WO2003086323A2

WO2003086323A2 - Compositions and methods for immunomodulation

Info

Publication number: WO2003086323A2
Application number: PCT/US2003/009714
Authority: WO
Inventors: Peter D. Gluckman
Original assignee: Neuronz Limited; Neuronz Biosciences, Inc.
Priority date: 2002-04-05
Filing date: 2003-03-28
Publication date: 2003-10-23
Also published as: WO2003086323A3; AU2003218460A8; AU2003218460A1

Abstract

This invention provides methods and kits for treating a mammal, especially a human, and more especially a preterm infant, having an immune system disorder, by increasing the effective concentration of a GPE-related compound in the central nervous system of the mammal. This increase may be achieved by administration to the mammal of an effective amount of a GPErelated compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug. The use of a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug in the manufacture of a medicament for treating an immune system disorder in a mammal, especially a human; and compositions containing a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug for treating an immune system disorder in a mammal, especially a human.

Description

COMPOSITIONS AND METHODS FOR MMUNOMODULATION

Related Application

This application claims priority to United States Provisional Patent Application Serial No: 60/370,788, filed April 5, 2002, incorporated herein fully by reference.

Field of the Invention

[0001] invention relates to immunomodulation for the treatment of immune system disorders. Specifically, this invention relates to the use of Gly-Pro-Glu (GPE) and related peptides to treat immune system disorders.

BACKGROUND TO THE INVENTION

The Immune System

[0002] The immune system is one of the primary defenses against disease bearing microbes and other foreign antigens in higher animals. An immune response is mediated by the action of specific immune cells which react to specific antigens. Potential antigens can be a variety of substances, often proteins, which are foreign to an individual's body. They are most frequently located on the outer surfaces of cells. Potential antigens can be found on pollen grains, tissue grafts, animal parasites, viruses and bacteria.

[0003] In humans, many potential antigens never pass the body's first line of defense and therefore never trigger the immune system. This line of defense consists of the skin, mucous membranes, tears and stomach acid. When pathogens or other foreign substances pass this line of defense the immune response is activated. [0004] The immune response is made up of a complex sequence of events.

It primarily depends on three major cell types - macrophages, thymus-derived lymphocytes (T-lymphocytes) and bone marrow-derived lymphocytes (B- lymphocytes). These interact with one another, either directly or via interleukins. In addition, the immune system is integrally connected with the complement, kinin, clotting and fibrinolytic systems, all of which are involved in inflammation.

[0005] All the cells of the immune system, tissue cells and white blood cells (or leukocytes) develop from pluripotent stem cells in the bone marrow. The production of white blood cells is through two main pathways of differentiation. The lymphoid lineage produces T-lymphocytes and B-lymphocytes while the myeloid pathway gives rise to monocytes and granulocytes. Monocytes circulate in the blood and migrate into organs and tissues to become macrophages. Monocytes, macrophages and granulocytes destroy pathogens and other potential antigens by phagocytosis, that is by engulfing and destroying the foreign material. In addition to this role, monocytes and macrophages are an important link to the acquired immune mechanism since they can process and present antigens as well as producing molecules that stimulate lymphocyte differentiation to eliminate the antigen.

[0006] There are two principal responses of the acquired immune mechanism, hmnoral and cellular, both of which react to antigens. Specialized white blood cells called lymphocytes are responsible for both humoral and cellular immunity. Lymphocyte precursor cells are made in the bone marrow of adult humans followed by migration to various organs or in the yolk sac of a developing fetus followed by migration into the fetus and then to various organs. In humans some of these precursor cells migrate to the thymus, which is a two-lobed, glandular appearing structure located in the upper chest just behind the sternum, where they are transformed into T-lymphocytes, which are involved in cellular immunity. In humans, the rest of the precursor cells migrate to the spleen where they are transformed into B-lymphocytes, which are involved in humoral immunity. The T- and B-lymphocytes are structurally indistinguishable although they function differently and can be distinguished through various chemical means. The mature lymphocytes circulate in the blood and can also be found in the lymph nodes as well as the spleen and thymus.

[0007] Humoral immunity forms a major defense against bacterial infections and is mediated by the B-lymphocytes that have receptors for particular antigens on their cell surfaces. When bacteria or viruses, for example, invade an organism, B-lymphocytes react to and combine with the antigens on the bacterial or viral surface and the lymphocyte is stimulated to divide. Its daughter cells are transformed into specialized cells called plasma cells. These cells produce and then secrete large quantities of antibodies into the general circulation. The antibodies are specific for the antigens which stimulated their production and react only with those antigens. Antibodies known as agglutinins cause several antigen-containing substances to agglutinate or clump together. This keeps the antigen containing substance from spreading to the tissues and allows the phagocytes to capture or the lymph nodes to filter the invading material. Other antibodies act by opening holes in bacterial cell walls, thereby killing the bacteria. These are known as lysins. Antibodies called antitoxins combine with toxins produced by bacteria and thereby neutralize them.

[0008] Once a pathogen invades the body and the immune response begins antibodies can be made in several hours. This initial reaction is called the primary response or primary immunization. However, during that time the pathogens have also been dividing and sometimes producing toxin, either of which results in various disease symptoms. It may take days or weeks before enough antibodies are made to eliminate all the pathogens but once they disappear the disease symptoms disappear as well. The lymphocytes, plasma cells, and antibodies remain and circulate in the blood so that if the same pathogens enter the body a second time the lymphocytes react immediately and start antibody production. The response of the sensitized lymphocytes is called the secondary response. The secondary response results in the production of higher levels of antibody than were produced during the primary response. So many antibodies are produced so rapidly that the microbes are unable to divide and cause disease. This type of humoral immunity is known as immediate hypersensitivity due to the fact that a previously exposed organism can respond within minutes to an antigen, as in the case of hay fever. Another example of immediate hypersensitivity would be anaphylactic shock, an extreme allergic reaction that sometimes occurs when an individual is exposed to an antigen to which he has been sensitized. At times, this humoral response to the antigen can result in death.

[0009] In cellular immunity, T-lymphocytes are activated when they encounter antigens on cells from another individual, as in the case of transplants, tumors or viruses. Like B-lymphocytes, T-lymphocytes are specific and each type reacts with only one antigen. The lymphocytes enlarge, divide, and produce lymphokines that participate in the attack on the foreign antigen. They also stimulate the phagocytic activity of macrophages. Although immunological memory exists as with humoral immunity the response is much slower. It may take as long as ten or twelve hours to develop a response in a previously sensitized individual and this type of cellular immunity is therefore known as delayed hypersensitivity. The allergic reaction to poison ivy, oak, and sumac and the red splotch seen in a positive tuberculin skin test are examples of cellular immune responses.

[0010] Circulating antibodies and cellular immune responses also play a role in the rejection of transplanted tissues and organs. Unless the donor is the identical twin of the recipient or is the individual himself, the recipient's lymphocytes recognize the transplant as "non-self and immediately respond by attacking it (transplant rejection). The exceptions to this situation are transplants to non-vascularized areas (privileged sites) such as the cornea of the eye, where lymphocytes do not circulate and therefore are not sensitized and do not prompt an immune response. It is currently difficult to suppress the immune reaction to prevent rejection of the transplant without severely damaging the patient in other ways. The patient must also be given massive doses of antibiotics because his own defenses against infection have been suppressed. Suppression of the immune system would thus be useful in preventing transplant rej ection.

[0011] Immunoproliferative disorders are characterized by the production of abnormally increased numbers of antibody-producing cells. Suppression of the immune system would be useful in minimizing or eliminating the effects of these disorders.

[0012] A normal functioning immune system distinguishes between the antigens of foreign invading organisms (non-self) and tissues native to its own body (self) so as to provide a defense against foreign organisms. Hence, central to the proper functioning of the immune system is the ability of the system to discriminate between self and non-self. When a patient's immune system fails to discriminate between self and non-self and starts to react against self antigens then an autoimmune disorder arises.

[0013] Autoimmune diseases may be classified as organ-specific or non- organ-specific depending on whether the response is primarily against either antigens localized to particular organs or widespread antigens. In organ-specific diseases, for example Hashimoto's thyroiditis, lesions are restricted because the antigen in the organ acts as a target for immunological attack. In non-organ- specific disease, for example systemic lupus erythematosus (SLE), complexes formed with the antigens involved are deposited systemically, particularly in the kidney, joints, and skin, thus giving rise to the more disseminated features of the disease.

[0014] Some forms of autoimmunity come about as the result of trauma to an area usually not exposed to lymphocytes such as neural tissue or the lens of the eye. When the tissues in these areas become exposed to lymphocytes their surface proteins can act as antigens and trigger the production of .antibodies and cellular immune responses, which then begin to destroy those tissues. Other autoimmune diseases develop after exposure of the individual to antigens which are antigenically similar to, that is, cross-react with, the individual's own tissue. Rheumatic fever is an example of this type of disease in which the antigen of the streptococcal bacterium that causes rheumatic fever is cross-reactive with parts of the human heart. The antibodies cannot differentiate between the bacterial antigens and the heart muscle antigens and cells with either of those antigens can be destroyed. Suppression of the immune system in these autoimmune diseases would be useful in minimizing or eliminating the effects of the disease.

[0015] Inmunodeficiencies may result from many etiologies including but not limited to a primary hereditary genetic abnormality (for example DiGeorge syndrome), accidental exposure (for example to radiation, heavy metals, or insecticides), therapeutic intervention (for example chemotherapy with glucocorticoids or radiation therapy) or acquired as a result of infection (for example with human immunodeficiency virus (HIN), cytomegalovirus (CMN), mycobacteria, or a parasite). Patients with immunodeficiencies would benefit from methods of stimulating the immune system.

[0016] Various forms of inflammation are characterized by activation of macrophages. Macrophages are thought to induce and maintain inflammatory processes mainly by producing various products, which by acting on other cells bring about the deleterious consequences of inflammation.

[0017] hnmunomodulators are capable of stimulating or suppressing the immune system of an animal depending on the status of the patient. More specifically, immunomodulators stimulate the immune system of the animal when it is weakened and suppress the immune system when it is hyperactive. There is both enormous utility and strong demand for an immunomodulator more potent than conventional immunomodulators and without serious side effects.

[0018] A premature infant is a baby who has been born 3 weeks or more before the due date. The cause of premature labor, whether or not preceded by premature rupture of the membranes, is usually unknown. However, maternal histories commonly show low socioeconomic status, inadequate prenatal care, poor nutrition, poor education, unwed state, and intercurrent, untreated illness or infection. Other risk factors include untreated maternal bacterial vaginosis and previous preterm birth. The premature infant is small, usually weighing less than 2.5 Kg, and tends to have thin, shiny pink skin through which the underlying veins are easily seen.

[0019] Prematurity remains the main cause of mortality and permanent disability among infants. Between five and ten percent of all infants are born prematurely, requiring physiologic, nutritional, endocrinal and immunological adaptation that can be detrimental to long-term development and growth. Additionally, organ injury often takes place during the neonatal period.

[0020] The immune response and premature infancy has recently become an important area of research related to a broad range of diseases. For example, infections such as sepsis or meningitis are about four times more likely to occur in premature infants than in full-terms. In these cases extremely premature babies usually need to be given antibiotics for long periods of time, often causing long-term problems. An immature immune system may also contribute to insults of the central nervous system (CNS), for example white matter neural damage, cerebral infarction or asphyxia. SUMMARY OF THE INVENTION

[0021] In its first aspect, this invention is a method for treating a mammal, especially a human, more especially a preterm infant, having an immune system disorder, comprising increasing the effective concentration of a GPE-related compound in the central nervous system of the mammal. This increase may be achieved by administration to the mammal of an effective amount of a GPE- related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug.

[0022] In another aspect, this invention is the use of a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug in the manufacture of a medicament for treating an immune system disorder in a mammal, especially a human, and more especially a preterm infant; and compositions containing a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug for treating an immune system disorder in a mammal, especially a human.

[0023] The present invention also aims to treat or prevent injury to premature infants through immunomodulation, and includes compositions and methods of immunomodulation for preterm (premature) infants.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows the effects of GPE on the proliferation of sensitized and naϊve lymph node (LN) cells stimulated with concanavalin A in vitro.

[0025] FIG. 2 shows the stimulation index (SI) for GPE on sensitized and naϊve LN cells stimulated with concanavalin A in vitro and incubated with different concentrations of GPE. [0026] FIG. 3 shows the effects of MBP on the proliferation of sensitized and naϊve LN cells.

DESCRIPTION OF THE INVENTION

Definitions

[0027] A "GPE-related compound" is GPE or a GPE analog.

[0028] A "GPE analog" is a small peptide (not more than 5 amino acids) or peptidomimetic (a compound where one or more of the amide bonds of such a peptide is replaced by a non-amide bond) that is capable of effective binding to mammalian central nervous system GPE receptors. Preferred GPE analogs are those capable of effectively producing an immunomodulatory effect substantially equivalent to that produced by GPE itself. GPE analogs include the dipeptides gly-pro (GP), pro-glu (PE), and cyc/ø-pro-gly (cPG), and the tripeptides and derivatives GPE amide, GPE stearate, gly-pro-D-glutamate (GP-D-E), gly-pro-thr (GPT), gly-glu-pro (GEP), glu-gly-pro (EGP), glu-pro-gly (EPG), pro-gly-glu (PGE), and pro-glu-gly (PEG).

[0029] A "prodrug" of a GPE-related compound is a compound comprising the GPE-related compound and a carrier linked to the GPE-related compound by chemical bond(s) that are cleaved by biological processes within a mammal when the prodrug is administered to the mammal, such as by the action of enzyme(s) present within the mammal. Prodrags include, for example, esters of the GPE-related compound, such as the l-[(ethoxycarbonyl)oxy]ethyl ester, and polypeptides that, when cleaved by a mammalian enzyme, yield the GPE- related compound. Suitable enzymes include an acid protease that generates des-(l-3) IGF-1 and GPE from IGF-1 (Yamamoto et al. (1994), Generation of des(l-3) insulin-like growth factor-I in serum by an acid protease, Endocrinology, 135(6): 2432-2439), proprotein and prohormone convertases (Seidah et al. (1999) Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides, Brain Research 848: 45-

62), serum proteases, trypsin (in a calcium magnesium-free solution), cathepsin-

D, and pepstatin-A.

[0030] "Treating a mammal having an immune system disorder" by increasing the effective concentration of a GPE-related compound in the central nervous system of the mammal includes immunomodulation, both the stimulation of the immune system of a mammal having a weakened immune system and the suppression of the immune system of a mammal having a hyperactive immune system.

[0031] An "effective amount" of a GPE-related compound, prodrug, or implant is that amount of such compound, prodrug, or implant that, when administered to a mammal having an immune system disorder, produces an increase in effective concentration of a GPE-related compound in the central nervous system of the mammal sufficient to promote treatment or immunomodulation in that mammal.

Description and preferred embodiments

[0032] In its first aspect, this invention is a method for treating a mammal, especially a human, having an immune system disorder, comprising increasing the effective concentration of a GPE-related compound in the central nervous system of the mammal. This increase may be achieved by administration to the mammal of an effective amount of a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug.

[0033] In another aspect, this invention is the use of a GPE-related compound, a prodrug thereof, or an implant containing cells that express the

GPE-related compound or prodrug in the manufacture of a medicament for treating an immune system disorder in a mammal, especially a human; and compositions containing a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug for treating an immune system disorder in a mammal, especially a human.

[0034] Preferably the immune system disorder is selected from autoimmune disorders, immunoproliferative disorders, immunodeficiency disorders, inflammatory disorders, hypersensitivity disorders, disorders related to transplantation, and disorders related to transfusion. More preferably the immune system disorder involves hyperactivity, suppression or deficiency of the immune system. Most preferably the immune system disorder involves abnormal proliferation, subnormal proliferation, hyperactivity, deficiency, suppression or destruction of white blood cells. Preferably the white blood cells are one or more of T-lymphocytes, B-lymphocytes, monocytes, macrophages, and granulocytes.

[0035] Preferably stimulation of the immune system occurs by one or more of stimulating proliferation of white blood cells and inhibiting the death of white blood cells. Preferably the white blood cells are one or more of T- lymphocytes, B-lymphocytes, monocytes, macrophages, and granulocytes.

[0036] Preferably suppression or inhibition of the immune system occurs by suppressing or inhibiting proliferation of white blood cells. Preferably the white blood cells are one or more of T-lymphocytes, B-lymphocytes, monocytes, macrophages, and granulocytes.

[0037] The preferred GPE-related compound is GPE.

[0038] The increase in the effective concentration of a GPE-related compound in the central nervous system of the mammal may be achieved by administration to the mammal of an effective amount of the GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrug. The administration may be either prophylactic (after identification of a mammal with a predisposing or precipitating factor for immune system disorder, for example, exposure to certain chemicals, but prior to a diagnosis of an immune system disorder), therapeutic (after diagnosis of an immune system disorder has been made), or both. Additionally, administration of a GPE-related compound may be continued following suppression or stimulation of the immune system to prevent relapse in the previously affected area.

[0039] The GPE-related compound or prodrug can be administered alone, or as is preferred, as a part of a pharmaceutical composition or medicament. In general, GPE compounds will be administered as pharmaceutical compositions by one of the following routes: directly to the central nervous system, oral, topical, systemic (e.g. transdermal, intranasal, or by suppository), parenteral (e.g. intramuscular, subcutaneous, or intravenous injection), by implantation and by infusion through such devices as osmotic pumps, trmsdermal patches and the like. Compositions may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulation, solutions, suspensions, elixirs, aerosols or any other appropriate compositions; and comprise at least GPE-related compound or prodrug in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions, may be found in such standard references as Gennaro, ed. (2000), "Remington: The Science and Practice of Pharmacy", 20^th ed., Lippincott, Williams & Wilkins, Philadelphia PA. Suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, .and the like, with isotonic solutions being preferred for intravenous administration.

[0040] The GPE or other GPE-related compound, or prodrug can be administered directly to the central nervous system. This route of administration can involve, for example, lateral cerebro ventricular injection, focal injection, or a surgically inserted shunt into the lateral cerebral ventricle.

[0041] An advantage of GPE and other GPE-related compounds is that they can be administered peripherally and have both peripheral and central nervous system effects. Thus, GPE and other GPE-related compounds and prodrags need not be administered directly to the central nervous system in order to have effect in the central nervous system. Any peripheral route known in the art can be employed. Two particularly convenient peripheral administration routes are by subcutaneous injection (e.g. dissolved in 0.9% sodium chloride) and by oral administration (e.g., in a tablet or capsule).

[0042] GPE and other GPE-related compounds and prodrags can also be administered by a sustained-release system. Suitable examples of sustained- release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained-release matrices include polylactides (US 3773919; EP 58481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al. (1983), Biopolymers, 22: 547-556), poly(2- hydroxyethyhnethacrylate) (Langer et al. (1981), J Biomed. Mater. Res., 15: 267-277) ethylene vinyl acetate (Langer et al., supra), or poly-D-(-)-3-hydroxy- butyric acid (EP 133988). Sustained-release compositions also include liposomally entrapped compounds. Liposomes containing the compound are prepared by methods known per se: DE 3218121; Hwang et al. (1980), Proc. Nat 'I Acad. Sci. USA, 77: 4030-4034; EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; JP 83-118008; US 4485045, US 4544545; and EP 102324. Ordinarily, the liposomes are of the small (from about 20 to about 80 nm diameter) unilamellar type in which the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted for the most efficacious therapy. Other sustained-release systems include implantable osmotic systems of the type described in US 5980508. The GPE-related compounds and prodrags may also be PEGylated to increase their lifetime in vivo, based on, e.g. the conjugate technology described in WO 95/32003.

Mechanical devices providing sustained infusion, such as those commonly used for the delivery of insulin, may also be used.

[0043] The effective concentration of GPE or other GPE-related compounds can also be increased by the use of an implant which is or includes a stable expression cell line which is capable of expressing the GPE-related compound in an active form within the body, or more particularly the central nervous system of the patient (Martinez-Serrano et al. (1998), Proc. Nat'l Acad. Sci. USA, 95: 1858-1863; Chen et al. (1995), J Neurosci., 15(4): 2819-2825). Cells such as astrocytes (Yoshimoto et al. (1995), Brain Res., 691: 25-36), fibroblasts (Chen et al. (1995), J. Neurosci., 15(4): 2819-2825; Frim et al. (1994), Proc. Nat'l Acad. Sci. USA, 91: 5104-5108), HiB5 cells (Martinez-Serrano et al. (1998), Proc. Nat'l Acad. Sci. USA, 95: 1858-1863), and baby hamster kidney cells (Tseng et al., (1997), J. Neurosci., 17(1): 325-333), either primary cells or cell lines, immortalized or not, and engineered to express the GPE-related compound may be implanted into the brain or elsewhere in the body, or encapsulated in biocompatible polymers, fibers or other materials and the cell- containing capsules then implanted into the brain or elsewhere in the body. Cells may be cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum and 1% penicillin/streptomycin prior to encapsulation and/or implantation. Cells to be encapsulated may be suspended in a solution of 1:1 culture medium:3% collagen at a density of 500,000 cells/μL. This cell suspension can then be encapsulated in capsules such as poly(ether-sulfone)fibers from AKZO-Fiber Nobel AG, Wupperthal, Germany). Capsules are preferably cultured for 4 days before implantation.

[0044] Engineering cells to express a GPE-related compound in active form may be achieved through the use of an expression vector. For example, for GPE, multiple copies of any DNA sequence specific for the amino acids methionine/glycine/proline/glutamic acid and a stop codon are linked together, either with or without additional DNA sequences specific for a stop codon between each 4 amino acid sequence, to form a complete sequence of between

30-50 nucleotides. Note that the start codon will always be ATG, the codon for methionine, whereas the stop codon may be TAA, TAG or TGA. This complete sequence comprises the expression vector for GPE. The expression vector as a whole will generally also include a promoter for the cell to be implanted, and may include selection markers and other DNA sequences common in the biotechnology field. This vector is then integrated into the genome of the cells to be implanted.

[0045] The calculation of the effective amount of GPE-related compound or prodrug to be administered will be dependent upon the route of administration and upon the nature of the condition which is to be treated, and will be routine to a persons of ordinary skill in the art. For a human, where the dose is administered centrally, a suitable dose range for GPE is between about 0.1 μg and about 400 μg per Kg of body weight per day; a preferred dose range is between about .5 μg/Kg/day and about 50 μg/Kg/day, and a more preferred dose range is from about 1 μg/Kg/day to about 25 μg/Kg/day. For peripheral administration, the doses are about 10-fold to 1000-fold higher; and suitable dose ranges will be readily determinable by comparing the activities of peripherally administered GPE with the activity of centrally-administered GPE in a suitable model and scaling the central GPE dose range above accordingly. Suitable dose ranges for other GPE-related compounds will be readily determinable by comparing the activities of the compounds with the activity of GPE in a suitable model and scaling the GPE dose range above accordingly; and suitable dose ranges for prodrags and implants will be determinable in the same manner.

[0046] The GPE-related compound or prodrug can be obtained from a suitable commercial source. Alternatively, the GPE-related compound or prodrag can be directly synthesized by conventional methods, such as the stepwise solid phase synthesis method of Merrifield et al., (1963) J. Amer.

Chem. Soc, 85: 2149-2156, or other appropriate methods known to those of ordinary skill in chemical/biochemical synthesis. Synthesis can also involve the use of commercially available peptide synthesizers such as the Applied

Biosystems model 430A.

[0047] If desired, a combination of GPE-related compounds may be administered. In addition, one or more GPE-related compounds may be co- administered with:

• treatments for immune system disorders, such as azathioprine, cyclophosphamide, mercaptopurine, methotrexate, prednisone, methylprednisolone, succinylacetone, rapamycin, D-penicillamine, lobenzarit, and antibiotics;

• treatments for stimulation of the immune system (including but not limited to concanavalin A);

• treatments for suppression of the immune system; • treatments for cancer, severe burns, complications of sepsis or multiple trauma; and/or

• other agents or growth factors, for example, transforming growth factor beta (TGF-/3).

"Co-administered" or "co-administration" includes not only administration within the same pharmaceutical composition, or administration at the same time, but also administration as part of the same course of treatment for the disease or disorder being treated, generally an immune system disorder but also including conditions in which the immune system is compromised, such as in cancers, severe burns, complications of sepsis or multiple trauma, or the like. Thus, co-administration may, for example, include administration of the GPE- related compound continuously such as by means of an intracerebroventricular shunt, and administration of azathioprine tablets orally once daily. [0048] The invention will now be described in more detail with reference to the following non-limiting example.

EXAMPLE

[0049] This experiment was approved by the University of Auckland Animal Ethics Committee and all efforts were made to minimize the suffering incurred and the number of animals used.

[0050] Immunized mice were prepared as follows: SJL/J Mice (10.2 weeks old) were immunized with 150 μg myelin basic protein (MBP) (Sigma Lot # 40K7011, Sigma-Aldrich Corporation, St. Louis, MO, USA) and 500 μg of the H37Ra strain of M. tuberculosis contained in 100 μl_ complete Freund's adjuvant, (50 μ.L in each flank, simultaneously) at day 0, and killed at day 7.

[0051] Stimulator cells were prepared as follows. Two spleens were obtained from non-immunized syngeneic mice, disrupted and a single cell suspension made. This was transferred to a clean centrifuge tube and debris was removed by underlaying the suspension with fetal calf serum and allowing the suspension to settle for 5 minutes. The cell layer was then transferred to a clean tube, washed once at 1700 rpm for 7 minutes, and then resuspended in 5 mL medium. Red blood cells were removed by suspending the cells in 0.184 M NH₄C1, spimiing down the cells, and resuspending the pellet quickly in 0.184 M NH₄C1 using 3 mL per spleen. The cells were incubated on ice for 10 min, then underlayered with approximately 1.5 mL fetal calf serum and centrifuged in the cold for 7 min at 1000 rpm. The supernatant and fetal calf serum were then removed, and the cells resuspended in 3 mL of fresh medium. The spleen cells were then inactivated by treatment with mitomycin C. Mitomycin C (400 μg/mL, 0.15 mL) was mixed rapidly with 3 mL cell suspension (1:20) using a syringe and a 27 G needle (mitomycin C final concentration 20 μg/mL), and incubated at 37 °C under 5% CO₂ for 30 min, after which the volume was made up to approximately 9 mL with medium. Following this, the cells were spun down at 4 °C and 1000 rpm for 7 min and then resuspended in fresh medium. The cell suspension was washed twice more and the stimulator cells resuspended at 2 x 10⁵ cells/ml in RPMI growth medium.

[0052] Responder cells were prepared as follows: An immunized mouse was killed, and the inguinal and axillary lymph nodes (LN) extracted and placed in a Petri dish. A single cell suspension of LN cells was made, washed, and resuspended at 2 x 10⁶ cells/mL in complete RPMI 1640 medium.

[0053] A stimulator/responder cell mix was prepared as follows: the stimulator and responder cells were mixed 1:1 and the resulting suspension was used for setting up plates. The final concentration of the cell types in this suspension was: 10⁵ stimulator cells/mL, and 10⁶ responder cells/mL, giving a stimulator/responder cell ratio of 1:10. Fifty microliters of this suspension contained 5000 stimulator and 50,000 responder cells.

[0054] A lymph node/spleen cell mix was prepared as follows: an immunized mouse was killed, and the inguinal and axillary LNs extracted and placed in a Petri dish. A single cell suspension of LN cells was made, washed, and resuspended at 2 x 10⁶ cells/mL in complete RPMI 1640 medium. The spleen was also extracted and a single cell suspension made by following the steps described above for the preparation of stimulator cells. The LN and spleen cell suspensions were mixed 1:1. The final concentration of LN and spleen cells in the cell mix was 10⁶ cells/mL.

[0055] For a standard proliferation assay, five different concentrations of MBP were used: 100, 50, 25, 12.5 and 6.25 μg/mL. 4 replicates were made for every concentration. The MBP stock concentration was 50 mg/mL in water. A solution was prepared at twice the intended final concentration in the assay wells by diluting 4 μL of this stock in 996 μL of complete RPMI 1640 medium, giving a concentration of 200 μg/mL. A 2-fold serial dilution was made in RPMI growth medium. [0056] A solution of concanavalin A (Con A) was made at 10 μg/mL in complete RPMI 1640 medium. Fifty microliters of Con A was added to the wells, giving a final concentration of 500 ng/well.

[0057] For the study of the action of GPE on Con A stimulated cells, the following concentrations of GPE were used: 0.1 nM, 1 nM, 0.01 μM, 0.1 μM, 1 μM, 0.01 mM, 0.1 mM, and 1 mM. A suspension of stimulator/responder cells (or LN/spleen mix) was made containing 10 μg/mL of Con A in RPMI 1640 growth medium.

[0058] Ninety-six well microtiter plates were set up as follows:

1. Rows A to D were used for 4 replicates. 2. To column 1, 100 μL of RPMI 1640 medium was added (blank control).

3. To columns 2 and 3, 50 μL of stimulator/responder (or LN/spleen) cells were added.

4. To columns 4 to 10, 50 μL of cells diluted in Con A were added.

[0059] Addition of GPE and controls : 1. To column 2, 50 μL of RPMI 1640 media was added (negative control).

2. To column 3, 50 μL of a suspension of Con A at 10 μg/mL was added (Con A proliferation control).

3. To columns 4 to 10 (GPE test wells), 50 μL of diluted GPE was added starting from the lowest dilution (0.1 nM).

[0060] The plates, containing cells cultured in a 100 μL volume, were then incubated in a humidified incubator with a 5% CO₂ atmosphere at 37 °C.

[0061] The Promega CellTiter 96^® aqueous non-radioactive cell proliferation assay kit (Promega, Inc., Madison WI, USA) was used to determine the degree of proliferation of lymphoid cells in vitro upon incubation with different compounds. The assay is based on the conversion of (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) into a formazan product that can be quantified colorimetrically.

[0062] On day 3 of incubation of the cells, solutions of MTS and phenazine methosulfate (PMS) were thawed either at room temperature or in a water bath for 10 minutes. MTS solution (2 mL) was removed and transferred to a clean test tube. PMS solution (100 μL) was added to the MTS solution, and the tube gently swirled to ensure complete mixing of the reagents, immediately before addition to the culture plate containing the cells.

[0063] The plates were then taken from the incubator and transferred to a cabinet II sterile hood. Twenty microliters of the combined MTS ΪMS solution were pipetted into each well. A multichannel pipettor was used to make sure that the generation of the formazan started at the same time in all wells. Following the addition, the plates were incubated for a further 4 hours to allow for color development, and read at 490 nm using a 96 well plate ELISA reader. A slight amount of spontaneous 490 nm absorbance occurs in culture medium incubated with combined MTS/PMS solution, typically 0.1 - 0.2 absorbance units after 4 hours of incubation. This degree of background absorbance was corrected for in the assays by the blank and negative control wells.

[0064] As seen in FIGS. 1 and 2, GPE and Con A demonstrated a synergistic effect on the proliferation of MBP-sensitized cells, reaching stimulation indexes (Sis) of 3.3 to 3.8 times that of the negative control, while Con A alone gave a SI of 2.2 [The stimulation index is the ratio of the absorbance of a "test" data point and the "negative" or background control within the same experiment. Following determination of mean absorbance values from quadruplicates, the SI is calculated for each data point by dividing the test absorbance by the background (or negative control) absorbance]. There was no significant dose response to different concentrations of GPE. GPE and Con A also demonstrated a synergistic effect on the proliferation of naϊve cells, with Sis of 2.5 to 3. As seen in FIG. 3, the proliferation of MBP-sensitized stimulator/responder cells showed a non-significant increase over naϊve cells.

[0065] Although this invention has been described with reference to particular embodiments, those persons skilled in the art will appreciate that variations and modifications may be made without departing from the scope of the invention.

INDUSTRIAL APPLICABILITY

Methods of this invention can be used in the pharmaceutical and medical industries to treat patients with immune system disorders. Kits of this invention can be used in the phannaceutical and medical industries for treating patients suffering from immune system disorders.

Claims

I CLAIM:

1. A method for treating a mammal having an immune system disorder, comprising increasing the effective concentration of a GPE-related compound in the central nervous system of the mammal.

2. The method as claimed in claim 1 wherein the mammal is human.

3. The method of claim 2 where the human is a preterm infant.

4. The method of claim 1 where the GPE-related compound is GPE.

5. The method of claim 1 where the GPE-related compound is a GPE analog.

6. The method of claim 5 where the GPE analog is selected from the group consisting of GP, PE, cPG, GP (E-amide), GPE stearate, GP(D-E), GPT, GEP,

EGP, EPG, PGE, and PEG.

7. The method of claim 1 where the immune system disorder is selected from the group consisting of autoimmune disorders, immunoproliferative disorders, immunodeficiency disorders, inflammatory disorders, hypersensitivity disorders, disorders related to transplantation, and disorders related to blood transfusion.

8. The method of claim 7 where the immune system disorder is an autoimmune disorder.

9. The method of claim 7 where the immune system disorder is an immunoproliferative disorder.

10. The method of claim 7 where the immune system disorder is an immunodeficiency disorder.

11. The method of claim 7 where the immune system disorder is an inflammatory disorder.

12. The method of claim 7 where the immune system disorder is a hypersensitivity disorder.

13. The method of claim 7 where the immune system disorder is a disorder related to transplantation.

14. The method of claim 7 where the immune system disorder is a disorder related to blood transfusion.

15. The method of claim 1 where the immune system disorder involves hyperactivity, suppression or deficiency of said immune system.

16. The method of claim 1 where the immune system disorder involves abnormal proliferation, subnormal proliferation, hyperactivity, deficiency, suppression or destruction of white blood cells.

17. The method of claim 16 where the white blood cells are T-lymphocytes.

18. The method of claim 16 where the white blood cells are B-lymphocytes.

19. The method of claim 16 where the white blood cells are monocytes.

20. The method of claim 16 where the white blood cells are macrophages.

21. The method of claim 16 where the white blood cells are granulocytes.

22. The method of claim 1 where the GPE-related compound treats the immune system disorder by stimulating the immune system of the mammal.

23. The method of claim 22 where the stimulation of the immune system comprises stimulating proliferation of white blood cells.

24. The method of claim 22 where the stimulation of the immune system comprises inhibiting the death of white blood cells.

25. The method of claims 23 or 24 where the white blood cells are T- lymphocytes.

26. The method of claims 23 or 24 where the white blood cells are B- lymphocytes.

27. The method of claims 22 or 23 where the white blood cells are monocytes.

28. The method of claims 23 or 24 where the white blood cells are macrophages.

29. The method of claims 23 or 24 where the white blood cells are granulocytes.

30. The method of claim 22 for the treatment of immunodeficiency disorders.

31. The method of claim 1 where the GPE-related compound treats the immune system disorder by suppressing or inhibiting the immune system of the mammal.

32. The method of claim 31 where the suppression or inhibition of the immune system comprises suppressing or inhibiting proliferation of white blood cells.

33. The method of claim 32 where the white blood cells are T-lymphocytes.

34. The method of claim 32 where the white blood cells are B-lymphocytes.

35. The method of claim 32 where the white blood cells are monocytes.

36. The method of claim 32 where the white blood cells are macrophages.

37. The method of claim 32 where the white blood cells are granulocytes.

38. The method of claim 31 for the treatment of autoimmune disorders, immunoproliferative disorders, inflammatory disorders, hypersensitivity disorders, disorders related to transplantation, disorders related to transfusion.

39. The method of claim 1 where the treatment is therapeutic.

40. The method of claim 1 where the treatment is prophylactic.

41. The method of claim 1 where the treatment is continued following suppression or stimulation of the immune system to prevent relapse in a previously affected area.

42. The method of claim 1 comprising administration to the mammal of an effective amount of a GPE-related compound, a prodrug thereof, or an implant containing cells that express the GPE-related compound or prodrag.

43. The method of claim 42 where the GPE-related compound, prodrug, or implant is administered directly to the cerebral ventricle of the mammal.

44. The method of claim 42 where the GPE-related compound, prodrug, or implant is administered peripherally to the mammal.

45. The method of claim 1 where the GPE-related compound or prodrag is administered in a pharmaceutical composition.

46. The method of claim 1 where the treatment is administered directly to the part(s) of the body where the immune system disorder is located.

47. The method of claim 1 where the immune system disorder is a disorder of white blood cells and where the treatment is administered directly to where the affected white blood cells are located.

48. The method of claim 47 where the white blood cells are T-lymphocytes.

49. The method of claim 47 where the white blood cells are B-lymphocytes.

50. The method of claim 47 where the white blood cells are monocytes.

51. The method of claim 47 where the white blood cells are macrophages.

52. The method of claim 47 where the white blood cells are granulocytes.

53. The method of claim 42 where the GPE-related compound is administered centrally in an amount from about 0.1 μg/Kg/day to about 400 μg/Kg/day.

54. The method of claim 53 where the GPE-related compound is administered centrally in an amount from about 0.5 μg/Kg/day to about 100 μg/Kg/day.

55. The method of claim 54 where the GPE-related compound is administered centrally in an amount from about 1 μg/Kg/day to about 25 μg/Kg/day.

56. The method of claim 42 where the GPE-related compound is co- administered with another treatment for the immune system disorder.

57. The method of claim 56 where the other treatment comprises administration of a compound selected from the group consisting of azathioprine, cyclophosphamide, mercaptopurine, methotrexate, prednisone, methylprednisilone, succinylacetone, rapamycin, D-penicillamine, lobenzarit, and antibiotics.

58. The method of claim 42 where the GPE-related compound is co- administered with an immunostimulant.

59. The method of claim 58 where the immunostimulant is concanavalin A.

60. The method of claim 42 where the GPE-related compound is co- administered with an immunosuppressant.

61. The method of claim 42 where the GPE-related compound is co- administered with a treatment for cancer, severe burns, complications of sepsis or multiple trauma.

62. The method of claim 42 where the GPE-related compound is co- administered with an anti-α-4 integrin antibody.

63. The method of claim 62 where the anti-o-4 integrin antibody is anti-MAdCAM-1.

64. A kit comprising a GPE-related compound formulated in a pharmaceutically acceptable buffer, a container for holding said GPE-related compoxmd formulated in a pharmaceutically acceptable buffer, and instructions for use of the kit in the treatment of an immune system disorder in a mammal.