs are zinc-dependent endopeptidases involved in the degradation of all components of the extracellular matrix. We have demonstrated recently that spacer-linked, oligopeptide conjugates of cytotoxic anthraquinones can serve as efficient substrates for human recombinant MMP-9, and that the position of the cleavage site may be manipulated by the appropriate choice of amino-acid sequence in the oligopeptide motif that also masks the toxicity of the active agent.6 MMP-9 expression has been described earlier in human MM and in the 5TMM model.7, 8, 9 These findings made it interesting to investigate whether MMP-9 is a candidate enzymatic target for prodrug therapy in MM. EV1 is a prodrug with an MMP-9 gly-nva cleavage site. Cleavage of the prodrug with MMP-9 results in the liberation of the active drug which is an inhibitor of topoisomerases with in vivo activity. EV1 was conjugated to fluorescein isothiocyanate (FITC) to afford EV1-FITC (Figure 1) and fluorescence could be detected when the prodrug is cleaved by MMP-9.

Figure 1
figure1

Chemical structure of EV1-FITC.

Materials and methods

Mice

C57BL/KaLwRij mice were purchased from Harlan CPB (Horst, The Netherlands), housed under conventional conditions and treated as approved by the Ethical Committee for Animal Experiments, VUB (License no. LA1230281).

5T33MM cell lines

The 5T33MMvv cell line originated initially from elderly C57BL/KaLwRij mice that spontaneously developed MM. The cells have since been propagated in young syngeneic mice by intravenous transfer of the diseased BM.10 The progression of the disease was followed up by protein electrophoresis of the serum samples.11 Mice were killed when a serum paraprotein concentration of 10 mg/ml was reached. BM was isolated from hind legs and vertebrae. MM cells were purified by Lympholyte M (Cedarlane, Hornby, Canada) gradient centrifugation at 1000 g for 20 min. These cells can be used for intravenous injection into syngeneic mice (0.5 × 106 cells/mouse) for continuation of the 5T33MM model or for in vitro experiments.

The 5T33MMvt cell line, obtained by the Radl group, resulted spontaneously from cultured 5T33MMvv cells and grows in vitro independent of BM stroma. The cells were cultured in RPMI1640 medium (Cambrex, Verviers, Belgium) supplemented with penicillin–streptomycin (Cambrex), Na pyruvate (Cambrex), glutamine (Cambrex), MEM (Cambrex) and 10% serum (Fetal clone I, Hyclone, UT, USA).

Synthetic chemistry

Thin layer chromatography (TLC) was performed on Kieselgel 60 F254-precoated aluminium plates; EV1-FITC and its metabolites were absorbed in the visible region. Preparative flash chromatography was performed using Kieselgel 60 silica gel (Merck). FITC, DIPEA and reagents were from Molekula (Poole, England). Mass spectrometric characterisation of compounds was carried out using a Bruker Esquire 3000+ ion trap instrument with electrospray ionisation (ESMS).

The synthesis of the spacer-linked anthraquinone-heptapeptide conjugate precursor of EV1-FITC, incorporating the D-ala-ala-ala-leu-gly-nva-pro sequence, together with intermediate conjugates from one to seven amino acids, formed via standard peptide coupling using Boc-protection-deprotection strategies, have been described earlier.6 The free heptapeptide conjugate was converted into EV1-FITC by the following procedure:

Synthesis of 1-[3-(fluoresceinylthioureido-D-alanyl-L-alanyl-L-alanyl-L-leucyl-glycyl-L-norvalyl-L-prolylamino)propylamino]anthracene-9,10-dione (EV1-FITC)

1-[3-(D-alanyl-L-alanyl-L-alanyl-L-leucyl-glycyl-L-norvalyl-L-prolylamino)propylamino]anthracene-9,10-dione trifluoroacetate6 (0.1 g; 0.10 mmol) was dissolved in DMF (15 ml), and DIPEA (76 μl; 4.4 equivalents) was added. The mixture was stirred at 25°C for 15 min, fluorescein isothiocyanate isomer 5 (FITC) (0.04 g; 1.1 equivalents) was added and stirring was continued for 24 h with protection from light.

The reaction mixture was transferred to a separating funnel, extracted with chloroform (2 × 20 ml), washed with saturated sodium bicarbonate (2 × 20 ml) and water (2 × 20 ml). The organic layer was dried (anhydrous MgSO4), filtered, evaporated to low volume in vacuo and applied to a silica gel 60 chromatography column (2.5 × 15 cm) eluted with a gradient of dichloromethane-methanol. Fractions containing the product (Rf 0.38; dichloromethane-methanol (9:1)) were combined and evaporated to dryness to afford an analytically pure sample of the title compound. Yield: 0.04 g (38%), Mp 128°C. ESMS(+) m/z 1271 (100%)(M+Na)+, 1249 (7%)(M+H)+, 882 (46%), 454 (40%), 413 (64%), 390 (82%), 47 (58%). M,1248 Da. Rt 21.4 min (HPLC).

Preparation of organ homogenates

BM cells isolated by flushing hind legs and vertebrae, and spleen cells isolated by crushing the organ, were treated with NH4Cl to lyse red blood cells and snap frozen. Lung, kidney and heart were isolated, washed with water and snap frozen. Cells and tissue samples were homogenized by a Polytron PT1200 homogenizer (Kinematica AG, Lucerne, Switzerland) in lysis buffer (50 mM Tris-HCl (pH 7.5), 50 U/ml penicillin, 50 μg/ml streptomycin) and incubated for 2 h at 4°C. Then, the homogenate was centrifuged at 14 000 rpm for 30 min at 4°C and the supernatant was collected. The protein content was measured with the BCA Pierce assay (Pierce, Rockford, USA).

Incubation of 5T33MM cells and organ homogenates with EV1-FITC

5T33MMvv cells were isolated as described in ‘5T33MM cell lines’ and cell purity was assessed by determining plasmacytosis on cytospins stained with May–Grünwald–Giemsa. The mean purity of MM cells used for the experiments is 85±5.3%. 5T33MMvt cells were washed twice in supplemented medium without serum. Organ homogenates (100 μg/ml) or 5T33MM cells (1 × 105 cells/well) were plated in a 96-well plate in the presence of 20 μ M EV1-FITC (total volume is 100 μl). In some experiments, 5T33MM cells or BM homogenates were incubated with an MMP-2/MMP-9 inhibitor, CTT (Biomol, Exeter, UK), a control peptide, STT (Biomol) or a serine proteinase inhibitor, aprotinin (Sigma, St Louis, MO, USA) 60 min prior to EV1-FITC addition. Fluorescence was measured at times indicated in the figure with a Victor Wallac fluorometer (PerkinElmer, Monza, Italy).

Gelatin zymography

Homogenates at a concentration of 100 μg/ml were analysed for MMP-9 expression by gelatin zymography as described previously.8

Incubation of BM and spleen cells with EV1-FITC

BM cells and spleen cells were isolated from age- and sex-matched naïve and 5T33MM-diseased mice and treated with NH4Cl to remove red blood cells. Cells (1 × 105/well) were plated in a 96-well plate in supplemented RPMI medium without serum in the presence of 20 μ M EV1-FITC. Fluorescence was measured at the times indicated in the figure.

Injection of EV1-FITC in mice

EV1-FITC, dissolved in DMSO, was injected intraperitoneally at 50 mg/kg (volume of 100 μl) in naïve and 5T33MM-diseased mice. Mice injected with DMSO were used as controls. After 2 h, mice were bled for paraprotein measurement in serum and killed. Hind legs were flushed and a piece of the spleen was crushed in supplemented RPMI medium without serum. Cells were counted, and 1.5 million cells in 100 μl medium were put in a 96-well plate and fluorescence was measured.

High performance liquid chromatography (HPLC)

HPLC was performed on an Agilent 1100 Series instrument fitted with a diode array detector to record absorbance in the UV range (analytical wavelengths 252 and 314 nm), and an autosampler. The system was fitted with a reverse phase semipreparative column (C18 HiChrom HIRPB-250A; 25 cm × 4.6 mm). Samples were eluted over 45 min with the mobile phases, A: 10% MeCN, 89.95% H2O, 0.045% TFA and B: 60% MeCN, 39.95% H2O, 0.02% TFA (solvent composition: 60%A+40%B (t=0); 5%A+95%B (t=25, 28 min); 60%A+40%B (t=30, 45 min)). 5T33MM homogenates of BM and spleen (protein at 500 μg/ml) were treated with EV1-FITC prodrug at 50 μ M at 37°C. Analyses were performed with a flow rate of 1.4 ml/min at a column temperature of 20°C. Compounds were dissolved initially in DMSO (final concentration 0.005% in the incubation medium). Homogenate-treated samples (EV1-FITC and metabolite standards) were injected onto the column in volumes of 20 μl. Homogenate-free control prodrug incubations were conducted in 0.01 M PBS at pH 7.4. In vitro prodrug metabolites were identified by comparison with authentic samples6 by HPLC-MS.

Results

Fluorescence release by 5T33MMvt and 5T33MMvv cells

The prodrug EV1 was conjugated to FITC wherein the intrinsic fluorescence of the FITC label is internally quenched by the anthraquinone chromophore when the oligopeptide motif remains intact and thus can be useful to detect MMP-9 cleavage of the prodrug. Since there is a differential MMP-9 expression between 5T33MMvt (no MMP-9 secretion) and 5T33MMvv (MMP-9 secretion) cells,8 we investigated the fluorescence release in time by adding EV1-FITC to the cells. Addition of EV1-FITC (20 μ M) resulted in a higher release of fluorescence with the MMP-9-producing 5T33MMvv cells than with the non-MMP-9-producing 5T33MMvt cells (Figure 2). To determine if this fluorescence release was caused by MMP-9 cleavage, 5T33MMvv cells were incubated with an MMP-2/MMP-9-specific inhibitor, CTT, 1 h before the addition of EV1-FITC. This resulted in a dose-dependent inhibition of fluorescence release compared with the control peptide STT (Figure 3a). However, the serine proteinase inhibitor aprotinin had no effect on the fluorescence release (Figure 3b). Similar results were obtained with human primary MM cells (data not shown). CTT has no effect on cell viability as determined by trypan blue staining.

Figure 2
figure2

Fluorescence release by 5T33MMvt and 5T33MMvv cells. EV1-FITC (20 μ M) was added to the cells and fluorescence was measured in time. Results represent the mean value±s.d. of three independent experiments performed in duplicate.

Figure 3
figure3

The effect of proteinase inhibition on fluorescence release. An MMP-2/MMP-9-specific inhibitor CTT (a), the control peptide STT (a) or a serine proteinase inhibitor aprotinin (b) was added to 5T33MMvv cells 1 h before addition of EV1-FITC (20 μ M). Fluorescence was measured in time. The mean value±s.d. of three independent experiments performed in duplicate are shown.

MMP-9 expression and fluorescence release in organ homogenates

In the 5T33MM model, it has been described that the tumour cells are only located in the BM and spleen.11 Homogenates of organs of a 5T33MM-diseased mouse were analysed for MMP-9 expression. MMP-9 was detectable in the tumour-bearing organs, BM and spleen, but not in the nontumour-bearing organs, lung, kidney and heart (Figure 4). EV1-FITC was added to the homogenates and fluorescence was measured after 2 h. More fluorescence was detected in homogenates from the BM and spleen than from the lung, heart and kidney (Figure 5). Preincubation of 5T33MM BM homogenates with the MMP-2/MMP-9 inhibitor, CTT, 1 h before EV1-FITC addition resulted in an inhibition of fluorescence release compared with the control peptide, STT (Figure 6a), while the serine proteinase inhibitor aprotinin had no effect on fluorescence release (Figure 6b).

Figure 4
figure4

Gelatin zymography of homogenates of organs of a 5T33MM-diseased mouse. Human fibrosarcoma HT1080 conditioned medium and control (homogenate buffer) were, respectively, positive and negative control. Mouse MMP-9 has a higher molecular mass than human MMP-9 and is thus higher located in the gel.

Figure 5
figure5

Fluorescence release measured after 2 h in organ homogenates of 5T33MM-diseased mice with 20 μ M EV1-FITC. Results represent the mean value±s.d. of three independent experiments performed in duplicate. Asterisk (*) indicates significant difference compared to BM (P<0.05, Mann–Whitney test). °Indicates significant difference compared to spleen (P<0.05, Mann–Whitney test).

Figure 6
figure6

The effect of proteinase inhibition on fluorescence release. CTT (a), STT (a) or aprotinin (b) was added to 5T33MM BM homogenates 1 h before EV1-FITC (20 μ M) addition. Results represent the mean±s.d. of duplicates. Results from one experiment, representative of three, are shown.

Comparison between normal and MM cells

The aim of the prodrug strategy is to achieve more selectivity for tumour vs normal cells. Therefore, BM and spleen cells were isolated from naïve and 5T33MM-diseased mice. EV1-FITC was added to the cells and fluorescence was measured in time. A higher fluorescence release was measured with cells of 5T33MM-bearing mice than with cells of naïve mice (Figure 7). Similar results were obtained by adding EV1-FITC to homogenates made from the cells (data not shown). 5T33MM-bearing animals and naïve animals were injected i.p. with EV1-FITC (50 mg/kg). After 2 h, BM and spleen cells were isolated and fluorescence was measured. A higher amount of fluorescence could be detected with the cells of the 5T33MM-bearing mice compared to the cells of the naïve mice (Figure 8). With cells isolated from mice injected with control (DMSO), no fluorescence could be detected.

Figure 7
figure7

Fluorescence release by BM and spleen cells isolated from a 5T33MM-bearing and a naïve mouse. EV1-FITC (20 μ M) was added to the cells and fluorescence was measured in time. Mean values±s.d. from duplicates from one experiment, representative for three, are presented.

Figure 8
figure8

Fluorescence release by BM and spleen cells isolated from mice injected i.p. with EV1-FITC (50 mg/kg). Results represent mean values±s.d. of five animals per group. Asterisk (*) indicates significant difference (P<0.05, Mann–Whitney test).

Prodrug in vitro metabolism in tumour homogenates

HPLC analysis of the in vitro metabolism (Figure 9) of EV1-FITC (labelled F (Rt 21.4 min)) in incubations with 5T33MM-derived spleen and BM homogenate samples showed that the active agent (spacer-linked proline-anthraquinone conjugate, labelled D (Rt 11.1 min)) and its principal metabolite (anthraquinone-spacer compound, B (Rt 8.4 min)) were efficiently released, whereas EV1-FITC was largely undegraded (at 24 h) upon incubation with homogenates of spleen from the undiseased mouse, or with heart or kidney tissue from either source (data not shown). Early time points determined that EV1-FITC was initially cleaved to the norvalylproline dipeptide conjugate (labelled E (Rt 12.3 min)), consistent with the MMP-9-mediated gly-nva cleavage site, previously noted for the non-FITC labelled precursor EV1 (Young et al. Br J Cancer 2003; 88 (Suppl 1): S27; abstract). Signals labelled A and C (Figure 9) were shown to be FITC-labelled peptide fragments by comparison with studies on the corresponding unconjugated FITC-heptapeptide (data not shown).

Figure 9
figure9

HPLC chromatogram of the in vitro metabolism of EV1-FITC (F) showing release of the active agent (D) and its principal metabolite (B) upon incubation (5 h exposure) with 5T33MM-derived spleen (lower trace) and BM (upper trace) homogenates; representative of three independent experiments.

Discussion

Several groups have reported the expression of MMPs in MM cells and bone marrow stromal cells (BMSCs): the production of MMP-2, MMP-7 and MMP-9 has been demonstrated in purified myeloma cells.7, 12, 13, 14 MMP-8 and MMP-13 could be detected in human MM cells and in 5T2MM-diseased BM cells.9, 15 BMSCs secrete MMP-1 and MMP-2. Endothelial cells from MM patients produce MMP-2 and MMP-9 in a higher amount than human umbilical vein endothelial cells.7, 16 MMP-1 production by BMSCs is upregulated in response to MM cells.7 In the 5T33MM mouse model, an in vivo upregulation of MMP-9 expression has been observed.8 Whilst the in vitro stroma-independent growing 5T33MMvt cells secrete no MMP-9, an enhanced MMP-9 secretion was observed when these 5T33MMvt cells were injected in naïve mice and harvested from the BM after disease development. Contact with BMECs is partly responsible for this increased MMP-9 expression.8, 17 Recently, we were able to demonstrate in the 5T2MM model that MMPs are involved in different processes of the MM disease including tumour growth, angiogenesis and osteolytic bone disease.9 The involvement of MMPs in MM and other tumour types makes these enzymes interesting therapeutic targets. Although MMP inhibitors were demonstrated to be very effective in animal models, clinical trials were quite disappointing.18, 19 A different approach is to use the increased MMP activity in cancer for targeted therapy that is, to exploit MMP proteolytic activity for the activation of nontoxic prodrugs. Prodrugs are agents that are transformed by a specific enzyme or specific tumour condition to result in an active drug. The purpose of this strategy is to improve the specificity and efficacy and to reduce systemic toxicity of pharmaceuticals. In this study, we investigated this approach in the 5T33MM mouse model by using a prodrug conjugated to fluorescein isothiocyanate, EV1-FITC. The active agent in the prodrug is comprised of the L-proline adduct of 1-(3-aminopropylamino)anthraquinone, code-named NU:UB31,20 shown earlier to be a dual inhibitor of DNA topoisomerase I and II, with in vivo activity against a range of experimental tumour models (Jackson et al. Br J Cancer 2002; 86 (Suppl 1): P 259; abstract). The anthraquinone chromophore was found to be an excellent quencher of the fluorescence of FITC in the derived heptapeptide prodrug EV1-FITC, as a result of resonance energy transfer.21 Since FITC fluorescence is suppressed until the carrier of the prodrug is cleaved off, it was simple to measure the amount of prodrug activated by detecting FITC release, in a manner comparable to the use of N-DNP-labelled fluorogenic peptides for assay of MMP activity.22 In this study, we were able to demonstrate more activation of the prodrug (as measured by a higher fluorescence release) in the MMP-9-producing 5T33MMvv cells and tumour-bearing organs (BM and spleen) compared to the MMP-9-negative 5T33MMvt cells and nontumour-bearing organs (lung, kidney, heart). The activation of the prodrug was consistent with MMP-9 activity, since the fluorescence release could be inhibited by an MMP-2/MMP-9 inhibitor and not by the serine proteinase inhibitor, aprotinin. The suppressive effects of CTT were incomplete. It is conceivable that other tumour-associated proteases, belonging to the endopeptidase class, could contribute to fluorescence release; however, the action of aminopeptidases or carboxypeptidases can be discounted because both the N- and C-termini in EV1-FITC are blocked by nonhydrolysable, FITC and anthraquinone groups, respectively. Importantly, the data obtained by using the MMP inhibitor show a concentration-dependent inhibition of fluorescence release consistent with inhibition of MMP-mediated peptide cleavage; however, the concentration of inhibitor required to achieve complete inhibition of fluorescence release was not determined. The results suggested that the prodrug can be activated to the tumour microenvironment using MMP-9 activity. This was further confirmed by the fact that BM and spleen cells isolated from 5T33MM-bearing animals could activate the prodrug to a higher extent compared to cells from healthy mice. In addition, BM and spleen cells isolated from 5T33MM-diseased animals injected with EV1-FITC demonstrated a higher fluorescence release than cells from healthy animals injected with EV1-FITC. The major determinant of the efficiency of a substrate of an MMP is known to be the amino acid in the P1′ position (using the Schecter and Berger nomenclature)23 of the scissile bond.24, 25 The unlabelled prodrug was recently shown to be cleaved at the glycine–norvaline junction with MMP-9 or homogenates of MMP-9-rich HT1080 human fibrosarcoma tissue, consistent with the straight-chain residue at the alpha carbon of norvaline occupying the S-1′ subsite on MMP-9, previously shown to be a deep hydrophobic pocket. For the labelled prodrug, EV1-FITC, it has been demonstrated that MMP-9-rich, 5T33MM-derived spleen and BM homogenates induce an analogous cleavage pattern to release the cytotoxic agent and its metabolite, detected by HPLC, thus maintaining sensitivity to endopeptidase action while resisting proteolytic degradation at the FITC-blocked N-terminus. It may be speculated that the FITC group may enhance MMP-9 binding due to an affinity of MMP-9 for conjugated aromatic systems in the P-5 position of substrates.26

In conclusion, this is the first study in myeloma that demonstrated that MMP-9 activity can be exploited to activate prodrugs and therefore target the tumour cells in the BM microenvironment. This strategy can especially be applied with chemotherapeutics known to be nonselective for cancer cells and that cause damage to normal tissues. For instance, the FITC-labelled peptide carrier could be coupled (by standard peptide chemistry) to the N-terminus of the commonly used chemotherapeutic in MM, melphalan, to afford a prodrug. In addition, the strategy may be extendable to other agents that target aspects of the MM–microenvironment interactions related to invasion and angiogenesis, to improve their effectiveness by achieving a higher local dose.

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Acknowledgements

This work was financially supported by the Stichting Emmanuel Vander Scheuren, Onderzoeksraad-Vrije Universiteit Brussel (OZR-VUB), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, Kom op tegen Kanker, Belgische Federatie tegen Kanker, Fortis, VIVA and VIS. Karin Vanderkerken is a postdoctoral fellow of the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (FWO-Vl). The financial support of BTG International plc (ADS and DM) and assistance of the EPSRC Mass Spectrometry Service Centre, Swansea, UK, are gratefully acknowledged.

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Correspondence to K Vanderkerken.

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Van Valckenborgh, E., Mincher, D., Di Salvo, A. et al. Targeting an MMP-9-activated prodrug to multiple myeloma-diseased bone marrow: a proof of principle in the 5T33MM mouse model. Leukemia 19, 1628–1633 (2005). https://doi.org/10.1038/sj.leu.2403866

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Keywords

  • matrix metalloproteinases
  • multiple myeloma
  • 5T33
  • prodrug
  • MMP-9

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