SCREENING METHOD FOR COMPOUNDS

Abstract: The present invention relates to screening assays and methods for the identification of drugs, in particular those which inhibit or induce inflammatory or allergic responses. The identification method comprises mixing a test compound with IFN- gamma and determining whether the test compound has conjugated or interacted with IFN- gamma .

Description: DESCRIPTION SCREENING METHOD FOR COMPOUNDS The present invention relates to screening assays and methods for the identification of drugs which inhibit or induce inflammatory or allergic responses. The prevalence of allergic diseases such as hayfever, asthma and eczema has increased steadily during the latter half of this century to reach epidemic proportions. This trend has been associated to a large extent to with Western lifestyle, as postulated by Cookson, W. Nature 402, suppl. B5-B11 (1999). Further, it has been postulated that childhood infections may protect against allergy by directing the immune response away from the allergic phenotype. During this time, when the prevalence of allergic diseases has been increasing, the use of antibiotics, and in particular β-lactam antibiotics, has increased dramatically. Allergic responses are mediated by a specific class of antibodies, namely the IgE class. IgE sensitises mast cells to release histamine and other biologically active amines and mediators in response to an allergen. IgE allergic responses are controlled by the action of cytokines, secreted by T helper cells. IgE responses are induced by interleukin-4 (IL-4) and interleukin-13 (IL-13) which are produced by type 2 T helper (Th2) cells, and are counteracted by interferon-γ (IFN-γ) produced by type 1 T helper (Thl) cells, as described by Pene, J. et. al. IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons α, γ and protaglandin E 2 Proc. Natl. Acad. Sci. USA 85, 6880 (1988). IFN -γ promotes inflammation and cell-mediated immune defence. IL-4 promotes antibody-mediated immunity and IgE mediated allergy. Furthermore, IFN -γ promotes further Thl cell activation and inhibits Th2 cell activation, whereas, IL-4 promotes further Th2 cell activation and inhibits Thl cell activation. It has been suggested that antibiotics, in particular penicillin, reduce the release of IFN-γ by Thl cells (Brander, C. et al Heterogenous T cell responses to β- lactam-modified self-structures are observed in penicillin - allergic individuals. J Immunol, 155, 2670-2678, 1995), and that penicillin reduces the release of IFN -γ by penicillin-specific-T cell clones (Padovan, E. et al, Penicilloyl peptides are recognised on T cell antigenic determinants in penicillin allergy. Eur. J. Immunol. 27, 1303-1307 (1997). In light of the fact that compounds such as penicillin have been linked with inducing allergic responses, it would be desirable if there was a test by which such compounds could be screened to ascertain whether they induce such responses. We have found that β-lactam benzyl penicillin conjugates to IFN-γ, blocking its activity rather than blocking its release. This blocking of activity may be important in counteracting the effect of IL-4 and IgE mediated allergic responses. Furthermore, we have found that β-lactam benzyl penicillin conjugates to IL-4 to a far lesser degree than to IFN-γ. Compounds which conjugate to IFN-γ, blocking its activity, could prevent IFN-γ from inhibiting Th 2 cell activation. Consequently, IgE responses would not be counteracted by the action of IFN-γ, since its activity is blocked, and an allergic response may be induced. It is an object of the present invention to provide a means for identifying compounds likely to cause or promote allergic responses. In accordance with a first aspect of the present invention, there is provided a method for identifying compounds which induce or promote allergic responses comprising: a) mixing a test compound with a IFN-γ, and b) determining whether the test compound has conjugated or interacted with IFN-γ The test compound may conjugate or interact chemically, structurally or functionally with IFN-γ or in any other way. According to the above mentioned method, test compounds which conjugate or interact with IFN-γ are identified as compounds which induce an allergic response. Compounds which conjugate with IFN-γ may inhibit its activity and thereby induce an allergic response by virtue of its inability to inhibit Th2 cell activation. Consequently, IgE responses induced by IL-4 would be permitted to occur outside the control of IFN-γ. Examples of those compounds which may be screened by this method include the β-lactam antibiotics, which comprise penicillins, cephalosporins, cephamycins, carbapenams and monobactams. Preferably, the mixing of the test compounds with a IFN-γ, in which compounds may conjugate or interact therewith, is carried out in a physiological or other appropriate aqueous buffer. The preferred methods for determining conjugation or interaction comprise immunodetection (with antibody), binding studies (with labelled compounds) or detection of any structural change in the IFN-γ protein which comprise electrophoretic mobility studies, spectroscopic properties or changes in immunodetection of the cytokine or biological activity. Test compounds which do not conjugate or interact with the cytokine are unlikely to produce or promote allergic responses and are therefore more likely to prove useful as a pharmaceutical, or a component thereof. In accordance with a second aspect of the present invention there is provided a compound identified according to the method described hereinabove for use as an active pharmaceutical substance. In accordance with a third aspect of the present invention, there is provided a method for identifying a compound for use as an active pharmaceutical substance comprising: a) mixing a test compound with IFN-γ, and b) determining whether the test compound has conjugated or interacted with IFN-γ The test compound may conjugate or interact chemically, structurally or functionally with IFN-γ or in any other way. As hereinabove described, IFN-γ promotes inflammation. Consequently, it would be desirable to identify compounds which conjugate or interact with IFN-γ which may be useful as anti-inflammatories. In accordance with a fourth aspect of the present invention there is provided a compound identified according to the method claimed herein above for use as an anti-inflammatory drug. The compound identified may be used for the treatment of rheumatoid arthritis, inflammatory lung disease and other IFN-γ mediated inflammatory disease. In accordance with a fifth aspect of the present invention there is provided a method of identifying a compound for use as an active substance in an anti- inflammatory drug comprising: a) contacting a test compound with IFN-γ, and b) determining whether the test compound has conjugated or interacted with IFN-γ The test compound may conjugate or interact chemically, structurally or functionally with IFN-γ or in any other way. A method demonstrating the present invention will now be shown by way of example only: A determination was carried out in accordance with the present invention with a β-lactam antibiotic, namely benzyl penicillin (BP), as a test compound. Cells for use in the examples were prepared as follows :- Peripheral blood mononuclear cells (PBMC) were purified from the blood of healthy volunteers. The HMC-1 mast cell line was grown in Iscove's modified Dulbecco's medium plus 5% FCS (IF5) and subcultured weekly at 1 : 10. Peripheral blood monocytes (PBM) were purified from PBMC using anti-CD 14- coated magnetic beads (MACS: Milteni Biotech, Bisley, UK), according to the manufacturer's instructions. The generation of PBMC supematants and RNA for the examples were prepared as follows: PBMC (lOVml) were incubated in RPMI-1640 medium containing TCH (ICN) with or without PHA (Sigma, 2.5μg/ml). At each time point, samples were centrifuged for 5 min at 400g, the supematants collected and analysed of IFN-γ by ELISA (IDS, Boldon, UK). SDS-PAGE and Western Blotting techniques for the examples were carried out as follows:- Carrier-free recombinant human IFN-γ (PeproTech, London, UK) was made to lOμg/ml and incubated overnight with or without BP (final concentrations 25,5,0.5mg/ml). At the end of the incubation period, 5x loading buffer was added and 20μl of each sample loaded onto SDS-polyacrylamide gels. The gels were then analysed by Western blotting, using a rabbit polyclonal anti-BP antibody. A comparative study was conducted using IL-4 in place of IFN-γ. Fig. 1 Western Blot illustrating the conjugation or interaction of a test compound, BP, to IFN-γ; and Fig. 2 Investigation of the effect of conjugation or interaction of the test compound, BP, on IFN-γ activity Fig. 3 Investigation of the effect of conjugation or interaction of the test compound, BP on IFN-γ activity. Table 1 Natural and recombinant IFN-γ in cell-free experiments Concentration of IFN-γ (pg/ml) detected Sample -BP +BP IFN-γ 200 pg/ml 192 13 IFN-γ 1000 pg/ml 1015 70 PBMC supernatant 1 152 0 PBMC supernatant 2 389 0 PBMC supernatant 3 >400 92 Purified recombinant IFN-γ or cell-free supematants from stimulated PBMC were incubated overnight with BP (500μg/ml), then assayed by ELISA, Supernatant 1, PBMC stimulated for 20h with IL-2; supernatant 2, PBMC stimulated for 43 h with IL-2; supernatant 3, PBMC stimulated for 43 h with PHA. As clearly demonstrated, BP reduces the amount of IFN-γ detected by immunoassay in recombant purified preparations and harvested cell supematants stimulated with IL-2 (lOμg/ml) or PHA (2.5 μg/ml) Fig. 1 illustrates a method by which conjugation or interaction of a test compound with a cytokine, namely IFN-γ, can be determined. Carrier-free recombinant human IFN-γ (PeproTech, London, UK) were made to lOμg/ml and incubated overnight with or without Benzyl Penicillin (BP) (final concentrations: 25, 5, 0.5 mg/ml). At the end of the incubation period, 5x loading buffer was added and 20μl of each sample loaded onto SDS-polyacrylamide gels. For all samples, aliquots were then analysed by Western blotting, using a rabbit polyclonal anti-BP antibody. A comparative studay was conducted using IL-4 in place of IFN-γ. Fig. 2 illustrates whether the effect of conjugation or interaction of the test compound, BP, with IFN-γ has an effect on IFN-γ activity; IFN-γ was tested for induction of MHC class II DR on human peripheral blood monocytes (PBM). IFN-γ (lμg/ml) was incubated overnight with various concentrations of BP. HMC-1 cells were pulsed with or without IFN-γ (lOOng/ml), BP, or BP-treated IFNγ, for 30 min and washed 3 times in PBS. The cells were resuspended in IFS and co-cultured (2xl0 5 ) overnight with PBM (2xl0 5 ) in a total volume of 200μl in polypropylene tubes (Falcon). Six tubes were set up for each HMC-1 treatment. At the end of culture, antibodies were added as follows: 1, none; 2, anti-DR (Caltag, supplied by TCS, Buckingham, UK); 3, anti-CD45 (Caltag) plus isotype control for anti-DR; 4, anti-DR and anti-CD45 (in triplicate. Samples 1, 2 and 3 were used to set voltage gains and compensation levels. CD45 positive cells (PBM) were gated in sample 4 and the median DR fluorescence intensity measured. Results are presented as the mean ± standard deviation of median fluorescence, for triplicate determinations, measured as increase in DR over control (HMC-1 pulsed without IFN-γ). As demonstrated, pre-incubation of IFN-γ with test compound, BP, reduced its bioactivity in a concentration dependent manner. Fig. 3 illustrates another example for assessing the effect of a test compound with IFN-γ activity. The effect of a test compound, BP, on IFN-γ mediated up- regulation of macrophage chemoattractant protein -1 (MCP-1) mRNA expression in the human A549 lung epithelial cell line. IFN-γ (lμg/ml) was incubated overnight with BP (20 or 10 mg/ml). Confluent A549 cells were incubated with IFN-γ, BP-treated IFN-γ (both diluted to final concentrations of 20, 2 and 0.2 ng/ml), BP or nothing. After 2h, RNA was extracted from the cells with TRIzol and analysed by RNase protection. RNase protection assays were carried out as follows :- T7 RNA polymerase was used to synthesis P radiolabelled antisense RNA probes, using cDNA templates HCK 3, according to the manufacturer's instructions (Pharmingen, Cambridge, UK). Probe solutions were hybridised to 10-20μg of total RNA at 56 °C overnight followed by digestion with RNAse A and Tl (Pharmingen). Samples were digested with proteinase K, phenol extracted and precipitated in ethanol. Each sample was loaded on to a 6% polyacrylamide urea gel, run at 38 mA in 0.5 x TBE (tris-borate EDTA buffer) alongside the undigested probe as size marker. Gels were exposed overnight to autoradiography film (KODAK XAR-5) at -70 °C with intensifying screens. Densitometric analysis was carried out using the "Image" programme (National Institutes of Health, USA). Fresh IFN-γ (lanes 7-9) and IFN-γ that had been incubated alone overnight at 37° (as control, lanes 1-3), induced a concentration dependent increase of MCP-1 mRNA. Pre-incubation of IFN-γ overnight with BP inhibited induction of MCP-1 mRNA (lanes 4-6). BP mixed with IFN-γ immediately before addition to the responder cells had a less marked inhibiting effect on the biological activity of the cytokine (lanes 10-12). Lane 13 shows unstimulated cells.

Claims: CLAIMS 1. A method for identifying compounds which induce or promote an allergic responses comprising: a) mixing a test compound with IFN -γ b) determining whether the test compound has conjugated or interacted with IFN -γ. 2. A compound identified according to the method of claim 1 for use as an active pharmaceutical substance. 3. A method for identifying a compound for use as an active pharmaceutical substance comprising: a) mixing a test compound with IFN -γ, and b) determining whether the test compound has conjugated or interacted with IFN -γ. 4. A compound identified according to the method of claim 3 for use as an anti-inflammatory drug. 5. A method of identifying a compound for use as an active substance in an anti-inflammatory drug comprising: a) contacting the test compound with IFN -γ, and b) determining whether the test compound has conjugated or interacted with IFN -γ.

Inventors: BROOKS BERNADETTE MARY, COLEMAN JOHN WILLIAM, BROOKS, BERNADETTE, MARY, COLEMAN, JOHN, WILLIAM
Applicants: BROOKS BERNADETTE MARY, COLEMAN JOHN WILLIAM, UNIV LIVERPOOL, BROOKS, BERNADETTE, MARY, COLEMAN, JOHN, WILLIAM, THE UNIVERSITY OF LIVERPOOL
Application year: 2001
Main classification: G01N33
IPC codes: A61K45, A61P29, A61P37, G01N33