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WO1996003993A2 - Modulation de l'activite fonctionnelle de thymocytes et de lymphocytes t - Google Patents

Modulation de l'activite fonctionnelle de thymocytes et de lymphocytes t Download PDF

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Publication number
WO1996003993A2
WO1996003993A2 PCT/US1995/009915 US9509915W WO9603993A2 WO 1996003993 A2 WO1996003993 A2 WO 1996003993A2 US 9509915 W US9509915 W US 9509915W WO 9603993 A2 WO9603993 A2 WO 9603993A2
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WIPO (PCT)
Prior art keywords
cgrp
apoptosis
thymocytes
cells
dhea
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PCT/US1995/009915
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English (en)
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WO1996003993A3 (fr
Inventor
Karen Bulloch
Bruce S. Mcewen
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The Rockefeller University
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Publication of WO1996003993A2 publication Critical patent/WO1996003993A2/fr
Publication of WO1996003993A3 publication Critical patent/WO1996003993A3/fr

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/225—Calcitonin gene related peptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention relates to modulation of cell mediated immune responses, and to modulation or inhibition of thymocyte cancers or thymomas.
  • CGRP calcitonin gene related peptide
  • thymocytes are "selected" by their ability to discriminate between self and non-self and to carry out certain functions that regulate antigen specific cellular and humoral responses in the periphery.
  • Pre T cells that enter and proliferate within the thymus 95 % are negatively selected and die by a process known as apoptosis (Scollay et al. , 1983, J. Immunol. 132: 1085-1088). Only five percent will be positively selected to emigrate to the periphery as functional T cells.
  • Apoptosis is an active process of gene-directed, non- inflammatory cell death (Kerr and Harmon, 1991, "Definition and Incidence of Apoptosis: An Historical Perspective", Apoptosis: The Molecular Basis of Cell Death, edited by L. David Tomei and Frederick O. Cope. Plainview: Cold Spring Harbor Laboratory Press, p. 5-29; Smith et al., 1994, "Multiple Gene Regulation of Apoptosis", Apoptosis II: The Molecular Basis of Apoptosis in Disease, edited by L. David Tomei and Frederick O. Cope. Plainview: Cold Spring Harbor Laboratory Press, pp. 43-87) which eliminates cells without causing an immune response.
  • T cells The mechanisms that constitute the education of T cells are not fully understood and it is clear that no single signal molecule functions alone to carry out the many complex events involved in this process.
  • the cells that migrate to and develop within the thymus do so in waves and consist of a dynamic heterogeneous population that is subject to the nervous, endocrine, and paracrine microenvironments of the thymus. Accordingly, there is a need in the art to identify factors that regulate these events.
  • CGRP is found in intrathymic nerves distributed in the corticomedullary boundaries adjacent to the vasculature with branches emanating into the cortical and medullary regions. Some fibers are invested in the arteries but the majority form varicosities among the trabeculae and the cells of the thymus. CGRP is also found in a discrete population of cells located in the medulla, at the cortico-medullary boundary, and in subcapsular and trabecular mast cells. The inventors have shown that this peptide at physiological doses markedly attenuates in vitro mitogen stimulated proliferation. Furthermore, the type I antagonist CGRP 8 .
  • the present invention is directed to a method for suppressing an immune response.
  • the method comprises administering to a subject suspected of suffering from a disease or disorder that involves an inappropriate immune response an amount of dehydroepiandrosterone (DHEA) effective to induce the activity of calcitonin gene related peptide (CGRP) locally in the area of the inappropriate immune response.
  • DHEA dehydroepiandrosterone
  • CGRP calcitonin gene related peptide
  • the induction of CGRP activity is sufficient to inhibit a functional activity of a helper (CD4+) T cell.
  • a particular advantage of the present invention is that it provides for modulating the magnitude of an immune response via CGRP-mediated regulation of helper T cell activity, without systemic administration of the potent dose of CGRP, which could induce unwanted or adverse side effects.
  • the disease or disorder is an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, Type I diabetes, thyroiditis, myesthemia gravis, and rheumatoid arthritis.
  • the DHEA may be administered systemically or locally to the site of the inappropriate immune response.
  • the invention in another aspect, relates to a method for enhancing an immune response comprising administering to a subject suspected of suffering from a disease or disorder that involves an ineffective immune response an amount of an inhibitor of calcitonin gene related peptide (CGRP), such as but not limited to the CGRP antagonist CGRP 8 . 3 , effective to enhance a functional activity of a helper (CD4+) T cell.
  • CGRP calcitonin gene related peptide
  • the invention provides for administering an amount of an inhibitor of dehydroepiandrosterone (DHEA) effective to enhance a functional activity of a helper (CD4+) T cell.
  • DHEA dehydroepiandrosterone
  • the invention provides a method for treating a thymic cancer or thymoma comprising administering to a subject suspected of suffering from a thymic cancer or a thymoma an amount of calcitonin gene related peptide (CGRP) effective to induce apoptosis of thymic cancer or thymoma cells.
  • CGRP calcitonin gene related peptide
  • the CGRP is administered locally to the site of the thymic cancer or thymoma.
  • the CGRP may be administered in a liposome. Liposomal administration of drugs has been found to reduce the systemic, adverse side effects of such drugs.
  • the invention provides a method for preventing tissue damage associated with excessive secretion of calcitonin gene related peptide (CGRP) in a hyper-response to an insult, comprising administering an amount of an inhibitor of CGRP effective to inhibit apoptosis of cells in the area of the tissue damage.
  • the inhibitor does not antagonize CGRP-mediated attenuation of helper T cell activity.
  • an amount of CGRP, or DHEA, or a combination thereof, effective to inhibit a functional activity of a helper T cell is administered with the antagonist of CGRP-induced apoptosis.
  • the present invention advantageously provides for regulation of the cellular and physiological events that accompany a serious insult, by simultaneously providing CGRP to inhibit inflammation mediated by infiltrating activated immune cells, and an agent that can inhibit CGRP-mediated apoptosis.
  • the insult may be severe brain trauma or a stroke leading to brain ischemia, or result from severing a limb, removing an organ for transplantation, myocardial infarct, or pulmonary embolism.
  • the inhibitor of CGRP-mediated apoptosis is administered in response to head trauma, particularly to avoid neural apoptosis associated with increased CGRP expression that follows trauma and other neural insult.
  • the invention further provides a method for identifying an agent capable of inhibiting CGRP-mediated apoptosis.
  • This method comprises culturing test thymocytes with about 10 "12 M to about 10 ⁇ 6 M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes for about 8 to about 24 hours; culturing control thymocytes with about 10 "12 M to about 10" 6 M CGRP for the same time as the test thymocytes in (a); and determining the extent of apoptosis of the thymocytes in each of the cultures.
  • a decrease in the extent of apoptosis of test thymocytes cultured with CGRP and the agent compared to the extent of apoptosis of the control thymocytes is indicative of the ability of the agent to inhibit CGRP-mediated apoptosis.
  • FIGURE 1 The suppressive effect of CGRP on Con A-stimulated thymocyte proliferation, as determined by tritiated thymidine incorporation, was reversed by CGRPg. 37 . This effect was found in six independent runs, each with n's of 5-6. One representative experiment using thymocytes from male mice is presented here. CGRP g 37 in the presence of Con A enhanced proliferation over Con A alone; the same effect was obtained in seven other experiments besides the ones shown here and in Figure 3. CGRP and CGRP g _ 37 in the absence of Con A had no effect on thymocyte proliferation. * p ⁇ 0.001 compared to Con A alone, using a one way ANOVA followed by Tukey's post hoc comparison. Error bars indicate the standard error of the mean.
  • FIGURE 2 Dose response effects of CGRP g 37 on Con A induced thymocyte proliferation. Con A + CGRP g . 37 enhanced thymocyte proliferation (hatched bars) is statistically greater than proliferation induced by Con A alone at the 2, 4 and 8 microgram/ml dose (open bars). " p ⁇ 0.001 , Student's t test. Error bars indicate the standard error of the mean. Male mice were a source of the thymocytes. FIGURE 3. Dose response effect of DHEA on Con A induced thymocyte proliferation. Con A was used at 4 micrograms/ml for all points. Male mice were the source of the thymocytes. One way analysis of variance (ANOVA) indicated a statistically significant treatment effect, p ⁇ 0.0001.
  • FIGURE 4 Effect of CGRP g _ 37 on DHEA-induced suppression of Con
  • CGRP g . 37 reversed the suppress ive effect of DHEA on Con A, whereas DHEA had no effect on thymocyte proliferation in the absence of Con A ( ** p ⁇ 0.001 versus DHEA + Con A one way ANOVA followed by Tukey's post hoc comparison).
  • the reversal of DHEA suppression by CGRP g 37 was found in 4 separate experiments besides the one shown here, each with n's of 5-6. Male mice were the source of the thymocytes.
  • FIGURE 5 Effects of DHEA, CGRP, and CGRP ⁇ on Con A-induced thymocyte proliferation in male mice. Compared to Con A alone, CGRP (10 ⁇ 9 M) significantly inhibited suppression of Con A stimulation of thymocyte proliferation ( * p ⁇ 0.05 using a one way ANOVA followed by Tukey's post hoc comparison). CGRPg.---* (10 6 M) reversed the effect of CGRP or CGRP plus DHEA f * p ⁇ 0.001). Error bars indicate the standard error of the mean.
  • B Effects of DHEA, CGRP, and CGRP g . 37 on Con A induced thymocyte proliferation in females.
  • the first bar represent results for background controls. Bar (a), (b), (e), (f), and (g) correspond to Figure 6; (c) Con A and CGRP; (d) CGRP 8 . 37 .
  • FIGURE 9 Percent and types (as indicated by CD markers for thymocytes) of total apoptotic cells in (A) control, (B) CGRP (10 " M), and (C) CGRP (10 "8 M) + CGRP g 37 (10 ⁇ 6 M) treated cultures at 8 hours.
  • the markers evaluated were CD3 (a), CD4/CD8 (b), and CD4 (c).
  • the amount of CD8 apoptotic cells is less than 1 % .
  • the percent of CD- labelled cells was adjusted to reflect the percent of apoptotic cells per group.
  • the experiment is representative of two individual determinations of the type of cells undergoing apoptosis. All apoptotic data points for both experiments fell well within the statistical range represented in Figure 7.
  • FIGURE 11 Photomicrograph of CGRP + cells (arrow) within the medulla and at the cortico-medullary junction of the thymus. Schematic insert represents regions of the thymus and the maturational steps that thymocytes undergo as they enter different regions of the thymus.
  • the present invention relates to the nascent understanding of the role of calcitonin gene related peptide (CGRP).
  • CGRP calcitonin gene related peptide
  • the invention relates to the discoveries that a neurosteroid agonizes CGRP-mediated immunomodulation locally; that an antagonist of CGRP can block CGRP's ability to inhibit T cell activation; and that CGRP induces apoptosis in certain cell types.
  • CGRP is a 37 amino acid peptide that is well characterized for its vasoactive properties: it is one of the most potent vasodilators known. This peptide has been found to be localized in immune tissues. For example, it is uniquely distributed in cells and nerves of the thymus, and is thought to play a special role in thymus immune function. Previous data have shown that this peptide prevents CD4 (most likely TH1) cells from being activated in the thymus gland and hence may serve as a regional endogenous regulator of the class of the immune response.
  • CGRP may play a role in the exiting of mature lymphocytes from the thymus.
  • the present invention is directed to regulating immune system function by regulating CGRP activity with agonists and antagonists of CGRP.
  • the present invention is directed in part to the discovery that CGRP mediates its effects through two different receptors, and that these receptors can be independently agonized and antagonized in order to modulate CGRP-mediated responses in vivo.
  • One effect of CGRP is to modulate activation of mature T cells. This effect appears to be modulated through CGRP type 1 receptor (CGRP Rl).
  • CGRP R2 CGRP type 1 receptor
  • a second effect, which operates via a different receptor termed CGRP type 2 receptor (CGRP R2), is to induce apoptosis, e.g. , in a selected subset of T cells.
  • the present invention thus provides for targeting a subset of cells for apoptosis, while preventing suppression of mature T cell activation, for example by providing CGRP (or an analog or derivative thereof, or an agent that stimulates production of endogenous CGRP) with an inhibitor of the CGRP Rl (e.g. , CGRP 8 . 37 ).
  • an agonist of the type 2 receptor that does not affect the type 1 receptor can be provided.
  • the invention provides for inhibiting CGRP-mediated apoptosis, for example by providing an inhibitor of CGRP R2 when CGRP is administered or its endogenous expression increased; however, this inhibitor will not inhibit CGRP-mediated inhibition of T cell activation.
  • a specific agonist of the type 1 receptor that is inactive at the type 2 receptor may be provided. In this treatment regimen, apoptosis and inflammation can both be avoided, e.g. , following severe brain trauma.
  • an "antagonist” is an agent that inhibits or prevents an activity of a molecule.
  • the inhibitory effect of an antagonist can be mediated by competitive inhibition or by non-competitive inhibition.
  • an antagonist of an activity of CGRP for use according to the invention may intercede at a point upstream or downstream of the events induced by CGRP, i.e. , it may not act directly on CGRP, but nevertheless blocks the effects of CGRP.
  • Such an antagonist may, for example, inhibit expression or cause down-regulation of a CGRP receptor, or activate an opposing cellular activity.
  • an "agonist” is an agent that promotes an activity of a molecule, i.e. , the opposite of an antagonist.
  • An agonist according to the invention may induce activity or expression of the molecule, or it may demonstrate the same effects as the molecule (i.e. , function analogous to a non-competitive and competitive inhibitor, respectively).
  • a CGRP type 1 receptor agonist produces signals associated with CGRP binding with a type 1 receptor, e.g. , thymocyte apoptosis or T cell suppression.
  • a CGRP type 1 receptor agonist has no effect on a CGRP type 2 receptor.
  • a CGRP type 2 receptor agonist produces signals associated with CGRP binding to a CGRP type 2 receptor.
  • a CGRP type 2 receptor agonist has no effect on a CGRP type 1 receptor.
  • CGRP agonists that are highly specific for a single CGRP receptor type are employed.
  • Use of a specific receptor agonist may produce the same effects as administration of CGRP with the antagonist of the other receptor: e.g. , use of a CGRP type 1 receptor agonist may produce the same effects as administration of CGRP with a CGRP type 2 receptor antagonist, and vice versa.
  • CGRP refers to calcitonin-gene related peptide.
  • the peptide may be from any animal species, preferably human, and including, but not limited to, other primates, murine, rat, canine, feline, etc.
  • the term further includes the polypeptide that may be obtained from animal sources, such as, but not limited to human, other primate, ovine, porcine, murine, or rat sources.
  • the term also refers to recombinant peptides expressed from genes encoding such peptide.
  • the recombinant peptide may have the same post-translational modifications as the native peptides, or may be differently modified, e.g.
  • polypeptide by expression in prokaryotic expression systems, in which the polypeptide will not be glycosylated and may contain an N-terminal methionine residue; expression in yeast expression systems, in which the polypeptide may be decorated with a yeast polysaccharide; or expression in a mammalian expression system, in which native or non-native glycosylation is possible.
  • yeast expression systems in which the polypeptide may be decorated with a yeast polysaccharide
  • mammalian expression system in which native or non-native glycosylation is possible.
  • analogs of the naturally occurring peptide e.g. , that contain conservative amino acid substitutions, or that contain one or more non-peptide bonds, are also contemplated.
  • fusion polypeptides containing N-terminal (or C-terminal) amino acids that represent cleavage sites, leader sequences, tags e.g.
  • CGRP hexa-Histidine tag
  • CGRP hexa-Histidine tag
  • Derivatives of the polypeptide i.e. , chemically modified forms of the natural peptide, or analogs thereof, such as those prepared by conjugation to a targeting molecule to target the polypeptide across the blood brain barrier (such as transferrin) , conjugation to a hydrophobic peptide or a fatty acid chain to facilitate transport across the blood brain barrier, phosphorylation, carboxymethylation, N-terminal acetylation, pegylation (particularly N-terminal pegylation) or other derivitizations, are also contemplated.
  • the polypeptide may be a truncated form of the natural peptide, or analog or derivative thereof, provided the truncated form demonstrates a functional activity of CGRP.
  • DHEA dehydroepiandrosterone
  • prasterone hydroxyandrost-5-en-17-one
  • dehydroisoandrosterone etc.; see The Merck Index, Tenth Edition, index number 7606
  • DHEA can be obtained from commercial sources, e.g. , Sigma, or can be prepared synthetically (see The Merck Index, supra).
  • a composition comprising "A” (where "A” is a single protein, peptide, steroid, etc.) is substantially free of “B” (where “B” comprises one or more contaminating molecule or molecules, not including racemic forms of "A") when at least about 75 % by weight of the proteins, peptide, steroid, etc. (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition be substantially free of contamination, and generally that such compositions contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al. , Immunology, Second Ed. , 1984, Benjamin/Cummings: Menlo Park, California, p. 384).
  • a primary challenge with an antigen alone, in the absence of an adjuvant will fail to elicit a humoral or cellular immune response.
  • Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • the adjuvant is pharmaceutically acceptable.
  • the present invention is based, in part, on the unexpected discovery concerning regulation of thymocyte activity and maturation in the thymus: a natural steroid dehydroepiandrosterone (DHEA) protects the thymic tissue by stimulating the release of CGRP within the thymus gland.
  • DHEA dehydroepiandrosterone
  • Prior studies indicate that DHEA has immunostimulatory activity, e.g. , induces IL-2 secretion by T lymphocytes (Daynes et al. , 1990, Eur. J. Immunol. 20:793-802). This suppressive action is only seen in the thymus, and has not been observed in the spleen. Suppression by DHEA is reversed by the CGRP antagonist CGRP g _ 37 .
  • the invention further relates to the unexpected observation that in addition to its modulatory role within the thymus, CGRP causes apoptosis of thymocytes. This effect may be mediated by the type 2 CGRP (CGRP R2) receptor since the antagonist CGRP 8 . 37 does not prevent or stimulate thymic cell apoptosis. Furthermore, DHEA within the assay time limitations also does not cause apoptosis.
  • the invention is further based on observations of the role of CGRP after neural injury.
  • the examples, infra describe that adrenalectomy (ADX) results in death and birth of granule cell neurons in the dentate gyrus (DG) of the hippocampal formation (HF).
  • DG dentate gyrus
  • HF hippocampal formation
  • DG dentate gyrus
  • HF hippocampal formation
  • ML inner third molecular layer
  • CA3c neurons termed "the Yellow Brick Road.
  • CGRP immunoreactivity is primarily found in large dense core vesicles (100 to 200 nm) contained in axons and in small axon terminals which form asymmetrical synapses within the neuropil of the inner third molecular layer and in a few cells within the granule cell layer.
  • the ADX increase of CGRP is not reversed by low doses of corticosterone that occupy mineralocorticoid receptors, nor is the Yellow Brick Road abolished by lesioning input pathways from the hypothalamus. Instead, it appears that the Yellow Brick Road is produced by CGRP elaborated within intrinsic neurons. Estrogen treatment, which stimulates synaptogenesis, causes formation of the Yellow Brick Road without ADX.
  • CGRP In ischemic brains (a stroke model), CGRP is expressed in the pyramidal cells of CA1 and a diffuse pattern similar to the cGRP band in the inner third of molecular layer of the DG in the stratum radiatum associated pathway which primarily arise from CA3 and hilar neurons. CGRP is also expressed in hilar neurons. Neurons in colchicine treated rats express dense CGRP-immunoreactivity both along the injection tract and within the target site (the DG). Colchicine has been shown in the brainstem to stimulate CGRP in motor neurons, which indicates that colchicine induces expression of CGRP as well as interferes with spinal formation. These data indicate a protective role of CGRP during reorganization of neural tissue, which could otherwise lead to an inflammatory immune response against apoptic cells and antigens associated with the remodelling process.
  • the invention is directed to administration of DHEA to induce CGRP activity locally. Local activation of CGRP avoids or reduces deleterious side effects that could accompany systemic administration of CGRP.
  • the invention further relates to administration of CGRP to induce apoptosis of undesired thymocytes, in particular thymic cancers or thymomas.
  • the invention accordingly relates to the identification of specific agonists of the CGRP type-1 receptor, to specific agonists of the type 2 receptor, and to antagonists of the latter activity of CGRP, i.e. , antagonists of CGRP-mediated apoptosis.
  • An antagonist of CGRP-mediated apoptosis would antagonize CGRP activity at the type 2 receptor.
  • Such antagonists are believed to be important to protect tissue from severe trauma or ischemic events.
  • administration of such an inhibitor of CGRP is particularly desired for brain trauma or stroke, where the injury results in excessive secretion of CGRP in hyper-response to the insult.
  • CGRP may have a protective role by preventing deleterious cellular immune responses and inflammation, excessive CGRP may be responsible for the programmed cell death associated with trauma or ischemia.
  • compositions comprising one or more of CGRP, DHEA, an antagonist of CGRP- or DHEA-mediated helper T cell attenuation, and an antagonist of CGRP-induced apoptosis.
  • compositions are pharmaceutical compositions, suitable for administration to a subject for treatment of a disease or disorder associated with an inappropriate immune response or apoptosis.
  • the methods of the present invention are applicable to treatment of animal subjects, more particularly mammals, and preferably humans. The methods can also be advantageously employed for the treatment ot non-human primate, canine, feline, bovine, ovine, caprine, equine, or non-domesticated animal subjects.
  • the component or components of a therapeutic composition of the invention may be introduced parenterally, orally, nasally, or rectally.
  • Parenteral administration includes, but is not limited to, intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. More preferably, where administration of CGRP is indicated to induce apoptosis of a tumor, it may be introduced by injection into the tumor or into tissues surrounding the tumor.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al.
  • the therapeutic compound can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al. , N. Engl. J. Med. 321:574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e. , the brain, thus requiring only a fraction of the systemic dose (see, e.g. , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor.
  • CGRP helper T cell activity in an immune response.
  • CGRP can suppress certain aspects of the cellular immune response while permitting and possibly even enhancing the humoral immune response.
  • CGRP is a potent vasodilator, and has additional functional activities, systemic administration of this peptide could have undesirable, deleterious side effects.
  • DHEA neurosteroid dehydroepiandrosterone
  • CGRP 8 . 37 CGRP 8 . 37
  • the present invention has important implications for treating diseases or disorders characterized by inappropriate cellular immune activation, particularly where such immune activation occurs in proximity to or in tissues that contain CGRP-producing cells. Since most tissues are likely to include CGRP-containing nerve fibers, the present invention relates to a wide range of therapeutic uses. For example, in cellular (autoimmune) diseases such as rheumatoid arthritis, Type I diabetes, thyroiditis, myesthemia gravis, and, in particular, the neurological disease multiple sclerosis, where the affected tissues include CGRP-containing cells or are innervated with CGRP-producing cells, or both, administration of DHEA can induce local CGRP activity. Local induction of CGRP activity will reduce the frequency and severity of systemic side effects of CGRP, since a local concentration effective to inhibit a functional activity of a T cell can be developed without requiring a systemic concentration of such potency.
  • autoimmune rheumatoid arthritis
  • Type I diabetes Type I diabetes
  • thyroiditis thyroiditis
  • the present invention provides for modulating the class of an immune response by introducing CGRP, or a CGRP type 1 receptor agonist or a molecule that induces CGRP; or an antagonist of CGRP, depending on whether the disease or disorder requires amplification or attenuation of cellular immune response.
  • CGRP CGRP type 1 receptor agonist or a molecule that induces CGRP
  • an antagonist of CGRP depending on whether the disease or disorder requires amplification or attenuation of cellular immune response.
  • An antagonist of CGRP can be a peptide antagonist, such as but not limited to CGRP 8 37 , an antibody that neutralizes the functional activity of CGRP, a soluble receptor that neutralizes the functional activity of CGRP, or a small molecule analog antagonist. It may also be possible to block CGRP attenuation of cellular immunity by providing an inhibitor of DHEA, such as but not limited to a DHEA antagonist, an antibody to DHEA, or a soluble receptor for DHEA.
  • the present invention contemplates administration of an antagonist of CGRP that can block CGRP-mediated attenuation of cellular immunity, thus allowing for a more robust cell-mediated immune response.
  • the immune protection/enhancement mechanisms reported in the literature for DHEA may very well be related to its interaction with CGRP on a regional level and thus offers strategies for regulating the pathophysiology of pathogenic and/or natural insults.
  • CGRP Induction of Apoptosis in Cancers
  • CGRP can be a specific agent for the treatment and management of certain kinds of cancers of the thymus or thymocytes, e.g. , thymomas.
  • the CGRP or type 2 receptor agonist is administered locally to the site of the tumor, in order to avoid excessive systemic exposure and undesirable side effects.
  • thymomas consist of transformed CD4+CD8+ cells; however, the invention relates to other thymic cancers made up of cells that are CGRP sensitive, particularly cells that bear the CGRP type 2 receptor.
  • this aspect of the invention extends to the treatment of any cancer or tumor in which the transformed cell has the receptor for CGRP that is involved in apoptosis, i.e. , the type 2 receptor.
  • the term "type 2 receptor” refers to a receptor for CGRP that mediates apoptosis, and to which CGRP 8 37 does not competitively bind or antagonize.
  • Tissue injury from an insult is frequently exacerbated by the endogenous responses to these events.
  • traumatic injuries and ischemia induce immune response mechanisms, which frequently escalate to inflammation.
  • Other factors released in response to insults to regulate endogenous responses e.g. , immune response, may overcompensate, and cause damage themselves.
  • insults of this sort may lead to disregulation of the endogenous response mechanisms, leading to further tissue injury.
  • CGRP may contribute to the apoptosis of cells in tissues affected by trauma or ischemia.
  • a hyper- response to severe insult such as observed in the brain's limbic system
  • an over- action of the peptide's natural function inhibition of cellular immune response to avoid a deleterious cellular response may occur.
  • the present invention contemplates administering a quantity of an inhibitor or antagonist of CGRP binding or activation via the CGRP receptor that mediates apoptosis, i.e. , the type 2 receptor as defined hereinabove, effective to inhibit or prevent apoptosis, but that does not impair the anti-inflammatory effect of CGRP mediated via the type 1 receptor.
  • the invention contemplates that the type 2 receptor antagonist will not inhibit CGRP binding to the type 1 receptor, which type 1 receptor activity is antagonized by CGRP 8 . 37 .
  • a CGRP antagonist of the receptor involved in apoptosis also antagonizes type 1 receptor activity
  • an amount of such an antagonist effective to inhibit CGRP- apoptosis receptor activity but ineffective to inhibit type 1 receptor activity may be administered to a subject in need of such treatment.
  • the present invention further provides for attenuating cell-mediated immune responses, i.e. , pathological inflammation, and apoptosis concurrently.
  • attenuating cell-mediated immune responses i.e. , pathological inflammation, and apoptosis concurrently.
  • CGRP specific for CGRP induction of apoptosis can provide a potent treatment for conditions characterized by cellular infiltrates, e.g. , during the cellular phase of experimental autoimmune encephalomyelitis (EAE), multiple sclerosis, or in brain cells within the hippocampus after head trauma or ischemia induced by stroke.
  • EAE experimental autoimmune encephalomyelitis
  • the CGRP can function to attenuate the cell-mediated immune response, as described above, and the specific type 2 receptor antagonist can prevent CGRP induced apoptosis.
  • the affected tissue is protected from the effects of CGRP and from inflammation.
  • administering In addition to treatment of trauma and stroke, administration of an inhibiter of CGRP-mediated apoptosis is desired to protect tissues and organs from ischemic and reperfusion injury. Frequently, although ischemia results in some tissue damage, activation of immune response mechanisms and onset of excessive opposing regulatory mechanisms (e.g. , production of profuse amounts of CGRP) during reperfusion of ischemic tissues damages the tissues to a greater extent than the ischemia. Thus, administration of an antagonist of CGRP-mediated apoptosis is desired in the treatment of myocardial tissue after a myocardial infarct, the lung after a pulmonary embolism, the brain after a stroke, and other conditions where the blood supply to an affected organ is limited or temporarily cut off.
  • an antagonist of CGRP-mediated apoptosis is desired in the treatment of myocardial tissue after a myocardial infarct, the lung after a pulmonary embolism, the brain after a stroke, and other conditions where the blood
  • CGRP-mediated apoptosis it may be desirable to bathe organs or severed limbs that are to be transplanted or reattached with fluid containing an antagonist of CGRP-mediated apoptosis. More preferably, for treatment of ischemia and maintenance of organs or tissues to be transplanted or reattached, a CGRP type 1 receptor agonist, or alternatively both an antagonist of CGRP-mediated apoptosis (type 2 receptor antagonist) and CGRP are administered. Administration of CGRP will inhibit the immune cell activation that can accompany reperfusion. Moreover, CGRP, as a vasodilator, may facilitate re-opening of the blocked artery causing an ischemic event.
  • CGRP is administered to inhibit cellular immune responses
  • the invention also contemplates administration of DHEA in place of or concurrently with CGRP.
  • administration of DHEA may be preferred, since this neurosteroid does not appear to induce apoptosis, and thus may obviate or reduce the need for an antagonist of CGRP-induced apoptosis. 6/03993
  • the present invention provides an assay for identifying an antagonist of CGRP- mediated apoptosis.
  • an antagonist can be identified by culturing test thymocytes with a concentration of about 10 ⁇ 12 to about 10 "6 M CGRP and an agent to be assayed for the ability to inhibit CGRP-mediated apoptosis of thymocytes, which agent is present at physiological concentrations, e.g. , about 10 "5 M or less, preferably less than or equal to about 10 "6 M, and more preferably about 10 "7 M.
  • Control thymocytes are cultured with an equal concentration or concentrations of CGRP, without the agent being assayed.
  • the extent of apoptosis of the test and control cultures is determined, e.g. , using a cell viability assay such as trypan blue exclusion or immunocytofluorography.
  • a decrease in the extend of apoptosis in the test culture indicates that the agent antagonizes CGRP-mediated apoptosis.
  • Such an agent can be further tested to determine whether it antagonizes CGRP type I receptor activity, i.e. , inhibits CGRP attenuation of helper T cell activation.
  • the agent will inhibit or antagonize CGRP-induced apoptosis, but not CGRP-mediated attenuation of T cell activation.
  • CGRP has been identified by immunocytochemistry in cell bodies and nerve fibers of the murine thymus. Receptors for CGRP have been characterized within the gland, and their activation by nanomolar levels of CGRP was found to suppress Con A stimulated proliferation of mature virgin CD4 thymocytes. This suppression is blocked by an antagonist for CGRP (CGRP 8 . 37 ). CGRP also inhibits the proliferation of Con A and antigen stimulated splenic T cells but to a lesser degree than that observed in the thymus.
  • the present Example was designed in part to determine if some or all of the CGRP induced suppression in the thymus was due to apoptosis.
  • Thymocytes were plated out with the appropriate reagent and evaluated for apoptosis using FACS analysis and propidium iodide to distinguish apoptotic cells.
  • Con A did not significantly induce apoptosis in thymocytes whereas CGRP alone (10 12 to 10 "7 M) p ⁇ 0.04 and in the presence of Con A induced a greater than 2 fold increase in apoptotic cells p ⁇ 0.008.
  • the antagonist, CGRP 8 . 37 did not cause apoptosis alone or in the presence of Con A, nor did it block programmed cell death caused by Con A or CGRP.
  • CGRP CGRP receptor not sensitive to the antagonist. Since CGRP-mediated inhibition of Con A induced proliferation of thymocytes and splenocytes is blocked by the antagonist, CGRP appears to mediate at least two separate functions on thymocytes via two different CGRP receptors.
  • DHEA and CGRP are naturally occurring substances that are reported to have both opposing and complementary effects on immune functions.
  • Example is directed to determining how they might work together to influence the mitogen-stimulated proliferation of thymocytes.
  • Con A-induced thymocyte proliferation assays CGRP and DHEA each inhibited proliferation.
  • CGRP 8 37 CGRP amino acids 8-37
  • the proliferative response was significantly greater than the Con A response alone, across a range of Con A doses.
  • CGRP g 37 blocked the inhibitory effect of DHEA.
  • CGRP 837 , CGRP, or DHEA taken alone or in combination, stimulated thymocyte proliferation in the absence of Con A.
  • CGRP affects the proliferation of CD4 + T cells, and thus may be a regional endogenous inhibitor of the proliferation of virgin mature T cells while they remain in the thymus. DHEA appears to act via endogenous CGRP on the thymus CD4 + T cell population.
  • mice Materials and Methods Animals. BALB/c ity-R male and female mice, 4-8 weeks old, were used in these experiments. The animals were bred and raised pathogen-free in the animal facilities at the VA Medical Center, La Jolla. They were fed commercial food and water ad libitum. Room lighting was controlled in a 12-h cycle. Male mice were used in the experiments except where designated. The mice were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NIH, 1985).
  • CGRP and CGRP 8 . 37 were obtained from Peninsula Laboratories, Belmont, California. Con A and DHEA were obtained from Sigma, St. Louis, Missouri. Rabbit anti-CGRP was obtained from Cambridge Research Biochemicals, Wilmington Delaware. Antibody specificity was determined by absorption of the activity with 1 x 10 "5 M CGRP. ABC rabbit staining kits were obtained from Vector Laboratories, Burlingame, California.
  • the cells were washed in supplemented RPMI (5 % FCS, 0.03 mg/ml gentamicin, 1 % nonessential amino acids, 1 % sodium pyruvate, 2 mM glutamine and 50 ⁇ M 2-mercaptoethanol).
  • RPMI RPMI
  • % FCS 0.03 mg/ml gentamicin, 1 % nonessential amino acids, 1 % sodium pyruvate, 2 mM glutamine and 50 ⁇ M 2-mercaptoethanol.
  • Proliferation Assay Cells were plated in Falcon 96 well flat bottom tissue culture plates at 3xl0 6 cells per well. The plates were pre-soaked in PBS for an hour to eliminate any plate toxicity. Dose response curves were generated for all reagents to determine optimal doses. Con A was added at concentrations of 2, 4, and 8 ⁇ g/ml; CGRP, CGRP 8 . 37 , and DHEA were used at 1 x 10 6 M to 1 x 10 13 M.
  • the final total volume per well was 0.25 ml.
  • the plates were incubated for 72 hours at 37°C in a humidified incubator containing 5 % C0 2 .
  • Quadruplicate wells were then pulsed with 1 ⁇ Ci per well of 3 H-thymidine (Amersham Corp. , specific activity 6.7 Ci/mmol) and incubated for an additional eighteen hours.
  • Cultures were harvested onto glass fiber filter paper, placed in vials with scintillation cocktail, and counted on a scintillation counter. The results were expressed as mean CPM of 3 H-thymidine incorporation ⁇ standard error of the means (SEMs).
  • the assays were repeated a minimum of three times. The significance of the results was determined by using a student T test or a one way analysis of the variance (ANOVA) followed by Tukey's post hoc test where indicated.
  • ANOVA one way analysis of the variance
  • DHEA has been reported to stimulated IL-2 production (Daynes et al. , 1990, Eur. J. Immunol. 20:793-802), it was tested in thymocyte proliferation assays, anticipating stimulation. However, the results showed that the addition of DHEA at 10 "6 M to 10 "13 M produced a shallow inhibition of Con A-induced thymocyte proliferation ( Figure 3). The addition of CGRP 8 . 37 to DHEA + Con A -treated cultures restored the response to the level of Con A alone, while DHEA had no effect on thymocyte proliferation in the absence of Con A ( Figure 4).
  • CGRP secreting cells have not yet been identified in the thymus, it is clear that the CGRP is not derived from the serum in the medium since the level of CGRP in fetal calf serum is below the femtomolar range. It is possible that CD4 + Th-1 cells produce CGRP and release it as their own autocrine feed-back mechanism to shut down IL-2 synthesis. Conceivably, DHEA could also induce CGRP secretion in Th-1 cells in conjunction with its ability to activate IL-2 synthesis as part of a feedback loop within the thymus (Daynes et al. , 1990, supra).
  • CGRP 8 . 37 elevated proliferation in 8 out of 8 independent experiments; 4) when CGRP and DHEA were added together to inhibit proliferation, as shown in Figure 5, CGRP g 37 restored proliferation to baseline in 1 experiment and elevated it above baseline in 3 experiments. Given the fact that these assays were conducted over a 4 day interval, there was no control over the amount of endogenous CGRP that may have been present and released.
  • DHEA is primarily synthesized by the testes in mice
  • an assay to determine if there was a difference between the proliferation of male and female thymocytes stimulated by Con A in the presence of DHEA was performed.
  • the results show a quantitative but not qualitative difference.
  • the quantitative differences between the male and female responses are not surprising due to the well-characterized differences between the immune responses of males and females (Ahmed et al. , 1985, J. Immunol. 134:204-209).
  • IL-2 driven proliferation of T cells requires prolactin, and CGRP is known to inhibit prolactin release (Elie et al., 1990, Neuropeptides 16: 109-113, Fahim et al., 1990, Neuroendocrinol. 51:688-693; Netti et al. , 1989, Neuroendocrinol. 49:242-247).
  • Physiological significance for thymus function An in vivo role for these in vitro observations seems quite plausible given the function of the thymus and the distribution of CGRP nerves and cells.
  • the thymus is a primary lymphoid organ.
  • T cells that can recognize foreign antigens and protect the body from infection both through cellular and humoral immunity.
  • T cells are necessary for the activation of both classes of the immune response in the periphery.
  • immunocompetent cells that leave the thymus, only a fraction of those meet with a specific antigen, clonally expand and are capable of carrying out their effector function.
  • apoptosis programmed cell death
  • the thymus which is not designed as an effector organ, cannot have mature T cells activated within its boundaries, particularly cytotoxic T cells.
  • Wang et al. (1992, supra) have presented data that indicates that CGRP affects CD4 + Th-1 cells.
  • the activated peripheral CD4 + Th-1 cell helps in the generation of antigen specific CD8 + cells through its production of IL-2. However, this normally takes place in secondary lymphoid tissue.
  • CGRP-immunoreactive nerves and cells in areas within the thymus that are rich in mature T cells offers such a mechanism.
  • CGRP containing cells within the medulla and the cortico-medullary boundary may suppress such accidental activation and proliferation of functional, educated T cells.
  • CGRP is one of the most potent vasodilator known (Brain et al., 1984, Nature 313:54-55). Its presence in nerves may slow cells in the perivascular space from premature activation while simultaneously permitting them to leave the thymus.
  • the in vitro suppression of T cell proliferation by DHEA through what appears to be a CGRP-related mechanism, may represent an in vivo neurosteroid/hormonal regulation within the thymus of inappropriate activation of mature T cells.
  • the neuroendocrine role of CGRP within the thymus is examined to determine if its attenuation of thymocyte proliferation is simply due to a quiescence of the CD4 population and/or a reduction in the available pool of cells via apoptosis that were destined to be CD4 antigen sensitive cells.
  • CGRP Chemicals. CGRP, CGRP 8 . 37 , and Con A were obtained as described in Example 2. Corticosterone was obtained from Steraloids, Wilton, NH.
  • Thymus and spleen cells were obtained as described in Example 2. The trabeculae and non-suspended clumps were allowed to sediment out.
  • Suspended cells were removed, centrifuged, resuspended and counted for viability using trypan blue exclusion.
  • results were expressed as mean CPM of 3 H-Thymidine incorporation +/- standard error of the means (SEMs). The significance of the results was determined by using a student T test or a one way analysis of the variance (ANOVA) followed by Tukey's post hoc test where applicable.
  • Apoptosis Cells were prepared as described above and harvested at 8 and 24 hours. Cultures treated with corticosterone and CGRP were subjected to dose response evaluations to determine the optimal dose for inducing apoptosis within the physiological range (10 "8 M for CGRP, and 10 "7 M for corticosterone) (data not shown). At the end of the incubation period, the cells were washed twice and resuspended in PBS. The cells were then fixed in 50% EtOH/PBS.
  • Thymocytes were prepared as described above and incubated for 8 and 24, hours. After the incubation period, cells were harvested, washed twice and resuspended in PBS, and counted using trypan blue exclusion to test for viability. Cells (10 6 ) were then labelled with antibodies to CD3, CD4, or CD8, using a 1/100 dilution of antibody. FITC labelled hamster anti-mouse CD3, rat anti-mouse CD4 and CD8, and PE labelled rat anti-mouse CD8 were used for double staining with the FITC labelled CD4 (Pharmingen San Diego, CA). The cells were incubated on ice for 45 minutes, washed, and resuspended in 0.5 ml 2% formaldehyde in PBS.
  • thymocytes Single and double stained thymocytes were analyzed on a Becton Dickinson FACStar PLUS flow cytometer (Becton Dickinson, Mt. View, CA). Ten million cells were analyzed per sample. Viable cells and non viable cells, as determined by their forward versus 90 degree scatter patterns, were examined separately for surface antigens. Data were evaluated using Multiplus Software from Phoenix Flow Systems.
  • the percent of CD-labelled cells was adjusted to reflect the percent of apoptotic cells per group. For example, if a culture condition had 10% total apoptotic cells, of which 50% were double positive, then the adjusted percentage of 5 % would reflect the actual number of apoptotic CD double positive cells.
  • Example 2 also shows that the ability of mature, virgin thymocytes to respond to the mitogen Con A is inhibited by CGRP and that this inhibition is mediated by a type 1 CGRP receptor that is sensitive to the antagonist CGRP 8 37 (Bulloch et al., 1995, supra). Furthermore, there is an increase in proliferation in cultures treated with the antagonist alone, which indicates that endogenous CGRP plays a role in suppressing activation and proliferation of the mature thymocytes within the gland.
  • CGRP CGRP induced apoptosis
  • CD4/CD8 double positive cells are approximately equal in number to the CD3 positive cells it is plausible, given that most double positive thymocytes are also positive for CD3, that the apototic CD4 positive cells in these cultures are the CD3 negative/CD4 positive immature thymocytes that occupy the outer cortical epithelial region of the gland ( Figure 11).
  • CGRP has another unique immune function in that it is capable of inducing apoptosis in a manner as potent as the endogenous glucocorticoid, corticosterone.
  • CGRP+ nerves and cells in areas within the thymus that are relatively rich in mature T cells suggests the endogenous role of this peptide within the thymic parenchyma is to contain accidental proliferation and activation of T cell responses that would be deleterious to the survival and function of the gland.
  • CGRP is a potent vasodilator (Brain et al., 1984, Nature 313:54-55), and its presence in nerves may protect the perivascular space from premature activation of immunocytes while simultaneously permitting the cells to exit the thymus.
  • CGRP can be viewed as an anti-autoimmune agent for the thymus.
  • CGRP may serve to regulate the type of immune response by restraining the T-cell cytotoxic response as well as by inducing apoptosis in other immune cells in different stages of their activation within environments crucial to the survival of the species.
  • the type of CGRP receptor involved in apoptosis still needs to be identified.
  • Biol Chem 267:21052-57 showed that CGRP inhibits 11-2 production in a cloned TH-1 cell, it is possible that clearance of cytotoxic T cells, after pathogens have been eliminated, may be linked in the later phases of this response to down regulation of IL-2 by CGRP in CGRP enriched regions and/or to the expression of the apoptotic CGRP receptor on these cells.
  • ADX results in cell death in granule cells of the dentate gyrus (DG).
  • DG dentate gyrus
  • HF hippocampal formulation
  • CGRP is increased in the HF after several types of insults, including ischemia and colchicine injection, in addition to ADX.
  • CGRP also suppresses cellular immune responses in specific organs such as the thymus, by inducing apoptosis in thymocytes, suppresses cell-mediated immunity and blocks macrophages from presenting antigen.
  • CGRP-ir CGRP-immunoreactivity
  • Electron microscopic analysis of the DG shows that CGRP-ir is primarily found in large, dense core vesicles (100-120 nm) contained in axons and in small axon terminals which form asymmetrical synapses with dendritic spines in the ITML and a few cells within the granule cell layer.
  • Fornix lesions did not reduce CGRP-ir in the HF of ADX rats, indicating that the CGRP is mostly derived from intrinsic neurons rather than the subcortical pathway.

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Abstract

La présente invention concerne la modulation des réponses immunitaires de cellules et la modulation ou l'inhibition de cancers des thymocytes ou thymomes. Selon un premier aspect, le présente invention concerne un procédé pour supprimer une réponse immune en administrant de la déshydroépiandrostérone (DHEA). Selon un autre aspect, l'invention concerne un procédé pour augmenter une réponse immune, consistant à administrer une certaine quantité d'un inhibiteur du peptide apparenté au gène de la calcitonine (CGRP) comme, par exemple l'antagoniste du CGRP qu'est CGRP8-37 ou une certaine quantité d'un inhibiteur de la DHEA. L'invention concerne également un procédé pour traiter un cancer du thymus ou thymone consistant à administrer une quantité de CGRP suffisante pour induire une apoptose des cellules du cancer du thymus ou thymome. L'invention concerne en outre un procédé pour empêcher des atteintes aux tissus, consistant à administrer une quantité d'inhibiteur du CGRP suffisante pour inhiber l'apoptose de cellules dans la région de l'atteinte aux tissus. L'invention concerne encore un procédé pour identifier un agent capable d'inhiber l'apoptose due au CGRP.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0845269A3 (fr) * 1996-11-29 1999-04-07 Weihan Wo Composition lyposomale contenant le peptide apparenté au gène de calcitonine et sa préparation
EP1033989A4 (fr) * 1997-11-07 2007-05-02 Procede pour attenuer des reactions allergiques induites par des mastocytes
WO2010075238A1 (fr) 2008-12-23 2010-07-01 Amgen Inc. Protéines de liaison au récepteur cgrp humain
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates
WO2019087161A1 (fr) * 2017-11-06 2019-05-09 Auckland Uniservices Limited Conjugués peptidiques à titre d'antagonistes du récepteur cgrp, leurs procédés de préparation et leurs utilisations

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US4518595A (en) * 1983-07-19 1985-05-21 The Jackson Laboratory Method for treating diabetes using DHEA compounds
US4628052A (en) * 1985-05-28 1986-12-09 Peat Raymond F Pharmaceutical compositions containing dehydroepiandrosterone and other anesthetic steroids in the treatment of arthritis and other joint disabilities
WO1991000737A1 (fr) * 1989-07-10 1991-01-24 Amylin Corporation Traitement de l'obesite et de l'hypertension essentielle ainsi que des troubles associes
AU675222B2 (en) * 1992-05-01 1997-01-30 University Of Utah, The Compositions and methods for regulating IL-6 production in vivo
SE9201425D0 (sv) * 1992-05-06 1992-05-06 Kabi Pharmacia Ab Method and means for preventing constriction of the pupil in the eye
WO1994008589A1 (fr) * 1992-10-09 1994-04-28 The Board Of Trustees Of The Leland Stanford Junior University Traitement du lupus erythemateux systemique a la deshydroepiandrosterone

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0845269A3 (fr) * 1996-11-29 1999-04-07 Weihan Wo Composition lyposomale contenant le peptide apparenté au gène de calcitonine et sa préparation
EP1033989A4 (fr) * 1997-11-07 2007-05-02 Procede pour attenuer des reactions allergiques induites par des mastocytes
US8168592B2 (en) 2005-10-21 2012-05-01 Amgen Inc. CGRP peptide antagonists and conjugates
WO2010075238A1 (fr) 2008-12-23 2010-07-01 Amgen Inc. Protéines de liaison au récepteur cgrp humain
EP3184546A1 (fr) 2008-12-23 2017-06-28 Amgen, Inc Protéines de liaison au récepteur cgrp humain
EP3569620A1 (fr) 2008-12-23 2019-11-20 Amgen Inc. Anticorps se liant au récepteur cgrp humain
EP4585264A2 (fr) 2008-12-23 2025-07-16 Amgen Inc. Proteines humaines de liaison au recepteur cgrp
WO2019087161A1 (fr) * 2017-11-06 2019-05-09 Auckland Uniservices Limited Conjugués peptidiques à titre d'antagonistes du récepteur cgrp, leurs procédés de préparation et leurs utilisations

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