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A series of N-arylated phenylalanine derivatives has been synthesized and has been shown to be potent inhibitors of the integrin VLA-4. N-phenyl and N-heteroaryl derivatives with hydrogen bond acceptors in the meta position demonstrated low nanomolar activity against VLA-4.
SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.
Two structural classes of dual alpha4beta1/alpha4beta7 integrin antagonists were investigated via solid-phase parallel synthesis. Using an acylated amino acid backbone, lead compounds containing biphenylalanine or tyrosine carbamate scaffolds were optimized for inhibition of alpha4beta1/VCAM and alpha4beta7/MAdCAM. A comparison of the structure-activity relationships in the inhibition of the alpha4beta7/MAdCAM interaction for substituted amines employed in both scaffolds suggests a similar binding mode for the compounds.
A library of 2302 small molecule beta-turn mimetics was screened for inhibition of the alpha 4 beta 1 integrin-CS1 splice variant binding interaction. Preliminary data revealed several active ligands, and validation with purified material culminated in the identification of some of the first small molecule ligands (1, IC50 = 5 microM, and 2, IC50 = 8 microM) to be reported for this class of integrins.
Considering that in the allogeneic situation the adhesion of recipient lymphocytes to donor endothelial cells initiates the cellular rejection, we questioned the possible occurrence of a similar process in the xenogeneic situation. The adhesion of human peripheral blood lymphocytes (PBL) to porcine aortic endothelial cells (PAEC) was thus studied in an in vitro porcine-to-human xenogeneic model. It was found that 25.9% of human PBL adhered to resting PAEC. Furthermore, this adhesion increased significantly when the PAEC were stimulated by the human cytokine TNF-alpha (tumor necrosis factor-alpha). The effect of human TNF-alpha was concentration- and time-dependent and was maximal (from 25.9% to 35.6%) with 100 U/ml during 6 h. Moreover, blocking experiments with monoclonal antibody (mAb) demonstrated the role of the PBL adhesion molecules LFA-1 and especially VLA-4. Indeed, an anti-CD11a mAb decreased PBL adhesion to resting PAEC by 17.1% and to TNF-alpha stimulated PAEC by 16.9%, whereas an anti-CD49d mAb decreased dramatically PBL adhesion to resting PAEC by 53.1% and to TNF-alpha stimulated PAEC by 41.0%. Finally, phenotypic analysis of the adherent PBL showed that 50.5% of adherent cells to resting PAEC were NK (natural killer) cells, whereas 50.7% of adherent cells to TNF-alpha stimulated PAEC were T lymphocytes, showing the preferential adhesion of NK cells to resting PAEC, and that the stimulation of the PAEC with human TNF-alpha affects predominantly T lymphocyte adhesion. These results indicate that human PBL could bind to xenogeneic PAEC and that this interaction could be a first step of a xenogeneic cellular rejection.
The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.
Administration of anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (mAb) plus anti-lymphocyte function associated antigen (LFA)-1 mAb induces tolerance in murine cardiac transplantation, while anti-vascular cell adhesion molecule (VCAM)-1 mAb plus anti-very late antigen (VLA)-4 mAb administration prolongs graft survival, but leads to tolerance only in some cases. BALB/c mice hearts were transplanted into C3H/He recipients. Each combination of anti-VCAM-1 plus anti-VLA-4 mAbs (100 microg each/day, i.p.) or anti-ICAM-1 plus anti-LFA-1 mAbs (50 microg each/day, i.p.) was administered for 5 days. For control study, third group mice received daily with FK506 administration (1 mg/kg/day). The cardiac allografts and recipients' spleens were harvested on day 7; the expression of cytokines were detected using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR. Th2 cytokines (IL-4 and IL-10) were markedly enhanced and Th1 cytokines (IFN-gamma and IL-2) were suppressed in recipients treated with anti-ICAM-1 mAb plus anti-LFA-1 mAb, while poor Th2 cytokine expression allowed persistent Th1 cytokine expression in recipient mice with anti-VCAM-1 mAb plus anti-VLA-4 mAb treatment. Both Th1 and Th2 cytokine expression was suppressed in FK506-treated mice. It is concluded that immunological tolerance and prolonged graft survival induced by blocking cell adhesion is regulated by different cytokine expression.
Design and synthesis of a library as potential VLA-4 antagonists has been accomplished, based around a proposed pharmacophoric model. Compounds possessing submicromolar potency were identified and structure-activity relationships were seen across the library. Further derivatisation produced compounds with IC(50)'s <10 nmol for inhibiting the VLA-4 mediated binding of fibronectin to RAMOS cells, providing an ideal starting point for a lead optimisation Programme.
alpha(4)beta(1) and alpha(4)beta(7) integrins are key regulators of physiologic and pathologic responses in inflammation and autoimmune disease. The effectiveness of anti-integrin antibodies to attenuate a number of inflammatory/immune conditions provides a strong rationale to target integrins for drug development. Important advances have been made in identifying potent and selective candidates, peptides and peptidomimetics, for further development. Herein, we report the discovery of a series of novel N-benzoyl-L-biphenylalanine derivatives that are potent inhibitors of alpha4 integrins. The potency of the initial lead compound (1: IC(50) alpha(4)beta(7)/alpha(4)beta(1)=5/33 microM) was optimized via sequential manipulation of substituents to generate low nM, orally bioavailable dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. The SAR also led to the identification of several subnanomolar antagonists (134, 142, and 143). Compound 81 (TR-14035; IC(50) alpha(4)beta(7)/alpha(4)beta(1)=7/87 nM) has completed Phase I studies in Europe. The synthesis, SAR and biological evaluation of these compounds are described.
The design and synthesis of novel integrin alpha(V)beta(3) antagonists based on a 1,5- or 2,5-substituted tetrahydrobenzaezpinone core is described. In vitro activity of respective compounds was determined via alpha(V)beta(3) binding assay, and selected derivatives were submitted to further characterization in functional cellular assays. SAR was obtained by modification of the benzazepinone core, variation of the spacer linking guanidine moiety and core, and modification of the guanidine mimetic. These efforts led to the identification of novel alpha(V)beta(3) inhibitors displaying potency in the subnanomolar range, selectivity versus alpha(IIb)beta(3) and functional efficacy in relevant cellular assays. A method for the preparation of enantiomerically pure derivatives was developed, and respective enantiomers evaluated in vitro. Compounds 31 and 37 were assessed for metabolic stability, resorption in the Caco-2 assay and pharmacokinetics.
OBJECTIVE: Adhesion molecules regulate the infiltration of leukocytes into the graft during rejection after lung transplantation. The first step of the adhesion cascade is mediated by selectins. Sialyl-LewisX is a ligand of P-selectin. The purpose of the study was to evaluate SLX, a synthetic oligosaccharide analog of Sialyl-LewisX, for anti-rejection prophylaxis after allogeneic and xenogeneic left lateral, orthotopic rat lung transplantation. METHODS: In groups A and B, allogeneic lung transplantation was performed using fully incompatible rat strains (donors: Dark-Agouti (RT1a); recipients: Lewis (RT11)). In group A (n = 10), recipients recieved 200 microg/d SLX i.v. on day 0-4. Group B rats (n = 10) served as untreated controls. The animals were sacrificed on days 5 and 10, respectively. In groups C and D, xenogenic lung transplantation was performed using Gold Syrian hamsters as donors and Lewis rats as recipients. In group C (n = 10), recipients received 200 microg/d SLX i.v. on day 0-4. Group D rats (n = 10) served as untreated controls. The animals were sacrificed on days 2 and 5, respectively. Rejection was graded by histology from 0 (no rejection) to 5 (necrosis). By immunhistology, alveolar, interstitial CD11a, CD18 and VLA-4 positive leukocytes were counted. RESULTS: Histologically, there were a lower grade of rejection (A: 2.7 +/- 0.6; B: 4.0 +/- 0.0; P < 0.05) and fewer CD11a positive leukocytes (A: 66 +/- 27; B: 186 +/- 73; P < 0.05) on day 5 in the SLX-treated allograft group compared to the untreated group. In xenotransplantation, SLX also reduced the grade of rejection (C: 3.3 +/- 0.5; D: 4.7 +/- 0.5; P < 0.05) and the number of CD11a positive leukocytes (C: 145 +/- 22; D: 176 +/- 20; P < 0.05) on day 2. CONCLUSIONS: It is concluded, that the administration of SLX significantly reduces allograft rejection. After discontinuation treatment with SLX unmodified rejection appeared. SLX also modifies xenograft rejection, but to a lesser extent, and xenograft necrosis appeared during treatment in this model.
The alpha integrin GPIIb is a marker of hematopoietic progenitors. Using a marking strategy based on Cre-loxP technology to trace the fate of GPIIb-expressing cells, we show that GPIIb is expressed during early definitive embryonic hematopoiesis. However, the marked fetal population is distinct from the hematopoietic cells that predominate in the adult, suggesting that at least two waves of progenitors arise concurrently or consecutively in the fetus. Furthermore, using an inactivated allele of gpIIb, we provide evidence for a functional role of GPIIb on progenitors. We observe an increase in hematopoietic progenitors in the yolk sac, fetal liver, and bone marrow, an effect which may, in part, be explained by loss of binding to fibronectin.
OBJECTIVE: Because recruitment and retention of lymphoid cells appear to be critical components of the pathogenesis of lichen planus, we have compared the expression and distribution of a panel of vascular adhesion molecules (ELAM-1, P-selectin, ICAM-1, VCAM-1, PECAM-1, CD34) and leukocyte adhesion molecule ligands (LFA-1, Mac-1, VLA4, L-selectin) in biopsies of this disease. STUDY-DESIGN: Frozen sections of 12 clinically and histologically confirmed cases of lichen planus and 9 normal control tissues were evaluated immunohistochemically with a standard 1-day avidin-biotin peroxidase technique. Staining intensity of vascular endothelium was evaluated semiquantitatively. Three microvascular zones or compartments were defined and evaluated separately. RESULTS: Generally, different staining patterns were observed in association with the various endothelium-associated adhesion molecules. In normal controls, PECAM was intensely expressed and VCAM-1 was weakly expressed. Intermediate staining was associated with ELAM-1, P-selectin, ICAM-1, and CD34. Staining within the three microvascular compartments frequently showed variations in intensity. In lichen planus, increased staining for ELAM-1, P-selectin, ICAM-1, and VCAM-1 was evident in one or more of the microvascular compartments. In the subepithelial vascular compartment where the infiltrate was the most dense, VCAM-1 appeared to show the greatest positive change. Almost all cells in the lichen planus infiltrates stained positive for ICAM-1, L-selectin, LFA-1, and VLA4, and large numbers of cells also exhibited VCAM-1, PECAM-1, and Mac-1 immunoreactivity. CONCLUSIONS: It appears that upregulation of ELAM-1, ICAM-1, and VCAM-1 (especially by endothelial cells in the subepithelial vascular plexus) could play a role in the pathogenesis of lichen planus. The expression of leukocyte receptors L-selectin, LFA-1, and VLA4 by most of the cells in the lichen planus infiltrate suggest that these molecules may be responsible for recruitment as well as retention in the active lichen planus lesion.
BACKGROUND: Kimura's disease is a rare but distinctive eosinophilic inflammatory disorder of unknown etiology; few reported case studies have focused on the immunopathologic background of this unique disease. OBJECTIVE: To define better the immunopathogenetic features of Kimura's disease, we attempted to quantitatively analyze values of cytokines and soluble interleukin-2 receptor (sIL-2R) in peripheral blood (PB), as well as perform surface immunophenotypic analysis of eosinophils from a Japanese patient with chronic relapsing Kimura's disease. RESULTS: Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and sIL-2R were elevated, and newly expressed antigens on eosinophils CD4, CD25, and HLA-DR were found to be involved in the pathophysiology of this disorder. CONCLUSIONS: Kimura's disease may be a disease in which activated lymphocytes release cytokines, and these released cytokines, such as GM-CSF and TNF-alpha cause eosinophil activation. These processes may be related to the pathogenesis of this disorder.
The integrin adhesion molecules are involved in the recruitment and activation of inflammatory cells at sites of inflammation in a variety of diseases. In the present study, we have investigated the effects of blocking monoclonal antibodies (mAbs) directed against CD49d (alpha(4) integrin), CD18 (beta(2) integrin) and the alpha sub-units of beta(2) integrin CD11a (LFA-1 integrin) and CD11b (Mac-1 integrin), on antigen (Ag)-induced acute bronchoconstriction and cellular recruitment in allergic rabbits in vivo. Inhaled Ag (Alternaria tenuis) challenge of neonatally sensitised rabbits caused an acute bronchoconstriction demonstrated by an increase in lung resistance (R(L)) and decrease in dynamic compliance (C(dyn)) and pulmonary inflammation characterised by an increase in bronchoalveolar lavage (BAL) inflammatory cells, particularly eosinophils, 24 h after challenge. Pre-treatment with the anti-CD49d mAb (Max-68P), significantly inhibited the Ag-induced acute bronchoconstriction in terms of R(L) and (C(dyn)). Treatment with the other anti-integrin mAbs had no effect on the acute bronchoconstriction after inhaled Ag challenge.Pre-treatment with the anti-integrin mAbs had differential effects in blocking the recruitment of inflammatory cells 24 h after inhaled Ag in the allergic rabbits. The data show that in the allergic rabbit model of asthma, VLA-4 (CD49d/CD29) only, is involved in the acute bronchoconstriction, suggesting an involvement of mast cell degranulation. Furthermore, eosinophil recruitment and activation appears to be mediated by a combination of VLA-4 (CD49d/CD29) and LFA-1 (CD18/CD11a). However in contrast, lymphocyte recruitment appears to be mediated by a combination of LFA-1 (CD18/CD11a) and Mac-1 (CD18/CD11b).
The aim of this work was to quantify by flow cytometry the main adhesion receptors on CD34+ cells. These cells were isolated from bone marrow (BM) or mobilized peripheral blood (PB). The proportions of CD34+/CD49d+ and CD34+/CD49e+ are weaker on PB cells, without quantitative expression variation. This phenotypic variation may induce CD34+ cells exist from BM into circulation, promoting the mobilization. The homing to the BM implicate the CD62L receptor, which expression was found more frequently and stronger on PB cells than on BM. The CD11b, CD18 and CD54 receptors are implicated in CD34+ cells adhesion to BM micro-environment. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. Moreover, CD54 receptor was more frequently expressed on PB cells. Quantitative analysis revealed that CD18 was more strongly expressed on BM than on PB cells. This quantitative variation could promote progenitor adhesion by interacting with stromal cells. Finally, quantitative expression of the main receptors on CD34+ cells provides an original option for studying CD34+ cells during the mobilization, the homing or the adhesion to BM micro-environment.
The determinants of the prevalence of CD8(+) T cells in the inflamed myocardium of Trypanosoma cruzi-infected patients and experimental animals are undefined. Using C3H/He mice infected with the Colombiana strain of T. cruzi, we found that the distribution of CD4(+)/CD8(-) and CD4(-)/CD8(+) T cells in the myocardium mirrors the frequency of cells expressing the CD62L(Low)LFA-1(High)VLA-4(High) activation phenotype among CD4(+)/CD8(-) and CD4(-)/CD8(+ )peripheral blood T cells. Consistently, vascular cell adhesion molecule-1-positive endothelial cells and a fine fibronectin network surrounding VLA-4(+) mononuclear cells were found in the inflamed myocardium. Further, interferon gamma (IFN-gamma) and IFN-gamma-induced chemokines (RANTES, MIG and CRG-2/IP-10), as well as JE/MCP-1 and MIP1-alpha, were found to be the dominant cytokines expressed in situ during acute and chronic myocarditis elicited by T. cruzi. In contrast, interleukin 4 mRNA was only detected during the chronic phase. Altogether, the results indicate that the distribution of T-cell subsets in the myocardium of T. cruzi-infected mice reflects the particular profile of adhesion molecules acquired by most peripheral CD8(+) T lymphocytes and point to the possibility that multiple IFN-gamma-inducible molecules present in the inflamed tissue contribute to the establishment and maintenance of T. cruzi-induced myocarditis.
In recent years antagonists of very late antigen-4 (VLA4, also known as integrin alpha(4) beta(1)) have shown great promise in treating inflammatory disorders in a number of animal models. The most advanced in this endeavor is a humanized anti-alpha(4) antibody, Antegren, which is in phase II clinical trials for multiple sclerosis. The first reported small-molecule VLA4 antagonist to advance into clinical trials is currently in phase I as an aerosol for treating asthma. A number of peptides, cyclic peptides and peptidomimetics have been disclosed and are in preliminary stages of development.