WO2007142754A2 - Procédés et compositions destinés à l'altération cognitive - Google Patents

Procédés et compositions destinés à l'altération cognitive Download PDF

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WO2007142754A2
WO2007142754A2 PCT/US2007/009838 US2007009838W WO2007142754A2 WO 2007142754 A2 WO2007142754 A2 WO 2007142754A2 US 2007009838 W US2007009838 W US 2007009838W WO 2007142754 A2 WO2007142754 A2 WO 2007142754A2
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tlr agonist
group
tlr
formulation
subject
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PCT/US2007/009838
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WO2007142754A3 (fr
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Dennis A. Carson
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The Regents Of The University Of California
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • A61K38/23—Calcitonins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to the use of agonists of a Toll-like receptor (TLR), particularly of TLR7, TLR8, or TLR9, to alter or modulate a cognitive function of a subject desirous or in need of such altered or modulated cognitive function, as well as to compositions and methods for administering such compounds to subjects.
  • TLR Toll-like receptor
  • Such alterations include reducing appetite (which can reduce food intake), enhancing alertness, and enhancing memory, among others.
  • innate immunity is heritable and involves the recognition of antigens by a small number of weakly specific "pattern-recognition" receptors (PRRs) expressed in macrophages, dendritic cells, and B lymphocytes.
  • PRRs weakly specific "pattern-recognition” receptors
  • the antigens recognized by PRRs are typically highly conserved, and include such molecules as lipopolysaccharides, peptidoglycans, lipoteichoic acids, and microbial nucleic acids.
  • PRRs include molecules that are secreted, such as lectins and various components of the complement system, as well as those that are expressed on or within cells to induce endocytosis or signaling.
  • the recognition of antigens by components of the innate immune system induces an immediate inflammatory response and triggers adaptive immunity, which is often not rapid enough to eradicate microorganisms as it involves cell proliferation, gene activation, and protein synthesis.
  • Dendritic cells are found in mammalian immune system, and they are vital in the defense against pathogens, as they initiate T cell responses and produce cytokines and other molecules that can regulate adaptive immunity. Dendritic cells normally occur at a low frequency in tissues that are in contact with the environment, for example, in the skin (i.e., Langerhans cells) and the lining of nose, lungs, stomach, and intestines. Especially when immature, they can also be found in blood. Once activated, they migrate to the lymphoid tissues where they interact with T cells and B cells to initiate and shape the particular immune response.
  • MDCs myeloid dendritic cells
  • PDCs plasmacytoid dendritic cells
  • DCs express pathogen recognition receptors such as Toll-like receptors (TLRs) and C-type lectins that recognize different fragments of pathogens, and subsequently activate or present pathogen fragments to T cells.
  • TLRs Toll-like receptors
  • C-type lectins that recognize different fragments of pathogens, and subsequently activate or present pathogen fragments to T cells.
  • TLRs Toll-like receptors
  • C-type lectins that recognize different fragments of pathogens
  • effector cells can, for example, prime T cells.
  • Recognition of pathogens by DCs is largely dependent on TLRs.
  • dendritic cells function as links between the innate and adaptive immune systems, and an effective defense against pathogens requires the host to discriminate between different pathogens to induce an appropriate response.
  • Different DC types express different patterns of recognition molecules.
  • Pathogen exposure causes various DC changes, including activating DC maturation, up-regulation of the expression of co- stimulatory molecules in DCs, and the production of various cytokine patterns characteristic of different T cell responses. Differential signaling can be mediated via different TLRs, which leads to activation of conserved host signaling pathways that control expression of various immune response genes.
  • Toll-like receptors are a class of type I transmembrane protein PRRs that recognize conserved structures in microbial pathogens and certain host molecules.
  • TLRs are expressed on macrophages and dendritic cells, and they recognize a specific pattern of pathogen components, including endotoxins (lipopolysaccharide).
  • Pathogen recognition by TLRs activates the innate immune system through signaling pathways and rapidly provokes inflammatory responses, such as inducing the production of cytokines.
  • PAMPs pathogen-associated molecular patterns
  • TLRs The structures recognized by TLRs (i.e., TLR ligands) tend to be somewhat invariant, at least in select regions. As a result, a relatively small number of receptors can recognize a large number of different microbes. As a result of the limited recognition capacity of TLRs, these receptors contribute little to a host's ability to distinguish between commensal and pathogenic microbes, as such organisms generally exhibit far more structural similarities than differences. For dedicated phagocytic cells (e.g., macrophages), this limited ability to distinguish commensal from pathogenic organisms is not particularly relevant, since any microbe they encounter is a potential threat to the host.
  • phagocytic cells e.g., macrophages
  • TLRl - TLRlO epithelial cells
  • TLR ligands include cell wall components, proteins, nucleic acids, and synthetic chemical compounds, all of which can activate DCs as immune adjuvants.
  • Each TLR type activates DCs in a similar, but distinct manner.
  • TLRs can be divided into subgroups according to their interferon (IFN) inducing ability.
  • IFN interferon
  • TLR2 does not induce IFN- ⁇ or IFN- ⁇ , but TLR4 can stimulate IFN- ⁇ production.
  • TLR3, TLR7, and TLR9 can induce both IFN- ⁇ and IFN- ⁇ . So far, the B-cell and PDC are the only immune cell subsets in humans identified as expressing TLR9.
  • the brain was considered to be a privileged organ from an immunological perspective due to the apparent inability of neurons to mount an immune response and process antigens. While this is now known to be only partly true, the CNS shows a well-organized innate immune response to systemic bacterial infection and cerebral injury. Indeed, TLR4 are constitutively expressed in the circumventricular organs (CVOs), choroid plexus, and leptomeninges. In CVOs, circulating LPS causes rapid transcriptional activation of the TLR2 gene, as well as a wide variety of genes encoding pro-inflammatory molecules. In contrast, across the CNS a delayed response to LPS occurs in cells located at CVO-microglia boundaries so even though pathogens may lack direct access to the brain parenchyma, they can trigger an innate immune reaction in cerebral tissue without damaging neuronal tissue.
  • CVOs circumventricular organs
  • choroid plexus choroid plexus
  • leptomeninges circulating LPS causes rapid transcriptional activation of the TLR
  • the present invention is based on the seminal observation that the immune system can be used modulate certain cognitive functions to achieve a desired effect, which, in effect, reverses the widely held view that the brain can modulate immune system activity, which itself stems from the theory of stress.
  • Recent advances in the study of the inter-relationships between the CNS and the immune system have revealed a vast network of communication pathways between these systems. Lymphoid organs are innervated by branches of the autonomic nervous system. Accessory immune cells and lymphocytes have functional membrane receptors for most neurotransmitters and neuropeptides, and their activation leads to changes in immune function, including cell proliferation and specific immune responses.
  • brain lesions and stressors can induce changes in immune system function, not all of which are mediated by the neuroendocrine system.
  • the communication pathways that link the brain and immune system are normally activated by signals from the immune system, and they serve to regulate immune responses. These signals originate from accessory immune cells such as monocytes and macrophages, and they are represented mainly by pro-inflammatory cytokines.
  • Pro-inflammatory cytokines produced at the periphery act on the brain via two major pathways: (1) a pathway allowing pathogen-specific molecular patterns to act on TLRs in those brain areas that are devoid of a functional blood- brain barrier, the so-called "circumventricular" areas; and (2) a neural pathway, represented by the afferent nerves that innervate site of infection and injury.
  • peripherally produced cytokines induce the expression of brain cytokines that are produced by resident macrophages and microglial cells. These locally produced cytokines diffuse throughout the brain parenchyma to act on target brain areas so as to organize the central components of the host response to infection (e.g., fever, neuroendocrine activation, and sickness behavior).
  • the inventor has discovered that inducing a similar pattern of cytokine expression in target brain areas can be used to achieve a desired cognitive alteration, such as appetite suppression, enhanced memory function and/or learning, etc., thereby affording new approaches to the treatment of various diseases or disorders as well as to altering one or more cognitive functions in desirable ways.
  • a desired cognitive alteration such as appetite suppression, enhanced memory function and/or learning, etc.
  • eating disorders represent an important disease class throughout the world, particularly in developed countries, as they cause, or contribute to, significant morbidity and mortality in millions of people annually in the U.S. alone.
  • These disorders include obesity, binge-eating disorder (BED), anorexia, and bulimia.
  • BED binge-eating disorder
  • Obesity, or excess adipose tissue is increasingly prevalent in developed societies.
  • hypernutrition i.e., overfeeding
  • reducing food intake for example, by suppressing appetite, would be beneficial in the treatment of obesity and many other diet-related disorders.
  • agents refers to an active ingredient delivered to achieve an intended therapeutic benefit.
  • combination therapy refers to a therapeutic regimen that involves the provision of at least two distinct therapies to achieve an indicated therapeutic effect.
  • a combination therapy may involve the administration of two or more chemically distinct active ingredients, or agents, for example, a TLR agonist and calcitonin.
  • a combination therapy may involve the administration of one or more TLR agonists, alone or in conjunction with another agent as well as the delivery of another therapy.
  • the active ingredients may be administered as part of the same composition or as different compositions.
  • the compositions comprising the different active ingredients may be administered at the same or different times, by the same or different routes, using the same of different dosing regimens, all as the particular context requires and as determined by the attending physician.
  • the drug(s) may be delivered before, during, and/or after the period the subject is in therapy.
  • a TLR agonist that modulates of TLR7 is a compound that increases or decreases, as the case may be, the signaling activity of TLR7 as compared to TLR7 signaling activity in the absence of the compound.
  • “Monotherapy” refers to a treatment regimen based on the delivery of one therapeutically effective compound, whether administered as a single dose or several doses over time.
  • a “patentable” composition, process, machine, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed. For example, with regard to novelty, non- obviousness, or the like, if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non-obviousness, etc., the claim(s), being limited by definition to “patentable” embodiments, specifically exclude the unpatentable embodiment(s). Also, the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity.
  • a "plurality” means more than one.
  • a “purine analog” refers to a synthetic (Le., non-naturally occurring) molecule derived from a purine.
  • a “derivative” is a chemical substance related structurally to another substance and theoretically derivable from it, and in general has the same basic structure as the parent compound. Thus, “derivative” includes metabolites of a compound of the invention that may result following administration of the compound, as well as to prodrug forms of a compound of the invention.
  • kits is used herein in various contexts, e.g., a particular species of chemotherapeutic agent. In each context, the term refers to a population of chemically indistinct molecules of the sort referred in the particular context.
  • a "subject” or “patient” refers to an animal in need of treatment that can be effected by molecules of the invention. Animals that can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine, equine, feline, ovine, porcine, and primate (including humans and non-humans primates) animals being particularly preferred examples.
  • the objects of this invention include the provision of patentable methods to ' alter a cognitive function in a subject (e.g., a mammal, particularly a human) in need or desirous of such alteration, for example, to suppress appetite in order to reduce food intake, to enhance alertness, and to enhance learning or memory function, as well as patentable compositions for use in performing these and other methods.
  • This is accomplished by administering to the subject an amount of a TLR agonist effective to modulate the target cognitive function, alone or in combination with a second agent.
  • TLR agonists include small molecules, oligonucleotides, nucleic acids, peptides, and polypeptides.
  • Preferred TLR agonists target TLR7, TLR8, and/or TLR9.
  • Second agents include calcitonin, neurotensin, amylin, a peptide activator of Protease-activated-receptor-2, nicotine, a sympathomimetic agent, and corticosteroids.
  • the invention concerns compositions that comprise such a compound (or conjugate that includes at least one such compound) and a carrier, which is preferably a pharmaceutically acceptable carrier when the composition is intended for use in humans.
  • the compositions can be liquid or dry in form.
  • the compositions are designed for local, as opposed to systemic, delivery, particularly to a mucosal surface, for example, of an intranasal membrane, a buccal surface, a gastrointestinal membrane, and a genitourinary membrane.
  • compositions are those formulated for mucosal delivery, particularly for delivery of a TLR agonist to cells within the nasal membranes, particularly to mast cells.
  • compositions of the invention relate to various uses for the compositions of the invention.
  • Preferred uses include the alteration of at least one cognitive function.
  • One such aspect relates to reducing food intake through the local delivery of a composition according to the invention.
  • Another aspect concerns enhancing a subject's alertness by the local administration of a composition according to the invention, while another aspect, memory can be improved by locally administering an effective amount of a TLR agonist to a subject's brain.
  • mice received 0.5 ⁇ mole 1 V136 at 7 am.
  • Control mice received saline containing 10%
  • FIG. 3 Mucosal administration of 1 V136 is more effective than systemic administration.
  • C Serum cytokine levels in i.n. or i.p. mice treated with 150 nmole 1V136. Data shown are given as mean ⁇ SD. * denotes p ⁇ 0.05 as compared to the control group, t denotes ⁇ 0.05 as compared to the i.n administration group.
  • FIG. 4 lV136-induced anorexia in cytokine- or cytokine receptor-deficient mice.
  • A-B DFNaR ko, IL-I receptor (R) ko, IL-6 ko, ob/ob, and wild type mice
  • i.p. received anti-TNF- ⁇ (aTNF ⁇ ) or isotype control mAbs (150 ⁇ g, respectively) 30 min. prior to i.n. treatment with 1V136.
  • Food intake (C) and body weight (D) were monitored for 24 hr. after the treatment.
  • E-F INDO (5 mg/kg) was injected 1 hr. prior to i.n. administration of 1 V136.
  • Food intake (E) and body weight (F) were monitored for 24 hr.
  • Mast cells are involved in lV136-induced anorexia.
  • C Cytokine levels in serum 2 hr. after treatments were assessed by Luminex assay.
  • D Histamine receptor antagonists (CIM or CM), or mast cell stabilizer (CROM), were i.p.
  • the present invention is based on the discovery that various cognitive functions (e.g., appetite) can be transiently modulated by the administration of small doses of a TLR agonist to a subject.
  • various cognitive functions e.g., appetite
  • the compounds of the invention stimulate components of the innate immune system.
  • the compounds of the invention act as agonists of Toll-like Receptors, particularly TLR7, TLR8, or TLR 9.
  • TLR7, TLR8, or TLR 9 At least ten mammalian TLR species and a number of naturally occurring and synthetic agonists have been identified.
  • TLR7 and TLR9 recognize and respond to imiquimod and immunostimulatory CpG single- stranded oligonucleotides (ISS-ODN), respectively.
  • ISS-ODN compounds can have naturally occurring or modified backbone chemistries (e.g., backbones having phosphodi ester and/or phosphorothioate linkages).
  • TLR9 activator Bacterial DNA, which is rich in unmethylated CpG dinucleotides sequences, is also a potent TLR9 activator.
  • Other synthetic TLR9 agonists include guanosine analogs.
  • the synthetic immunomodulator R-848 (resiquimod) activates both TLR7 and TLR8. While TLR stimulation initiates a common signaling cascade (involving the adaptor protein MyD88, the transcription factor NF-kB, and pro-inflammatory and effector cytokines), certain cell types tend to produce certain TLRs. For example, TLR7 and TLR9 are found predominantly on the internal faces of endosomes in dendritic cells
  • TLR7 ligaand is RNA, especially viral or bacterial RNA enriched in uridine sequences, such as influenza mRNA.
  • TLR8 is found predominantly in human blood monocytes. Mast cells are known to express significant levels of TLR7, and such cells are found in the cell layers that comprise the nasal membranes in animals, including humans.
  • modulation of, particularly enhancing, TLR activity by contacting cells that express TLR7, TLR8, or TLR 9with an agonist compound is believed to locally stimulate an innate immune response, which, in turn, leads to alterations in neurons proximate to the region(s) of such local stimulation.
  • TLRs and compounds that activate them include TLR2 (activated by, for example, lipoproteins, peptidoglycans, and zymosan), TLR3 (which is expressed in membranes of dendritic cells and of cells from colic mucosa and can be activated by, for example, double-stranded RNA molecules, including poly-A:poly-U, poly-I:poly-U, and poly-I:poly-C), TLR4 (activated by, for example, lipopolysaccharide, heat shock proteins, etc.), and TLR5 (activated by, for example, flagellin).
  • TLR2 activated by, for example, lipoproteins, peptidoglycans, and zymosan
  • TLR3 which is expressed in membranes of dendritic cells and of cells from colic mucosa and can be activated by, for example, double-stranded RNA molecules, including poly-A:poly-U, poly-I:poly-U, and poly-
  • TLR agonists act as TLR agonists, and are particularly agonists of TLR7, TLR8, or TLR 9.
  • a TLR agonist is a compound that stimulates signaling from the particular TLR.
  • a TLR7 agonist stimulates signaling from TLR7 in a suitable in vitro or cell-based assay.
  • TLR agonists include small molecules, oligonucleotides, peptides, and polypeptides.
  • Representative compounds known to stimulate TLR activity include purine analog agonists of TLR7, the TLR8 agonist resiquimod, and synthetic CpG oligonucleotide agonists of TLR9.
  • TLR agonist activity is assessed using any suitable assay.
  • putative TLR7/8/9 agonists may be screened by their ability to induced production of particular cytokines (e.g., interferon-alpha, interleukin-6, interleukin-8, etc.) from plasmacytoid dendritic cells, a blood mononuclear cell population.
  • cytokines e.g., interferon-alpha, interleukin-6, interleukin-8, etc.
  • TLR 7 and TLR9 agonists are specifically characterized by the ability to induce high levels of type I interferons (alpha and beta) in human plasmacytoid dendritic cells, but not in human monocytes depleted of such cells, for example,
  • type I interferons alpha and beta
  • the TLR specificity for a particular TLR agonist can be confirmed, if desired, for example, by using HEK293 cells transfected with an expression vectors encoding for the appropriate TLR, and using a NF-kB luciferase reporter gene as a readout.
  • TLR7 agonist compounds useful in practicing this invention are TLR7 agonist compounds.
  • Representative examples of such compounds include those described in commonly owned provisional patent application serial number 60/xxx,xxx (serial number to be assigned), filed on an even date herewith, entitled, "Purine Analogs", and having attorney docket number UCR-4010-PV, the disclosure of which is hereby incorporated by reference in its entirity.
  • Other preferred TLR agonists are described in commonly owned, pending provisional patent application serial number 60/710,337, filed on 22 August 2005, and U.S. patent number 6,329,381.
  • TLR agonist compounds conjugated one or more different chemical entities, such as one or more other TLR agonists (of the same or different chemical species), a targeting moiety or other defined chemical entity, as well as to whole cells or other lipid vesicles.
  • Conjugates can be formed by covalent or non-covalent linkage between the respective active ingredients. Covalent linkages are preferably formed by way of linker molecules.
  • linker molecules refer to any molecular group useful for linking at least two distinct chemical entities, e.g., a purine analog and a targeting moiety or specific binding molecule.
  • each of the reactants contain a chemically complementary reactive group.
  • complementary reactive groups are amino and carboxyl groups to form amide linkages, carboxy and hydroxy groups to form ester linkages, amino and alkyl halides to form alkylamino linkages, thiols and thiols to form disulfides, or thiols and maleimides or alkylhalides to form thioethers. Hydroxyl, carboxyl, amino, and other functionalities may be introduced by known methods when not already present.
  • one or more of reactive complementary groups can be "protected", in which event the protected reactive group must be “deprotected” prior to performing the chemistry needed to effect the particular linkage chemistry.
  • Any suitable protection/deprotection scheme can be employed in a particular circumstance.
  • any suitable molecular group can be used as a linker, although which molecular group is suitable for a particular situation may vary, although it is easily within the skill of those in the art to select or prepare an appropriate molecular group with suitable chemically complementary reactive groups to perform the desired linkage. Regardless of the molecular group selected in a particular circumstance, it preferably provides for stable covalent linkage between the different chemical entities to form a conjugate according to the invention.
  • a covalent linkage should be stable relative to the solution conditions under which the linker and linking groups are subjected.
  • linkers of any suitable length or arrangement can be employed, although linkers that contain about 4-80 carbons, preferably from about 10-70 carbons, more preferably about 10-50 carbons, and even more preferably from about 10-30 carbons or about 10 to 20 carbons, are preferred.
  • Linkers may also contain one or more heteroatoms (e.g., N, O, S, and P) in the molecular linking groups, particularly from 0-10 heteroatoms.
  • the molecular linking group may be branched or straight chain.
  • conjugates may be formed directly between a purine analog and targeting moiety or specific binding molecule, in which case a linker is not employed.
  • a substitutent of the purine analog and a substituent of the specific binding molecule are typically derivatized to provide the complementary reactive groups (on e or more which may, if appropriate, be protected) necessary to perform a suitable chemistry to link the different chemical entities.
  • non-covalent linkages between active ingredients of a conjugate may also be employed to form a conjugate according to the invention.
  • non-covalent linkages include intermolecular interactions mediated by electrostatic forces, hydrophobicity, etc.
  • members of a high affinity binding pair can be used to link two or more molecules.
  • high affinity binding pairs include antibodies and antigens, biotin and streptavidin, and cell surface or intracellular receptors and their respective ligands.
  • a “specific binding molecule” refers to a molecule that binds to a target analyte (e.g., a tissue- or cell-type-specific cell surface receptor) and does not substantially bind to any other molecule present in the sample.
  • a target analyte e.g., a tissue- or cell-type-specific cell surface receptor
  • substantially binding is meant that binding occurs to an extent sufficient to affect the desired result, i.e., delivery of the conjugate to the target tissue or cell, although a small amount of non-specific binding may occur.
  • the specific binding molecule can be an antibody or an antibody fragment (e.g., the Fab region of an antibody), a ligand for a receptor, a receptor or receptor fragment that binds a ligand, or a member of a high-affinity binding pair (e.g., a biotin-streptavidin pair).
  • a "derivative” is a chemical substance related structurally to another substance and theoretically derivable from it, and in general has the same basic structure as the parent compound.
  • different positions on a purine analog can be selected as conjugation sites for the linker and specific binding molecule. Conjugation of linker and specific binding molecule at these sites does not substantially adversely affect the ability of the attached purine analog to induce an innate immune response.
  • an innate immune response is not "substantially adversely affected” if the conjugate retains at least about 10%, preferably at least about 50%, more preferably at least about 75%, and even more preferably at least about 90% of the ability of the unconjugated form of the purine analog to induce an innate immune response in any suitable assay, for example, a in vitro cytokine induction assay.
  • the specific binding molecule is an antibody or antibody fragment.
  • antibody refers to an immunoglobulin, whether natural or partially or wholly synthetically produced, including derivatives that maintain specific binding ability.
  • the term also covers any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.
  • An antibody may be monoclonal or polyclonal, and can be a member of any immunoglobulin class (or combination of classes), including any of the human classes: IgG, IgM, IgA, IgD, IgG, and IgE.
  • An "antibody fragment” is any derivative of an antibody that contains less than the complete heavy and light immunoglobulin chains. Preferably, an antibody fragment retains at least a significant portion of the antigen binding domain of at least a heavy or light immunoglobulin chain. Examples of antibody fragments include, but are not limited to, Fab, Fab 1 , F(ab') 2 , scFv, Fv, dsFv diabody, and Fd fragments.
  • Antibodies and antibody fragments can be produced using any suitable technique, including production from hybridomas.
  • Antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, or they can be recombinantly produced from one or more nucleic acid molecules that encode the particular antibody fragment sequence(s). Alternatively, antibody fragments can be wholly or partially synthetically produced.
  • antibody fragments include single chain antibody fragments, as well as fragments comprising multiple chains, which preferably are linked together, for instance, by disulfide linkages.
  • Antibody fragments can also be multimolecular complexes.
  • a functional antibody fragment typically comprises at least about 50, and often more than about 200, amino acid residues.
  • a “Fab” fragment is essentially equivalent to that obtained by digestion of immunoglobulins (typically IgG) with the enzyme papain. Fab fragments are preferably recombinantly produced.
  • the heavy chain segment of the Fab fragment is an "Fd” fragment.
  • An “Fab”' fragment is an antibody fragment essentially equivalent to that obtained by reduction of the disulfide bridge or bridges joining the two heavy chains in a F(ab')2 fragment.
  • Fab 1 fragments are also preferably recombinantly produced.
  • An “Fv” fragment consists of one V L and one VH domain held together by non-covalent interactions.
  • the term “dsFv” refers to an Fv with an engineered intermolecular disulfide bond to stabilize a V L - V H pair.
  • a "F(ab')2" fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins (typically IgG) by pepsin digestion at pH 4.0-4.5, and is
  • Single-chain Fvs are recombinant antibody fragments consisting of only a variable light chain (VL) and variable heavy chain (V H ) covalently linked to each other. Either V L or V H may be an amino-terminal domain.
  • the interchain linkage may be accomplished via any suitable linker that connects the two domains without significant steric interference. Typically, such linkers are comprised primarily of stretches of glycine and serine residues with some glutamic acid or lysine residues interspersed for solubility.
  • “Diabodies” are dimeric scFvs. The components of diabodies typically have shorter peptide linkers than most scFvs and they show a preference for associating as dimers.
  • Active fragments of antibodies preferably include the Fv region of an antibody. Active fragments of antibodies can be made using methods known in the art, such as proteolytic digestion of samples including antibodies. Antibodies may be polyclonal or monoclonal, and include humanized antibodies, unless otherwise specified.
  • a preparation of antibodies can be crude, such as can be prepared from cell culture or whole blood or serum or plasma, or can be partially purified, such as by crude separation methods such as molecular weight purification or ammonium sulfate precipitation, or can be substantially purified, such as by affinity chromatography for a class of antibody, subclass of antibody, or by binding with a particular antigen or epitope.
  • Methods for antibody preparation, production, and purification are known in the art, such as provided by Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor (1988).
  • the invention also contemplates conjugates wherein the purine analog is conjugated to an amino acid or peptide.
  • amino acid comprises the residues of the natural amino acids (e.g., AIa 5 Arg, Asn, Asp, Cys, GIu, GIn, GIy, His, HyI, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and VaI) in D or L form, as well as unnatural amino acids ⁇ e.g., phosphoserme, phosphothreonine, phosphotyrosine, hydroxyproline, g ⁇ mm ⁇ -carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, ⁇ -methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and
  • the term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (Ci-C 6 )alkyl, phenyl or benzyl ester or amide; or as an -methylbenzyl amide).
  • a conventional amino protecting group e.g., acetyl or benzyloxycarbonyl
  • natural and unnatural amino acids protected at the carboxy terminus e.g., as a (Ci-C 6 )alkyl, phenyl or benzyl ester or amide; or as an -methylbenzyl amide.
  • Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T.W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981, and references cited therein).
  • amino acid can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine.
  • peptides these are typically polymers of amino acid residues, which may be linked by peptide bonds as occur in proteins in nature, by synthetic linkages, or combinations of these.
  • Peptides include those that embody antigenic determinants (such as may be bound by an antibody or a T cell receptor) or structures useful for purification.
  • TLR Agonists Other compounds having TLR signal stimulating activity may also be employed.
  • any in vitro biochemical or cell-based assay for assessing TLR activity can be used to screen compounds for TLR agonist activity.
  • Preferred assays for this purpose are those amenable to high-throughput screening.
  • Secondary screens for example, a cell-based assay from which pro-inflammatory cytokine expression can be assessed
  • Compounds that may be screened for TLR- binding activity include small molecules, nucleic acids (including synthetic oligonucleotides), peptides, peptidomimetics, polypeptides, and proteins.
  • any chemical entity can be screened for TLR -modulating activity. Those found to stimulate TLR activity and an innate immune response may be used in practicing the invention.
  • the present invention also includes other forms of the compounds of the invention, including prodrug forms.
  • a “prodrug” is a compound that contains one or more functional groups that can be removed or modified in vivo to result in a molecule that can exhibit therapeutic utility in vivo.
  • a “polymorph” refers to a compound that has an identical chemical composition (i.e., it is of the same compound species) as compared to another compound but that differs in crystal structure.
  • the compounds of the invention can be synthesized by any suitable method.
  • small molecules may be synthesized using suitable chemistries adapted to produce the desired compound.
  • Synthetic procedures can be solution-based chemistries, or they may be solid state.
  • U.S. patent no. 6,329,3Sl 5 which provides synthetic procedures for a particularly preferred TLR7 small molecule agonist.
  • commonly owned, pending provisional patent application serial number 60/xxx,xxx, above serial number to be assigned, filed on an even date herewith, entitled, "Purine Analogs", and having attorney docket number UCR-4010-PV) and 60/710,337, filed on 22 August 2005.
  • Solid state chemistries are particularly useful in the synthesis of oligonucleotides and peptides. Recombinant methods are preferred in the production of polypeptides.
  • a compound useful in the practicing the invention can be prepared as an acid salt or as a base salt, as well as in free acid or free base forms.
  • certain of the compounds of the invention may exist as zwitterions, wherein counter ions are provided by the solvent molecules themselves, or from other ions dissolved o ⁇ suspended in the solvent.
  • salt refers to a cationic salt formed at any acidic (e.g., carboxyl) group, or an anionic salt formed at any basic (e.g., amino) group.
  • Preferred cationic salts include the alkali metal salts (such as, for example, sodium and potassium), alkaline earth metal salts (such as, for example, magnesium and calcium), and organic salts.
  • Preferred anionic salts include the halides (such as, for example, chloride salts). When intended for administration to a subject, such salts should be appropriate for such use.
  • pharmaceutically acceptable means suitable for use in humans, whereas “veterinarily acceptable” means suitable for use in non-human animals, particularly non-human mammals.
  • pharmaceutically acceptable salt refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable.
  • the compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases.
  • Pharmaceutically acceptable salts see, e.g., Berge, et al. ((1977) /. Pharm. ScL, vol. 66, 1).
  • examples of pharmaceutically acceptable salts of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine.
  • inorganic acids for example hydrochloric acid, hydrobromic acid, sulfuric acid,
  • Preferred carboxylic acid salts are those of hydrophilic amines, such as glucamine or N-(Ci-C4)alkylglucamine (see, Adger et al. (U.S. Pat. No. 5,811,558)).
  • Salts also include those from inorganic bases, such as ammonia, hydroxyethylamine and hydrazine. Suitable organic bases include methylamine, ethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, ethylenedi amine, hydroxyethylamine, morpholine, piperazine, and guanidine.
  • non-toxic pharmaceutically acceptable salts non-toxic salts formed with nontoxic, pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases.
  • the salts include those derived from inorganic acids.
  • Inorganic acids useful for producing inorganic salts include hydrochloric acid, hydrobromic acid, hydroiodidic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids useful for deriving organic salts include acetic acid, aspartic acid, butyric acid, propionic acid, glutamic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, palmitic acid, pectinic acid, picric acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, lactic acid, mandelic acid, nicotinic acid, benzenesulphonic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, succinic acid, tartric acid, and the like.
  • basic nitrogen- containing groups can be derivatized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides, and iodides; and arylalkyl halides such as benzyl and phenethyl bromides and others.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl, lauryl, myristyl
  • Compounds of the invention that also contain acidic groups are capable of forming base salts with various cations, particularly pharmaceutically acceptable cations, for example, in situ during the final isolation and purification of a compound.
  • Such salts include the alkali metal or alkaline earth metal salts.
  • Suitable base salts are formed from bases that form non-toxic salts.
  • Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include by way of example only, sodium, potassium, lithium, aluminum, ammonium, calcium, zinc, and magnesium salts, with sodium and potassium salts being particularly preferred.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl) amines, tri (substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkeny
  • amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
  • a compound of the invention may exist as an internal salt, or zwitterion. They may form acceptable salts (e.g., a pharmaceutically acceptable salt) with acids and bases. Such zwitterions and salts are included within the scope of the invention. Such salts, as well as other salts, can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable organic acid. For example, a pharmaceutically acceptable salt of a compound of the invention may be readily prepared by mixing together solutions of compound and the desired acid or base, as appropriate.
  • Stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum production of yields of the desired final product.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • Salts may also be prepared by ion exchange, such as by equilibrating a solution containing the particularly purine analog with an appropriate ion exchange resin. Ion exchange may also be used to convert one salt form, such as a salt with an acid or base that is not pharmaceutically acceptable, to another salt form. Such methods are generally well known in the art.
  • compositions As described throughout this specification, the compounds of the invention are useful as therapeutic agents.
  • the compounds will generally be formulated so as to be amenable to administration to a subject by the chosen route.
  • a further aspect of this invention concerns compositions, particularly pharmaceutical or veterinary compositions, comprising a TLR agonist, particularly an agonist of TLR7, TLR8, and TLR9, such as, for example, a compound represented by Formula 1, or an acceptable salt, base, or prodrug form thereof, formulated together with one or more non-toxic acceptable carriers, preferably pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier and “physiologically acceptable carrier” refer to molecular entities and compositions that are physiologically tolerable and do not typically produce an unintended allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a subject.
  • pharmaceutically acceptable carriers are used.
  • composition(s) used in the practice of the invention may be processed in accordance with conventional methods of pharmaceutical compounding techniques to produce medicinal agents ⁇ i.e., medicaments or therapeutic compositions) for administration to subjects, including humans and other mammals, i.e., "pharmaceutical” and “veterinary” administration, respectively.
  • medicinal agents ⁇ i.e., medicaments or therapeutic compositions
  • subjects including humans and other mammals, i.e., "pharmaceutical” and “veterinary” administration, respectively.
  • pharmaceutically acceptable carrier Typically, a compound such as a TLR agonist is combined as a composition with a pharmaceutically acceptable carrier.
  • composition(s) may also include one or more of the following: preserving agents; solubilizing agents; stabilizing agents; wetting agents; emulsifiers; sweeteners; colorants; odorants; salts; buffers; coating agents; and antioxidants.
  • the drugs used in the practice of trie invention are typically prepared as free acids or bases, which are then preferably combined with a suitable compound to yield a pharmaceutically acceptable salt.
  • the compounds, and their respective acid or base salts can be formulated into liquid, preferably aqueous, formulations for storage and administration, as well as dried formulations that may, for example, be used as powders for intranasal administration or be reconstituted into liquid form just prior to administration to a subject.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by. dissolving, dispersing, etc. the particular active compound and optional pharmaceutical adjuvants in an aqueous carrier.
  • Aqueous carriers include water (particularly water for injection into humans), alcoholic/aqueous solutions, and emulsions and suspensions.
  • Preferred pharmaceutically acceptable aqueous carriers include sterile buffered isotonic saline solutions.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • Nonaqueous solvents may also be included, although when included they preferably comprise less than about 50%, more preferably lass than about 25%, and even more preferably less about 10%, of the total solvent volume of the solution.
  • non-aqueous solvents examples include propylene glycol, ethanol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • the pharmaceutical and veterinary compositions of the invention, whether dry or liquid, are preferably formulated for intranasal administration.
  • the composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, antioxidants, antimicrobials, pH buffering agents and the like, for example, sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, antioxidants, antimicrobials, pH buffering agents and the like, for example, sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.
  • the composition or formulation to be administered will, in any event, contain a quantity of the active compound in an amount effective to alleviate the symptoms of the subject being treated.
  • compositions of the invention may also be formulated for targeted delivery of the active ingredient to a subset of tissues or cells in a subject.
  • targeted delivery is accomplished by formulating a compound of the invention with a targeting moiety.
  • moieties include lipids, liposomes, and ligands for molecules that bind, or are bound by, other molecules in vivo.
  • a composition is comprised of "substantially all" of a particular compound, or a particular form a compound (e.g., an isomer) when a composition comprises comprises at least about 90%, and preferably at least about 95%, 99%, and 99.9%, of the particular composition on a weight basis.
  • a composition comprises a "mixture" of compounds, or forms of the same compound, when each compound (e.g., isomer) represents at least about 10% of the composition on a weight basis.
  • a “liquid composition” refers to one that, in its filled and finished form as provided from a manufacturer to an end user (e.g., a doctor or nurse), is a liquid or solution, as opposed to a solid.
  • solid refers to compositions that are not liquids or solutions.
  • such solids include dried compositions prepared by lyophilization, freeze-drying, precipitation, and similar procedures.
  • compositions may also be prepared in a solid form (including granules, powders or suppositories).
  • the compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, dragees, and granules.
  • Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • the active compound may be admixed with at least one inert excipient such as a sugar (e.g., sucrose, lactose, mannitol, and sorbitol) or a cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Such dosage forms may also comprise additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents.
  • Tablets and pills can additionally be prepared with one or more suitable coatings, such as an enteric coating Examples include concentrated sugar solutions, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be also added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent(s).
  • sustained-release materials have been established and are known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • compositions which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in any suitable conventional manner.
  • compositions that help to solubilize the TLR agonist as well as provide mucoadhesive (i.e., adhere well to mucosa) proprerties so as to result in high bioavailability of the particular TLR agonist at the desired site of action.
  • such may include one or more monoglycerides and/or oils, as well as an emulsifier.
  • Preferred monoglycerides are saturated or unsaturated monoglycerides having about 10 to 22 carbon atoms in the hydrocarbon chain.
  • Examples include monoolein, monopalmitolein, monomyristolein, monoelaidin, monoerucin, and a mixture of monoglycerides semi-synthesized from triglycerides of vegetable or animal oil.
  • An oil if included, may be a triglyceride, an iodinated oil, or a vegetable or animal oil.
  • Triglycerides include saturated or unsaturated triglycerides having 2 to about 20 carbon atoms in the hydrocarbon chain. Representative examples include triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, and triolein.
  • Iodized oils include iodized poppy seed oil such as
  • Vegetable oils include soybean oil, cottonseed oil, olive oil, poppyseed oil, linseed oil, and sesame oil.
  • Animal oils include squalane and squalene.
  • Emulsifiers include phospholipids (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), derivatives of the foregoing, and polymeric lipids containing a hydrophilic polymer conjugated to qa lipid headgroup), non- ionic surfactants (e.g., a poloxamer such as polyoxyethylene-polyoxypropylene copolymer, a sorbitan ester (e.g., Span), a polyoxyethylene sorbitan (e.g., Tween), and a polyoxyethylene ether (e.g., Brij)), anionic surfactants (e.g., PS, phosphatidic acid (PA), derivatives of the foregoing, and sodium dodecyl sulfate (SDS)), cationic surfactants (e.g., l,2-dioleyl-3- trimethylammonium propane (DOTAP),
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • Preferred compositions include those for nasal delivery.
  • Preferred compositions for liquid nasal delivery include those that comprise a liquid nasal carrier, i.e., a liquid vehicle (e.g., solution, emulsion, or suspension) designed for delivery of a TLR agonist to the nasal mucosa of a subject, and can include one or more diluents suitable for application to the nasal mucosa.
  • a liquid nasal carrier i.e., a liquid vehicle (e.g., solution, emulsion, or suspension) designed for delivery of a TLR agonist to the nasal mucosa of a subject
  • Suitable diluents include aqueous or non-aqueous diluents or combinations thereof.
  • Examples of aqueous diluents include saline, water, water for injection (WFI), dextrose, combinations thereof.
  • Illustrative non-aqueous diluents include alcohols, particularly polyhydroxy alcohols such as propylene glycol, polyethylene glycol, glycerol, and vegetable and mineral oils.
  • the aqueous and/or non-aqueous diluents can be added in various concentrations and combinations to form solutions, suspensions, oil-in-water emulsions, or water-in-oil emulsions.
  • a liquid nasal carrier can be present in any suitable amount, for example, about 10% to about 99%, about 20% to about 98%, about 30% to about 97%, by weight.
  • Liquid nasal carriers can also optionally include one or more pharmaceutically acceptable excipients, i.e., a substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the particular composition.
  • pharmaceutically acceptable excipients i.e., a substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the particular composition.
  • Illustrative excipients include antioxidants (e.g., butylated hydroxytoluene, butylated hydroxyanisole, potassium metabisulfite, and the like), adjuvants, surfactants, co-solvents, adhesives, agents to adjust pH and/or osmolality, preservatives, thickening agents, sweetening agents, flavoring agents, taste masking agents, colorants, buffering agents, and penetration enhancers.
  • antioxidants e.g., butylated hydroxytoluene, butylated hydroxyanisole, potassium metabisulfite, and the like
  • adjuvants e.g., butylated hydroxytoluene, butylated hydroxyanisole, potassium metabisulfite, and the like
  • adjuvants e.g., butylated hydroxytoluene, butylated hydroxyanisole, potassium metabisulfite, and the like
  • surfactants e.g.
  • a preservative may also be included, typically in quantities sufficient to preserve the composition but low enough not to cause irritation of the nasal mucosa upon administration.
  • Suitable preservatives include benzalkonium chloride, methyl-, ethyl-, propyl-, or butylparaben, benzyl alcohol, phenylethyl alcohol, benzethonium, and combinations of such compounds.
  • buffering agents i.e., compounds that reduce pH changes
  • Buffering agents include salts include, for example, a bicarbonate or carbonate salts of a Group IA metal, an alkaline earth metal buffering agent, an aluminum buffering agent, a calcium buffering agent, a sodium buffering agent, and a magnesium buffering agent.
  • buffering agents include alkali (sodium and potassium) or alkaline earth (calcium and magnesium) carbonates, phosphates, bicarbonates, citrates, borates, acetates, phthalates, tartrates, succinates, and the like, such as sodium or potassium phosphate, citrate, borate, acetate, bicarbonate, and carbonate.
  • compositions of the invention may also include one or more compounds that promote rapid clearance of an active agent (e.g., a TLR7 agonist) from the systemic circulation in order to minimize undesired systemic effects.
  • an active agent e.g., a TLR7 agonist
  • examples of such compounds are those that enhance hepatic and/or renal function or metabolism.
  • compounds that enhance p- glycoprotein activity or hepatic metabolism may be used.
  • a formulation of the invention may also optionally comprise one or more surfactants, compounds that increase viscosity (e.g., methylcellulose, carboxymethylcellulose sodium, ethylcellulose, carrageenan, carbopol, and/or combinations thereof), one or more sweeteners and/or flavoring agents (for example, to mask any bitter or bad taste that may occur if the pharmaceutical composition drips back into the mouth after intranasal administration).
  • surfactants e.g., methylcellulose, carboxymethylcellulose sodium, ethylcellulose, carrageenan, carbopol, and/or combinations thereof
  • sweeteners and/or flavoring agents for example, to mask any bitter or bad taste that may occur if the pharmaceutical composition drips back into the mouth after intranasal administration.
  • compositions of the invention can be prepared in any suitable manner.
  • the compositions are prepared by mixing a TLR agonist, alone or together with one or more other active agents, with a liquid nasal carrier and one or more optional excipients at room temperature under aseptic conditions.
  • the mixture can be prepared under non-aseptic conditions and then sterile filtered, autoclaved, or otherwise sterilized and packaged in a delivery device.
  • the therapeutic compositions of the invention are preferably made in the form of a dosage unit containing a given amount of a desired therapeutic agent (e.g., a TLR7 agonist) and a carrier (i.e., a physiologically acceptable excipient).
  • an "effective amount" of a TLR agonist or other agent is an amount intended to elict the desired modulation of the target cognitive function.
  • suitable dosages may range from about several nanograms (ng) to about several milligrams (mg) of active ingredient per kilogram body weight.
  • compositions of the invention have many applications, particularly in the context of altering a cognitive function of a subject through the administration of an amount of a composition of the invention effective to achieve the intended result.
  • they may be used to reduce food intake, and thus they can be used to treat disorders and diseases such as obesity and binge-eating disorder.
  • disorders and diseases such as obesity and binge-eating disorder.
  • central nervous system effects of these compositions they also have application in the context of altering other cognitive functions.
  • representative examples of cognitive functions other than appetite control that can be modulated include sustained or transient focused attention, divided attention, selective attention, working memory, intermediate term memory, perceptual and cognitive speed, language comprehension, expression, and degree of alertness while performing assigned tasks.
  • BED Binge eating disorder
  • BED Binge eating disorder
  • This disorder is characterized by bouts of over-eating, analogous to binge-drinking, and differs from chronic obesity, as BED is an intermittent syndrome. People afflicted with BED frequently eat large amounts of food while feeling a loss of control over their eating.
  • a preferred method to reduce a subject's food intake is to suppress his/her appetite.
  • to "reduce food intake” means to reduce the amount of food a subject will consume of its own volition over time when presented with excess food.
  • a subject's appetite is "suppressed” when it does not desire to ingest as much food as it would absent suppression.
  • the compounds and compositions of the invention can also be used to alter cognitive functions other than appetite. For or example, they can be used to alter a subject's sustained or transient focused attention, divided attention, selective attention, working memory, intermediate term memory, perceptual and cognitive speed, language comprehension, expression, and degree of alertness while performing assigned tasks.
  • Cognitive functions include sustained or transient focused attention, divided attention, selective attention, working memory, intermediate term memory, perceptual and cognitive speed, language comprehension, expression, and degree of alertness while performing assigned tasks.
  • Psychometric tests typically measure cognitive function (immediate and short-term memory and pattern recognition, for example) by providing the subject with a series of sensory stimuli, and measuring the subject's ability to consciously and voluntarily respond to and, in some tests, to remember, the stimuli.
  • Representative examples of such protocols include the series of tests commercially available from The Psychological Corporation, a division of Harcourt Brace Jovanovich Publishers, New York, N. Y, and the Wechsler Adult Intelligence Scale (WAIS).
  • Preferred cognitive testing protocols employ visual and/or auditory stimuli, and in general entail providing a subject a series of images or sounds and measuring the subject's ability to remember and respond to those stimuli. See, e.g., U.S. patent no. 6,964,638.
  • an automated test is used to assess a subject's cognitive state.
  • a subject is tested on one or more occasions, as needed or prescribed, while performing a brief cognitive task battery as his or her brain waves are recorded.
  • Changes in the subject's neurocognitive function are determined by combining measures of task performance and brain wave measures according to a formula previously determined from a normative reference group of subjects.
  • a computer can be used to show a patient a series of tasks (or tests), receive the subject's responses, and analyze those responses to assess one or more parameters of cognitive function.
  • Automated, as opposed to manual, testing is preferred, in order to increase objectivity, speed, and efficiency, as well as facilitate repeated testing over time of a single subject, as well as change over time in a subject's responses.
  • ERPs event-related brain potentials
  • EEG electroencephalographic
  • ERP waveforms reflect a variety of cognitive functions including those related to language comprehension, memory, and attention/vigilance.
  • ERPs can be detected, recorded, and analyzed to assess even subtle cognitive activity.
  • the use of ERPs is based on predicted differences in waveforms that result from well-known components (e.g., P300 and N400). See, e.g., U.S. patent no. 6,993,381.
  • EEG electroencephalogram
  • fMRI functional magnetic resonance imaging
  • PET positron emission tomography
  • brain-imaging techniques such as fMRI can be used to assess in real time changes in brain activity upon exposure to a composition according to the invention.
  • compositions of the invention can be administered via any suitable route to achieve local or targeted delivery of the TLR agonist (and other active agents, if present in the formulation).
  • Administration routes include oral administration, buccal administration, mucosal administration, nasal administration, sublingual administration, transdermal administration, parenteral administration (e.g., intraperitoneal injection, subcutaneous injection, intramuscular injection), and oral administration.
  • the composition may be of any suitable form, including, for example, a capsule, tablet, lozenge, pastille, powder, suspension, or liquid, among others.
  • Liquid formulations may be administered by inhalation or injection.
  • Such formulations typically include the active ingredient with suitable carriers, such as saline, dextrose, or water.
  • Injections are typically given parenterally.
  • parenteral includes infusion (including continuous or intermittent infusion) and injection via a subcutaneous, intravenous, intramuscular, intrasternal, or intraperitoneal route.
  • Injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the injectable preparation may also be a sterile injectable solution or suspension in a n ⁇ n-toxic parenterally acceptable diluent or solvent.
  • Suitable vehicles and solvents that may be employed are water for injection, Ringer's solution, and isotonic sodium chloride solution, among others.
  • sterile, fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Suppositories for rectal administration can be prepared by mixing the active ingredient(s) with a suitable non-irritating excipient such as cocoa butter and/or polyethylene glycols that are solid at ordinary temperatures but liquid at physiological temperatures.
  • a suitable topical dose of a composition may be administered one to four times daily. The dose may also be administered with intervening days during which no dose is applied.
  • Suitable compositions for topical delivery often comprise from 0.001% to 10% w/w of active ingredient, for example, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes), and drops suitable for administration to the eye, ear, or nose.
  • one aspect of the invention relates to methods for administering a compound of the invention to a subject in need of or desirous of treatment.
  • Such methods involve locally administering an effective amount, particularly a therapeutically effective amount, of such a compound to a subject.
  • Subjects are preferably animals, particularly mammals, and most preferably humans.
  • local administration to a subject involves administration of the compound to a mucosal surface, for example, to an intranasal membrane, a buccal surface, a gastrointestinal lining, and a vaginal surface.
  • nmol nanomole
  • Administration can be via a single dose, or as multiple doses.
  • Particularly preferred dosage forms are solid or dry formulations (e.g., as powders, aerosols, etc.), although liquid formulations can also be employed.
  • Such a compound may be administered alone, or in conjunction with one or more other active agents, which may be included in the same formulation as a compound represented by Formula 1.
  • the various active agents, including the compound represented by Formula 1 are formulated as separate compositions, which compositions may be administered substantially contemporaneously or at different times.
  • the TLR agonist is part of composition, which includes at least a carrier and an amount of such compound sufficient to achieve the desired effect for which it is to be administered in at least a subset of the population of subjects to whom it is administered ⁇ i.e., and "effective amount" of the particular compound or other active agent, as the case may be).
  • an effective amount of a TLR agonist is at least about 1 nanomole (nmol) of the compound.
  • an "effective amount” may be expressed as a mass (e.g., 100 nanograms (ng) , 10 micrograms (urn), etc.) or mass per unit of surface area (100 ng per square meter of body surface area (ng per m 2 )), body weight (1 ug per kilogram of body weight (ug/kg), etc.
  • a “therapeutically effective amount” refers to an amount of an active ingredient sufficient to effect treatment when administered to a subject in need of such treatment.
  • a “therapeutically effective amount” is one that produces an objective response in evaluable patients. Determination of therapeutically effective dosages of a composition comprising a purine analog according to the invention may be readily made by those of ordinary skill in the art.
  • the therapeutically effective amount will vary depending upon the particular subject and condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skill in the art. It will be appreciated that in the context of combination therapy, what constitutes a therapeutically effective amount of a particular active ingredient may differ from what constitutes a therapeutically effective amount of the active ingredient when administered as a monotherapy.
  • treatment means any treatment of a disease or disorder, including preventing or protecting against the disease or disorder (that is, causing the clinical symptoms not to develop); inhibiting the disease or disorder (Le., arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder ⁇ i.e., causing the regression of clinical symptoms).
  • prevention and “suppressing” a disease or disorder since the ultimate inductive event or events may be unknown or latent.
  • the term “prophylaxis” will be understood to constitute a type of “treatment” that encompasses both "preventing” and “suppressing”.
  • the term “protection” thus includes “prophylaxis”.
  • the methods of the invention involve both monotherapy and combination therapy.
  • the invention envisions the administration of two or more active agents, one of which is a TLR agonist, alone or in conjunction with another form of therapy or treatment intended to modulate a target cognitive function, for example, psychotherapy.
  • a TLR agonist of the invention is preferably administered intermittently, on an "as needed" basis, as opposed to chronically, in order to minimize the risk of a subject developing tolerance to the particular TLR agonist.
  • the subject receives a TLR agonist only on those occasions immediately prior to when binge eating is expected or is likely to occur.
  • a composition that includes the TLR agonist is administered just prior to the time when an enhanced degree of mental alertness is desired or required.
  • the risk of developing tolerance to a particular TLR agonist may be minimized by administering different TLR agonists, including agonists of different TLRs, over time.
  • microbes contain signature molecules that activate the innate and adaptive immune systems in an infected host, and an innate immune response is generally viewed as the first, or earliest, host defense mechanism against microbial infection.
  • Panels of TLR species have been identified as essential receptors that recognize particular pathogen- associated molecular patterns (PAMPs). At least ten human and twelve murine TLR species have been identified. Kaisho, T. & Kakira, S. (2006), J Allergy Clin Immunol, vol. 117: 979- 87.
  • TLRs are expressed on a range of cell types, including antigen-presenting cells (e.g., macrophages and dendritic cells) and other immune cells (e.g., neutrophils and T/B cells).
  • Naturally occurring agonists for TLRs have been identified, and include components from various microbes, such as bacterial cell walls components (LPS is a naturally occurring TLR4 ligand, and Lipoprotein is a naturally occurring TLR2 ligand), viral RNA (a naturally occurring TLR3 ligand), and bacterial DNA (a naturally occurring TLR9 ligand).
  • LPS is a naturally occurring TLR4 ligand
  • Lipoprotein is a naturally occurring TLR2 ligand
  • viral RNA a naturally occurring TLR3 ligand
  • bacterial DNA a naturally occurring TLR9 ligand
  • SM360320 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine
  • SM 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine
  • 1V136 induces high levels of type 1 DFN in human peripheral blood leukocytes (PBL) and inhibits HCV replication in human hepatocytes expressing TLR7.
  • PBL peripheral blood leukocytes
  • LPS lipopolysaccarides
  • TLR7 agonist IV 136 reduced food-intake and enhanced body weight loss in mice.
  • Anorexic behavior induced by this TLR7 agonist was transient and involved hypothermia, and appetite returned to normal within about twenty-four hours after treatment.
  • Mast cells were also identified as being at least partially involved in the observed anorexic behavior and hypothermia induced by this TLR7 agonist.
  • mice Female C57BL/6 (B6) mice, 5-6 weeks of age, and gene deficient mice, i.e., Lep ob /Lep ob (B6 background), WBB6F1-W/Wv (WAVv-/-) and its wild type (WAVv+/+), TLR2 knockout (ko), and TLR4 ko, were purchased from The Jackson Laboratory (Bar Harbor, ME). TLR2 ko and TLR4 ko mice were bred at the University of California, San Diego (San Diego, CA).
  • 1V136350320 (1V136) was synthesized as described (Lee, et al. (2006), supra), dissolved with DMSO (10OmM), and stored -80 0 C until use.
  • 1 V150 a derivative of 1V136, was synthesized as described (Lee, et al. (2006), supra).
  • Anti-TNF- ⁇ antibodies (clone: G281-2626), isotype control (Rat IgGl k chain), or Futhan (Nafamostat mesilate, a tryptasae inhibitor) were purchased from BD Pharmingen (San Diego, CA).
  • Indomethacin (UNDO), chlorpheniramine maleate (CM, a Hl antagonist), cimetidine (CJM, a Hl/H2 antagonist), and the mast cell stabilizer cromolyn sodium (CROM) were purchased from Sigma Chemical (St. Louis, MO).
  • LPS from Escherichia coli, serotype 0111:B4, L-2630, Sigma was dissolved in isotonic, pyrogen-free saline. AU reagents were diluted with saline before administration to mice.
  • mice were individually housed in new clean cages.
  • mice On the day of the experiments, the mice were weighed and lightly anesthetized with isoflurane gas. The mice intranasally (i.n.) received various concentrations of 1V136 in 30- 50 ⁇ l saline, after which they were returned to their cages. Each cage was stocked with a pre- weighed amount of food. All treatments were performed 7-9 am in the morning. Compounds were freshly prepared before administration. LPS was dissolved in saline and 2 ⁇ g per mouse was injected intraperitoneally (i.p.). Joppa, et al. (2005), Peptides, vol. 26: 2294-301.
  • anti-TNF- ⁇ monoclonal antibodies were administered i.p. 45 min. prior to the i.n. administration of 1 V136. Control treatments consisted of the equivalent volume of vehicle only.
  • HSTDO indomethacin
  • INDO indomethacin
  • Plal V136a or serum was isolated and stored at -20 0 C until measured by Multiplexed Biomaker Immunoassays for Luminex (LINCO Research, St Charles, MI).
  • Pro-inflammatory cytokines such as ILIb, IL-6, and TNF- ⁇ , which are induced by LPS (a natural TLR4 ligand), are known to cause anorexia. Dantzer, R. (2004), supra. Accordingly, the levels of various cytokines, including IL-6 and TNF- ⁇ (Table 1 , below), were measured after administration of 1 V136 and compared to the levels of these cytokines induced by intraperitoneal LPS (2 ⁇ g) administration. This dose of LPS was selected based on previous studies that indicated it was sufficient to induce anorexia. Mucosal administration of 1V136 induced the highest levels of these cytokines 2 hr. after administration, and these levels rapidly declined by 6 hr.
  • LPS a natural TLR4 ligand
  • Mucosal surfaces have unique lymphoid- and mucus-associated cells, including mucosa] dendritic cells and mast cells, which differ from the systemic lymphoid tissues such as antigen presenting cells (APCs) in spleen. Boyaka, et al. (2003), Curr Pharm Des, vol. 9: 1965-72.
  • the anorexic effects of mucosal (intranasal or gavage) 1V136 administration were investigated and compared to the effects observed following systemic (i.p.) administration (Figure 3). At the higher dose, delivery of 1 V136 by each of the three routes significantly induced anorexic behavior (Figure 3, A and B, black bar).
  • IFN ⁇ is known to be induced by TLR7 (Lee, et al. (2006), supra), and directly exerts its effects on the endocrine system by activating neuro-secretory hypothalamic neurons. Dafny, N. & Yang, P. B. (2005), Eur J Pharmacol, vol. 523: 1-15. IFN ⁇ also regulates the hypothalamic-pituitary-adrenocortical axis and modulates food intake regulation. Dafny, N. & Yang, P. B. (2005), supra. Thus, various gene-deficient mice or, alternatively, neutralized antibodies, were used to study the mechanism of lV136-induced anorexia.
  • 1V136 induced anorexic behavior in IFN receptor (R) ko, IL-IR ko, IL-6 ko, or leptin deficient (ob/ob) mice at similar levels to the anorexic behavior seen in the wild-type control ( Figure 4 C and D).
  • Anti-TNF- ⁇ mAb was used to neutralized systemic TNF-oc induced by 1 V136.
  • 150 ⁇ g of anti-TNF- ⁇ mAb or control mAb were injected 30 min. prior to the administration of 1V136. This dose was selected based on previous studies. Treatment with anti-TNF- ⁇ mAb did not alter the effects on food intake and body weight loss by 1V136 ( Figure 4, C and D).
  • Prostaglandin E2 produced in inflammatory processes may diffuse to the central nervous system and may be involved in anorexia/hypophagia.
  • PGE2 Prostaglandin E2
  • COX cyclooxygenase
  • INDO a non-selective COX inhibitor
  • TLRs are expressed on variety of immune cells, i.e., T cells, B cells, macrophages, dendritic cells, and mast cells, to trigger innate and adaptive immune responses.
  • TLR7 is expressed on mature mast cells and connective tissue type mast cells, but not on immature mast cells ⁇ Matsushima, 2004 #883 ⁇ . TLR7 increases pro-inflammatory cytokine and chemokine production by connective tissue type mast cells. Matsushima, et al. (2004), J Immunol, vol. 173: 531-41.
  • 1V136 was intranasally administered to mast cell-deficient mice (W/Wv-/-) or littermate control mice (WAVv+/+), and food intake and weight change were measured. T/B cell deficient mice (Rag -/-) were used as controls. The observed reduction in food intake by mice that received 1V136 was modest as compared to control mice ( Figure 5 A), while 1V136 significantly enhanced weight loss in mast cell-deficient mice.
  • 1 V136 induced a similar level of reduction in food intake and weight loss in Rag ko mice, as compared to control mice. 1V136 induced similar levels of IL-6 and TNF-cc in mast cell-deficient mice as compared to C57BL/6 mice or wild type control (Figure 5 C).
  • Histamine is a signature mediator released by mast cells deregulation. Some of the histamine receptors have been shown contributed to the behavior change in animals. Accordingly, the histamine antagonists chlorpheniramine maleate (CM, Hl antagonist), cimetidine (CIM, H1/H2 antagonist), and mast cell stabilizer cromolyn sodium (CROM) were injected i.p. prior to IV 136 administration ( Figure 5, D). CM, CIM, or CROM alone did not change any food intake, and none of these antagonists mast reversed the anorexic effects of 1V136 ( Figure 5, D).
  • Protease-activated receptor 2 PAR2 is expressed in neurons innervating the rodent mucosa, and can be activated by mast cell tryptase to induce inflammation.
  • Sickness behavior during inflammation or infection can include fever, anorexia, and even lethality.
  • Administration of LPS, TNF- ⁇ , or EL-Ib is known to induce fever (hyperthermia) in rodents.
  • hyperthermia may be an effect induced by 1V136
  • the temperatures of experimental animals treated with 1V136 150 and 500 nmole, administered i.n.
  • IVl 36 treatment resulted in the opposite effect - transient hypothermia, although this effect was diminished in mast cell-deficient mice ( Figure 6, B).
  • the lowest body temperatures were observed at 1-3 hr. post-administration, and temperatures returned to normal after 7 hr.
  • CM or CIM histamine receptor antagonists
  • CROM mast cell stabilizer
  • Microbial infections are often accompanied by sickness behavior that includes fever, anorexia, and lethargy. These reactions are believed to occur as a result of active and adaptive immune responses that, in part, temporarily suspend, normal 09838
  • TLR7 ligand 1V136 TLR7- MyD88 signaling pathway. This transient anorexia was accompanied by hypothermia, in contrast to LPS -induced anorexia, with which hyperthermia is associated.
  • LPS is an immune-stimulating cell wall component in gram-negative bacteria, and has been used to model sickness behavior by acute inflammatory responses.
  • LPS Systemic or CNS administration of LPS is known to induce anorexia for up to 24 hr. post-administration, and is accompanied with high levels of IL-Ib, IL-6, and TNF- ⁇ , each of which alone can also could anorexia when given by systemic or central administration.
  • TLR7 ligand 1V136 had several significant similarities and differences from that induced by LPS, including (1) induced anorexia accompanied by increased expression of pro-inflammatory cytokines, although deletion in any of these cytokines did not alter the TLR7 -induced anorexia ( Figure 4); (2) tolerance to repeated administration of 1 V136, which, when given repeatedly over time, ceased to induce anorexia, indicating that the anorexia induced by 1V136 was accompanied by a tolerance profile similar to LPS tolerance; (3) no difference between genders in the appearance of anorexia induced by IVl 36, whereas LPS-induced anorexia exhibits gender differentiation; (4) the absence of a role for Leptin in lV136-induced anorexia, whereas in LPS-induced anorexia, Leptin plays a significant role (Figure 4); and (5) Hyperthermia accompanies LPS-induced anorexia while TLR7 anorexia displays induction of hypotherm
  • Anorexia is a complex response controlled by a number of signals from the immune, neural, metabolic, and endocrine systems. Because pro-inflammatory cytokines and endocrine hormones are known to regulate LPS-induced anorexia, and 38
  • TLR7 -mediated anorexia During the process to identify the responsible molecule(s) involved in TLR7 -mediated anorexia, it was discovered that mast cell deficient mice displayed less anorexia, but not associated body weight loss. Mast cells are known to express various kinds of TLR and contribute innate immune reactions by secreting chemokines to recruit immune cells to sites of infection. TLR7 is expressed in mature mast cells at higher levels than in phenotypically immature mast cells.
  • Mast cells are a major source of histamines known to be involved in such mammalian regulatory processes as food-intake, sleep patterns, and thermoregulation. Histamine receptor Hl and H2 agonists are known to abolish hypothermia induced by histamine. Here, it was found that the peripheral administration of Hl or H2 histamine receptor antagonists or a mast cell stabilizer was not involved in 1V136- induced anorexia. Also, PAR2 is expressed in neurons in the rodent mucosa and is activated by mast cell tryptase to induce inflammation. Futhan, a tryptase inhibitor, did not affect the food intake, indicating that PAR2 activated by mast cell tryptase was not involved in the anorexic behavior associated with IV 136 treatment.
  • Hyperthermia is a hallmark symptom of an acute inflammatory response.
  • Proinflammatory cytokines such as TNF- ⁇ , IL-Ib, and IL-6 (which are also known as endogenous pyrogens), cause hyperthermia in most cases of systemic inflammation.
  • mucosal administration of a TLR7 agonist temporarily induced hypothermia.
  • hypothermia induced by the TLR7 agonist IVl 36 was different, as the TLR7 agonist-induced hypothermia was temporary and not followed by hyperthermia ( Figure 6) or mortality.
  • mucosal administration IVl 36 did not induce hypothermia in mast cell-deficient mice (WAVv), indicating that mast cells are involved in TLR7-mediated hypothermia.
  • TLR agonists have been widely utilized as adjuvants for immunotherapy in the treatment of cancer and infectious disease, or as anti-inflammatory agents in the treatment of asthma.
  • TLR7-mediated anorexic effects appear temporary, and recovery is quickly attained, for example, within 24 hr. of treatment. Further, TLR7 -mediated anorexic effects are independent of leptin and adiponectin levels in serum.
  • TLR agonists including TLR7 agonists such as 1 V136, can be used in the treatment of diseases and disorders that have a cognitive component, particularly those that occur intermittently and/or do not persist for long periods, such as binge-eating disorder, bulimia, etc.
  • Bone marrow was isolated from the femora and tibia of C57BL/6 mice.
  • Cells were plated on non-tissue culture-treated petri dishes and cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin (all from Invitrogen, San Diego, CA), and 30% L929 cell-conditioned media.
  • FBS fetal bovine serum
  • L-glutamine penicillin/streptomycin
  • L929 cell-conditioned media 30% L929 cell-conditioned media.
  • Cells were grown at 37°C, 5% CO2 for 7 days without replacing the medium. Macrophages were then harvested by gentle scraping, counted, and re-plated under various conditions

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Abstract

L'invention concerne des procédés et compositions destinés à altérer des fonctions cognitives dont l'appétit. De tels procédés consistent à administrer une quantité efficace d'un composé capable de parvenir à l'effet désiré, à savoir un agoniste de TLR, seul ou en combinaison avec d'autres composés ou compositions. Dans le contexte de la suppression de l'appétit, les agonistes de TLR peuvent être administrés pour réduire la ration alimentaire d'un sujet, seul ou en combinaison avec d'autres composés ou compositions qui affectent la satiété. Les troubles qui peuvent être traités comprennent l'hyperphagie boulimique, la boulimie et l'hypernutrition associée aux vacances.
PCT/US2007/009838 2006-05-31 2007-04-23 Procédés et compositions destinés à l'altération cognitive WO2007142754A2 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JUNG ET AL.: 'TLR4 but not TLKR2, signals autoregulatory apoptosis by cultured microglia: a critical role of ifn-beta as a decision maker' JOURNAL OF IMMUNOLOGY vol. 174, 2005, pages 6467 - 6476 *
ZHANG ET AL.: 'Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR 7/8 agonists' JOURNAL OF NEUROIMMUNOLOGY vol. 164, 2005, pages 154 - 160 *

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