Abstract:
This invention relates to the use of naturally occurring mammalian intermediary metabolites, Tcell ligands or Tcell receptor ligands, preferably glycosylceramides, for the treatment or prevention of immune mediated or immune related diseases or disorders. Specifically, the present invention provides compositions and methods for the treatment or prevention of pulmonary, respiratory or airway diseases or disorders such as asthma.

Description:
REFERENCE TO RELATED PATENT APPLICATIONS  
       [0001]     This application is a continuation-in-part of U.S. patent application Ser. No. 11/378,941, filed on Mar. 17, 2006, entitled “Glucocerebroside Treatment of Liver Disorders,” which is a continuation-in-part of U.S. patent application Ser. No. 10/675,980, filed on Sep. 30, 2003, entitled “Glucocerebroside Treatment of Disease,” which is a continuation-in-part of U.S. patent application Ser. No. 10/375,906, filed on Feb. 27, 2003, entitled “Regulation of Immune. Responses by Manipulation of Intermediary Metabolite Levels.” The contents of the aforementioned patent applications are hereby incorporated by reference, in their entireties. 
     
    
     FIELD OF THE INVENTION  
       [0002]     This invention relates to the use of a naturally occurring, mammalian intermediary metabolites or T cell receptor ligands, preferably a glycosylceramide, for the treatment or prevention of immune mediated or immune related diseases or disorders. Specifically, the present invention provides compositions and methods for the treatment or prevention of pulmonary, respiratory or airway diseases or disorders such as asthma.  
         [0003]     All patents, patent applications, patent publications, scientific articles, and the like, are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.  
       BACKGROUND OF THE INVENTION  
       [0004]     Various methods have been described for the treatment of immune related or immune mediated disorders or diseases, infectious diseases, metabolic disorders and different types of cancer in mammalian subjects. One of these methods involves the modulation of immune responses in a subject. This includes the down regulation of the immune response system using procedures or combinations of procedures for producing and applying a new and unexpected immune modulation termed selective immune down regulation (SIDR). Immunological modulation is an artificially induced variation in a subject&#39;s immune system in response to the introduction of reagents, procedures and processes. These procedures have been described in detail in U.S. patent application Ser. No. 08/808,629, filed on Feb. 28, 1997, U.S. patent application Ser. No. 10/377,628, filed on Mar. 4, 2003, U.S. application Ser. No. 10/377,603, filed on Mar. 4, 2003, U.S. patent application Ser. No. 09/447,704, filed on Feb. 28, 1997, U.S. application Ser. No. 10/385,440, filed on May 9, 2001, and U.S. application Ser. No. 09/356,294, filed on Jul. 16, 1999. Each of the foregoing patents is incorporated by reference in its entirety in the present application and may further be used in conjunction with the present invention.  
         [0005]     Other methods describe the use of educated or treated cells in the treatment of a variety of diseases. Specifically, the methods are directed to the manipulation of the NKT cell population in a subject that results in the modulation of the Th1/Th2 balance toward anti-inflammatory or pro-inflammatory cytokine producing cells. A detailed description of these inventions have been disclosed in U.S. patent application Ser. No. 10/451,811, entitled “Educated NKT Cells and Their Uses in the Treatment of Immune-Related Disorders” by Yaron Ilan et al., filed on Jun. 25, 2003, PCT Application No. IL01/01197, filed on Dec. 24, 2001, and U.S. application Ser. No. 10/375,906, filed on Feb. 27, 2003. Each of the foregoing patents is incorporated by reference in its entirety in the present application and may further be used in conjunction with the present invention.  
         [0006]     The present invention provides a new method for the treatment or prevention of pulmonary, respiratory and airway diseases or disorders such as asthma, in mammalian subjects, and preferably, in human subjects. This method involves the administration of a mammalian intermediary metabolite to a subject. In a preferred embodiment the mammalian intermediary metabolite is a T cell ligand or a T cell receptor ligand.  
         [0007]     The mammalian intermediary metabolite, the T cell ligand or the T cell receptor ligand may comprise a lipid, a polar lipid, or a conjugated biomolecule. The conjugated biomolecule may in turn comprise a glycolipid, a sulfated glycolipid, a lipoprotein, an apolipoprotein, a glycoprotein other than an antibody, a cytokine, or a hormone. A glycolipid may comprise a monosaccharide ceramide, a disaccharide ceramide or a polysaccharide ceramide, with a β-linkage between the saccharide and ceramide portions. Examples of β-linked monosaccharide ceramides can include but not be limited to a glucosylceramide, or a galactosylceramide and an example of a disaccharide ceramide can include but not be limited to lactosylceramide. It is also understood that any derivatives of the foregoing as well as any analogs other than α-linked analogs may also find use.  
         [0008]     Glucosylceramide is a naturally occurring glycolipid consisting of ceramide, to which glucose is attached. A ceramide, which is a sphingosine and a fatty acid, is the structural unit common to all sphingolipids. Sphingolipids have a variety of cellular functions. These include membrane structural roles and cell signaling participation. (Sullard et al., 2000 Journal of Mass Spectrometry 35: 347-353.) Glucosylceramide is made by the enzyme glucosylceramide synthase which attaches the two molecules together. (see FIG. 1 and FIG. 2). An example of a glucosylceramide includes glucocerebroside, or a glucocerebroside analog or derivative.  
         [0009]     An example of a pulmonary, respiratory or airway disease or disorder is asthma. Asthma is a respiratory disease caused by an inappropriate response to usually innocuous environmental stimuli. Exposure to the stimulant can lead to a series of adverse reactions such as inflammation of the bronchial passages, increased levels of mucus secretions and difficulty in breathing. Cellular markers for asthma include increased levels of eosinophils, CD+T4 lymphocytes and IgE producing B cells. Cytokine markers can include increased levels of IL4, IL5 and IL13. Although the exact pathway that leads to asthma remains unknown, induction of the allergenic response in asthma seems to involve a participatory role of NKT cells, since mutants lacking this class of T-cell do not develop airway hyper-reactivity in model systems (Lisbonne et al., 2003 J. Immunol. 171; 1637-1641, Akbari et al., 2003 Nat. Med. 9; 582-588).  
         [0010]     Knowledge of a role of NKT cells in this system has led to the investigation of potential effects by α-galactosylceramide, since it is well known that this compound can induce dramatic effects upon NKT cells. Treatment of animals with α-galactosylceramides was first thought to produce a killing effect upon NKT cells since shortly after its administration, they seemed to completely disappear from the circulatory system (Matsuda et al., 2000 J. Exp Med, 192; 741-754 and Fujii et al., 2002 Nat Immunol 3; 867-874). It was later discovered that this effect was not a selective lethality effect upon NKT cells but rather that there was a loss of the marker used to identify these cells. When a different marker was used, the NKT cells were seen to still be present and furthermore, in a reversal of the earlier conclusions, there was stimulation and expansion of the NKT population after exposure to α-galactosylceramide (Crowe et al., 2003 J Immunol, 171; 4020-4027 and Wilson et al., 2003 Proc Nat Acad Sci USA 100; 10,913-10,918).  
         [0011]     Effects of α-galactosylceramide on the treatment of disease have been described in a number of different systems. For example, α-galactosylceramide has been used for enhancing immune responses as a treatment for anti-tumor and anti-pathogen activities and it has also been used to quell deleterious immune responses that are observed in diabetes and experimental autoimmune encephalomyelitis (Furlan et al., 2003 Eur J Immunol 33; 1830-1838 and Singh et al., 2001 J Exp Med 194; 1801-1811).  
         [0012]     Since NKT cells had previously been implicated in the development of asthma and α-galactosylceramide has been shown to be an effective immune modulator of NKT activity, α-galactosylceramide was investigated as a candidate for the treatment of asthma in a number of different laboratories. The results of these studies demonstrated strong effects upon animal models of asthma but a layer of complexity was revealed where it was found that there could be profoundly different effects, i.e. administration of α-galactosylceramide gave relief in some situations and exacerbated deleterious effects in other circumstances. Some understanding of these conflicting results may be achieved by dividing the series of experiments into two categories:  
         [0000]     1) test animals that were never exposed to an allergen prior to administration of α-galactosylceramide; and  
         [0000]     2) test animals that were sensitized to a particular allergen and given α-galactosylceramide afterwards.  
         [0013]     In a series of experiments where animals were sensitized to Ovalbumen (OVA), a surrogate for an asthma allergen, and then later challenged with nasal administration of OVA, the administration of α-galactosylcerebroside to the test animals either 2 hours prior to or during the course of the sensitization procedure (the first group format) led to exacerbation of the markers, signs and symptoms for asthma when the animals were later presented with the OVA challenge (Bilenki et al., 2004 Eur J Immunol. 34; 345-354, Kim et al., 2004 J. Allerg Clin Immunol 114; 1332-1338, Morishima et al., 2005 Eur J Immunol 35; 2803-2814). In fact, in one of these references (Kim et al., 2004) there was no reactivity to the challenge unless α-galactosylcerebroside accompanied the OVA during the sensitization step. Indeed, it was found that the intranasal (but not intravenous) administration of the α-galactocylcerebroside itself to otherwise naïve animals could lead to asthma like symptoms (Meyer et al., 2006 Proc Nat Acad Sci USA 103; 2782-2787). Under these circumstances it appears that α-galactocylcerebroside acted as an adjuvant in enhancing an immune response to an allergen. This has been previously seen in other systems where α-galactosylcerebroside has been used as an adjuvant in immunization procedures for malaria vaccines (Gonzales-Aseguinolaza et al., 2002 J Exp. Med. 195; 617-624) as well as immunization against influenza and adenovirus (Ko et al., 2005 J. Immunol 175; 3309-3317).  
         [0014]     In a series of experiments from the second group format, where sensitization of animals with OVA was carried out first and at a later time α-galactosylcerebroside was administered at the time of the challenge with OVA, the results were very different form the first group. Under these circumstances, α-galactosylcerebroside acted more like a toleragen, reducing the severity of the response to the allergen (Morishima, 2005 op. cit., Hachem et al., 2005 Eur J Immunol 35; 2793-2802 and Matsuda et al., 2005 Am J. Respir. Cell Mol. Biol. 31; 22-31). It would seem that the alteration of the directionality of the α-galactosylcerebroside effect from the first group may be due to an inflammatory response already being established in the test animal at the time of the α-galactosylcerebroside administration. This viewpoint is also supported by the results with administration of α-galactosylcerebroside alone. As described above, administration of this compound led to induction of asthma like symptoms, but when this experiment was continued and a second dose of was administered, the induction of asthma-like symptoms was strongly reduced (Meyers, 2005 op. cit.). This is potentially a demonstration of the dual effects of α-galactosylcerebroside acting as an immunogen in the first administration and then acting as a toleragen in the second administration when an immune response had already been established.  
         [0015]     On the other hand, this particular compound has been shown to have deleterious side effects that may counterbalance its potential application to therapeutic treatment of asthma. For instance, α-galactosylcerebroside has been shown to induce hepatic damage (Osman et al., 2000 Eur J Immunol 30; 1919-1928, Nakagawa et al., 2001 J. Immunol 166; 6578-6584).  
       SUMMARY OF THE INVENTION  
       [0016]     This invention relates to the use of a naturally occurring mammalian intermediary metabolite, for the treatment or prevention of pulmonary, respiratory or airway diseases or disorders in mammalian subjects. In a preferred embodiment, the disease being treated or prevented is asthma and the mammalian intermediary metabolite is a T cell ligand or T cell receptor ligand.  
         [0017]     This invention further provides a process for treating or preventing pulmonary, respiratory or airway diseases or disorders in a mammalian subject comprising administering to said subject an effective amount of a mammalian intermediary metabolite, wherein said metabolite is a T cell ligand, a T cell receptor ligand, a lipid, a polar lipid, a conjugated biomolecule, a glycolipid, a sulfated glycolipid, a lipoprotein, an apolipoprotein, a glycoprotein other than an antibody, a cytokine, a hormone, a monosaccharide ceramide, a disaccharide ceramide, a polysaccharide ceramide, a non-α-linked glycosylceramide, a β-glycolipid, a β-glycolipid derivative, an analog of a β-linked glycolipid other than an α-linked glycolipid, β-glycosylceramide, a β-glycosylceramide derivative or an analog of a β-linked glycosylceramide other than an α-linked glycosylceramide.  
         [0018]     Another aspect of the present invention provides for the treatment or prevention of a disease or disorder in a mammalian subject comprising the ex vivo treating or educating of cells obtained from the mammalian subject. The cells are treated or educated with an effective amount of an intermediary metabolite, wherein said metabolite is a T cell ligand, a T cell receptor ligand, a lipid, a polar lipid, a conjugated biomolecule, a glycolipid, a sulfated glycolipid, a lipoprotein, an apolipoprotein, a glycoprotein other than an antibody, a cytokine, a hormone, a monosaccharide ceramide, a disaccharide ceramide, a polysaccharide ceramide, an α-glycosylceramide, a non-α-linked glycosylcerebroside, a β-glycolipid, a β-glycolipid derivative, an analog of a β-linked glycolipid other than an α-linked glycolipid, β-glycosylceramide, β-glycosylceramide derivative or an analog of a β-linked glycosylceramide other than an α-linked glycosylceramide. The treated or educated cells are then re-administered to the subject.  
         [0019]     The present invention also relates to the treatment or prevention of a disease in a mammalian subject comprising the re-administration of treated or educated cells to the subject, and the direct administration to said subject of an effective amount of an intermediary metabolite, wherein said metabolite is a T cell ligand, a T cell receptor ligand, a lipid, a polar lipid, a conjugated biomolecule, a glycolipid, a sulfated glycolipid, a lipoprotein, an apolipoprotein, a glycoprotein other than antibodies an antibody, a cytokine, a hormone, a monosaccharide ceramide, a disaccharide ceramide, a polysaccharide ceramide, an α-glycosylcerebroside, a non-α-linked glycosylcerebroside, a β-glycolipid, a β-glycolipid derivative, an analog of a β-linked glycolipid, β-glycosylceramide, β-glycosylceramide derivative or an analog of a β-linked glycosylceramide.  
         [0020]     Numerous other aspects and embodiments of the present invention are described in further detail below. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0021]     The present invention provides methods for the treatment or prevention of a pulmonary, respiratory or airway disease or disorder in a mammalian subject by the administration of an effective amount of a mammalian intermediary metabolite to a subject. The mammalian intermediary metabolite includes, but is not limited to a T cell ligand, a T cell receptor ligand, a lipid, a polar lipid, a conjugated biomolecule, a glycolipid, a sulfated glycolipid, a lipoprotein, an apolipoprotein, a glycoprotein other than an antibody, a cytokine, a hormone, a monosaccharide ceramide, a disaccharide ceramide, a polysaccharide ceramide, a glucosylceramide, a galactosylceramide, a glycosylceramide, a glycosylceramide derivative, a glycosylceramide analog other than an α-linked glycosylceramide, a sphingosine, a sphingolipid, a ceramide, a β-glucosylceramide, a β-glucosylceramide derivative, a β-glucosylceramide analog other than an α-linked glucosylceramide a β-galactosylceramide, a β-galactosylceramide derivative, a β-galactosylceramide analog other than an α-linked galactosylceramide, a β-lactosylceramide, a β-lactosylceramide derivative, or a β-lactosylceramide analog other than an α-linked lactosylceramide. In a preferred embodiment of the invention, the mammalian subject is a human being.  
         [0022]     In a preferred embodiment of the invention, the mammalian subject is a human being, the pulmonary and/or respiratory disease is asthma, and the mammalian intermediary metabolite is a β-glycosylceramide,  
         [0023]     The β-glycosylceramide and its analogs or derivatives may be administered by oral, intravenous, intraperitoneal, intramuscular, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, by inhalation or any combination thereof. It has already been disclosed in related applications that this class of mammalian intermediary metabolites has dual properties. The administration of β-glucosylceramide can enhance immune surveillance as shown in studies with dietetic augmentation where its inclusion in food resulted in suppression of tumor growth with chemically treated (Schmelz et al., 1999) and genetically tumor-prone mice (Symolon et al., 2003). On the other hand, β-glycosylceramides can have immune suppressive abilities in situations where immune responses are actually disadvantageous to the test animal. An example of this beneficial effect has been seen with treatment of conA-induced hepatitis (Margalit et al., 2005 Am J Physiol Gastroint Liver Physiology 289; G917-G925).  
         [0024]     This duality of potentiation has previously been seen with α-galactosylceramide and we have previously discussed this in the context of treating animal models for asthma where one format led to exacerbation of a reaction and a second format led to alleviation of symptoms. In terms of translating the results of animal model laboratory experiments with α-galactosylceramide into therapies for asthma patients, the second group format is more reflective of potential protocols for amelioration of asthma and its symptoms. In common with the second group of animal models, sensitization to some environmental antigens has already taken place in these individuals and it is because an immune response has already been induced that places them in need of a therapeutic intervention to alleviate symptoms. Thus, it is apparent that the hypersensitivity to these antigens that is already induced could allow α-galactosylceramide to provide beneficial results when administered to asthma patients.  
         [0025]     However, this substance is foreign to mammalian cells and as such, its interactions with mammalian components are of an artificial nature. Furthermore, as described previously, it can induce asthma on its own. Additionally, this compound is known to induce hepatic cell damage. Thus some embodiments of the present invention avoid the use of α-galactosylceramide and use β-glycosylceramides, thereby permitting appropriate immunomodulatory effects on asthma while avoiding consequences of a foreign appearing compound.  
         [0026]     The sugar group of the glycosylceramides can be a monosaccharide such as glucose or lactose (glycosylceramides and lactosylceramides) or a disaccharide such as galactose (galactosylceramides). Intermediary metabolites such as glycosylceramides can be purified and isolated from natural sources or they can be synthesized artificially.  
         [0027]     On the one hand, purification from a natural source allows an inexpensive source of the reagent to be used for the present invention. Also, it should be pointed out that numerous biological molecules that are mammalian intermediary metabolites are synthesized in other biological systems besides mammalian cells. Thus, the same identical molecule may be found in mammalian cells, non-mammalian eukaryotic cells and even in prokaryotes such as bacteria or yeast. In principle, these non-mammalian sources may also be used to provide desirable intermediary metabolites. As such, in the present invention, an intermediary metabolite is defined as a mammalian intermediary metabolite strictly in terms of whether it is a molecule that is naturally present in a mammalian cell and not the particular source from which it is isolated in order to be used in a therapeutic procedure. However, one drawback of the use of a biological systems approach is that these natural sources of glycosylceramides frequently consist of a large family of similar species that vary in the length of their carbon chains and placements of double bonds. Thus isolation of a single species may be problematic with some sources. On the other hand, some sources such as soy beans display only a single species, thereby allowing almost a pre-purified supply.  
         [0028]     On the other hand, directed synthesis of a particular glycosylceramide offers the advantage that no mammalian or other cells are needed and a series of synthetic steps should culminate in a single specific species of β-glycosylceramide. Side products of the various reactions in these steps should usually be chemically differentiated enough that the desired products will be readily separated, leading to a final product with a selected length for each carbon chain as well as the presence of a double bond at any desired site in the chains. The use of synthetic routes will also allow the use of glycosylceramide analogs in the present invention, where substitutions may be used for various components. An example of this approach would be synthesis of an analog where the oxygen joining the sugar to the ceramide portion is replaced by a carbon or sulfur atom.  
         [0029]     A third route is also possible that combines synthetic and natural sources, where a particular desired intermediary metabolite is present in a mammalian cells but there are no convenient alternative non-mammalian sources. In this case one or more of the genes in the pathway for synthesis of the desired intermediary metabolite can be cloned and inserted into a bacterial or yeast expression vector. As long as there is sufficient amount of an appropriate precursor in the host cells, the vector can allow the production of the desired intermediary mammalian metabolite in a non-mammalian host.  
         [0030]     The present invention describes a method for treating a disease where regulatory, immune-regulatory or NKT cells are obtained from the subject to be treated, or from another subject, and are educated or treated ex vivo. The cells are treated or educated by the presence of intermediary metabolite, antigens or epitopes, and antigen presenting cells, or any combination thereof. The treated or educated cells are then re-administered to the subject. The cells may be administered to the subject by adoptive transfer.  
         [0031]     In addition to the method described above involving the ex vivo treatment or education of cells, the present invention also provides for a method where the ex vivo treatment or education is accompanied by the method of directly administering to the subject to be treated, by a variety of ways, an effective amount of the intermediary metabolite, antigen presenting cells, and antigens or epitopes, or any combination of the above. The disease may also be treated by only the direct administration of an effective amount of the intermediary metabolite, antigen presenting cells, and antigens or epitopes, or any combination of the above.  
         [0032]     A therapeutic composition for the use in the treatment of the disease may comprise an effective amount of the intermediary metabolite, antigen presenting cells, and antigens or epitopes, or any combination of the above.  
         [0033]     The treatment of a disease in any of the described methods results in a change in the number or function of regulatory, immune-regulatory or NKT cells. This change encompasses a reduction, inhibition, or decrease in the number or function of the cells. This inhibition may be caused by the competitive displacement of activating elements from the CD1d molecule. A change may also include a stimulation or increase in the number or function of the cells. This stimulation may be caused by increased binding of the activating elements from the CD1d molecule.  
         [0034]     The treatment of a disease may also result in a change in the cytokine responses. Any cytokine in the immune system may be involved in these responses. The change could result in a pro-inflammatory or an anti-inflammatory response. There may also be a pro-inflammatory, and an anti-inflammatory response since certain cytokines may increase and others may decrease, simultaneously.  
         [0035]     Another result of the treatment of a disease is an alteration of the regulatory, immune-regulatory or NKT cell distribution in the subject. This change may also be accompanied by a change in the peripheral/intrahepatic T cell ratio. A further result may also include a change in intrahepatic CD8+ T cell trapping. There may be an increase or a decrease in the intrahepatic trapping. The result may also include a change in intrasplenic T cell trapping, where said change could be an increase or decrease.  
         [0036]     Also provided in the present invention are two in vitro screening assays for an analog or derivative of an intermediary metabolite which is administered to the subject to treat a disease. The first assay involves providing regulatory, immune-regulatory or NKT cells from the subject being treated or another subject, antigen presenting cells, and an analog or derivative of the intermediary metabolite in vitro. If a decrease in the regulatory, immune-regulatory or NKT cell proliferation is identified, then that specific analog or derivative is a treatment for disease.  
         [0037]     The second assay involves providing in a first test tube, regulatory, immune-regulatory or NKT cells and BSA; in a second test tube, regulatory, immune-regulatory or NKT cells and the analog or derivative of an intermediary metabolite; in a third test tube, regulatory, immune-regulatory or NKT cells, antigen presenting cells and BSA; and in a fourth test tube, regulatory, immune-regulatory or NKT cells, antigen presenting cells and the analog or derivative of the intermediary metabolite. If the least amount of regulatory, immune-regulatory or NKT cell proliferation is found in the fourth test tube, then that specific analog or derivative is a treatment for the disease.  
         [0038]     In a preferred embodiment of the present invention, when administration takes place by oral means there is minimal interference with digestion and absorption of a mammalian intermediary metabolite, or an analog or derivative thereof, including but not limited to mammalian intermediary metabolites such as a lipid, a conjugated biomolecule, a polar lipid, a glycolipid, a glycosylceramide, lipoprotein, apolipoprotein, cytokine, or hormones, a monosaccharide ceramide, a glucosylceramide, a galactosylceramide, a disaccharide ceramide, a lactosylceramide, a sphingosine, a sphingolipid, a ceramide, a lipoprotein, an apolipoprotein, a cytokine, a hormone, a T cell ligand, a T cell receptor ligand, or a glycoprotein other than an antibody, in the mammalian subject. Specifically, the mammalian subject has been without food and/or water for a certain amount of hours prior to the administration of the aforesaid molecules, treatment of the mammalian subject or the manipulation of cells in the mammalian subject. When carrying out oral administration, the intermediary metabolite may be prepared synthetically or it may have been derived from a natural source; in the latter case the intermediary metabolite has undergone one or more purification steps to separate the intermediary metabolite from other substances that may have been present.  
         [0039]     When treating cells ex vivo and readministering them to a patient, it is it is a subject of the present invention that non-mammalian intermediary metabolites may also find use in appropriate dosages. In a similar fashion, the screening method that has been described may also be used with non-mammalian intermediary metabolites. It is also a subject of the present invention that although a single intermediary metabolite may be used for treatment, there may also be benefits achieved by the use of a mixture that contains more than one intermediary metabolite.