WO1995013808A1 - Procede d'inhibition selective de la transduction du signal de l'interleukine-2 (il-2) - Google Patents

Procede d'inhibition selective de la transduction du signal de l'interleukine-2 (il-2) Download PDF

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WO1995013808A1
WO1995013808A1 PCT/US1994/013202 US9413202W WO9513808A1 WO 1995013808 A1 WO1995013808 A1 WO 1995013808A1 US 9413202 W US9413202 W US 9413202W WO 9513808 A1 WO9513808 A1 WO 9513808A1
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cell
compound
cells
substituted
compounds
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Glenn C. Rice
Stuart L. Bursten
Jack W. Singer
William T. Tino
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Cell Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine

Definitions

  • the invention provides methods for preventing accumulation of intracellular myristylated phosphatidic acid (myrPA) and selectively inhibiting interleukin-2 (IL-2) signal transduction that results in immunosuppressive activity useful for treatment of graft versus host disease, autoimmune diseases, certain T-cell or B-cell leukemias and lymphomas, and for transplant recipients.
  • myrPA myristylated phosphatidic acid
  • IL-2 interleukin-2
  • Interleukin-2 is a cytokine that is considered to be an important mediator of T-cell cytotoxicity, and particularly LAK (lymphocyte activated killer) cell activity.
  • IL-2 is the primary T-cell growth factor and is a key determinant in the magnitude of an immune response. Binding of IL-2 to its high affinity receptor on activated T-cells is essential for T-cell proliferation and a "normal" immune response. This is especiallv true in vivo where T-cells are present at different stages of activation. The mechanism of IL-2 signal transduction, after binding of IL-2 to its cell surface receptor, is not well understood.
  • IL-2 signaling is thought to have more than one component and may involve indirect signaling through upregulation of an IL-2 receptor and cell proliferation on cells expressing IL-2 cell surface receptors.
  • IL-2 receptor does not contain a tyrosine kinase motif or possess endogenous kinase activity.
  • IL-2 does not appear to act through protein kinase C to increase cellular Ca 2+ concentration, or to activate a classical guanine nucleotide binding protein (Otani et al., Proc. Natl. Acad. Sci. USA 89:2787, 1992).
  • the proliferation of mature resting T-cells is initiated by the antigen receptor.
  • Signal transduction is accomplished by antigen interacting with a T-cell receptor complex (TCR) and inducing expression of IL-2, expression of IL-2 receptor, and cell proliferation.
  • TCR T-cell receptor complex
  • the antigen is displayed in the context of a major histocompatibility antigen on the surface of antigen-presenting cells.
  • Immunosuppressive drugs that are available include cyclosporin A (CsA) and FK506. Both of these immunosuppression drugs are thought to exert their activity through intracellular binding to immunophilins (either cyclophilin for CsA or FK binding protein for FK506) (Tandinh et al., FASEB 6:3410, 1992).
  • the target for the immunophilin complex is thought to be the protein phosphatase calcineurin (Liu et al., Cell 66:807, 1991).
  • CsA and FK506 both interfere with T-cell receptor signaling, without affecting direct IL-2 signaling.
  • CsA The end effect of treatment with CsA or FK506 is an inhibition of T-cell proliferation due to inhibition of TCR signal transduction mechanisms.
  • CsA also is thought to inhibit T-cell activation by inhibiting Ca + calmodulin-dependent activation of IL-2 gene transcription (Furman et al., Proc. Natl. Acad. Sci. USA 89:3686, 1992 and O'Keefe et al., Nature 357:692, 1992).
  • IL-2 is produced, CsA is unable to inhibit the progression of the T-cell cycle.
  • Rapamycin is a synthetic molecule that also inhibits 1L-2 -mediated T-cell proliferation and it is being investigated for clinical use as an immunosuppression compound.
  • Glucocorticoids are among the most potent and widely used immunosuppressive agents, particularly for the treatment of allograft rejection and autoimmune and allergic diseases. However, the mechanism by which they suppress immune inflammatory responses have not been clearly defined. Glucocorticoids affect distribution, growth, differentiation and function of neutrophils, monocytes and lymphocytes by expression of specific genes (Boumpas et al., Clin. Exp. Rheumatol. 9:413, 1991).
  • Glucocorticoids have been shown to inhibit RNA synthesis, production of cytokines including IL-2, interleukin-4 (IL-4) and interferon gamma (IFN- ⁇ ), and proliferation and function of T- cells. However, it has not been established whether this pleotrophic inhibitory effect is mediated through the IL-2 receptor and/or through TCR. In one study, the glucocorticoid dexamethasone inhibited IL-2-dependent tyrosine phosphorylation and inhibited preactivated T-cells by down-regulating signal transduction through the IL-2 receptor (Paliogianni et al., J. Immunol. 151:4081, 1993).
  • CsA and FK506 are structurally unrelated, both compounds share several mechanistic similarities on a cellular and molecular level. Corticosteroids and antimetabolites can inhibit lymphocyte proliferative responses in vitro and serve as potent immunosuppressants in vivo. However, CsA and FK506 are selective for certain kinds of activation events based on what appears to be a subset of a Ca-associated signal transduction pathway that is prominent in lymphocytes but is also present in other cell types. IL-2 production appears to play a central role in the action of CsA and FK506, especially with regard to IL-2 gene transcription.
  • CsA and FK506 can block transcriptional activity mediated by the transcriptional factors NF-AT, Oct-1 and NF- ⁇ B due possibly to interference with synthesis or transport of these factors into the nucleus or it may be due to inhibition of their functional activation.
  • Pharmacologically relevant concentrations of CsA inhibit the release of granule-associated serine esterase from murine cytotoxic lymphocytes (CTL) triggered with anti-CD3 or by a combination of Ca ionophore and PMA (Trenn et al, J. Immunol. 42:3796, 1989).
  • exocytosis takes place 4-5 hours after activation, is dependent on the presence of extracellular Ca, but is not inhibited by cycloheximide or actinomycin D.
  • CsA must be added within the first hour after activation.
  • FK5 * ⁇ and CsA inhibition of lymphocyte signal transduction events is dependent upon the mode of cellular activation. In general, only activation pathways that cause a measurable rise in intracellular Ca are sensitive to CsA/FK506.
  • One of the best documented examples is a lack of inhibition of 1L-2 production by CsA and FK506 when human lymphocytes are activated via the CD28 pathway (June et al, Mol. Cell Biol. 7:4472, 1987; Kay et al., Immunol Lett. 23: 155, 1989; and Lin et al., Cell. Immunol. 133:269, 1991).
  • IL-2 signaling is a pleotropic event with, most likely, many parallel events or a multiplicity of sequential events.
  • IL-2 signal transduction was investigated in an inducible B lymphoma.
  • the lymphoma cells respond to IL-2 by proliferating and differentiating into antibody- secreting cells, with both responses blocked by IL-4.
  • IL-2 stimulated (in a dose- dependent fashion) a rapid hydrolysis of an inositol-containing glycolipid to yield a myristylated diacyglycerol and inositol phosphate-glycan, which was inhibited by IL-4.
  • IL-2 and IL-4 signals are transduced by different pathways that intersect at an early point.
  • the authors suggest that IL-4 exerts its inhibitory action through preventing the activation of a Glycan PI (PjG)-specific phospholipase C.
  • PjG Glycan PI
  • Cano et al. (EurJ. Immunol. 22: 1883, 1992) speculated that IL-2 stimulated a transient increase in phosphatidic acid (PA) in cloned T lymphocytes through activation of phospholipase D activity, but did not increase (in direct contrast to Eardley and Koshland, infra) diacylglycerol levels.
  • PA phosphatidic acid
  • PA was able to induce c-myc RNA transcription in CTLL-2 cells as well as IL-2 receptor (CD25) expression on the cell membrane with equal potency at saturating doses of IL-2.
  • T-cell mediated immune responses to foreign antigens requires cells in several lineages.
  • Foreign antigen is presented to naive T-cells by antigen presenting cells which express protein antigens attached to the MHC complex.
  • T-cells respond to this through the T-cell receptor complex (CD3) and also require signals through accessory cell surface receptors such as CD28.
  • Inhibition of T-cell responses to antigen by drugs can be achieved by interruption of signaling through the CD3 complex, through inhibition of accessory pathways such as CD28, or through interruption of signaling by the T-cell proliferation factor, IL-2.
  • CsA and FK506 interrupt signaling through the CD3 complex by inhibiting calcineurin activation of transcription factors.
  • IL-2 signaling is a pleotropic event that can be inhibited in numerous ways, each with different clinical consequences.
  • Conventional immunosuppressive therapies exhibit dose-related side affects.
  • the invention resulted from studies of IL-2 signal transduction and compounds that have a specific affect on IL-2 signal transduction (e.g., the accumulation of myrPA) but do not have undesirable side effects observed in conventional therapies.
  • the present invention provides a method for preventing accumulation of myrPA, which occurs in response to IL-2 signaling and for inhibiting interleukin-2 (IL-2) signal transduction that results in immunosuppressive activity useful for treatment of various immune diseases, such as * for example, graft versus host disease, autoimmune diseases and certain T-cell leukemias.
  • the method comprises administering a compound that inhibits signal transduction through cellular accumulation of myrPA, preferably alkyl and alkyl and/or nonarachidonyl PA.
  • the compound is an organic molecule that specifically inhibits generation of myrPA.
  • Most preferred compounds of this method are small organic molecules that can mimic binding to a complex of enzymes, which play a role in mediating signal amplification, thereby resulting in a diminution of intracellular phosphatidic acid (PA) levels in response to an inflammatory stimulus.
  • Most preferred compounds useful in the inventive method for treating immune diseases include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures a compound having a straight or branched aliphatic hydrocarbon structure of formula I:
  • n is an integer from one to four and m is an integer from four to twenty.
  • Rj an .2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2) W R5- If R ] or R2 is - (CH2) W R5, w may be an integer from one to twenty and R5 may be an hydroxyl, halo, Cj.g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle.
  • R ⁇ and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocyle.
  • R3 may be either hydrogen or C1.3.
  • Preferred compounds may have one of Rj or R2 and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms. This R1/R3 or R2 R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six.
  • Figure 1 shows assay results from a thymocyte proliferation.
  • Murine thymocytes were induced to proliferate in response to a sub-mitogenic dose of ConA and IL-2.
  • This assay is a model of T-cell activation and does not distinguish between ConA-activated or IL-2 activated pathways.
  • Representative compounds, useful in the inventive method nos. 2576, 2575, 2571, 2573— see table below for chemical name and structure) inhibit this proliferative response.
  • This assay is an indicator of immunosuppressive activity.
  • Figure 2 shows the effect of CsA on thymocyte proliferation in the assay of figure 1.
  • FIGS 3 and 4 show the results of a CT-6.1 cell proliferation assay.
  • CT-6.1 cells are an IL-2 -dependent, murine T-cell clone that proliferate in response to IL-2.
  • This assay is a predictive model for IL-2 induced proliferation and signaling.
  • Representative compounds, useful in the inventive method nos. 2583, 2584, 2576, 2574 and 2571-see table below for corresponding chemical name and structure) inhibit IL-2 induced proliferation of this cell line.
  • CsA did not inhibit an IL-2 induced response.
  • FIGs 5, 6, and 7 show the results of a splenocyte proliferation assay with several inventive compounds.
  • Murine splenocytes were activated with a monoclonal antibody directed against the CD3 complex (T-cell receptor). This resulted in a polyclonal T-cell proliferative response.
  • Representative compounds useful in the inventive method nos. 2576, 2558, 2586, 2583, 2584, 2574 and 2571 ⁇ see below for chemical names and structures
  • CsA inhibited this response.
  • This assay is predictive of immunosuppressive activity, but does not distinguish between CD3 activated pathways and IL-2 activated pathways.
  • Figures 8 and 9 show the results of a thymocyte proliferation assay.
  • Murine thymocytes were incubated overnight with ConA in the presence or absence of CsA of representative compounds useful in the inventive method (compounds nos. 2558, 2510, 2523, 2534, 2519, 2559, 2570, 1920, 2562 and 2514). The cells were washed and recultured with IL-2. The results show that CsA inhibited the ability of thymocytes to respond to IL-2 (presumably by interfering with upregulation of the IL-2 receptor). Conversely, illustrative compounds nos. 2558, 2510 and 2523 did not inhibit this aspect of T-cell activation. This assay shows, in part, that the immunosuppressive activity of the inventive method, as represented by the compounds tested, differs from that of CsA. Also, as shown in figure 9, all representative compounds tested inhibited IL-2 induced proliferation of ConA activated thymocytes.
  • Figure 10 shows the results of a splenocyte proliferation assay.
  • Murine splenocytes were incubated overnight with anti-CD3 monoclonal antibody in the presence or absence of CsA or representative compounds useful in the inventive method (nos. 2583 , 2510 and 2514).
  • the cells were washed, re-cultured, and stimulated with IL-2.
  • CsA inhibited anti-CD3 mediated responsiveness to IL-2 (presumably by inhibiting upregulation of the IL-2 receptor).
  • the compounds tested did not inhibit this aspect of T- cell activation.
  • Figure 11 shows the results of a splenocyte proliferation assay.
  • Murine splenocytes were incubated overnight with anti-CD3 monoclonal antibody in the absence of any compound.
  • the cells were washed and re-cultured with IL-2 in the presence or absence of CsA or representative compounds useful in the inventive method (nos. 2584, 2562, 2510, 2514, 2576, 2558, 2586 and 2573).
  • All representative compounds tested inhibited IL-2 -induced proliferation of anti-CD3 activated splenocytes.
  • CsA did not inhibit this response.
  • Figure 12 shows the results of a thymocyte proliferation assay.
  • Murine thymocytes were incubated with ConA and IL-1 to induce thymocyte proliferation.
  • CsA and representative compounds of the inventive method nos. 2576, 2584, 2510 and 2514, all inhibited this proliferative response. This assay is indicative of immunosuppressive activity.
  • Figure 13 shows levels of IL-2 release from ConA/IL-1 activated thymocytes.
  • Supernatants from thymocyte cultures used in the assay of fig. 12 were analyzed in an ELISA assay for the presence of murine IL-2.
  • CsA inhibited IL-2 release from the stimulated thymocytes, while representative compounds nos. 2576, 2584, 2583, 2510 or 2514 did not inhibit this release.
  • Figure 14 shows levels of IL-4 release in an assay using ConA/IL-1 activated thymocytes.
  • Supernatants were analysed in an ELISA assay for murine IL-4.
  • CsA significantly inhibited IL-4 release.
  • Representative compounds useful in the inventive method (specifically, compounds nos. 2576, 2584, 2583, 2510 or 2514) showed a differential effect on IL-4 release. Higher compound concentrations resulted in an inhibition of IL-4 release while lower concentrations showed little effect.
  • Different sub- populations of murine T-cells secrete different cytokines.
  • TH1 cells secrete IL-2, IFN- gamma, and GM-CSF, while TH2 cells secrete IL-4, IL-5 , IL-6, and IL- 10.
  • Figure 15 shows the results of an assay for IFN- ⁇ release from ConA/IL-1 activated thymocytes. Supernatants were analyzed in an ELISA assay for murine IFN- ⁇ levels. CsA inhibited IFN- ⁇ release. Representative compounds useful in the inventive method (compounds nos. 2576, 2584, 2583, 2510 and 2514) also inhibited IFN- ⁇ release at low concentrations. These data show that the representative compounds tested have a different effect on IFN- ⁇ release than on IL-2 release. Both cytokines (IL-2 and IFN- ⁇ ) are secreted by the same sub-population of T-cells. Therefore, these data indicate that different regulatory pathways exist for synthesis and secretion of IL-2 and IFN- ⁇ .
  • Figure 16 shows immunosuppressive activity of CsA and representative compounds useful in the inventive method (specifically, compounds nos. 2576, 2583, 2584 and 2510) in inhibiting anti-CD3 induced T-cell proliferation.
  • Figure 17 shows IL-2 release from anti-CD3 activated splenocytes.
  • Supernatants from the splenocyte cultures in the assay of fig. 16 were assayed for the presence of murine IL-2.
  • CsA had a potent effect on inhibition of IL-2 secretion.
  • representative compounds tested exhibited dose dependent inhibition of IL-2 release.
  • Figure 18 shows IL-4 release from anti-CD3 activated splenocytes.
  • Supernatants from the splenocyte cultures from the assay in fig. 16 were assayed for the presence of murine IL-4. Both CsA and the representative compounds tested inhibited IL-4 release.
  • Figure 19 shows IFN- ⁇ release from Anti-CD3 activated splenocytes.
  • Supernatants from the splenocyte . ultures from the assay in fig. 16 were assayed for murine IFN- ⁇ levels. The results a_e similar to those seen for IL-4.
  • Inhibition of the release of various immunoregulatory cytokines are predictive of immunosuppressive activity. These cytokines play a key role in the generation of an immune response and maintaining the magnitude of that response. Inhibition of their secretion by these representative compounds, evidence that the inventive method is useful in preventing graft rejection and treating various autoimmune diseases, such as lupus, rheumatoid arthritis, multiple sclerosis, and myasthenia gra vis.
  • Figure 20 shows the result of an experiment with representative compound no. 2576, investigating antigen specific T-cell anergy.
  • Murine splenocytes were stimulated with an alloantigen target cell in the presence or absence of compound no. 2576. Following five days in culture the cells were harvested, washed, re-cultured, and re- stimulated with the original priming antigen and anti-CD3 monoclonal antibody.
  • This figure shows that the secondary response of responding cells to the priming antigen was inhibited if the cells were cultured with the representative compound during the primary response.
  • the polyclonal response to anti-CD3 was not affected, indicating that compound no. 2576 did induce a general state of non-responsiveness nor was it toxic to the responding cells.
  • 2576 is capable of inducing antigen-specific tolerance in T-cells.
  • This specific type of immunosuppressive activity is useful in transplantation biology, specifically, for example, for the prevention of graft rejection.
  • These data further demonstrate that the representative compound, for use in the inventive method, induces tolerance to grafted tissue. Such a method is useful for treating autoimmune diseases.
  • Figures 21 and 22 show the results of a B-cell proliferation assay: Murine splenocytes were stimulated with anti-IgM antibodies and murine IL-4, which specifically activates B-cell proliferation. Figures 21 and 22 show that representative compounds nos. 2573, 2567, 2575, 2559, 2583, 2584, 2576, 2574 and 2571 inhibit this proliferative response. Inhibition of B-cell proliferation is useful for treating antibody-mediated diseases, such as lupus, rheumatoid arthritis, and myasthenia gravis.
  • antibody-mediated diseases such as lupus, rheumatoid arthritis, and myasthenia gravis.
  • FIGs 23-25 show the results of a murine mixed tumor lymphocyte reaction (MLR) assay.
  • MLR murine mixed tumor lymphocyte reaction
  • Murine splenocytes were stimulated with an alloantigen target cell in the presence or absence of representative compounds nos. 2576, 2571, 2573, 2510, 2562, 2514, 2583, 2584 and 3509.
  • Thes compounds inhibited the proliferative response to alloantigen.
  • This assay is an in vitro model of a graft rejection-type response. Inhibition of this response is indicative of immunosuppressive activity, specifically in the area of transplantation biology.
  • Figures 26-29 show mass spectra printouts of CT-6.1 cell serum, which were first treated with or without compounds useful in the inventive method prior to exhaustively hydrolyzing the resulting serum to obtain a corresponding lipid profile.
  • figure 26 shows results from CT-6.1 control cells (no stimulation).
  • Figure 27 shows results from cells stimulated with IL-2 for 15 seconds.
  • Figures 28 and 29 show results from the CT-6.1 cells stimulated with IL-2 for 15 seconds and also treated with either 100 nM (figure 28) or 1 ⁇ M (figure 29) of representative compound no. 2576.
  • Figures 3 ⁇ and 30B show a flow cytometry analysis of anti-CD3-mediated upregulation of ⁇ .L-2 receptor alpha subunit (CD25), with CsA ( Figure 30A) or with a representative compound effective in the inventive method, compound no. 2583 ( Figure 30B).
  • Figure 31 illustrates that IL-2 induces myristylated phosphatidic acid in CT-6.1 cells and that a conventional therapy, CsA, does not exhibit an ability to inhibit the DL-2-induced increase in myr-PA, but that the inventive method does.
  • Figure 32 illustrates that myristylated DAG is not increased following IL-2 stimulation, confirming that representative compound no. 2576 effectively blocks accumulation of myrPA by blocking PIG-PLD catalyzed hydrolysis of GPI
  • Figures 33 is a representative HPLC chromatogram, illustrating separation of various lipid classes from a lipid sample. A "PA peak" is shown as "D-2.”
  • Figures 34A-C are FAB/MS spectra from analysis of a corresponding HPLC-separated PA fraction, showing reproducible appearance of the myrPA species, shown by peaks labeled no. 577/578, 603/605 and 627/629. upon stimulation with IL-2.
  • Figure 35 is also an FAB/MS spectrum confirming elimination of corresponding myrPA peaks after treatment with representative compound no. 2576 (table below).
  • Figure 36 illustrates data obtained in an in vivo mouse hemolytic model, which show that representative compound no. 3578 inhibits an immune response in mice sensitized witii sheep red blood cells.
  • Figure 37 is a bar graph showing no appreciable decrease in splenic cellularity in the in vivo mouse hemolytic model, confirming no toxicity by administering compounds in the inventive method.
  • the inventive method using illustrative compounds disclosed, as prototypes of specific IL-2 signaling inhibitors which inhibit the increase in myrPA that follows IL-2 stimulation, inhibit hydrolysis of PjG (by inhibiting a specific PjGPLD) and subsequent generation of myrPA, preferably alkyl and nonarachidonyl species.
  • the invention inhibits IL-2 driven proliferation and thus a cell mediated immune response to antigen.
  • the illustrated compounds Unlike compounds which effect CD3 signaling (probably CsA and FK506), the illustrated compounds have minimal effects on de novo expression of IL-2 by activated lymphocytes. Since the illustrated compounds can suppress a cell mediated immune response and the generation of effector cells (i.e., have immune suppression activity), the illustrated compounds have therapeutic utility to suppress the response to endogenous or exogenous antigens. Therefore, compounds of this class are useful for the treatment of and prevention of clinical symptoms of various autoimmune disorders, generally involving T-cell mediation. Such autoimmune disorders include, but are not limited to diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosis, insulin-dependent diabetes mellitus (IDDM), and psoriasis.
  • autoimmune disorders include, but are not limited to diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosis, insulin-dependent diabetes mellitus (IDDM), and psori
  • the illustrated compounds have therapeutic utility to treat or prevent various immune mediated diseases (such as glomerulonephritis of various types and inflammatory bowel diseases), suppress graft versus host disease (such as following bone marrow transplantation), treat certain T-cell leukemias, and prevent the rejection of donor organs (e.g., kidneys, hearts, livers, pancreas, bowel and lung), following transplantation.
  • various immune mediated diseases such as glomerulonephritis of various types and inflammatory bowel diseases
  • suppress graft versus host disease such as following bone marrow transplantation
  • treat certain T-cell leukemias e.g., kidneys, hearts, livers, pancreas, bowel and lung
  • Anergy is defined as a prolonged state of T-cell "unresponsiveness" due to T-cell anitgen recognition without co-stimulation, or by blocking proliferation induced by co- stimulation. This later mechanism of T-cell anergy arises, for instance, by blocking a T- cell's ability to proliferate in response to IL-2.
  • Anergy is generally considered to be a type of tolerance to antigen activation and is predictive of those agents that could induce tolerance in vivo. This is important in transplantation and organ rejection, as well as other autoimmune diseases such as scleroderma, rheumatoid arthritis, lupus, and diabetes related autoimmunity.
  • the illustrated compounds can induce anergy, and therefore, are effective for treating or preventing autoimmune diseases, such as scleroderma, rheumatoid arthritis, lupus and IDDM, and useful for preventing organ rejection in various transplantation procedures.
  • immunosuppressive compounds whose mechanism of action is specific for inhibiting IL-2 signaling, will cause apoptosis of immune cells stimulated by antigen. Stimulation of CD3 with a concurrent block in IL-2 signaling can also lead to clonal deletion or tolerance.
  • the inventive method avoids these disadvantages, as exemplified by the administration of representative compounds, which inhibit the secondary response to antigen even after the compound is no longer present without blocking the cross linking response of the CD3 complex by a monoclonal antibody. This illustrates that specific tolerance to an antigen can be achieved without inhibiting the general immune response to antigens.
  • T-cell helper function specific for a given antigen
  • B-cells are stimulated by IL-2. Therefore, inhibiting IL-2 signaling decreases expansion of antibody producing clones and thereby suppress antibody production.
  • inventive method specifically permits IL-2 signaling and therefore is useful for treating antibody mediated immune disorders without undesirable side effects of conventional therapies, including, but not limited to, idiopathic thrombocytopenic purpera, autoimmune hemolytic anemia, myasthenia gravis, Goodpastures Syndrome, acute glomerulonephritis and acute rheumatic fever.
  • IL-2 signaling inhibition is therefore useful in the treatment or prevention of clinical symptoms for numerous disease states involving T-cell activation and hyperproliferation.
  • autoimmune diseases may be, for example, lupus, scleroderma, rheumatoid arthritis, multiple sclerosis, glomerular nephritis as well as potential malignancies.
  • ATL Adult T-cell leukemia
  • IL-2R high affinity IL-2 receptors
  • B-cell, monocytic and granulocytic leukemias express elevated levels of IL-2 receptor alpha subunit (IL-2R- ⁇ ).
  • IL-2R- ⁇ IL-2 receptor alpha subunit
  • IL-2R- ⁇ is also expressed on Reed-Sternberg cells of patients with Hodgkin's disease and on malignant cells of patients with true histiocytic lymphoma. Similarly, a proportion of patients with cutaneous T-cell lymphomas express IL-2R- ⁇ on the malignant cells. Finally, all patients with human T-cell lymphotrophic virus I (HTLN-1) associated adult T-cell leukemia (ATL) cells constitutively express large numbers of IL-2R- ⁇ .
  • HTLN-1 human T-cell lymphotrophic virus I
  • ATL adult T-cell leukemia
  • IL-2 signaling in these malignancies provide a selective means for irradication of proliferation and survival and may be used as a selective means for irradication of malignant cells expressing IL-2R, and particularly IL-2R- ⁇ . Therefore, the inventive method, which inhibits IL-2 signaling, is useful for treating those malignancies whose malignant cell express large amounts of IL- 2R and particularly, IL-2R- .
  • IL-2 Human IL-2 has been shown to induce lung microvascular injury and pulmonary edema in humans.
  • IL-2 induces platelet activating factor (PAF) in IL-2 treated rats, and PAF mediates the IL-2 induced lung injury (Rabinovich et al., Clin. Invest. 89: 1669-1673, 1992.).
  • PAF platelet activating factor
  • inventive method treats or prevents immune disorders by administering a compound capable of inhibiting cellular accumulation of myristylated PA
  • compounds useful in the inventive method must block IL-2 signal transduction by inhibiting an accumulation of myristylated PA, which directly results from IL-2 signaling.
  • a procedure has therefore been developed to verify whether a compound is useful in the inventive method.
  • a predictive assay for determining compounds useful in the inventive method utilizes in vitro techniques that model the IL-2 signaling phenomenon.
  • cells cultures that are dependent on IL-2 are the most preferred cells.
  • IL-2 dependent cells include, CT-6.1 and BCLj cells.
  • the cells are stimulated with IL-2, with or without a selected compound, according to conventional methods. After incubation of IL-2 dependent cells with the IL-2 (and selected compound) for a determined period, the cells are immersed in ice cold methanol to stop any cellular signaling reactions. Once any cell signaling activity has been initiated, allowed to amplify for a determined period and then interrupted according to the procedure defined above, PAs must first be separated from other lipids found in serum by chemically extracting the lipids and then subsequently using high performance liquid chromatography (HPLC) to separate and detect PAs.
  • HPLC high performance liquid chromatography
  • Chemical extraction can be accomplished, for example, by the method of Bligh et al. (Canadian J. Biochem. Physiol. 37:914-917, 1959) or that of Folch et al. (J. Biochem. 226:497-509, 1957). Briefly, the method of Bligh et al. involves organically extracting lipids from biological tissue homogenates or fluids. One volume of sample and three volumes of methanol: chloroform (2:1) are vigorously shaken for 2 minutes. One volume of chlorofoim is added and the resulting preparation is shaken vigorously for 30 seconds, after which, one volume of water is added and the mixture shaken vigorously for 30 seconds. The mixture is filtered and the upper aqueous layer is discarded. The lower organic layer will contain a mixture of lipid classes.
  • lipids are extracted from biological tissue homogenates or body fluids.
  • One volume of sample plus 20 volumes of chloroform:methanol (2:1) are vigorously shaken for 2 minutes.
  • the mixture is filtered and an amount of 0.1 N KC1, equal to 20% of the extraction mixture volume, is added and the resulting mixture is shaken vigorously for an additional 2 minutes.
  • the aqueous and organic phases are allowed to separate.
  • the upper aqueous layer is discarded.
  • the lower organic layer contains a mixture of lipid classes.
  • Free fatty acids and neutral lipids can be separated from phospholipids by normal phase (silica) HPLC by modifying the method of Van Kessel et al. (Biochim et Biophys Acta 486:524-530, 1977). This method involves separation of lipids into their major classes by HPLC.
  • a 5 micron, 25 cm x 0.45 cm silica HPLC column is connected to a binary solvent delivery system, followed with a UN detector. The organic layer containing the lipid sample is injected on the column and a solvent gradient is run at 1.0 ml/minute.
  • the solvent gradient is hexane:isopropanol:water in the proportions 3:4:0.75, run isocratically for 3 minutes, which is then ramped to hexane:isopropanol:water in the proportions 3:4: 1.4 in 15 minutes, and then the sample is run isocratically at the same proportions for 15 min. Detection is made at 206 nm.
  • the PAs run at about 6-8 minutes when run at 1 ml/min and in hexane:isopropanol (3:4).
  • Figure 33 illustrates a representative HPLC chromatogram, illustrating separation of lipids from the lipid sample. PA peaks are shown labeled as, for example, PA1 and PA2.
  • this assay identifies myrPA species in particular according to one of the following procedures. Having isolated the lipids by class using the foregoing HPLC procedure, fast atom bombardment mass spectrometry (FAB/MS) of the HPLC-isolated fractions is conducted to verify that a compound is useful in the inventive method by specifically inhibiting accumulation of myrPA in response to stimulus with IL-2. In this procedure, FAB/MS spectra are acquired using a VG 70 SEQ tandem hybrid instrument of EBqQ geometry (VG Analytical Altrincham, UK).
  • FAB/MS fast atom bombardment mass spectrometry
  • the instrument is equipped with a standard unheated VG FAB ion source and a standard saddle-field gun (Ion Tech, Middlesex, UK), producing a beam of Xe atoms at 8 keV and 1mA.
  • the mass spectrometer is adjusted to a resolving power of 1,000, and spectra are obtained at 8 keV and a scan speed of lOs/decade.
  • the HPLC fraction samples are applied to the FAB target as solutions of known concentrations.
  • 2-Hydroxyethyl disulfide is used as a matrix in the positive-ion FAB/MS, and triethanolamine is used as a matrix in the negative FAB/MS.
  • the foregoing procedure is used to correlate the specific blocking of IL-2 signaling by using compounds which reduce or eliminate accumalated levels of myrPA, formed in response to stimulus by IL-2.
  • this procedure permits identification of compounds which are useful in treating, inter alia, autoimmune disorders according to the inventive method.
  • the illustrated inventive method comprising administration of a specific compound, inhibits T-cell activation and proliferation, although by a distinctly different mechanism than either CsA or FK506.
  • the illustrated compounds interfere with direct IL-2 signaling without affecting T-cell receptor (TCR) signaling.
  • TCR T-cell receptor
  • Signal blocking with the illustrated compounds not only inhibits T-cell proliferation in response to an antigen, but also occupies the TCR in the absence of IL-2 responsiveness, resulting in a desirable, prolonged state of unresponsiveness, such as anergy (Mueller et al., Annu. Rev. Immunol. 7:445-480, 1989).
  • rapamycin is a synthetic molecule which inhibits T-cell proliferation via IL-2 signaling, specifically by inhibiting p70S6 kinase activity (Kuo et al, Nature 358:70-73, 1992).
  • the illustrated compounds do not inhibit IL-2 -induced p70S6 kinase activity, as shown in mouse CT-6.1 cells. Therefore, the illustrated compounds inhibit IL- 2-induced proliferative responses via a different mechanism than rapamycin.
  • TCR T-cell receptor complex
  • IL-2 is the primary T-cell growth factor, and is a key determinant in the magnitude of the immune response.
  • the mechanism of immune suppression by CsA and FK506 is thought to involve intracellular binding to immunophilins.
  • the target for the immunophilin complex is thought to be the protein phosphatase calcineurin.
  • both CsA and FK506 interfere with T-cell receptor signaling, without affecting direct IL-2 signaling.
  • the end effect of treatment with CsA or FK506 is inhibition of T-cell proliferation due to inhibition of the TCR signal transduction mechanisms.
  • the inventive method is also capable of inhibiting T-cell activation and proliferation, but by a distinctly different mechanism than either CsA or FK506.
  • the illustrated compounds interfere with direct IL-2 signaling without affecting TCR signaling. Blocking signaling not only inhibits T-cell proliferation in response to antigen, but TCR occupancy in the absence of IL-2 responsiveness leads to a prolonged state of unresponsiveness, i.e., anergy.
  • IL-2 signal transduction involves, at least in part, breakdown of inositol phosphate-glycan (PjG) and the induction of the lipid second messenger molecules myristylated diacyglycerol (mDG) and myrPA.
  • IL-2 signaling in T lymphocytes results in hydrolysis of glycan-PI (PjG) through both or either Pl-gly can-directed phospholipase C (PjGPLC) and/or Pl-glycan-directed phospholipase D (PjGPLD).
  • PjGPLC results in formation of 1,2-dimyristoyl or 1-myristoyl 2-palmitoyl DG (diacylglycerol), whereas activation of PjGPLD results in formation of 1,2- dimyristoyl, 1-myristoyl 2-palmitoyl, or 1-o-tetradecanyl 2-palmitoleoyl PA species.
  • PjGPLD can hydrolyze the glycan-PI, whereas PjGPLC is ineffective.
  • the predominant products of IL-2 activation are the disclosed PA species, as opposed to DG species synthesized by PLC.
  • the rate of synthesis of glycan-PI in cells using this moiety as a signaling molecule is relatively high, resulting in rapid resynthesis of glycan-PI after its hydrolysis for signaling (i.e., in ⁇ 30 seconds after complete hydrolysis of the fraction). Therefore, any blockade of glycan Pi-hydrolysis by the inventive method in T lymphocytes that contain acyl moieties substituted to the inositol group of the phosphatide strongly suggest that PjG- PLD is being blocked or inhibited in these cells.
  • the control glycan PI contains 1-o-tetradeca- enyl 2-palmitoleoyl PA (M+H/Z 603), a docosanoyl (22:0 acyl) group adduced to be from the 2'-o-inositol position (M+H/Z 339), and the breakdown products of the glycan moiety at 577 (containing inositol, glucosamine, and mannose) and 744 (containing an additional mannose moiety).
  • IL-2 When stimulated with IL-2 for 15 seconds, as seen in Figure 28, this fraction is greatly decreased in mass, with resulting proportionate loss of each of the component moieties. In the presence of 100 nM of representative compound no.
  • the representative compound no. 2576 belongs to a class of low molecular weight synthetic compounds designed to inhibit the IL-2 induced breakdown of PjG and subsequent generation of myrDG and myrPA in lymphoid cells. Using HPLC and FAB/MS in CT-6.1 cells, representative compound no.
  • inventive method induces antigen-specific T-cell anergy in a primary mixed tumor-lymphocyte culture (MTLC) using responding C57BL/6 spenocytes (H-2b) and 2PK3 B-cell tumor cells (H2d). Following a five day co-culture with representative compound no. 2576 and antigen, splenocytes were unable to respond to subsequent exposure to the primary antigen, but could respond normally to anti-CD3 stimulation. Also, lysates from IL-2 stimulated CT-6.1 cells showed that the inventive method, represented by administrating compound no.
  • compounds useful for treating or preventing any of the foregoing diseases or indications are identified by their ability to block intracellular myrPA accumulation.
  • compounds useful in the present invention are amino alcohol or chiral primary or secondary alcohol compounds, preferably substituted by a heterocyclic moiety.
  • Preferred heterocyclic moieties may be, for example, a substituted or unsubstituted xanthine, uracil or thymine.
  • the compounds which are effective in carrying out the inventive method can be administered to a human or other subject in a conventional dosage form prepared by combining the compound which inhibits intracellular accumulation of myrPA with a conventional pharmaceutically acceptable carrier or diluent according to known pharmaceutical formulation techniques.
  • Most preferred compounds of this method are small organic molecule that can mimic binding to a complex of enzymes, which play a role in mediating signal amplification, thereby resulting in a diminution of intracellular PA levels in response to an inflammatory stimulus.
  • Most preferred compounds useful in the inventive method for treating, inter alia, autoimmune diseases include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof, the compound having a straight or branched aliphatic hydrocarbon structure of formula I:
  • n is an integer from one to four and m is an integer from four to twenty.
  • R ⁇ and R2 are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or -(CH2) W R5- If Rj or R2 is - (CH2) W R5 , w may be an integer from one to twenty and R5 may be an hydroxyl, halo, C j_g alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle.
  • R ⁇ and R2 may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocyle.
  • R3 may be either hydrogen or C 1.3.
  • Preferred compounds may have one of R] or R? and R3 that form a substituted or unsubstituted linking carbon chain, having from one to four carbon atoms. This 1/ 3 or R2/R3 linking chain will join the O and N in a cyclic structure, an integer sum equal to n + a number of carbon atoms in the linking carbon chain being less than six.
  • a total sum of carbon atoms comprising R ] or R2, (CH2) n and
  • R4 is a terminal moiety comprising a substituted or unsubstituted, oxidized or reduced ring system, the ring system having a single ring or two to three fused rings, a ring comprising from three to seven ring atoms.
  • R5 may be hydroxy, chloro, fluoro, bromo, or ⁇ . alkoxy, or a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to seven carbon atoms, more preferably, a mono-, di- or tri-substituted carbocycle or heterocycle.
  • (CH2) m may be unsubstituted, or more preferably, (CH2) m is substituted by a halogen atom, an hydroxyl group, or substituted or unsubstituted Cn .jQ) alkoxyl, C ⁇ .JQ) alkyl, C(2-10) alkenyl or C ⁇ .J O) alkynyl group.
  • Substituents of the R1/R3 or R2 R3 linking chain may include, without limitations C ⁇ .4) alkyl, C -4 alkenyl, hydroxyl, carbonyl, amino, thio, thiol, thiocarbonyl and imino group or a single atom, such as, for example, chlorine, bromine, fluorine and oxygen
  • the terminal moiety ring system may be saturated, but alternatively, preferred compounds have a ring system terminal moiety having at least one unsaturated carbon-carbon double bond.
  • ring system substituents may include, but are not intended to be limited to, Cn - 4) alkyl, C ⁇ _4) alkenyl, hydroxyl, carbonyl, amino, thio, thiol, thiocarbonyl and imino group or a single atom.
  • Single atoms corresponding to ring substituents may include, but are not intended to be limited to, chlorine, bromine, fluorine and oxygen.
  • the compounds may have ring systems, in which all ring atoms are carbon atoms.
  • Preferred compounds, in which all ring atoms are carbon atoms may have ring systems that include, but are not intended to be limited to, one of the following groups: phenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, biscyclooctyl, indanyl, indenyl, decalinyl, resorcinolyl, tetralinyl, ⁇ -tetralonyl, 1-indanonyl, cyclohexanedionyl or cyclopentanedionyl.
  • ring systems that include, but are not intended to be limited to, one of the following groups: phenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
  • At least one ring atom of the ring system may be other than carbon.
  • Non-carbon ring atoms may include, for example, atoms such as nitrogen, oxygen, sulfur and phosphorus.
  • Terminal moiety ring systems which have at least one ring atom that is other than carbon may include, for example, ring systems that have three or four atoms in at least one ring of the system.
  • Preferred ring systems having at least one non-carbon atom and at least one ring which has three or four atoms may include, but are not intended to be limited to: azetidinedionyl; azetidinonyl; azetidinyl; aziridinonyl; aziridinyl; azirinyl; diaziridinonyl; diaziridinyl; diazirinyl; dioxetanyl; dioxiranyl; dithietanyl; episulfonyl; lactamyl; lactonyl; oxathietanyl; oxathiiranyl; oxaziranyl; oxaziridinyl; oxaziridinyl;
  • preferred compounds may have terminal moieties that have at least one ring having at least five ring atoms, at least one of the at least five ring atoms being other than carbon.
  • the at least five-atom ring system may include, but is not intended to be limited to, one of the following substituted or unsubstituted groups: adeninyl; alloxanyl; alloxazinyl; anthracenyl; anthrenyl; azapinyl; azapurinyl; azinyl; azolyl; barbituric acid; biotinyl; chromylenyl; cinnolinyl; coumarinyl; coumaronyl; depsidinyl; diazepinyl; diazinyl; diazocinyl; dioxadiazinyl; dioxanyl; dioxenyl; dioxepinyl; dioxinonyl; dioxolan
  • a ring system may include one of the following: acridinyl; acridonyl; alkylpyridinyl; anthraquinonyl; ascorbyl; azaazulenyl; azabenzanthracenyl; azabenzanthrenyl; azabenzophenanthrenyl; azachrysenyl; azacyclazinyl; azaindolyl; azanaphthacenyl; azanaphthalenyl; azapyrenyl; azatriphenylenyl; azepinyl; azinoindolyl; azinopyrrolyl; benzacridinyl; benzazapinyl; benzofuryl; benzonaphthyridinyl; benzopyranonyl; benzopyranyl; benzopyronyl; benzoquinol
  • the ring system may include: adeninyl; alloxanyl; alloxazinyl; anthranilyl; azabenzanthrenyl; azabenzonaphthenyl; azanaphthacenyl; azaphenoxazinyl; azapurinyl; azinyl; azoloazinyl; azolyl; barbituric acid; benzazinyl; benzimidazolethionvl: benzimidazolonyl; benzimidazolyl; benzisothiazolyl; benzisoxazolyl; benzocinnolinyl; be- ⁇ azocinyl; benzodioxanyl; benzodioxolanyl; benzodioxolyl; benzopyridazinyl; I .
  • Terminal ring systems having at least one ring having three hetero atoms may include, but are not intended to be limited to, one of the following ring systems: azabenzonaphthenyl; benzofuroxanyl; benzothiadiazinyl; benzotriazepinonyl; benzotriazolyl; benzoxadizinyl; dioxadiazinyl; dithiadazolyl; dithiazolyl; furazanyl; furoxanyl; hydrotriazolyl; hydroxytrizinyl; oxadiazinyl; oxadiazolyl; oxathiazinonyl; oxatiiazolyl; pentazinyl; pentazolyl; petrazinyl; polyoxadiazolyl; sydononyl; tetraoxanyl; tetrazepinyl; tetrazinyl; tetrazolyl; thiadiazinyl; thiadiazoliny
  • the most preferred ring systems include, for example, dimethylxanthinyl, methylxanthinyl, phthalimidyl, homophthalimidyl, methylbenzoyleneureayl, quinazolinonyl, octylcarboxamidobenzenyl, methylbenzamidyl, methyldioxotetrahydropteridinyl, glutarimidyl, piperidonyl, succinimidyl, dimethoxybenzenyl, methyldihydrouracilyl, methyluracilyl, methylthyminyl, piperidinyl, dihydroxybenzenyl, or methylpurinyl, even more preferably, methylxanthinyl, dimethylxanthinyl or a derivative thereof.
  • the most preferred compounds may also have a ring-system terminal moiety that has at least one substituent bonded to at least one ring of the ring system, the at least one substituent being bonded to a carbon ring atom of the at least one ring by an sp bond, in which the carbon ring atom is adjacent to a hetero atom of the ring.
  • ring-system terminal moieties, having at least one hetero atom may be linked to -(CH2) m of formula I by a bond between the at least one hetero atom and -(CH2) m -
  • Compounds useful in the inventive method may also include resolved enantiomers and/or diastereomers, hydrates, salts, solvates and mixtures thereof that have a straight or branched aliphatic hydrocarbon structure of formula II:
  • n, m, R3, and R4 are defined as provided in formula I above.
  • R and R7 are hydrogen, a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length, or -(CH2) x Rg, at least one of R or R7 being - (CH2) X R8-
  • x is an integer from zero to fourteen and Rg is a moiety having a general structure as provided in formula III
  • m, R and R4 are defined as provided in formula I above.
  • Z is N or CH and p is an integer from zero to four.
  • R9 is I i or a straight or branched chain alkane, alkene or alkyne of up to twenty carbon atoms in length.
  • Preferred compounds useful in the inventive method include both __ and S enantiomers and racemic mixtures of the following compounds: Compound No. Compound Name Chemical Structure
  • the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables.
  • a compound which inhibits myrPA, according to the inventive methodk is administered in an amount sufficient to treat or prevent the disease.
  • the route of administration of the compound e.g., the compounds discussed and exemplified above
  • parenteral includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, transdermal, opthalmic, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the daily parenteral dosage regimen will preferably be from about 0.01 mg/kg to about 25 mg/kg of total body weight, most preferably from about 0.1 mg/kg to about 4 mg/kg.
  • each parenteral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 400 mg.
  • the compounds are generally active when given orally and can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
  • a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule.
  • a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • suitable pharmaceutical carrier(s) for example, aqueous gums, celluloses, silicates or oils
  • the daily oral dosage regimen will preferably be from about 0.01 mg/kg to about 40 mg/kg of total body weight.
  • each oral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 1000 mg.
  • the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment (i.e., the number of doses of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof given per day and duration of therapy) can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • Example 1 This example illustrates the inventive method by administration of representative small molecule amino alcohol compounds and their resulting effect on different mechanisms of immunosuppression activity, as compared with the mechanisms of two widely-studied immunosuppressants, CsA or FK506.
  • Figure 8 shows proliferation assay results from mouse thymocytes preincubated overnight with ConA ("priming step"), washed, and restimulated with IL-2 without the presence of drug. On day 4, the cells were pulsed with tritiated thymidine and allowed to incubate for an additional 4 hours. The cells were harvested and incorporated tritiated thymidine was determined in a liquid scintillation counter.
  • pre-incubation with ConA "primes" the thymocytes by stimulating the CD3 receptor in a manner that is similar to that of antigen recognition.
  • antibody directed against CD3 can be substituted for ConA.
  • the "priming step” e.g. CD3 signaling
  • the thymocytes did not proliferate in response to IL-2.
  • an amino alcohol-substituted compound specifically, representative compound no. 2558
  • Representative compound no. 2558 did not inhibit the "priming” step and is thus an immunosuppressant compound capable of achieving the different mechanism of action of the inventive method.
  • Example 2 This example illustrates the effect of various immunosuppressive compounds useful in the inventive method on IL-2 receptor (alpha chain CD25) expression on mouse splenoyctes.
  • IL-2 receptor is not expressed on resting T-cells, but is rapidly induced by certain events, such as antigen recognition, treatment with ConA, or antibodies to CD3 (anti-CD3).
  • IL-2 receptor expression is required for IL-2 dependent proliferation.
  • Murine splenocytes were stimulated with anti-CD3 (10 ug/ml) with or without CsA (20 ⁇ M) or representative compound no. 2583 (1 ⁇ M). Following overnight incubation, the splenocytv-.
  • Figures 30A and 30B show frequency histograms of measurements for IL-2 receptor density of 20,000 cell samples. As expected, media control had a low level of fluorescence, while stimulation with anti-CD3 stimulated large increases in IL-2 receptor expression.
  • Figure 30A shows Co-incubation with CsA inhibited CD3 stimulated IL-2 receptor expression
  • Figure 30B shows incubation with representative compound no. 2583 at a concentration that blocks 90% of proliferation had no effect on receptor expression.
  • CsA and the inventive method, as illustrated by this representative compound affect immune cells by different mechanisms of action.
  • Example 4 This example illustrates that several representative compounds, useful in the inventive method, inhibit direct IL-2 induced proliferation in a CT-6.1 cells.
  • IL-2 was removed from the medium for 24 hours prior to stimulation.
  • One hour prior to B -2 stimulation either CsA or a representative compound no. 2584, 2576 or 2571 were added at various concentrations.
  • the cells were stimulated with IL-2 and tritiated thymidine incorporation was measured 48 hrs. later. Background counts were less than 5000 cpm.
  • Figures 8, 9, 10 and 11 show that CsA and the compounds have divergent effects on IL-2 induced proliferation. CsA does not inhibit proliferation, even at very high concentrations.
  • the representative compounds, administering according to the inventive method inhibit direct IL-2 induced CT-6.1 proliferation.
  • Example 5 Example 5
  • a B-cell tumor, 2PK3 (H-2d) was used as a stimulating cell to C57BL/6 splenocytes (H-2b), the responding cell.
  • Mixed cultures were incubated for 5 days with either media only or with 1 ⁇ M of representative compound no. 2576. At 5 days, the cells were washed and resuspended with either media, the original antigen (2PK3) or anti-CD3. Tritiated thymidine was added and it incorporation was measured 24 hours later.
  • the cultures treated according to the inventive method responded to anti-CD3, as did the untreated cultures.
  • those cultures which were incubated with representative compound no. 2576 for 5 days had a decreased response to the primary antigen, 2PK3.
  • the C57BL/6 splenocytes were inhibited from responding to the original antigen by this representative compound, but responded normally to anti-CD3 stimulation.
  • CT- 6.1 an IL-2 dependent T-cell clone
  • FBS fetal bovine serum
  • the cells were starved overnight at a density of 500,000 cells per mL in RPMI medium containing 0.5% fetal bovine serum.
  • the cells were then harvested, washed, and re-cultured in RPMI- 1640+0.5% FBS at a concentration of 10 million cells per mL.
  • CT-2576 or CsA was added to the cells at a final concentration of 10 nM or 100 nM, respectively, and the cells were incubated for an additional 1 hour. Controls, untreated with drug, were also set up. Following the one hour incubation, murine IL-2 was added to the cultures at a final concentration of 20 Units/mL. Aliquots of cells containing 10 million CT-6.1 cells were removed at various times following IL-2 stimulation, cells were fixed and processed for lipid analysis. Lipids were extracted using a modified Folch method. Neutral lipids and phospholipids were separated by HPLC and analyzed. Experiments for HPLC were performed as follows: at sequential times, aliquots were removed and lipids extracted.
  • HPLC analysis is performed with a Waters ⁇ -Porasil silica column (0.45 x 25 cm).
  • a mobile phase consisted of a gradient of 1-9% water in hexane:isopropanol (3:4, v/v) run at a flow rate of 1 mL/minute. This separation method was found to give adequate separation of DG, PA, PE, PI, PS, PC, glycosylated PI, and sphingomyelin.
  • FIG. 31 illustrates that IL-2 induces myrPA in CT-6.1 cells, as represented by the curve labeled "IL-2 Control.” Also shown in figure 31 is experimental data obtained by using CsA and a representative compound of the present method. CsA does not exhibit an ability to inhibit the IL-2-induced increase in myrPA, thus having little significance in effecting myrPA accumulation, as shown by the curve "IL-2-CsA.” However, the inventive method is capable of inhibiting a rapid increase in myrPA at approximately 100 seconds following stimulation with IL-2. Comparing the curves labeled "IL-2 Control” and "IL-2 + 2576" illustrates the substantial effect the inventive method has on completely blocking accumulation of myrPA.
  • figure 32 illustrates that myristylated-DAG is not increased following IL-2 stimulation.
  • This result indicates that GPI hydrolysis is catalyzed by GPI-PLD rather than GPI-PLC in CT-6.1 cells, and that treatment with representative compound no. 2576 effectively blocks accumulation of myrPA by blocking PIG-PLD catalyzed hydrolysis of GPI.
  • This example shows how an immune response may be inhibited by administering a compound useful in the inventive method to mice sensitized with sheep red blood cells (SRBC), illustrating an in vivo effect of the inventive method.
  • SRBC sheep red blood cells
  • representative compound no. 3578 was administered to CD-I (ICR) female mice (Charles River), 10-14 weeks of age, which were sensitized intraperitoneally with 1.25 x 10 3 sheep red blood cells (SRBC) in 0.2 mL saline. Procedurally, the mice were divided into groups of 5-8. Compound no. 3578 was administered in either 25 or 12 ml kg doses, p.o., twice daily, commencing on the day of sensitization and continued daily through day 3. Control mice received a coequal volume of vehicle.
  • ICR CD-I
  • SRBC sheep red blood cells
  • mice On the fourth day after sensitization, the mice were sacrificed, each spleen excised, a splenocyte suspension prepared by homogenization in 4 mL Hank's Buffered salt solution (HBSS), and a nucleated cell count (WBC) determined by Coulter counter.
  • HBSS Hank's Buffered salt solution
  • WBC nucleated cell count
  • the data show that representative compound no. 3578 inhibited plaque formation by 31%, when administered at a dose of 12 mg/kg and at 91% when administered at a dose of 25 mg/kg.
  • the inventive method inhibits an immune response by administration of a compound which inhibits accumulation of myrPA, resulting from triggering an immune response.
  • spleens were removed and enumerated.
  • no appreciable decrease in splenic cellularity was determined at either concentration of compound administered.

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Abstract

L'invention concerne un procédé d'inhibition de la transduction du signal de l'interleukine-2 (IL-2), ce qui entraîne une activité immunosuppressive utile dans le traitement de la réaction du greffon contre l'hôte, des maladies auto-immunes, de certaines formes de leucémies et de lymphomes impliquant les lymphocytes T ou B, ainsi que pour les patients greffés. Ce procédé consiste à administrer un composé qui inhibe la transduction de signal par inhibition de l'accumulation d'acide phosphatidique myristylé (myrPA) survenant dans les cellules, en réponse à l'émission du signal de l'IL-2.
PCT/US1994/013202 1993-11-15 1994-11-15 Procede d'inhibition selective de la transduction du signal de l'interleukine-2 (il-2) WO1995013808A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12895/95A AU1289595A (en) 1993-11-15 1994-11-15 Method for selectively inhibiting il-2 signal transduction

Applications Claiming Priority (2)

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US15261193A 1993-11-15 1993-11-15
US08/152,611 1993-11-15

Publications (1)

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WO1995013808A1 true WO1995013808A1 (fr) 1995-05-26

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EP0739203A1 (fr) * 1994-01-14 1996-10-30 Cell Therapeutics, Inc. Methode de traitement d'affections dues a la proliferation cellulaire en reponse aux pdgf, egf, fgf et vegf
WO1998034601A2 (fr) * 1997-02-10 1998-08-13 Cell Therapeutics, Inc. Procede destine a inhiber une replication virale intracellulaire
WO1999003479A1 (fr) * 1997-07-16 1999-01-28 Modus Biological Membranes Ltd. Composition immuno-modulatrice a base de lipides
WO2001054721A1 (fr) * 2000-01-25 2001-08-02 Pharma Pacific Pty Ltd Antagonistes de reponse immunitaire th-1 employant la cytokine dans le traitement de maladies auto-immunes
US7939076B2 (en) 2003-04-23 2011-05-10 Medarex, Inc. Methods for the therapy of Inflammatory Bowel Disease using a type-1 interferon antagonist
US8460668B2 (en) 2004-06-21 2013-06-11 Medarex, Inc. Interferon alpha receptor I antibodies and their use

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US5288721A (en) * 1992-09-22 1994-02-22 Cell Therapeutics, Inc. Substituted epoxyalkyl xanthines
US5340813A (en) * 1992-11-09 1994-08-23 Cell Therapeutics, Inc. Substituted aminoalkyl xanthine compounds
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CELLULAR IMMUNOLOGY, Volume 131, No. 1, issued November 1990, IOANNIDES et al., "Inhibition of IL-2 Receptor Induction and IL-2 Production in the Human Leukemic Cell Line Jurkat by a Novel Peptide Inhibitor of Protein Kinase C", pages 242-252. *
EUROPEAN JOURNAL OF IMMUNOLOGY, Volume 22, issued July 1992, CANO et al., "Regulation of Interleukin-2 Responses by Phosphatidic Acid", pages 1883-1889. *
MEDLINE ABSTRACT, #91102808, issued 1991, KAHAN et al., "Synergistic Interactions of Cyclosporine and Rapamycin to Inhibit Immune Performances of Normal Human Peripheral Blood Lymphocytes in Vitro"; & TRANSPLANTATION, 51 (1), 232-239. *
THE JOURNAL OF IMMUNOLOGY, Volume 151, No. 8, issued 15 October 1993, PALIOGIANNI et al., "Novel Mechanism for Inhibition of Human T-Cells by Glucocorticoids. Glucocorticoids Inhibit Signal Transduction Through IL-2 Receptor", pages 4081-4089. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985926A (en) * 1993-11-01 1999-11-16 Cell Therapeutics, Inc. Method for inhibiting intracellular viral replication
EP0739203A4 (fr) * 1994-01-14 2000-12-20 Cell Therapeutics Inc Methode de traitement d'affections dues a la proliferation cellulaire en reponse aux pdgf, egf, fgf et vegf
EP0739203A1 (fr) * 1994-01-14 1996-10-30 Cell Therapeutics, Inc. Methode de traitement d'affections dues a la proliferation cellulaire en reponse aux pdgf, egf, fgf et vegf
WO1998034601A2 (fr) * 1997-02-10 1998-08-13 Cell Therapeutics, Inc. Procede destine a inhiber une replication virale intracellulaire
WO1998034601A3 (fr) * 1997-02-10 1998-10-29 Cell Therapeutics Inc Procede destine a inhiber une replication virale intracellulaire
EA002364B1 (ru) * 1997-07-16 2002-04-25 Модус Байолоджикал Мембрейнс Лтд. Применение иммуномодулирующей композиции на основе липидов
WO1999003479A1 (fr) * 1997-07-16 1999-01-28 Modus Biological Membranes Ltd. Composition immuno-modulatrice a base de lipides
US6288047B1 (en) 1997-07-16 2001-09-11 Modus Biological Membranes Ltd. Lipid-based immune modulator composition
WO2001054721A1 (fr) * 2000-01-25 2001-08-02 Pharma Pacific Pty Ltd Antagonistes de reponse immunitaire th-1 employant la cytokine dans le traitement de maladies auto-immunes
US7939076B2 (en) 2003-04-23 2011-05-10 Medarex, Inc. Methods for the therapy of Inflammatory Bowel Disease using a type-1 interferon antagonist
US8828393B2 (en) 2003-04-23 2014-09-09 Medarex, L.L.C. Methods for the therapy of inflammatory bowel disease using a type-1 interferon antagonist
US8460668B2 (en) 2004-06-21 2013-06-11 Medarex, Inc. Interferon alpha receptor I antibodies and their use
US9453077B2 (en) 2004-06-21 2016-09-27 E. R. Squibb & Sons, L.L.C. Interferon receptor 1 antibodies and their uses
US10385133B2 (en) 2004-06-21 2019-08-20 E.R. Squibb & Sons, L.L.C. Interferon receptor 1 antibodies and their uses
US11072664B2 (en) 2004-06-21 2021-07-27 E.R. Squibb & Sons, L.L.C. Interferon receptor 1 antibodies and their uses

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