CCR1-specific non-peptide antagonist: efficacy in a rabbit allograft rejection model.

The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1alpha and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca(2+) flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33+/-1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9+/-2.1 and 16.0+/-1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor.

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1alpha (MIP-1alpha) binding to mouse CCR1 at concentrations up to 10 microM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca(2+) stimulated by CCR1 agonists, but had no effect on N-formyl-Met-Leu-Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1alpha (SDF1alpha)-induced Ca(2+) mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Discovery of novel non-peptide CCR1 receptor antagonists.

Ligands for the CCR1 receptor (MIP-1alpha and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1alpha and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Identification and characterization of small molecule functional antagonists of the CCR1 chemokine receptor.

The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods.

The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.

The centrosome moves out of a nuclear indentation in human lymphocytes upon activation.

Using a beta-tubulin specific antibody, centrosomes were labeled in paraformaldehyde fixed human lymphocytes. Cells were kept in suspension to preserve the three-dimensional (3D) morphology as much as possible. The centrosome was generally identified as the focus of the microtubule array. Resting (G0) and phytohemagglutinin activated cells in G1 stage were taken for 3D analysis of the centrosome position, using confocal microscopy and 3D analysis software. Measurements were performed in relation to the nuclear center and the periphery of the propidium iodide stained area ("nuclear envelope"). The distribution of the distances between the centrosome and the nuclear center revealed that in most resting cells the centrosome was located at the basis of a nuclear indentation. Upon activation, however, the centrosome appeared to move out of the indentation during transition from G0 to G1 stage.

The nuclear position of pericentromeric DNA of chromosome 11 appears to be random in G0 and non-random in G1 human lymphocytes.

The nuclear topography of pericentromeric DNA of chromosome 11 was analyzed in G0 (nonstimulated) and G1 [phytohemagglutinin (PHA) stimulated] human lymphocytes by confocal microscopy. In addition to the nuclear center, the centrosome was used as a second point of reference in the three-dimensional (3D) analysis. Pericentromeric DNA of chromosome 11 and the centrosome were labeled using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence. To preserve the 3D morphology of the cells, these techniques were performed on whole cells in suspension. Three-dimensional images of the cells were analyzed with a recently developed 3D software program (Interactive Measurement of Axes and Positioning in 3 Dimensions). The distribution of the chromosome 11 centromeres appeared to be random during the G0 stage but clearly non-random during the G1 stage, when the nuclear center was used as a reference point. Further statistical analysis of the G1 cells revealed that the centromeres were randomly distributed in a shell underlying the nuclear membrane. A topographical relationship between the centrosome and the centromeres appeared to be absent during the G0 and G1 stages of the cell cycle.

Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy.

Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.

Detection of migrated allogeneic oligodendrocytes throughout the central nervous system of the galactocerebrosidase-deficient twitcher mouse.

Galactocerebrosidase-deficient oligodendrocytes of 'twitcher' (twi/twi) mice degenerate prematurely. Transplantation of normal bone marrow cells has been shown to alleviate symptoms and to prolong survival time. However, characteristic ataxia ('twitching') is not cured. In an attempt to improve further the condition of twitcher mice, allogeneic foetal liver cells were transplanted as a source of normal haemopoietic stem cells and supplemented with intracerebral transplantation of foetal brain cells. A reliable method was developed to detect donor-type cells in brain tissue. Bacteriophage lambda transgenic foetal mice were used as donors of both foetal liver and brain cells. Integrated copies of lambda DNA in donor cells were detected by in situ hybridization with biotinylated probes, which were then stained using streptavidin alkaline phosphatase. This technique was combined with immunohistochemistry to distinguish donor-type oligodendrocytes from macrophages. Immunoperoxidase staining with an antiserum to carbonic anhydrase-II produced dark perikarya of oligodendrocytes. The results demonstrated that local foetal brain cell grafts resulted in a wide dissemination of donor-type oligodendrocytes throughout the twitcher brain. The addition of a foetal brain cell graft to haemopoietic cell transplantation resulted in significantly prolonged survival of twitcher mice.

Syntheses of tolrestat analogues containing additional substituents in the ring and their evaluation as aldose reductase inhibitors. Identification of potent, orally active 2-fluoro derivatives.

A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.

Flow cytometric measurement of rRNA levels detected by fluorescent in situ hybridization in differentiating K-562 cells.

The flow cytometric detection of fluorescent in situ hybridization (FISH) performed on intact cells in suspension is a recently described method (Bauman et al. 1989). We studied the application of this method for monitoring cellular differentiation. The amount of rRNA which is taken for a good indicator of growth in size, the rate of protein synthesis and the G0 G1 transition was followed by FISH. For this purpose biotinylated single stranded RNA probes obtained by transcription from a 2.1 kb BglII-EcoRI fragment of the human 28S ribosomal RNA gene subcloned into plasmid pGEM2 were used. K-562 leukaemic cells, used as targets, were induced to differentiate by dimethyl sulfoxide, phorbol myristate acetate and hemin. In the last two cases the cell cycle analysis, growth kinetics, cellular morphology and immunophenotyping indicated differentiation into monocytic and erythroid direction, respectively. The differentiation was accompanied by a rapid increase followed by a decrease to the base level of rRNA. This was not observed in the uninduced exponentially growing control cells. Based on our results, we propose that the FC-FISH detection of the rRNA level is a valuable method to distinguish between cell subpopulations. We propose that using other probes, FC-FISH will become useful to monitor different cellular processes.

The use of E. coli exonuclease III to generate single stranded DNA in BrdUrd cell-cycle analysis permits simultaneous detection of cell surface antigens.

An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2'-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed. Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method. Enzymatic denaturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution. After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E. coli exonuclease III was used to generate stretches of single stranded DNA. This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies. The enzymatic denaturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone marrow cells. With this new method it is possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated.

Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes.

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov-Smirnov test. The difference between the two patterns was at a P less than 0.01 significance level.

Spatial topography of a pericentromeric region (1q12) in hemopoietic cells studied by in situ hybridization and confocal microscopy.

A fluorescent in situ hybridization procedure with a chromosome 1-specific (1q12) repetitive satellite DNA probe was used to label the 1q12 regions of the chromosomes 1 in spherical and polymorphic hemopoietic cell nuclei. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The 1q12 regions of chromosome 1 were measured to be closely associated with the nuclear envelope in isolated nuclei of unstimulated diploid human lymphocytes. The relative positions to each other in the periphery of these spherical nuclei could not be distinguished from a random distribution pattern. In the diploid and tetraploid polymorphic nuclei of cells of the promyelocytic leukemia cell line HL60 these pericentromeric sequences were also associated with the nuclear surface.

Three-dimensional reconstruction of pericentromeric (1q12) DNA and ribosomal RNA sequences in HL60 cells after double-target in situ hybridization and confocal microscopy.

A fluorescent in situ hybridization procedure was applied to simultaneously label intranuclear pericentromeric (1q12) sequences of the chromosomes 1 and cytoplasmic ribosomal RNA sequences in whole cells of the promyelocytic HL60 cell line. For this purpose biotinated chromosome 1-specific (1q12) repetitive satellite DNA and 28S ribosomal ssRNA probes were used. The entire procedure was performed in suspension to preserve nuclear morphology. The result was studied by three-dimensional analysis, as provided by a scanning laser confocal microscope. The intracellular positions of both cytoplasmic rRNA and intranuclear centromere 1 DNA could easily be distinguished. This approach could be useful as a framework for the study of the 3-D localization of genes and gene transcripts.

Flow cytometric detection of beta-globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization.

The novel method for flow cytometric detection of cellular RNA species in suspended cells by fluorescent in situ hybridization (FC-FISH) was applied in the evaluation of beta-globin expression in murine haemopoietic tissues. Normal murine bone marrow cells and regenerating bone marrow cells obtained after lethal irradiation and bone marrow transplantation as well as murine 15 d fetal liver were examined. Furthermore, spleens and bone marrow of phenylhydrazine-induced anaemic mice were studied. Biotinylated sense- and antisense single strand RNA probes, obtained by transcription of a 510 nucleotides murine beta-globin cDNA sequence subcloned into the pGEM1 plasmid were used as hybridization probes. For detection of the hybrids formed, avidin-FITC was used. Only the antisense beta-globin probe gave strongly positive fluorescence signals in a defined population of cells in each of the tissues examined, whereas the sense probe did not give signals higher than control samples. Melting characteristics of the hybrids showed the specificity of the in situ hybridization reaction. Forward light scatter distributions, reflecting cell size of the positive cells were as expected from erythroid cells. Within the erythrocyte subpopulation both beta-globin-negative and -positive cells were detected. The percentages of positive cells determined flow cytometrically correlated with the percentages observed in May-Grünwald/Giemsa stained preparations. Differences observed in fluorescence intensity between positive cells of different organs were no larger than about a factor of two, indicating a rather constant beta-globin mRNA content over the entire differentiation range. An exception was 15 d fetal liver, which was shown biochemically to contain about eight times more beta-globin RNA and which had a 2.4 times higher fluorescence intensity. We estimate that the sensitivity of the present method is such that as little as 500 copies per cell of a specific mRNA of 1 kb length would be detectable.