Inhibition of Chronic and Acute Skin Inflammation by Treatment with a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.071074
ISSN1525-2191
AutoresCornelia Halin, Hermann Fahrngruber, Josef G. Meingassner, Guido Bold, Amanda Littlewood-Evans, Anton Stuetz, Michael Detmar,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoAlthough vascular remodeling is a hallmark of many chronic inflammatory disorders, antivascular strategies to treat these conditions have received little attention to date. We investigated the effects of a newly identified vascular endothelial growth factor (VEGF) receptor tyrosine-kinase inhibitor, NVP-BAW2881, on endothelial cell function in vitro and its anti-inflammatory activity in different animal models. NVP-BAW2881 inhibited proliferation, migration, and tube formation by human umbilical vein endothelial cells and lymphatic endothelial cells in vitro. In a transgenic mouse model of psoriasis, NVP-BAW2881 reduced the number of blood and lymphatic vessels and infiltrating leukocytes in the skin, and normalized the epidermal architecture. NVP-BAW2881 also displayed strong anti-inflammatory effects in models of acute inflammation; pretreatment with topical NVP-BAW2881 significantly inhibited VEGF-A-induced vascular permeability in the skin of pigs and mice. Furthermore, topical application of NVP-BAW2881 reduced the inflammatory response elicited in pig skin by UV-B irradiation or by contact hypersensitivity reactions. These results demonstrate for the first time that VEGF receptor tyrosine-kinase inhibitors might be used to treat patients with inflammatory skin disorders such as psoriasis. Although vascular remodeling is a hallmark of many chronic inflammatory disorders, antivascular strategies to treat these conditions have received little attention to date. We investigated the effects of a newly identified vascular endothelial growth factor (VEGF) receptor tyrosine-kinase inhibitor, NVP-BAW2881, on endothelial cell function in vitro and its anti-inflammatory activity in different animal models. NVP-BAW2881 inhibited proliferation, migration, and tube formation by human umbilical vein endothelial cells and lymphatic endothelial cells in vitro. In a transgenic mouse model of psoriasis, NVP-BAW2881 reduced the number of blood and lymphatic vessels and infiltrating leukocytes in the skin, and normalized the epidermal architecture. NVP-BAW2881 also displayed strong anti-inflammatory effects in models of acute inflammation; pretreatment with topical NVP-BAW2881 significantly inhibited VEGF-A-induced vascular permeability in the skin of pigs and mice. Furthermore, topical application of NVP-BAW2881 reduced the inflammatory response elicited in pig skin by UV-B irradiation or by contact hypersensitivity reactions. These results demonstrate for the first time that VEGF receptor tyrosine-kinase inhibitors might be used to treat patients with inflammatory skin disorders such as psoriasis. Angiogenesis and lymphangiogenesis have important roles in tumor growth and metastasis.1Folkman J Angiogenesis.Annu Rev Med. 2006; 57: 1-18Crossref PubMed Scopus (1178) Google Scholar, 2Cao Y Opinion: emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis.Nat Rev Cancer. 2005; 5: 735-743Crossref PubMed Scopus (261) Google Scholar Vascular remodeling also accompanies many inflammatory and autoimmune disorders, such as renal transplant rejection, rheumatoid arthritis, inflammatory bowel disease, and the chronic inflammatory skin disease, psoriasis.3Kerjaschki D Regele HM Moosberger I Nagy-Bojarski K Watschinger B Soleiman A Birner P Krieger S Hovorka A Silberhumer G Laakkonen P Petrova T Langer B Raab I Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates.J Am Soc Nephrol. 2004; 15: 603-612Crossref PubMed Scopus (413) Google Scholar, 4Bainbridge J Sivakumar B Paleolog E Angiogenesis as a therapeutic target in arthritis: lessons from oncology.Curr Pharm Des. 2006; 12: 2631-2644Crossref PubMed Scopus (32) Google Scholar, 5Danese S Sans M de la Motte C Graziani C West G Phillips MH Pola R Rutella S Willis J Gasbarrini A Fiocchi C Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 6Leong TT Fearon U Veale DJ Angiogenesis in psoriasis and psoriatic arthritis: clues to disease pathogenesis.Curr Rheumatol Rep. 2005; 7: 325-329Crossref PubMed Scopus (45) Google Scholar Vascular endothelial growth factor (VEGF)-A levels are elevated in inflamed tissue associated with these conditions.7Koch AE Harlow LA Haines GK Amento EP Unemori EN Wong WL Pope RM Ferrara N Vascular endothelial growth factor. A cytokine modulating endothelial function in rheumatoid arthritis.J Immunol. 1994; 152: 4149-4156PubMed Google Scholar, 8Detmar M Brown LF Claffey KP Yeo KT Kocher O Jackman RW Berse B Dvorak HF Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (654) Google Scholar, 9Kanazawa S Tsunoda T Onuma E Majima T Kagiyama M Kikuchi K VEGF, basic-FGF, and TGF-beta in Crohn's disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation.Am J Gastroenterol. 2001; 96: 822-828PubMed Google Scholar Epidermal VEGF-A expression is strongly up-regulated8Detmar M Brown LF Claffey KP Yeo KT Kocher O Jackman RW Berse B Dvorak HF Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (654) Google Scholar and blood and lymphatic vessels are increased10Kunstfeld R Hirakawa S Hong YK Schacht V Lange-Asschenfeldt B Velasco P Lin C Fiebiger E Wei X Wu Y Hicklin D Bohlen P Detmar M Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (275) Google Scholar in human psoriatic skin lesions, indicating the involvement of VEGF-A in psoriasis pathogenesis. Furthermore, specific genetic polymorphisms of the gene encoding VEGF-A are correlated with psoriasis severity.11Young HS Summers AM Bhushan M Brenchley PE Griffiths CE Single-nucleotide polymorphisms of vascular endothelial growth factor in psoriasis of early onset.J Invest Dermatol. 2004; 122: 209-215Crossref PubMed Scopus (136) Google Scholar A variety of strategies have been pursued to inhibit neovascularization by blocking VEGF-A binding to VEGF receptors (VEGFRs)—particularly to VEGFR-2. VEGFR-2 is believed to be the major mediator of VEGF-A's (lymph)angiogenic activity in blood vascular and lymphatic endothelial cells.12Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7989) Google Scholar Thus far however, these approaches have primarily focused on the development of cancer therapeutics. The most successful anti-angiogenic strategy to date has been the development of a monoclonal antibody directed against VEGF-A (bevacizumab, Avastin).1Folkman J Angiogenesis.Annu Rev Med. 2006; 57: 1-18Crossref PubMed Scopus (1178) Google Scholar, 13Kerr DJ Targeting angiogenesis in cancer: clinical development of bevacizumab.Nat Clin Pract Oncol. 2004; 1: 39-43Crossref PubMed Scopus (80) Google Scholar Several other agents designed to block VEGF-A signaling are in late stages of preclinical and clinical development; these include antibodies that target VEGFR-2,14Lu D Shen J Vil MD Zhang H Jimenez X Bohlen P Witte L Zhu Z Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.J Biol Chem. 2003; 278: 43496-43507Crossref PubMed Scopus (172) Google Scholar soluble VEGFR-2 decoy receptors,15Rudge JS Thurston G Davis S Papadopoulos N Gale N Wiegand SJ Yancopoulos GD VEGF trap as a novel antiangiogenic treatment currently in clinical trials for cancer and eye diseases, and VelociGene-based discovery of the next generation of angiogenesis targets.Cold Spring Harb Symp Quant Biol. 2005; 70: 411-418Crossref PubMed Scopus (49) Google Scholar and small-molecule inhibitors of VEGFR tyrosine kinase (TK) activity.16Underiner TL Ruggeri B Gingrich DE Development of vascular endothelial growth factor receptor (VEGFR) kinase inhibitors as anti-angiogenic agents in cancer therapy.Curr Med Chem. 2004; 11: 731-745Crossref PubMed Scopus (125) Google Scholar, 17Morabito A De Maio E Di Maio M Normanno N Perrone F Tyrosine kinase inhibitors of vascular endothelial growth factor receptors in clinical trials: current status and future directions.Oncologist. 2006; 11: 753-764Crossref PubMed Scopus (245) Google Scholar Surprisingly, anti-(lymph)angiogenic substances have received little attention as potential therapeutics for chronic inflammatory conditions. In particular, the efficacy of small-molecule inhibitors of VEGFR TK activity has only been evaluated in a few in vivo studies of chronic inflammation.18Grosios K Wood J Esser R Raychaudhuri A Dawson J Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor. PTK787/ZK222584, causes significant anti-arthritic effects in models of rheumatoid arthritis.Inflamm Res. 2004; 53: 133-142Crossref PubMed Scopus (73) Google Scholar The potentially low cost of production of these organic molecules, as compared with those of anti-VEGF-A biopharmaceuticals, as well as their potential for oral or topical administration, make these compounds attractive for the treatment of patients with debilitating chronic inflammatory conditions. Furthermore, simultaneously interfering with all VEGFR-mediated signaling pathways with small-molecule TK inhibitors may be advantageous to antibody-mediated blockade of only one particular VEGF isoform or of one particular VEGFR. We investigated the in vitro and in vivo activity of the recently identified VEGFR TK inhibitor NVP-BAW2881, which inhibits human and mouse VEGFR TK activity at nanomolar concentrations. This compound blocked VEGF-A-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (LECs) in vitro. In a mouse model of psoriasis, both oral and topical administration of NVP-BAW2881 strongly reduced psoriasis-like inflammation in ear skin. Topical NVP-BAW2881 also effectively reduced VEGF-A-induced vascular permeability in the skin of mice and domestic pigs, whose skin more closely resembles human skin. Furthermore, the compound given topically significantly inhibited acute skin inflammation in pigs, namely contact hypersensitivity (CHS) reactions and UV-B (UVB)-induced erythema. These findings indicate that orally or topically applied VEGFR TK inhibitors might be used to treat patients with psoriasis and other inflammatory skin disorders. NVP-BAW2881 is a low-molecular weight compound developed for the VEGFR TK inhibitor program at Novartis. For in vitro functional assays, NVP-BAW2881 was dissolved in a 1 mmol/L stock solution of dimethyl sulfoxide. For topical treatment of mice and pigs, the compound was applied at 0.1% to 0.5% in a mixture of ethanol (30%) and propylene glycol (70%). NVP-BAW2881 was dissolved in polyethylene glycol-200 and orally administered to mice in a dose of 25 mg/kg/day in 10 ml/kg. The IC50 values of NVP-BAW2881 for various kinases were determined in a scintillation proximity assay, using recombinant glutathione-S-transferase-labeled kinases as substrates, as previously described.19Garcia-Echeverria C Pearson MA Marti A Meyer T Mestan J Zimmermann J Gao J Brueggen J Capraro HG Cozens R Evans DB Fabbro D Furet P Porta DG Liebetanz J Martiny-Baron G Ruetz S Hofmann F In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase.Cancer Cell. 2004; 5: 231-239Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 20Traxler P Allegrini PR Brandt R Brueggen J Cozens R Fabbro D Grosios K Lane HA McSheehy P Mestan J Meyer T Tang C Wartmann M Wood J Caravatti G AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity.Cancer Res. 2004; 64: 4931-4941Crossref PubMed Scopus (318) Google Scholar Cellular autophosphorylation assays were performed as described previously.19Garcia-Echeverria C Pearson MA Marti A Meyer T Mestan J Zimmermann J Gao J Brueggen J Capraro HG Cozens R Evans DB Fabbro D Furet P Porta DG Liebetanz J Martiny-Baron G Ruetz S Hofmann F In vivo antitumor activity of NVP-AEW541-A novel, potent, and selective inhibitor of the IGF-IR kinase.Cancer Cell. 2004; 5: 231-239Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar Human dermal LECs were isolated from neonatal human foreskins as described21Kajiya K Hirakawa S Ma B Drinnenberg I Detmar M Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (259) Google Scholar and cultured in LEC medium containing endothelial basal medium (Cambrex, Verviers, Belgium), supplemented with 20% fetal bovine serum (Gibco, Paisley, UK), antibiotic antimycotic solution, l-glutamine (2 mmol/L), hydrocortisone (10 μg/ml), and N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (2.5 × 10−2 mg/ml; all from Fluka, Buchs, Switzerland). Cells were passaged up to 10 times. HUVECs (Cambrex) were cultured in LEC medium supplemented with endothelial cell growth supplement (Cambrex) for up to 8 passages. HUVECs were transferred to LEC medium 3 days before functional assays were performed. HUVECs or LECs (1.2 × 103) were seeded into fibronectin-coated 96-well plates. After 24 hours, the cells were transferred into LEC medium containing 2% fetal bovine serum and incubated for an additional 24 hours. Cells (eight wells/condition) were incubated with medium alone (control), 20 ng/ml VEGF-A (Biological Resources Branch, National Cancer Institute, Washington DC), or a combination of 20 ng/ml VEGF-A and 1 nmol/L to 1 μmol/L NVP-BAW2881. Proliferation was also assayed in LECs incubated with 500 ng/ml VEGF-C (Cys156Ser; R&D Systems, Abingdon, UK). The dimethyl sulfoxide concentration was adjusted to 0.1% in all wells. After 72 hours, cells were incubated with 5-methylumbelliferylheptanoate for subsequent fluorescent quantification of viable cells, using a SpectraMax Gemini electron microscope (Bucher Biotec AG, Basel, Switzerland) as described.22Detmar M Tenorio S Hettmannsperger U Ruszczak Z Orfanos CE Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro.J Invest Dermatol. 1992; 98: 147-153Abstract Full Text PDF PubMed Scopus (105) Google Scholar Chemotactic cell migration toward 20 ng/ml VEGF-A was assayed in FluoroBlok transwell chambers (BD Falcon) as described.23Hong YK Lange-Asschenfeldt B Velasco P Hirakawa S Kunstfeld R Brown LF Bohlen P Senger DR Detmar M VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.FASEB J. 2004; 18: 1111-1113Crossref PubMed Scopus (263) Google Scholar Cells were incubated in endothelial basal medium containing 0.2% bovine serum albumin for 10 minutes at 37°C in the presence or absence of NVP-BAW2881 (10 nmol/L or 1 μmol/L). Lower chambers (3 to 5 per condition) were filled with 500 μl medium, VEGF-A (20 ng/ml) and NVP-BAW2881 (0 nmol/L, 10 nmol/L or 1 μmol/L). Only medium was added to control wells. Cells (105 in 100 μl) that were exposed to the various concentrations of NVP-BAW2881 were seeded into the upper chambers and incubated for 3 hours at 37°C. Cells that had migrated to the bottom side of the filter plate were stained with Calcein AM (Molecular Probes). Fluorescence intensity (proportional to the number of transmigrated cells) was measured using a SpectraMax Gemini electron microscope. Tube formation assays were performed as described.24Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (566) Google Scholar LECs or HUVECs were grown to confluence in fibronectin-coated 24-well plates. Each well (3 to 4 per condition) was overlaid with 0.5 ml of neutralized isotonic bovine dermal collagen type I (Invitrogen, Palo Alto, CA; 1 mg/ml for LECs, 1.2 mg/ml for HUVECs) containing VEGF-A (20 ng/ml) and NVP-BAW2881 (0 nmol/L, 10 nmol/L, or 1 μmol/L). Controls wells were overlaid with collagen only. The wells were incubated for 20 hours at 37°C and representative images were captured (four per well). The total length of tube-like structures per image was measured using IPlab software (BD Biosciences). Female hemizygous K14/VEGF-A TG mice (FVB background) that overexpress mouse VEGF-A164 in the epidermis under control of the human keratin 14 promoter25Xia YP Li B Hylton D Detmar M Yancopoulos GD Rudge JS Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis.Blood. 2003; 102: 161-168Crossref PubMed Scopus (315) Google Scholar were bred and housed in the animal facility of ETH. Female hairless SKH1 mice were purchased from Charles River Laboratories, Inc. (Austria). Young castrated male domestic pigs (German Edelschwein × Landrace; Josef Semrad, Münichstal, Austria) weighed 12 to 18 kg at the time of experimentation. Experiments were performed in accordance with the animal protocols approved by the “Kantonales Veterinäramt Zürich” (animal protocol Nr. 123/2005) and the “Landesregierung Wien” (study protocols MA 58-05120/05, MA 58-03893/2005/5, MA 58-03896/2005/5, and MA 58–03894/20057). A contact hypersensitivity response was induced in the ear skin of 8-week-old female K14/VEGF-A TG mice as described.10Kunstfeld R Hirakawa S Hong YK Schacht V Lange-Asschenfeldt B Velasco P Lin C Fiebiger E Wei X Wu Y Hicklin D Bohlen P Detmar M Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (275) Google Scholar Mice (10/group) were anesthetized by i.p. injection of medetomidine (0.2 mg/kg) and ketamine (80 mg/kg) and sensitized by topical application of 2% oxazolone (4-ethoxymethylene-2 phenyl-2-oxazoline-5-one; Sigma, St. Louis, MO) in acetone/olive oil (4:1 v/v) to the shaved abdomen (50 μl) and to each paw (5 μl). Five days after sensitization (day 0), the right ear was challenged by topical application of 10 μl oxazolone (1%) on each side. Starting on day 7, once-daily oral doses of 25 mg/kg NVP-BAW2881 or twice-daily topical doses of 0.5% NVP-BAW2881 were administered for 14 days. Control groups were given vehicles alone. The ear thickness was measured every other day using calipers. On day 21, mice were sacrificed and the weight of each ear and of its draining retro-auricular lymph node (LN) was determined. Female hairless SKH1 mice (three per group, two experiments performed) were treated topically on both flanks with 100 μl of 0.5% NVP-BAW2881 or vehicle alone. To avoid oral uptake, mice were housed individually and fitted with ruffs. After 2 hours, mice were given i.v. injections of 200 μl Evans Blue solution (0.5% in saline). Ten minutes later, 100 ng VEGF-A (in 50 μl PBS) were injected intradermally on both sites; and 25 minutes later, mice were sacrificed and the test sites were excised using a biopsy punch (8 mm diameter; Stiefel, Austria). Alternatively, 1.0 ml of 0.5% NVP-BAW2881 was applied to test areas of approximately 75 cm2 on the right ventral abdomen of domestic pigs (n = 6). Contralateral sites were treated with vehicle only. The solutions were applied 30, 7, and 3 hours before intradermal injection of VEGF-A. At time 0 hours, animals were sedated with ketamine and xylazine (16 and 4 mg/kg, i.m.) and 2% Evans Blue solution was injected i.v. (2 ml/kg body weight). Ten minutes later, 10 ng VEGF-A in 50 μl PBS was injected at four sites of the test areas. After 30 minutes, animals were sacrificed and 8-mm punch biopsies were taken from the injection sites. To analyze VEGF-A-induced extravasation of Evans Blue, samples were incubated in 0.5 ml formamide at room temperature for 48 hours. Subsequently, the concentration of Evans Blue in the supernatant was measured photometrically at 650 nm. In additional experiments, female hairless SKH1 mice (5 per group) were topically treated with 100 μl 1% NVP-BAW or vehicle alone on both flanks. Two hours later, 250 μl of a 0.5% Evans Blue solution was injected i.v. Extravasation was induced 10 minutes later at two sites of the pretreated areas either with 50 ng platelet-activating factor (PAF) (Novabiochem, Merck, Darmstadt, Germany) or with 25 μg histamine (Sigma), injected intradermally in 50 μl volumes. 30 minutes later, the mice were sacrificed and from the three injection sites in each mouse 8 mm punch biopsies were collected for photometrical measurement of extracted Evans Blue, as described above. In 10 animals, a total of 26 focal erythemas (approximately 4 cm2) were elicited on the shaved dorsolateral back with UVB (72 mJ/cm2), generated with TL20W lamps (Philips). Contralateral test sites were treated with 50 μl of 0.5% NVP-BAW2881 or vehicle immediately after irradiation, and again 3 and 6 hours later. After 6 and 24 hours, test sites were examined by reflectometry (Chromameter CR 400, Minolta) using a* values (L*a*b*system) for skin redness and with laser Doppler flowmetry (PF 5000, Perimed) for measurement of microperfusion.26El-Gamal S Hoffmann K Steinert P Gassmüller J Altmeyer P Objective assessment of human skin reaction to sun and UV-B.in: Frosch PJ Kligman AM Noninvasive Methods for the Quantification of Skin Functions. Springer, Berlin1993: 133-144Crossref Google Scholar, 27Duteil I Queille-Roussel C Czernielewski J Assessing treatment of psoriasis and eczema by noninvasive methods.in: Frosch PJ Kligman AM Noninvasive Methods for the Quantification of Skin Functions. Springer, Berlin1993: 223-240Crossref Google Scholar In addition, erythema was scored at each site: 0 (absent), 1 (scarcely visible, small spotted), 2 (mild, large spotted), 3 (pronounced, confluent), and 4 (severe or livid discoloring, homogenous redness). Animals (n = 8) were sensitized by applying 2, 4-dinitro fluorobenzene (DNFB, 10%, dissolved in dimethyl sulfoxide: acetone: olive oil [1:5:3, v/v/v]) onto the basis of both ears and onto both groins (100 μl/site). Eight days later, the animals were challenged with 15 μl of a 1% DNFB solution on eight test sites on both sides on the dorsolateral back. After 0.5 and 6 hours, contralateral test sites were treated with NVP-BAW2881 (0.1 or 0.5%) or vehicle. Test sites (in total 16 treated with 0.1%, 16 with 0.5%, and 32 with vehicle) were clinically examined 24 hours after challenge, when inflammation peaked. The changes were scored on a scale from 0 to 4, allowing a combined maximal score of 12 per designated site (Table 1).28Meingassner JG Grassberger M Fahrngruber H Moore HD Schuurman H Stutz A A novel anti-inflammatory drug. SDZ ASM 981, for the topical and oral treatment of skin diseases: in vivo pharmacology.Br J Dermatol. 1997; 137: 568-576Crossref PubMed Scopus (184) Google ScholarTable 1Scoring System Used to Evaluate CHS-Induced Skin Inflammation in Domestic PigsScoreErythema/IntensityErythema/ExtentInduration0AbsentAbsentAbsent1Scarcely visibleSmall spottedScarcely palpable2MildLarge spottedMild hardening3PronouncedConfluentPronounced hardening4Severe (or livid discoloring)Homogenous rednessPronounced and elevated hardeningErythema intensity, erythema extent, and induration of the skin were examined by optical inspection. Each parameter was scored on a scale from 0 to 4, allowing a combined maximal score of 12 per site examined. Open table in a new tab Erythema intensity, erythema extent, and induration of the skin were examined by optical inspection. Each parameter was scored on a scale from 0 to 4, allowing a combined maximal score of 12 per site examined. On day 21 after challenge, mice were sacrificed and their ears and auricular LNs were collected and weighed. Tissues were embedded in optimal cutting temperature compound (Sakkura Finetek, Torrance, CA) and frozen on dry ice, and 6-μm cryostat sections were cut. H&E staining was performed as described.10Kunstfeld R Hirakawa S Hong YK Schacht V Lange-Asschenfeldt B Velasco P Lin C Fiebiger E Wei X Wu Y Hicklin D Bohlen P Detmar M Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (275) Google Scholar Immunofluorescence was performed as described29Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1510) Google Scholar using the following antibodies: anti-mouse LYVE-1 (Angiobio, Del Mar, CA), MECA-32, anti-mouse CD31, anti-mouse CD45, anti-mouse CD11b (all from BD Biosciences), anti-mouse keratin 6, keratin 10, and loricrin (all from Covance Research Products, Berkeley, CA). Alexa488- or Alexa594-coupled secondary antibodies and Hoechst 33342 were purchased from Molecular Probes (Invitrogen, Basel, Switzerland). CD45, LYVE-1, or MECA-32-labeled ear sections from three mice per treatment group were examined on an Axioskop 2 mot plus microscope (Carl Zeiss, Feldbach, Switzerland), equipped with an AxioCam MRc camera (Zeiss) and a Plan-APOCHROMAT 10_/0.45 objective (Zeiss). Images of three individual fields of view were acquired per section. Computer-assisted analysis of digital images was performed using the IP-LAB software (Scanalytics, Fairfax, VA), as previously described.30Oura H Bertoncini J Velasco P Brown LF Carmeliet P Detmar M A critical role of placental growth factor in the induction of inflammation and edema formation.Blood. 2003; 101: 560-567Crossref PubMed Scopus (143) Google Scholar To quantify leukocyte infiltration, the percentage of ear tissue (covering the entire field of view, between the cartilage backbone and the epidermis) that stained positive for CD45 was determined. Furthermore, the average size of lymphatic vessels present in the entire field of view was determined. To identify changes in the blood vascular compartment, the percentage of tissue that stained positive for MECA-32 in the upper dermis (ie, the region located up to 120 μm below the epidermis) was determined. The latter parameter was chosen because chronically inflamed ears of control-treated VEGF-A TG mice presented numerous sponge-like, tortuous blood vessels in the upper dermis, making it difficult to identify single vessels and to quantify microvessel density. Data are shown as mean ± SE (SEM) and were analyzed with a Student's t-test (paired or unpaired, depending on the assay). Differences were considered statistically significant when P < 0.05. NVP-BAW2881 is a low molecular weight compound developed for the VEGFR TK inhibitor program at Novartis. In biochemical assays, NVP-BAW2881 was shown to primarily target the VEGFR TK family with IC50 values in the low nanomolar range. The IC50 value for this compound against human VEGFR-2 (hVEGFR2, also termed KDR) was 37 nmol/L (Table 2). NVP-BAW2881 was highly selective for VEGFR, although it also demonstrated activity against Tie2 (IC50 = 650 nmol/L) and RET (IC50 = 410 nmol/L). The IC50 values of NVP-BAW2881 toward a wide panel of other kinases were >10 μmol/L (data not shown).Table 2NVP-BAW2881 Inhibits Human and Mouse VEGFR TKs at Nanomolar ConcentrationsReceptorhVEGFR1hVEGFR1hVEGFR3mVEGFR2IC50 (μM)0.82 ± 0.0190.037 ± 0.080.42 ± 0.0150.165 ± 0.064The IC50 values of NVP-BAW2881 toward different human and mouse VEGFRs were determined in a scintillation proximity assay, using recombinant fusions of glutathione S-transferase (GST) with VEGFR tyrosine kinases. Incorporation of 33P into the peptidic substrates was quantified. IC50 values of a wide panel of other kinase tested were >10 μmol/L. Open table in a new tab The IC50 values of NVP-BAW2881 toward different human and mouse VEGFRs were determined in a scintillation proximity assay, using recombinant fusions of glutathione S-transferase (GST) with VEGFR tyrosine kinases. Incorporation of 33P into the peptidic substrates was quantified. IC50 values of a wide panel of other kinase tested were >10 μmol/L. In cellular assays, NVP-BAW2881 inhibited VEGF-A-induced phosphorylation of VEGFR-2 in HUVECs and in VEGFR-2-transfected Chinese hamster ovary cells, with IC50 values of 2.9 and 4.2 nmol/L, respectively (Table 3). NVP-BAW2881 also inhibited RET (NIH3T3 cells stably expressing a constitutively active RET mutant), platelet-derived growth factor receptor (PDGFR)β (in PDGFRβ expressing A31 mouse embryonic fibroblasts), and c-Kit (GIST882 cells expressing an activating KIT mutation) TK activities, with IC50 values of 45, 47, and 74 nmol/L, respectively. The compound showed moderate potency against cytoplasmic Bcr-Abl kinase (IC50 of approximate 0.6 μmol/L) while it was not effective (IC50 values >10 μmol/L) against a range of other kinases (data not shown). NVP-BAW2881 was also profiled in a panel of BaF3 cell lines, rendered interleukin-3-independent by transduction with constitutively active tyrosine kinases. The compound inhibited proliferation of the BaF3 cell line Tel-VEGFR-2 with an IC50 of 1 nmol/L, Tel-PDGF-Rβ (13 nmol/L) and PTC3-RET (152 nmol/L), and with weaker activity against Bcr-ABL (4750 nmol/L)
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