WO1996010090A1

WO1996010090A1 - A high capacity screen for immunoregulants

Info

Publication number: WO1996010090A1
Application number: PCT/US1995/012315
Authority: WO
Inventors: Robert C. Boltz, Jr.
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1994-09-29
Filing date: 1995-09-25
Publication date: 1996-04-04
Anticipated expiration: 1997-03-29

Abstract

A process for obtaining reproducible intracellular calcium concentration measurements for immunoregulants, which depolarize the membrane potential of human T cells by blocking potassium channel Kv1.3. A method for analyzing compounds for activity as immunoregulants using the reproducible intracellular calcium concentration measurement in a high capacity screening technique.

Description

TITLE OF THE INVENTION

A HIGH CAPACITY SCREEN FOR IMMUNOREGULANTS

BACKGROUND OF THE INVENTION

Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves ophthalmopathy. Although the underlying pathogenesis of each of these conditions may be quite different, they have in cominon the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such self-reactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.

Similarly, following a bone-marrow or an organ transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.

One end result of an autoimmune or a rejection process is tissue destruction caused by inflammatory cells and the mediators they release. Anti-inflammatory agents such as NSAID's and corticosteroids act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.

Cyclosporin A, which was approved by the US FDA in 1983, is currently the leading drug used to prevent rejection of transplanted organs. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. Though cyclosporin A is effective in fighting transplant rejection, it is nephrotoxic and is known to cause several undesirable side effects including kidney failure, abnormal liver function and gastrointestinal discomfort.

Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K+ channels, in their role as modulators of the plasma membrane potential in human T-lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In regulating membrane potential, K⁺ channels play a role in the regulation of intracellular Ca⁺⁺ homeostasis, which has been found to be important in T-cell activation. The screening for inhibitors of the human T- lymphocyte K⁺ channels is underdeveloped, due to the paucity of high capacity screens.

Functional voltage-gated K⁺ channels can exist as multimeric structures formed by the association of either identical or dissimilar subunits. This phenomena is thought to account for the wide diversity of K⁺ channels found in different tissues. Despite the rapid advances in the molecular biology of K+ channels, subunit compositions of native K⁺ channels and the physiologic role that particular channels play are, in most cases, still unclear. To address this issue of finding compounds which selectively inhibit Kvl.3, the screen identifies compounds which have the same biological inhibition profile as MgTX. The present screen utilizes a phenonmenon which is dependent on Kvl .3 block observed with MgTX.

The Kv 1.3 channel is a voltage-gated potassium channel that is found in neurons, blood cells, osteoclasts and T-lymphocytes. The Chandy and Cahalan laboratories proposed a hypothesis that blocking the Kγl.3 channel would illicit an immunosuppressant response. (Chandy et al., J. Exp. Med. 160, 369, 1984; Decoursey et al., Nature, 307, 465, 1984). However, the K⁺ channel blockers employed in their studies were non-selective. Until the present invention, no high capacity fluorescent screen for the Kγl.3 channel exists. Although a laboratory (Price et al., Proc. Natl. Acad. Sci. USA, 86, 10171 , 1989) showed that charybdotoxin would block Kyi .3 in human T cells, charybdotoxin was subsequently shown to inhibit four different K+ channels (K_v1.3 and three distinct small conductance Ca^+ activated K+ channels) in human T lymphocytes, limiting the use of this toxin as a probe for the physiological role of K_v 1.3 (Leonard et al., Proc. Natl. Acad. Sci. USA, 89, 10094, 1992). Since MgTX is a selective K_v 1.3 inhibitor, it is useful to show that blocking of K_v1.3 will inhibit T cell activation (Lin et al., J. Exp. Med , 177, 637, 1993).

A thirty-nine amino acid peptide, Margatoxin (MgTX), has been purified to homogeneity from venom of the scorpion Centruroides margaritatus. The gene encoding MgTX is constructed, and this gene is expressed in E. coli to produce recombinant MgTX. MgTX is a potent and selective inhibitor of a voltage-dependent K⁺ channel present in human lymphocytes. MgTX exhibits immunosuppressant activity with human T-lymphocytes, and is useful as an immunosuppressant, in modeling nonpeptidyl K⁺ channel blockers, and in establishing biochemical assays based on ligand binding or other protocols with which to screen for other novel modulators of voltage dependent K⁺ channels in lymphocytes and other tissues including the brain. As an immuno¬ suppressant, MgTX-like compounds would be useful in the treatment of autoimmune diseases, as well as to prevent the rejection of foreign organ transplants and/or related afflictions, diseases and illnesses.

The fluorescent screen of the present invention represents a unique tool with which to identify chemical species which block the function of Kyi .3. This channel has been identified as the major voltage- dependent K⁺ conductance in peripheral human T-lymphocytes. Human T-lymphocytes contain, in addition to K_v1.3, several distinct small- conductance Ca2+-activated K⁺ channels. ChTX also blocks these channels. Thus, ChTX-like compounds are not selective inhibitors of K_v1.3. MgTX has recently been demonstrated to depolarize human T cells (Leonard et al., Proc. Natl. Acad. Sci. U. S. A. 89, 10094, 1992) and to prevent activation and proliferation of these cells mediated by Ca2⁺- dependent pathways (Lin et al., J. Exp. Med., 177, 637, 1993). Compounds which biochemically behave like MgTX should be specific blockers of the K_v1.3 channel.

Venom of the new world scorpion Centruroides margaritatus was determined to contain an activity selectively directed against voltage- dependent K+ channels: it inhibited binding of [ ¹²⁵I]ChTX to K_v1.3 channels in rat brain synaptosomal membranes, but not to Maxi-K channels in smooth muscle sarcolemma. MgTX is structurally related to other known K⁺ channel blocking peptides, but is distinguished by its potent and selective blockade of Kγl .3. Given these properties, MgTX represents a useful tool for studying the physiologic role of K 1.3.

MgTX inhibits T lymphocyte activation through calcium activated pathways (Lin C.S., R.C. Boltz, J.T. Blake, M. Nguyen, A. Talento, P.A. Fischer, M.S. Springer, N.H. Sigal, R.S. Slaughter, M.L. Garcia, G.J. Kaczorowski, and G.C Koo. 1993. Voltage-gated potassium channels regulate calcium-dependant pathways involved in human T lymphocyte activation. J Exp Med 177:637.). MgTX has also been shown to block the Kv 1.3 channel resulting in an average depolarization of the membrane potential from a resting potential of -50 m V to -30 millivolts(Leonard R.J., M.L. Garcia, R.S. Slaughter, and J.P. Reuben. 1992. Selective blockers of voltage-gated K⁺ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin. Proc. Natl. Acad. Sci. USA 89: 10094.). Depolarization of T lymphocytes with extra-cellular potassium has also been shown to inhibit calcium influx (Hess S.D., M. Oortgiesen, and M.D. Cahalan.1993 Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. .J.Immunol. 150: 2620.) and activation(Freedman B.D., M.A.Price, and C.J. Deutsch. 1992 Evidence for voltage modulation of IL-2 production in mitogen stimulated human peripheral blood lymphocytes. J. Immunol. 149: 3784; Gelfand E.W., R.K. Cheung, G.B. Mills, and S.Grinstein. 1987. Role of membrane potential in the response of human lymphocytes to phytohemagglutinin. J. Immunol. 138: 527.).

It has been postulated that the effect of MgTX on T cell activation was due to its ability to depolarize the T cell and thereby affect the calcium transient. However, this depolarization by MgTX was thought not be sufficient to inhibit activation. It has also been shown that blocking this channel with MgTX results in an initial depolarization which is followed by a second depolarization upon T-cell receptor cross linking. The instant invention utilizes the direct correlation of MgTX induced suppression of the calcium transient with T cell activation.

Previously it has been shown that pre-incubation with MgTX results in an inhibition of the intra-cellular calcium transient in human T cells activated via cross linking of cell surface molecules (Lin C.S., R.C. Boltz, J.T. Blake, M. Nguyen, A. Talento, P.A. Fischer, M.S. Springer, N.H. Sigal, R.S. Slaughter, M.L. Garcia, G.J. Kaczorowski, and G.C Koo. 1993. Voltage-gated potassium channels regulate calcium- dependant pathways involved in human T lymphocyte activation. J Exp Med 177:637.).

It has been observed that within the heterogeneity of the calcium response, the MgTX reduction in the Ca^"1^ transient was in the peak region of the curve. It appeared to be due to a more noticeable reduction in cells exhibiting high [Ca⁺⁺]i rather than an overall suppression of the whole population. It has been inferred that a sub- population exhibiting the maximal initial [Ca⁺⁺]j was affected by MgTX.

Rabinovitch et al. have demonstrated that CD4 cells have a greater mean calcium response than CD8 cells(Rabinovitch P.S., C.H. June, A. Grossmann, and J.A. Ledbetter. 1986. Heterogeneity among T cells in intra-cellular free calcium after mitogen stimulation with PH A or anti- CD3. simultaneous use of indo-1 and immunofluorescence with flow cytometry. J Immunol 137: 952.). The possibility existed that the observed reduction in the mean calcium transient was a result of a preferential effect on the CD4 or other subsets.

The application of combined [Ca⁺⁺]i measurements with indo- 1 and cell surface markers of human T lymphocytes has been demonstrated (Rabinovitch P.S., C.H. June, A. Grossmann, and J.A. Ledbetter. 1986. "Heterogeneity among T cells in intra-cellular free calcium after mitogen stimulation with PHA or anti-CD3. simultaneous use of indo- 1 and immunofluorescence with flow cytometry." J. Immunol 137: 952; Rabinovitch P.S., and C.H. June. 1990. "Measurement of intra-cellular ionized calcium and membrane potential." In Flow Cytometry and Cell Sorting, Second Edition, M. Melamed, T. Lidmo, M. Mendelsohn , eds. John Wiley & Sons, Inc. New York, p. 651.).

This invention combines cell surface markers with [Ca++]i measurement to directly follow inhibition of calcium transients by K 1.3 blockers in T cell subsets. Alternatively, digital imaging microscopy, can be utilized to follow the effects of Kyi .3 blockers on calcium concentration of the single lymphocytes.

SUMMARY OF THE INVENTION

A process for screening for immunoregulant compounds that modulate T cell activation which comprises measuring the effect of the immunoregulant compound on intracellular calcium concentrations. The screen utilizes the newly observed phenomenon of reproducible inhibition of the calcium transient following T cell receptor crosslinking in susceptible T cell subset(s) effected by depolarization of the T cell membrane potential by Kv 1.3 blockers . The screen utilizes multiparameter fluorescence flow cytometry to follow this inhibition on only these susceptible T cell subsets. Alternatively, susceptible T cells purified by magnetic cell sorting can be followed using fluorescent calcium indicator dyes in a 96 well confocal fluorescent plate reader .

BRIEF DESCRIPTION OF THE FIGURE

Figure 1. Mean Intracellular Calcium Transient Time Course of Human T Cells in the presence of MgTX effected by anti-CD3 Cross-Linking. Shown are media control, MgTX inhibited and Potassium inhibited Human T Cells.

Margatoxin inhibits the Ca⁺2 transient. This pattern is reflective of compounds which block Kvl.3. This can be utilized for screening for Kv l.3 blockers using unseparated human T cells with discriminating antibodies with flow cytometry or on separated sensitive - 7 -

subsets with a confocal fluorescence spectrofluorimeter. Figure 1 shows the control, MgTX and potassium inhibited Ca transients from this experiment.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a process for screening for immunoregulant compounds that modulate T cell activation which comprises measuring the effect of the immunoregulant compound on intracellular calcium concentrations.

An embodiment of this invention is the process for screening for immunoregulant compounds that modulate T cell activation of whole

T cell preparations which comprises the steps of:

(a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 1 to about 10 μM per 2 million T cells per mL;

(b) tagging a resistant T cell subset of the fluorescent-stained whole T cell preparation with one or two fluorescent antibody cell surface markers;

(c) loading the fluorescent-tagged and stained T cell preparation into an analytical instrument capable of measuring fluorescence;

(d) measuring a baseline calcium dye fluorescence of the fluorescent-tagged and stained T cell preparation;

(e) adding the immunoregulant compound to the fluorescent- tagged and stained T cell preparation;

(f) measuring the immunoregulant compound induced calcium dye fluorescence of the fluorescent-tagged and stained T cell preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes; (g) adding anti-CD3 antibody or a biotinylated anti-CD3 to the fluorescent-tagged and stained T cell preparation containing the immunoregulant compound; (h) incubating the antibody or biotinylated anti-CD3 with the fluorescent-tagged and stained T cell preparation containing the immunoregulant compound for about 3 to about 10 minutes; (i) adding a crosslinker antibody or avidin to the anti-CD3 antibody or biotinylated anti-CD3 labeled, fluorescent- tagged and stained T cell preparation containing the immunoregulant compound; (j) incubating the crosslinker antibody or avidin with the anti-CD3 antibody or biotinylated anti-CD3 labeled, fluorescent- tagged and stained T cell preparation containing the immunoregulant compound for about 2 minutes; and

(k) measuring the integrated anti-CD3 induced calcium dye fluorescence of the antibody crosslinked or avidin crosslinked anti-CD3 labeled, fluorescent-tagged and stained T cell preparation containing the immunoregulant compound.

A second embodiment of this invention is the process for screening for immunoregulant compounds that modulate T cell activation of whole T cell preparations which comprises the steps of: (a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 1 to about 10 μM per 2 million T cells per mL;

(b) tagging a resistant T cell subset of the stained whole T cell preparation with one or two magnetic antibody cell surface markers;

(c) removing the magnetically-tagged resistant T cell subset from the stained whole T cell preparation;

(d) loading the magnetically-depleted, stained T cell preparation into an analytical instrument capable of measuring fluorescence;

(e) measuring a baseline calcium dye fluorescence the magnetically-depleted, stained T cell preparation;

(f) adding the immunoregulant compound to the magnetically - depleted, stained T cell preparation; (g) measuring the immunoregulant compound induced calcium dye fluorescence of the magnetically-depleted, stained T cell preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes;

(h) adding anti-CD3 antibody or a biotinylated anti-CD3 to the magnetically-depleted, stained T cell preparation containing the immunoregulant compound;

(i) incubating the anti-CD3 antibody or biotinylated anti-CD3 with the magnetically-depleted, stained T cell preparation containing the immunoregulant compound for about 3 to about 10 minutes;

(j) adding a crosslinker antibody or avidin to the anti-CD3 antibody or biotinylated anti-CD3 labeled, magnetically- depleted, stained T cell preparation containing the immunoregulant compound;

(k) incubating the crosslinker antibody or avidin with the anti-CD3 antibody or biotinylated anti-CD3 labeled, magnetically- depleted, stained T cell preparation containing the immunoregulant compound for about 2 minutes; and (1) measuring the integrated anti-CD3 induced calcium dye fluorescence of the antibody crosslinked or avidin crosslinked, anti-CD3 labeled, magnetically-depleted, stained T cell preparation containing the immunoregulant compound.

A third embodiment of this invention is the process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations which comprises the steps of:

(a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 2 to about 10 μM per 2 million T cells per mL;

(b) loading the stained whole T cell preparation into an analytical instrument capable of measuring fluorescence;

(c) measuring a baseline calcium dye fluorescence the stained whole T cell preparation; (d) adding the immunoregulant compound to the stained whole T cell preparation;

(e) measuring the immunoregulant compound induced calcium dye fluorescence of the stained whole T cell preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes;

(f) adding anti-CD3 antibody or a biotinylated anti-CD3 to the stained whole T cell preparation containing the immunoregulant compound;

(g) incubating the antibody or biotinylated anti-CD3 with the stained whole T cell preparation containing the immunoregulant compound for about 3 to about 10 minutes;

(h) adding a crosslinker antibody or avidin to the anti-CD3 antibody or biotinylated anti-CD3 labeled, stained whole T cell preparation containing the immunoregulant compound;

(i) incubating the crosslinker antibody or avidin with the anti-CD3 antibody or biotinylated anti-CD3 labeled, stained whole T cell preparation containing the immunoregulant compound for about 2 minutes; and

(j) measuring the integrated anti-CD3 induced calcium dye fluorescence of the antibody crosslinked or avidin crosslinked anti-CD3 labeled, stained whole T cell preparation containing the immunoregulant compound.

All analytical instruments capable of measuring fluorescence are within the scope of this invention, and include but are not limited to for example a flow cytometer, digital imaging microscope, confocal microscope, spectrofluorimeter, including a confocal spectrofluorimeter. Additionally, the scope of this invention includes analytical instruments capable of measuring fluorescence which have a computer capable of analyzing only the fluorescence on antibody negative or susceptible cells, in cases where the T-cell subsets are unseparated. The analytical instrument capable of measuring fluorescence is configured to measure the fluorescence either continuously or at discrete time windows. Preferrably, the analytical instrument capable of measuring fluorescence is configured to measure the fluorescence at 1 to about 3 discrete time windows. The discrete time window is defined as a time period ranging from about 1 second to about 1 hour.

The calcium indicator dyes commonly used are largely fluoescent indicators of the non-fluorescent calcium chelator B APTA, [See R. Tsien in Methods in Cell Biology, Vol. 30, D.L. Taylor and Y.L. Wang, Eds., Academic Press (1989) pp 127-156.] and which comprises: indo-1 , fura-2, fura dextran, indo dextran, quin-2 mag-fura-2, mag-fura-5, mag-indo-1, fluo-3 and rhod-2, calcium green, calcium orange and calcium crimson, calcium green dextrans. magnesium green, fura red. See R. Haugland, "MOLECULAR PROBES, Handbook of Fluorescent Probes and Research Chemicals, Set 20: Calcium Indicators, Chelators and Ionophores" (1992) ppl 13-128, for a complete list of calcium indcator dyes. The preferred indicator dyes are: indo-1 and fura-2.

The calcium indicator dye concentration used in the instant process is in the range of about 1 to about 10 μM per 2 x 10^ T cells per mL for the cell subsets, and preferably, about 1 μM per 2 x 10^ T cells per mL. The calcium indicator dye concentration used in the instant process for non subset cells is in the range of about 2 to about 10 μM per 2 x lθ6 T cells per mL, and preferably, about 4 to about 6 μM per 2 x lθ6 T cells per mL. The critical consideration is the intracellular dye concentration which relates to the ratio of dye concentration to the total integrated cell volume. It should be noted that the process steps (b) and (c) of the second embodiment, describe the process of tagging the T cell preparation with the magnetic antibody cell surface marker(s) [step (b)] and removing the magnetically-tagged T cells from the T cell preparation [step (c)]. These steps can precede process step (a) of the second embodiment which describes the staining of the T cell preparation with a fluorescent calcium indicator dye.

A preferred embodiment of this invention is the process which utilizes an analytical instrument selected from: a flow cytometer and a confocal microscope; when the calcium indicator dye is indo-1. Another preferred embodiment of this invention is the process which utilizes an analytical instrument selected from: a digital imaging microscope or spectrofluorimeter; when the calcium indicator dye is fura-2.

Also within the scope of this invention is the utilization of a dual fluorescent calcium indicator dye, such as indo-1 or fura-2 for enhanced sensitivity in measurements. A ratiometric measurement of the intracellular calcium concentration can be obtained. Other single fluorescent calcium indicator dyes include fluo-3 and rhod. Alternatively, the dual fluorescent calcium indicator dyes, indo-1 or fura- 2, can be used and a single flourescent emission measured with a single flourescence spectrofluorimeter.

Analytical Calcium Calcium Calcium Instruments Indicator Indicator Indicator Capable Of dyes — dyes — dyes — Measuring antibody magnetically non-subsets Fluorescence subsets separated High subsets Chelator

Level

No indo-1 indo-1 spectrofluorimeter fura-2 fura-2 fluo-3 fluo-3 confocal No indo- 1 indo-1 spectrofluorimeter fura-2 fura-2 fluo-3 fluo-3 indo-1 indo-1 indo-1 flow cytometer fura-2 fura-2 fura-2 fluo-3 fluo-3 fluo-3 indo-1 indo-1 indo-1

Digital Imaging Microscope fura-2 fura-2 fura-2 fluo-3 fluo-3 fluo-3 indo-1 indo-1 indo-1

Confocal Microscope fura-2 fura-2 fura-2 fluo-3 fluo-3 fluo-3 - 13 -

Fura-2 is a ratiometric dye and can be used also with a two laser system to obtain a ratiometeric calcium measurement or it can be used as a single emission calcium indicator.

The invention relates to a method for screening compounds which are either competitive or allosteric modulators of peptide binding whose effect on early activation events, specifcally membrane potential and calcium transient is similar to the activity seen with Margatoxin and is therefore useful in identifying compounds which suppress the immune system in a subject in need of such treatment comprising the administration to a subject in need of such treatment of a nontoxic immunosuppressant amount of an inhibitor.

Compounds screened using this methodology are viewed to be active as immunoregulants when about a 30% or greater inhibition of the baseline substracted peak value of the calcium transient is observed. See Figure 1.

The high capacity screen of this invention utilizes cell surface markers on the susceptible T cell subsets with whole T cell preparations using fluorescent antibodies to cell surface markers and taking measurements on one of the following instruments: a multiparameter fluorescence flow cytometer, a multiparameter fluorescence confocal microscope or a multiparameter fluorescence digital imaging microscope.

The high capacity screen of this invention can also utilize magnetically separated susceptible T cell subsets to achieve reproducible results with human cells with measurements taken on one of the following instruments: a multiparameter fluorescence flow cytometer, a multiparameter fluorescence confocal microscope, a multiparameter fluorescence digital imaging microscope, confocal spectrofluorimeter or a spectrofluorimeter. This reproducibility is not possible on whole T cells because of individual variations in the percent of T cells which are unresponsive to Kvl.3 blockers.

The high capacity screen of this invention can also utilize unseparated T cells loaded with a high concentration calcium chelator dye which renders all T cells susceptible to Kyi .3 blockers. This allows for screening a whole (unseparated) T cell preparation in a reproducible manner.

Specifically, the method of this invention is useful in screening for compounds that possess Ky 1.3 channel inhibitory activity. Kyi.3 channel inhibitors are useful in the treatment and prevention of the resistance to transplantation or transplantation rejection of organs or tissues (such as heart, kidney, liver, lung, bone marrow, cornea, pancreas, intestinum tenue, limb, muscle, nervus, medulla ossium, duodenum, small -bowel, medulla ossium, skin, pancreatic islet-cell, etc. including xeno transplantation), graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosis, nephrotic syndrome lupus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes mellitus, type II adult onset diabetes, uveitis, nephrotic syndrome, steroid- dependent and steroid-resistant nephrosis, Palmo-planter pustulosis, allergic encephalomyelitis, glomerulonephritis, etc., and infectious diseases caused by pathogenic microorganisms, as well as being useful to screen for compounds useful in treating inflammatory, proliferative and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses such as: psoriasis, psoriatic arthritis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, acne, Alopecia areata, eosinophilic fasciitis, and atherosclerosis.

Kyi .3 channel inhibitors are also useful in the treatment of respiratory diseases, for example: sarcoidosis, fibroid lung, idiopathic interstitial pneumonia, and reversible obstructive airways disease, including conditions such as asthma, including bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma, particularly chronic or inveterate asthma (for example late asthma and airway hyper- reponsiveness), bronchitis and the like. Kyi .3 channel inhibitors may also be useful for treating hepatic injury associated with ischemia. Ky l .3 channel inhibitors may also useful in the treatment of certain eye diseases such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystorphia epithelialis corneae, corneal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' ophthalmopathy, severe intraocular inflammation, and the like. A K 1.3 channel blocker is also useful for treating multidrug resistance of tumor cells, (i.e. enhancing the activity and/or sensitivity of chemotherapeutic agents), preventing or treating inflammation of mucosa or blood vessels (such as leukotriene B4-mediated diseases, gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel disease, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), necrotizing enterocolitis), or intestinal lesions associated with thermal burns, cytomegalovirus infection, particularly HCMV infection.

Further, Ky i.3 channel blockers are also useful for treating or preventing renal diseases including interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases selected from multiple myositis, Guillain-Barre syndrome, Meniere's disease and radiculopathy; endocrine diseases including hyperthyroidism and Basedow's disease; hematic diseases including pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis and anerythroplasia; bone diseases including osteoporosis; respiratory diseases including sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin diseases including dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases including arteriosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis; collagen including scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis; periodontal disease; nephrotic syndrome; hemolytic-uremic syndrome; and muscular dystrophy. Further still, Kyi .3 channel blockers, may be used in the treatment of diseases including intestinal inflammations/allergies such as Coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; and food-related allergic diseases which have symptomatic manifestation remote from the gastrointestinal tract, for example migraine, rhinitis and eczema.

Kyi .3 channel blockers may also be useful for the treatment and prevention of hepatic diseases such as immunogenic diseases (e.g. chronic autoimmune liver diseases including autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g. necrosis caused by toxins, viral hepatitis, shock or anoxia), B-virus hepatitis, non-A/non-B hepatitis and cirrhosis.

The following example is given for the purpose of illustrating the present invention and shall not be construed as being limitations on the scope or spirit of the invention.

EXAMPLE 1

Materials and Methods

Reagents: MgTX was purified from the venom of the scorpion Centruroides margaritatus as described by M. Garcia-Calvo. See Garcia-Calvo M., R.J. Leonard, J. Novick, S.P Stevens., W. Schmalhofer, G.J Kaczorowski., and M.L. Garcia. "Purification, characterization, and biosynthesis of margatoxin, a component of Centruoides margaritatus venom that selectively inhibits voltage-dependant potassium channels" . J Biol Chem 268.5: 18866-18874 (1993). All antibody preparations (biotin-anti-CD3, PE-anti-CD4 and PE-anti-CD8) were obtained from Becton Dickinson. The biotin-anti-CD3 was diluted 1 :10 with media for use. Avidin (#A9890 Sigma Chemical Company, Saint Louis, MO) was dissolved in phenol -red -free RPMI 1640 medium (GIBCO, Grand Island NY) at 1 mg/ml, filter sterilized and maintained at 4° C until diluted 1 :5 with medium for use.

Cell Preparations: Purified T cells were prepared by a modified "E- rosetting " method [Lin C.S., R.C. Boltz., J.J. Siekierka, and N.H. Sigal. "FK-506 and Cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes." Cellular Immunology 133: 269 (1991).] from lymphocyte-rich leucopaks. The purified cells were washed in lymphocyte cell culture medium, RPMI 1640 medium (GIBCO, Grand Island NY) and maintained in serum supplemented RPMI at 37° C for use within 24 hrs. The cells were washed in HEPES-buffered, phenol-red- free RPMI prior to staining. All subsequent fluorescent procedures were performed in this media. The T cells were separated into CD4 enriched (CD8-) and CD8 enriched (CD4-) populations using a MACS magnetic cell separator (Miltenyi Biotec GmbH, Bergish-Gladbach, Germany) according to product instructions.

Measurement of [Ca⁺⁺]_}

Flow Cytometry: T-cells were loaded at a concentration of 2 x 10⁶ to about 10 x 10⁶ cells/ml with 1 to about 10 μM indo-1, a calcium indicator dye, (Molecular Probes) in phenol red free RPMI for 45 minutes at 37° C in the dark. The cells were then washed by centrifugation and re-suspended in an equal volume of RPMI and incubated a second 45 minutes. The cells were washed and re-suspended at 5 x 10⁶ cells/ml.

The cells were maintained in the dark until use or additional staining.

When cells were stained for subset discrimination, either anti-CD4 or anti-CD8 was added to 200 μL of 5 x 10⁶ cells/ml, washed and re- suspended at 1 x 10⁷ cells/ml. Initial flow cytometric [Ca^j_j determinations were performed as previously described in Lin C.S., et al. Cellular Immunology 133: 269 (1991) . Multiparameter studies were carried out at 37° C on a FACStar Plus/Consort VAX (Becton Dickinson Immunocytometry Systems, San Jose, CA) equipped with a Zero Time Module (CYTEK Corp. Freemont, CA). Additions for of the inhibitor or mock inhibitor (media) were at 30 seconds, biotinylated anti-CD3 at 200 seconds, and avidin at 400 seconds. Cell data was accumulated as a single continuous, 8 parameter list-mode file with 10 time indicators/sec. Mean time course plots were calculated from the list mode data using KINPRO (Becton Dickinson Immunocytometry Systems, San Jose, CA). Digital Imaging Microscopy: For example, the digital imaging microscope components were purchased through Perceptics Corporation (Knoxville, TN). The components of system used in this study were an inverted Axiovert 35 microscope (Zeiss. Thornwood NY) fitted with a frame-transfer cooled-CCD camera (CH220 with a TC312 1024 x 512 masked chip, Photometries, Tucson AZ). Excitation filters, 340 DF 10 and 380 DF 13 (Omega Optical.Inc, Brattleboro VT), were controlled via a control device through an RS 232 connection (Ludl Electronic Products, Hawthorne, NY). System control, image recording and analysis was through TCL Image software (TNO, Delft Netherlands) operating on Apple Macintosh Ilx. Command macros were written and tested in-house for acquisition and analysis of image pairs.

Two adjacent chambers of each Lab-Tek 8 chambered cover-glass (NUNC Inc., Naperville EL) were coated with 0.8μg/cm² Cell-Tak (Collaborative Research In. Bedford MA) following the product usage instructions. The chambers were stored at 4° C until the day of the experiment. The chambers were equilibrated at room temperature prior to plating of the cells.

The cells were loaded with 1 to about 10 μM fura-2 (Molecular Probes) [Tsien R.Y., T.J. Rink, M. and Poenie. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitataion wavelengths. Cell Calcium 6: 155-157, 1985.] following the same procedure used above for indo-1 but were re- suspended at a concentration of 1 x 10⁶/ml. The cells were maintained in the dark until plating. At least 2.5 hrs prior to time course acquisition, 150μL of the suspension was plated in the Cell-Tak coated chambers. The # 1 cover-glass bottom permits sufficient passage of UV to excite the calcium monitoring dye fura-2. Ten minutes prior to the data acquisition, the chamber was placed in the heating stage and the addition pipets filled and placed in the heating block. Both were warmed to 37° C.

An external heating unit maintained the temperature of the cells in the disposable NUNC chamber as previously described in Boltz, R.C.D., G. Kath, B. Uhrig, J. McKeel, and C. Quinn. "A disposable- chamber temperature-regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital-imaging microscopy"; accepted for publication Cytometry 17:2 October, 1994. Pre-warming of addition solutions was accomplished in a separately regulated addition block. Prior to the initiation of the calcium time course, disposable Eppendorf tips were filled with the appropriate solutions and placed in the addition block for pre-heating to 37o C. During the experiment, additions were made by gently pushing the remote syringe plunger. By using addition volumes equal to that of the cell suspension, complete mixing was achieved. In order to measure changes in [Ca^ , fura image pairs were accumulated and stored every 10 seconds. Computer clock time was stored with each image. Each time course was stored on a REO-650 erasable optical disc (Pinnacle Micro, Irvine CA). Briefly, for analysis, cells were defined and numbered with a fluorescence threshold mask. [Ca⁺⁺]i for each time point was calculated by dividing, pixel by pixel, background-subtracted 340 by a masked background-subtracted 380 raw image. The area normalized integrated intensity of each cell was placed in an EXCEL type spreadsheet in the appropriate cell column in a row designated with the experiment time calculated from the computer clock time. The individual time course of the single cells was displayed using StatView II (Abacus Concepts, Berkeley CA).

Claims

WHAT IS CLAIMED IS:

1. A process for screening for an immunoregulant compound that modulates T cell activation which comprises measuring the effect of the immunoregulant compound on intracellular calcium concentration.

2. A process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations which comprises the steps of: ° (a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 1 to about 10 μM per 2 million T cells per mL;

(f) measuring the immunoregulant compound induced calcium dye fluorescence of the fluorescent-tagged and stained T cell ⁵ preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes;

(g) adding anti-CD3 antibody or a biotinylated anti-CD3 to the fluorescent-tagged and stained T cell preparation containing the immunoregulant compound; ⁰ (h) incubating the antibody or biotinylated anti-CD3 with the fluorescent-tagged and stained T cell preparation containing the immunoregulant compound for about 3 to about 10 minutes; (i) adding a crosslinker antibody or avidin to the anti-CD3 antibody or biotinylated anti-CD3 labeled, fluorescent- tagged and stained T cell preparation containing the immunoregulant compound;

(j) incubating the crosslinker antibody or avidin with the anti-CD3 antibody or biotinylated anti-CD3 labeled, fluorescent- tagged and stained T cell preparation containing the immunoregulant compound for about 2 minutes; and

3. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 2, wherein the analytical instrument capable of measuring fluorescence comprises: a flow cytometer, digital imaging microscope, or confocal microscope.

4. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 3, wherein the analytical instrument capable of measuring fluorescence further comprises a computer capable of analyzing only the fluorescence on antibody negative or susceptible cells.

5. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 3, wherein the analytical instrument capable of measuring fluorescence is configured to measure the integrated fluorescence continuously or at discrete time windows.

6. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 5, wherein the analytical instrument capable of measuring fluorescence is configured to measure the integrated fluorescence at 1 to about 3 discrete time windows.

7. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 3, wherein the fluorescent calcium indicator dye comprises: indo- 1 , fura-2, fluo-3 and rhod-2, fura dextran, indo dextran, quin-2 mag-fura-2, mag-fura-5, mag-indo-1 , calcium green, calcium orange, calcium crimson, calcium green dextrans, magnesium green, and fura red.

8. The process for screening for an immunoregulant compound that modulate T cell activation of whole T cell preparations, as recited in claim 7, wherein the analytical instrument capable of measuring fluorescence comprises: a flow cytometer or a confocal microscope; and the fluorescent calcium indicator dye is indo-1.

9. The process for screening for an immunoregulant compound that modulate T cell activation of whole T cell preparations, as recited in claim 8, wherein the fluorescence measurement comprises utilizing the simultaneous acquisition of two different emissions of indo- 1 to attain a ratiometric determination of the relative calcium concentration.

10. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 7, wherein the analytical instrument capable of measuring fluorescence comprises: a digital imaging microscope; and the fluorescent calcium indicator dye is fura-2.

11. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 10, wherein the fluorescence measurement comprises utilizing the alternating excitations of fura-2 to attain a ratiometric determination of the relative calcium concentration.

12. The process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations, as recited in claim 2, wherein the concentration of fluorescent calcium indicator dye is about 1 μM per 2 x 10^ T cells per mL.

13. A process for screening for an immunoregulant compound that modulates T cell activation of T cell preparations which comprises the steps of: (a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 1 to about 10 μM per 2 million T cells per mL;

(e) measuring a baseline calcium dye fluorescence of the magnetically-depleted, stained T cell preparation;

(f) adding the immunoregulant compound to the magnetically - depleted, stained T cell preparation; (g) measuring the immunoregulant compound induced calcium dye fluorescence of the magnetically-depleted, stained T cell preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes; (h) adding anti-CD3 antibody or a biotinylated anti-CD3 to the magnetically-depleted, stained T cell preparation containing the immunoregulant compound;

(k) incubating the crosslinker antibody or avidin with the anti-CD3 antibody or biotinylated anti-CD3 labeled, magnetically- depleted, stained T cell preparation containing the ⁵ immunoregulant compound for about 2 minutes; and

(1) measuring the integrated anti-CD3 induced calcium dye fluorescence of the antibody crosslinked or avidin crosslinked, anti-CD3 labeled, magnetically-depleted, stained T cell preparation containing the immunoregulant ⁰ compound.

14. A process for screening for an immunoregulant compound that modulates T cell activation of whole T cell preparations which comprises the steps of: ⁵ (a) staining the whole T cell preparation with a fluorescent calcium indicator dye at a concentration of about 2 to about 10 μM per 2 million T cells per mL; (b) loading the stained whole T cell preparation into an analytical instrument capable of measuring fluorescence; ° (c) measuring a baseline calcium dye fluorescence the stained whole T cell preparation; (d) adding the immunoregulant compound to the stained whole T cell preparation; (e) measuring the immunoregulant compound induced calcium dye fluorescence of the stained whole T cell preparation containing the immunoregulant compound after about 30 seconds to about 5 minutes;