Classifications

• • 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
• • 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
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to methods and compounds for modulating mast cell activity by inhibiting PI3K ⁇ .
• mast cell activity includes, but is not limited to, mast cell degranulation, mast cell migration, mast cell proliferation, and the expression and secretion of cytokines, chemokines, and growth factors by mast cells.
• the methods and compounds of the invention may be used to treat or prevent conditions associated with such mast cell activity that is undesirable.
• Phosphoinositide 3-kinase is a signaling enzyme that plays key roles in many cellular activities, including cellular growth, remodeling, and apoptosis [Wymann and Pirola, Biochem Biophys Acta. 1998;1436:127-150; Anderson et al, J Biol Chem. 1999;274:9907-9910; Rameh et al, JBiol Chem. 1999;274:8347-8350; Cantrell, JCell Sci. 2001;114:1439-1445; Coelho and Leevers, J Cell Sci. 2000;113:2927-2934; Vanhaesebroeck et al, Ann. RevBiochem.
• PI3K also plays roles in many other cellular processes, such as malignant transformation, growth factor signaling, inflammation, and immunity. See Rameh et al, J. Biol Chem, 274:8347-8350 (1999) for a review. Such diverse activities may be attributed at least in part to PDK's lipid and protein kinase activity.
• Class I PI3-kinases are the most extensively investigated class and contain two subunits, one of which plays primarily a regulatory/adaptor role (p85 ⁇ , ⁇ , p55 ⁇ or plOl isoform) and the other that maintains the catalytic role of the enzyme (pi 10a, ⁇ , ⁇ , or ⁇ isoform) [Wymann and Pirola, supra; Anderson et al, supra; Rameh et al, supra; Cantrell, supra; Coelho and Leevers, supra; Vanhaesebroeck et al, supra.] Identification of the pi lO ⁇ isoform of PI3-kinase is described in Chantry et al [JBiol Chem, 272:19236-41 (1997)].
• PI3Ks achieve intracellular signaling at least in part by catalyzing the addition of a phosphate group to the inositol ring of phosphoinositides [Wymann, et al, supra].
• One target of these phosphorylated products is the serine/threonine protein kinase B (P B or Akt).
• Akt subsequently phosphorylates several downstream targets, including the Bcl-2 family member Bad and caspase-9, thereby inhibiting their pro-apoptotic functions [Datta et a , Cell 91:231-41 (1997); Cardone et /.,
• Akt has also been shown to phosphorylate the forkhead transcription factor FKHR [Tang et al, J Biol. Chem., 274:16741-6 (1999)].
• FKHR forkhead transcription factor
• many other members of the apoptotic machinery as well as transcription factors contain the Akt consensus phosphorylation site [Datta et al, supra], Phosphorylation of Akt has been widely used as an indirect measure of
• PI3 -kinase activity in multiple cell types, including endothelial cells, [Shiojima, et al. , Circ. Res., 90: 1243-1250 (2002); Kandel et al. , Exp. Cell Res., 253:210-229 (1999); Cantley et al, Science 296:1655-1657 (2002)].
• PI3K activity is required for growth factor mediated survival of various cell types [Fantl et al, Ann. Rev. Biochem. 62:453-81 (1993); Datta et al, Genes & Dev. 13:2905-27 (1999)].
• PI3K nonselective phosphoinositide 3-kinase
• LY294002 and wortmannin have been shown to enhance destruction of tumor vasculature in irradiated endothelial cells [Edwards et al, Cancer Res. 62:4671-77 (2002)] and partially inhibit mast cell degranulation [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003)].
• LY294002 and wortmannin do not distinguish among the four members of class I PI3Ks, however.
• the IC 50 values of wortmannin against each of the various class I PI3Ks are in the range of 1-10 nM.
• the IC 50 values for LY294002 against each of these PI3Ks is about 1 ⁇ M [Fruman, et al,
• mast cells play diverse and significant roles. For example, mast cells are involved in mediatmg first line immune responses of the innate immune system seen in response to allergens or parasitic or bacterial infections.
• Mast cells also contribute to activation and recruitment of other inflammatory cells, such as neutrophils and T cells, to bring about second line immune responses required for an adaptive immune response.
• CD34 + mast cells circulate in the blood as committed precursor cells and fully mature in specific tissue sites.
• Mast cell development and maturation requires mast-cell growth factor, also known as stem cell factor (SCF), steel, or KIT ligand [Gurish et al., J. Exp. Med. 194:F1-F5 (2001)].
• SCF stem cell factor
• KIT ligand KIT ligand
• the interaction of KIT receptor with its ligand drives mast cell proliferation and differentiation (Feger et al, Int. Arch. Allergy Immunol. 127:110-14 (2002)].
• Mast cells are activated through crosslinking of the high affinity Fc ⁇ RI
• IgE receptor on the cell surface by antigen-bound IgE and to a lesser extent through crosslinking of the Fc ⁇ RI receptor by IgG [Tkaczyk et al., Lit Arch Allergy Immunol. 133:305-15 (2004)].
• Activation through Fc ⁇ RI is typically seen in acute allergic reactions and other types of hypersensitivity reactions, leading to the stimulation of additional immune cells and a full blown immune response.
• Mast cells contain metachromatic granules which store a variety of inflammatory mediators that are released upon mast cell activation.
• mediators include: histamine and serotonin; prostaglandin D2; proteolytic enzymes, such as tryptase that can destroy tissue or cleave complement components or coagulation components; heparin or chondroitin sulfate, which are anticoagulants; chemotactic factors, such as eosinophil chemotactic factor of anaphylaxis (an important regulator of eosinophil function) and neutrophil chemotactic factor.
• these mediators are released into the cellular environment causing acute and immediate immune responses such as vascular permeability and recruitment of lymphocytes.
• mast cell activity is necessary and desirable in healthy individuals. Unwanted mast cell activity, or excessive proliferation of otherwise normal or abnormal mast cells, may be a component of a wide variety of disease states and/or their symptoms, however.
• mast cell activity and/or proliferation it is often desirable, from a therapeutic or preventative standpoint, to reduce or eliminate mast cell activity and/or proliferation.
• numerous immune mediated diseases involve the release by mast cells of cytokines, chemokines and other factors. Cytokines, chemokines, and other factors recruit additional immune cells such as lymphocytes, including neutrophils and T cells, to sites of inflammation. This may lead to numerous immune mediated diseases.
• mast cell activity such as degranulation and tryptase protein have recently been localized to cerebrospinal fluid of patients with multiple sclerosis, an autoimmune disease typically thought to be mediated by T cell activity [Rozniecki et al., Ann Neurol 37:63-66 (1995)].
• mast cell deficient mice (W/W v ) induced to develop an experimental model of multiple sclerosis demonstrate delayed development of MS-like symptoms [Secor et al., J. Exp. Med. 191:813-22 (2000)].
• Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation of the joints and the presence of inflammatory cells in the synovial fluid of the joints, leading to a painful and debilitating disease.
• Mice lacking mast cells show resistance to induction of arthritis-like symptoms after infusion of antibodies to a cytoplasmic enzyme [Lee et al., Science 297:1689-1693 (2002)].
• mast cells accumulate in the extremities of mice affected by collagen-induced arthritis and degranulate during the disease [Woolley et al., Arthritis Res. 2:65-74 (2000)], indicating that mast cells may play a role in mediatmg inflammation and recruiting additional cells to the joints of patients suffering from rheumatoid arthritis.
• a significant population of mast cells resides in the skin. Bullous pemphigoid, an autoimmune disease of the skin exhibiting autoantibodies to cell junction proteins has also been shown to depend on mast cell activation. [Chen et al., JClin Invest. 108:1151-58 (2001)].
• W/W v mice deficient in mast cells did not develop bullous pemphigoid, although autoantibodies and complement proteins were present in the skin similar to normal mice, W/W v mice lacked neutrophil recruitment to the skin. Also, evidence of mast cell mediators have been detected in patients with bullous pemphigoid [Wintroub et al., New Eng. J. Med. 298:417-21 (2001)].
• Other immune disorders in which mast cells are thought to play a role include Sjogren's syndrome [Konttinen et al., Rheumatol Int. 19:141-7 (2000)], chronic urticaria [Napoli et al., Curr Allergy Asthma Rep.
• mast cell disease encompasses a heterogeneous group of clinical disorders characterized by the proliferation and accumulation of mast cells in a variety of tissues, most often the skin, but also in the skeletal, hematopoietic, gastrointestinal, cardiopulmonary, and central nervous systems.
• Mastocytosis is characterized by excess proliferation of mast cells, distributed in a predictable pattern throughout the skin (e.g., cutaneous mastocytosis and urticaria pigmentosa), bone marrow, gastrointestinal tract, lymph nodes, liver and spleen [Brockow et al, CWT. Opin. Allergy Clin. Immunol 1:449-54 (2001)].
• Mastocytosis is classified as either familial or sporadic, the latter being further subdivided into either cutaneous or systemic.
• Systemic mastocytosis is still further classified into indolent (chronic) mastocytosis and aggressive mastocytosis, as well as mast cell leukemia.
• CM hematologic disorder
• AHD hematologic disorder
• Cutaneous mastocytosis (CM) demonstrates typical clinical and histological skin lesions and absence of definitive signs (criteria) of systemic involvement.
• Most patients with CM are children and present with maculopapular cutaneous mastocytosis (for example, urticaria pigmentosa, UP).
• Other less frequent forms of CM are diffuse cutaneous mastocytosis (DCM) and mastocytoma of skin.
• SM Systemic mastocytosis
• MC pathologic mast cells
• Mast cell leukemia is a 'high-grade' leukemic disease defined by increased numbers of MC in bone marrow smears (greater than or equal to 20%) and peripheral blood, absence of skin lesions, multiorgan failure, and a short survival. In typical cases, circulating MC amount to greater than or equal to 10% of leukocytes (classical form of MCL).
• Mast cell sarcoma is a unifocal tumor that consists of atypical MC and shows a destructive growth without (primary) systemic involvement. This high-grade malignant MC disease has to be distinguished from a localized benign mastocytoma in either extracutaneous organs (extracutaneous mastocytoma) or skin. Additionally, mutations in the KIT receptor leading to mast cell hyper- proliferation have been found in patients with acute myeloid leukemia (AML)
• AML acute myeloid leukemia
• treatment regimens for mast cell related conditions typically employ non-specific treatment regimens developed for other proliferative or immune disorders (e.g., histamine receptor blockers, prostaglandin blockers, steroids), resulting in incomplete treatment, treatments which are not effective, or treatments that cause numerous immunosuppressive side-effects.
• a significant drawback to many therapies that may be used to treat conditions associated with undesirable mast cell activity is the non-specific inhibition of many cellular tyrosine kinases in mast cells and other cell types that are targeted by the treatment.
• kinase inhibitor therapeutics for treating mast cell proliferative disorders were originally developed to inhibit kinases such as platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor (VEGFR), or the Bcr/Abl mutation. These potential therapeutics proved ineffective at treating all forms of mastocytosis.
• PDGF-R platelet-derived growth factor receptor
• VEGFR vascular endothelial growth factor receptor
• Bcr/Abl mutation the Bcr/Abl mutation
• the present invention provides methods for effectively treating or preventing a condition, and/or a symptom of a condition, associated with or caused, at least in part, by undesirable mast cell activity .
• the methods of the invention are particularly useful in treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells.
• the invention provides a method for inhibiting an activity of mast cells, comprising administering to an individual a selective inhibitor of phosphoinositide 3 -kinase delta (PI3K ⁇ ) in an amount effective to inhibit mast cell activity.
• PI3K ⁇ phosphoinositide 3 -kinase delta
• the mast cell activity being inhibited is mast cell migration, mast cell proliferation, mast cell degranulation, or expression of or secretion of cytokines, chemokines, or growth factors from mast cells.
• the cytokine is TNF- ⁇ .
• the cytokine is LL-6.
• the chemokine being inhibited is eotaxin, MlPl- ⁇ , MlPl- ⁇ , MDC-1, MCP-1, or lymphotactin.
• the invention provides methods of reducing or preventing lymphocyte infiltration to a site of inflammation in a condition associated with undesirable mast cell activity comprising the step of administering to an individual a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) in an ⁇ amount effective to reduce or prevent lymphocyte infiltration to said site of inflammation in an amount effective to reduce lymphocyte recruitment signaling by mast cells in said individual.
• PI3K ⁇ phosphoinositide 3-kinase delta
• a condition associated with undesirable mast cell activity is any condition caused by or involving the underlying effects of any undesirable mast cell activity.
• the invention provides methods for treating or preventing a condition associated with undesirable mast cell activity in an individual, comprising the step of administering a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) in an amount effective to treat or prevent a condition associated with undesirable mast cell activity.
• a selective inhibitor of phosphoinositide 3-kinase delta PI3K ⁇
• the selective PI3K ⁇ inhibitor inhibits mast cell activity. Examples of such mast cell activity include mast cell migration, mast cell proliferation, mast cell degranulation, or expression or secretion of cytokines, chemokines, or growth factors from mast cells.
• the methods of the invention encompass treating or preventing conditions (or symptoms associated with conditions) mediated by immunoglobulin receptor cross-linking on mast cells
• Mediated by immunoglobulin crosslinking or “Ig-mediated” refers to the ability of Ig bound to receptors on mast cells to initiate, or facilitate, a condition associated with undesirable mast cell activity.
• Immunoglobulins which activate mast cells include IgG and IgE.
• the condition associated with undesirable mast cell activity is an IgE-mediated condition.
• the condition is an IgG-mediated condition.
• the condition is mediated by other stimuli such cytokines, chemokines or other growth factors.
• Conditions, and symptoms of conditions amenable to treatment or prevention by methods according to the invention include, but are not limited to, asthma, allergic reactions, or autoimmune diseases.
• the allergic reaction is type I hypersensitivity, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, or allergic asthma.
• Type I hypersensitivity reactions are reactions in which antigens (allergens) combine with specific IgE antibodies that are bound to membrane receptors on tissue mast cells and blood basophils.
• the antigen-antibody reaction causes the rapid release of potent vasoactive and inflammatory mediators, (e.g., histamine, tryptase, leukotrienes and prostaglandins) and later release of proinflammatory cytokines (e.g., interleukin-4 and interleukin-13).
• the mediators produce vasodilation, increased capillary permeability, glandular hypersecretion, smooth muscle spasm, and tissue infiltration of other inflammatory cells.
• Exemplary type I hypersensitivity disorders include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergic asthma, some cases of urticaria and GI food reactions, and systemic anaphylaxis.
• the condition is an autoimmune disease.
• the autoimmune disease contemplated by the invention may be multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, Sjogren's syndrome, chronic urticaria, thyroid eye disease, vasculitis, and peritonitis.
• the invention provides for a method of the invention wherein the
• PI3K ⁇ selective inhibitor is administered in an amount effective to inhibit Akt phosphorylation in said mast cells.
• selective PI3K ⁇ inhibitor and variants thereof such as “PI3K ⁇ selective inhibitor” and “selective inhibitor of PI3K ⁇ ” as used herein refer to a compound that inhibits the PI3K ⁇ isozyme more effectively than other isozymes of the PI3K family.
• a "selective PI3K ⁇ inhibitor” compound is understood to be more selective for PI3K ⁇ than compounds conventionally and generically designated PI3K inhibitors, e.g., wortmannin or LY294002.
• wortmannin and LY294002 are deemed “nonselective PI3K inhibitors.”
• the relative efficacies of compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC50.”
• IC50 determinations can be accomplished using . conventional techniques known in the art. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study.
• the experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used.
• concentration of the inhibitor that shows 50% enzyme activity is taken as the IC50 va e -
• other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC90, etc.
• a "selective PI3K ⁇ inhibitor” alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC50) with respect to PI3K ⁇ that is at least 10-fold, in another aspect at least 20-fold, and in another aspect at least 30-fold, lower than the IC50 value with respect to any or all of the other Class I PI3K family members.
• IC50 inhibitory concentration
• the term selective PI3K ⁇ inhibitor can be understood to refer to a compound that exhibits an IC50 with respect to PI3K ⁇ that is at least 50-fold, in another aspect at least 100-fold, in an additional aspect at least 200-fold, and in yet another aspect at least 500-fold, lower than the IC50 with respect to any or all of the other PI3K Class I family members.
• the term selective PI3K ⁇ inhibitor refers to an oligonucleotide that negatively regulates pi lO ⁇ expression at least 10-fold, in another aspect at least 20-fold, and in a further aspect at least 30-fold, lower than any or all of the other Class I PI3K family catalytic subunits (i.e., pi 10a, pi lO ⁇ , and pi lO ⁇ ).
• a PI3K ⁇ selective inhibitor is administered to an individual in an amount such that the inhibitor retains its PI3K ⁇ selectivity, as described above. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to "about” or “approximately” another particular value.
• Another embodiment includes from the one particular value and/or to the other particular value.
• values are expressed as approximations, by use of the antecedents such as "about” or "at least about,” it will be understood that the particular value forms another embodiment.
• Any selective inhibitor of PI3K ⁇ activity including but not limited to small molecule inhibitors, peptide inhibitors, non-peptide inhibitors, naturally occurring inhibitors, and synthetic inhibitors, may be used. Suitable PI3K ⁇ selective inhibitors have been described in U.S. Patent Publication 2002/161014 to Sadhu et al, the entire disclosure of which is hereby incorporated herein by reference.
• compounds of any type that selectively negatively regulate p 11 O ⁇ expression i. e.
• the invention provides for the use of antisense oligonucleotides which negatively regulate pi lO ⁇ expression via hybridization to messenger RNA (mR A) encoding pi 10 ⁇ .
• oligonucleotides that decrease pi lO ⁇ expression may be used in the methods of the invention.
• the methods of the invention may be applied to cell populations in vivo or ex vivo. "In vivo" means within a living individual, as within an animal or human.
• the methods of the invention may be used therapeutically in an individual, as described infra.
• the methods may also be used prophylactically.
• "Ex vz ' vo" means outside of a living individual.
• Examples of ex vivo cell populations include in vitro cell cultures and biological samples including but not limited to fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art.
• Exemplary biological fluid samples include blood, cerebrospinal fluid, and saliva.
• Exemplary tissue samples include tumors samples and biopsies of tissue.
• the invention may be used for a variety of purposes, including therapeutic and experimental purposes.
• the invention may be used ex vivo to determine the optimal schedule and/or dosing of administration of a PI3K ⁇ selective inhibitor for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. Other ex vivo uses for which the invention may be suited are described below or will become apparent to those skilled in the art.
• Mast cell activity refers to those biological activities carried out by mast cells which may be modulated by the compounds useful in the methods of the invention.
• Examples of these activities include cell migration, proliferation, activation, degranulation, expression of or secretion of chemokines, cytokines or other growth factors, and modulation of cell signaling pathways, for example, modulation of AKT phosphorylation.
• modulation of mast cell activity may be used herein.
• Modulation of mast cell activity as used herein refers to the reduction, inhibition, prevention, promotion or increase of one of the above listed activities of mast cells upon administration of a selective PI3K ⁇ inhibitor.
• a selective PI3K ⁇ inhibitor may inhibit the enzyme itself, may inhibit any downstream signaling effect of the PI3K ⁇ enzyme, or inhibit any further downstream activity of a mast cell.
• mast cell activity means mast cell activity that deviates from the normal, proper, or expected course.
• undesirable mast cell degranulation may include degranulation during allergic reaction causing hypersensitivity of the individual, while undesirable migration may include movement of mast cells into or out of tissue sites having unfavorable biological effects.
• Undesirable mast cell proliferation may include cell proliferation mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
• "Inhibiting undesirable mast cell activity” means to slow or stop the rate at which undesirable mast cell activity takes place. This may result either from a decreased rate of mast cell receptor activation, decreased inflammatory mediator or growth factor release, decreased cellular replication, or an increased rate of cell death. Cell death may occur by any mechanism, including apoptosis and mitotic catastrophe. Use of the methods in accordance with the present invention may result in partial or complete inhibition of undesirable mast cell activity,.
• Preventing undesirable mast cell activity means that the methods of the present invention may be used prophylactically to prevent or inhibit undesirable mast cell activity before it occurs, or to prevent or inhibit the recurrence thereof.
• the invention may be used in vivo or ex vivo where no undesirable cell activity has been identified or where no undesirable cell activity is ongoing, but where undesirable cell activity is suspected or expected, respectively.
• the invention may also be used in all its embodiments wherever undesirable cell activity has been previously treated to prevent or inhibit recurrence of the same.
• a “therapeutically effective amount” or “amount effective” means an amount effective to inhibit or reverse development of, to alleviate the existing symptoms of, to prolong survival of, or to cure the individual being treated.
• the "therapeutic index” is the dose ratio between toxic or undesired effect and therapeutic, or desired, effects, and is expressed as the ratio of LD 50 to ED 50 , which are defined below.
• An increase in the therapeutic index refers to a reduction in the amount of therapeutic necessary to reach a desired effect or to increase the effectiveness of the therapeutic administered.
• the treatment methods of the invention are useful in the fields of human medicine and veterinary medicine.
• the individual to be treated may be a mammal, preferably human, or another animal.
• individuals include but are not limited to farm animals including cows, sheep, pigs, horses, and goats; companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks, and geese.
• the invention further provides methods wherein the selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) s is administered in a plurality of doses.
• a plurality of doses includes administration of the inhibitor or other agent in more than one dose.
• the invention further provides that the selective PI3K ⁇ inhibitor is administered in a regimen which includes administering one or more additional therapeutic compounds commonly utilized in treatment of a condition associated with undesirable mast cell activity, including at least one immunomodulatory agent or other agent as appropriate to the condition or symptom being treated or prevented.
• the invention provides a method of reducing or preventing mast cell activity in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) and an immunomodulatory agent.
• the combination therapy may be administered in a single composition or each agent, such as the inhibitor and immunomodulatory agent, may be administered as a separate composition.
• each agent may be administered in a plurality of doses as necessary.
• the invention provides a method of reducing or preventing lymphocyte infiltration to a site of inflammation in an individual having a condition associated with undesirable mast cell activity, comprising administering to said individual a therapeutically effective amount of a combination therapy comprising a selective inhibitor of phosphoinositide 3-kinase delta (PI3K ⁇ ) and a immunomodulatory agent.
• Immunomodulatory agents contemplated by the invention include glucocorticoids or corticosteroids, immunosuppressants, antihistamines, aminosalicylates, steroid hormones, non-steroidal anti-inflammatory drugs (NSAIDs), .
• glucocorticoids are chosen from the group consisting of cortisone, dexamethosone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and budesonide.
• NSALDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as Vioxx ® (rofecoxib) and Celebrex® (celecoxib), and salicylate.
• Suitable analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hygrochloride.
• immunosuppressants include azathioprine (6-mercaptopurine (6-MP)), cyclophosphamide, cyclosporine, methotrexate, or penicillamine. Also contemplated are Xolair® (omalizumab), leukotriene antagonists, or other drugs commonly used for allergy or asthma. Methods of the invention may include administering formulations comprising an inhibitor of the invention with a particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
• methods of the invention may comprise administering an inhibitor with one or more of TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.
• compositions in accordance with the invention may also include other known angiopoietins, for example Ang- 1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular endothelial growth factor (VEGF).
• angiopoietins for example Ang- 1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, and/or vascular endothelial growth factor (VEGF).
• VEGF vascular endothelial growth factor
• Growth factors for use in pharmaceutical compositions of the invention include angiogenin, bone morpho genie protein- 1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein- 14, bone morphogenic protein- 15; bone morphogenic protein receptor IA, bone morphogenic protein receptor LB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor ⁇ , cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2 ⁇ , cytokine-induced neutrophil chemotactic factor 2 ⁇ , ⁇ endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial- derived neutrophil attractant,
• Immunomodulatory agents used for treatment can be administered in a plurality of doses. It is contemplated that the agents are administered in the combination methods according to the invention at a low dose, that is, at a dose lower than conventionally used in clinical situations where the agent or therapy is administered alone, because the PI3K ⁇ selective nature of the inliibitors of the invention increases the therapeutic index (i.e., the specificity) of the inventive combination therapies. Lowering the dose of the agent or therapy administered to an individual decreases the incidence of adverse effects associated with higher dosages, and can thereby improve the quality of life of a patient undergoing treatment. Further benefits include improved patient compliance, and a reduction in the number of hospitalizations needed for the treatment of adverse effects.
• methods of the invention are advantageous in that they permit treatment at higher doses of the PI3K ⁇ selective inhibitor(s) than nonselective inhibitors such as LY294002 and wortmannin, further maximizing the therapeutic efficacy of the inventive methods
• methods may include administering an inhibitor with one or more other agents which either enhance the activity of the inhibitor or compliment its activity or use in treatment. Such additional factors and/or agents may produce a synergistic effect with an inhibitor of the invention, or to minimize side effects.
• Methods of the invention contemplate use of a selective PI3K ⁇ inhibitor compound having formula (I) or pharmaceutically acceptable salts and solvates thereof:
• A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms, and at least one ring of the system is aromatic;
• X is selected from the group consisting of C(R D )2, CFTjCHRb, and
• R a is selected from the group consisting of hydrogen, Ci .galkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylCi ⁇ alkyl, C ⁇ _ 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
• R b is selected from the group consisting of hydrogen, Ci _6alkyl, heteroCi _3alkyl, Ci ⁇ alkyleneheteroCi ⁇ alkyl, arylheteroCi _3alkyl, aryl, heteroaryl, arylC ⁇ _3alkyl, heteroarylC ⁇ _3alkyl, C ⁇ _3alkylenearyl, and C
• R c is selected from the group consisting of hydrogen, C ⁇ . ⁇ alkyl, C3_ gcycloalkyl, aryl, and heteroaryl; and,
• alkyl is defined as straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups.
• the hydrocarbon group can contain up to 16 carbon atoms, for example, one to eight carbon atoms.
• alkyl includes "bridged alkyl,” i.e., a C6-C 16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl.
• cycloalkyl is defined as a cyclic C 3 -C 8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.
• alkenyl is defined identically as “alkyl,” except for containing a carbon-carbon double bond.
• Cycloalkenyl is defined similarly to cycloalkyl, except a carbon-carbon double bond is present in the ring.
• alkylene is defined as an alkyl group having a substituent.
• C 1-3 alkylenearyl refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group.
• heteroC 1-3 alkyl is defined as a C 1-3 alkyl group further containing a heteroatom selected from O, S, and NR a .
• arylheteroC salkyl refers to an aryl group having a heteroC ⁇ -3 alkyl substituent.
• halo or halogen is defined herein to include fluorine, bromine, chlorine, and iodine.
• aryl alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, e.g., phenyl or naphthyl.
• an "aryl” group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, phenyl, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino.
• exemplary aryl groups include phenyl, naphthyl, biphenyl, tetrahydronaphthyl, chlorophenyl, fluorophenyl, aminophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, carboxyphenyl, and the like.
• arylC 1-3 alkyl and “heteroarylC 1-3 alkyl” are defined as an aryl or heteroaryl group having a C 1-3 alkyl substituent.
• heteroaryl is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, and amino.
• heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.
• Het is defined as monocyclic, bicyclic, and tricyclic groups containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.
• Het groups include 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine, piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine, piperidine, 1,4-dithiane, and 1,4-dioxane.
• methods of the invention contemplate use of a PI3K ⁇ selective inhibitor compound having formula (II) or pharmaceutically acceptable salts and solvates thereof:
• R 8 is selected from the group consisting of hydrogen, Ci _6alkyl, halo,
• X 1 is selected from the group consisting of CH (i.e., a carbon atom having a hydrogen atom attached thereto) and nitrogen;
• R a is selected from the group consisting of hydrogen, Ci .galkyl, C3_ gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylCj_3alkyl, C _ 3alkylenearyl, heteroaryl, heteroarylC 1.3 alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
• R c is selected from the group consisting of hydrogen, Ci .galkyl, C3., gcycloalkyl, aryl, and heteroaryl; and,
• methods of the invention include use of a selective inhibitor of PI3K ⁇ compound having formula (III) or pharmaceutically acceptable salts and solvates thereof:
• R a is selected from the group consisting of hydrogen, Ci _6alkyl, C3.. gcycloalkyl, C3_gheterocycloalkyl, C ⁇ _3alkyleneN(R c )2, aryl, arylC 1.3 alkyl, C 3alkylenearyl, heteroaryl, heteroarylCi _3alkyl, and Ci _3alkyleneheteroaryl; or two R a groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
• methods of the invention embrace use of a PI3K ⁇ selective inhibitor selected from the group consisting of 2-(6-aminopurin-9-ylmethyl)- 3-(2-chlorophenyl)-6,7-dimethoxy-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-6-bromo-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-o- ylmethyl)-3-(2-chlorophenyl)-7-fluoro-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-6-chloro-3-(2-chlorophenyl)-3H-quinazolin-4-one; 2-(6-aminopurin-9- ylmethyl)-3-(2-chlorophenyl)-5-fluoro-3H-quinazolin-4-one; 2-(6-amino
• the methods can be practiced using a racemic mixture of the compounds or a specific enantiomer.
• the S-enantiomer of the above compounds is utilized. Therefore, the present invention includes all possible stereoisomers and geometric isomers of the aforementioned compounds.
• “Pharmaceutically acceptable salts” means any salts that are physiologically acceptable insofar as they are compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Some specific preferred examples are: acetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate.
• the invention contemplates an article of manufacture comprising a phosphoinositide 3-kinase delta (PI3K ⁇ ) selective inhibitor and a label indicating a method of use according to any one of the methods of the invention.
• the invention provides for use of a composition comprising at least one PI3K ⁇ selective inhibitor in the manufacture of a medicament for treating or preventing a condition associated with undesirable mast cell activity.
• Administration of prodrugs are also contemplated.
• prodrug refers to compounds that are rapidly transformed in vivo by hydrolysis to, for example, a compound having a structural formula described herein. Prodrug design is discussed generally in Hardma et al.
• prodrugs which, following a biotransformation, become more physiologically active in their altered state.
• Prodrugs therefore, encompass pharmacologically inactive compounds that are converted to biologically active metabolites.
• prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the resultant product.
• the prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life.
• prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate.
• the resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound.
• High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.
• PI3K ⁇ selective inhibitors include compounds that selectively negatively regulate pllO ⁇ mRNA expression more effectively than they do other isozymes of the PI3K family, and that possess acceptable pharmacological properties.
• Polynucleotides encoding human pi lO ⁇ are disclosed, for example, in Genbank Accession Nos. AR255866, NM 005026, U86453, U57843 and Y10055, the entire disclosures of which are incorporated herein by reference. See also, Vanhaesebroeck et al, Proc. Natl. Acad. Sci. 94:4330-4335 (1997), the entire disclosure of which is incorporated herein by reference.
• mRNA messenger RNA
• antisense oligonucleotides at least 5 to about 50 nucleotides in length, including all lengths (measured in number of nucleotides) in between, which specifically hybridize to mRNA encoding pi lO ⁇ and inhibit mRNA expression, and as a result pi 1 O ⁇ protein expression, are contemplated for use in the methods of the invention.
• Antisense oligonucleotides include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e. ' , make the oligonucleotide more resistant to nuclease degradation, particularly in vivo.
• antisense oligonucleotides that are perfectly complementary to a region in the target polynucleotide possess the highest degree of specific inhibition antisense oligonucleotides that are not perfectly complementary, i.e., those which include a limited number of mismatches with respect to a region in the target polynucleotide, also retain high degrees of hybridization specificity and therefore also can inhibit expression of the target mRNA.
• the invention contemplates methods using antisense oligonucleotides that are perfectly complementary to a target region in a polynucleotide encoding pi lO ⁇ , as well as methods that utilize antisense oligonucleotides that are not perfectly complementary (i.e., include mismatches) to a target region in the target polynucleotide to the extent that the mismatches do not preclude specific hybridization to the target region in the target polynucleotide.
• Methods for designing and optimizing antisense nucleotides are described in Lima et al, (JBiol Chem ;272:626-38. 1997), Kurreck et al, (Nucleic Acids Res. ;30:1911-8. 2002) and U.S. Patent No. 6,277,981 , which are incorporated herein by reference.
• antisense compounds are described in International Patent Publication WO 01/05958, which is incorporated herein by reference.
• the invention further contemplates methods utilizing ribozyme inhibitors which, as is known in the art, include a nucleotide region which specifically hybridizes to a target polynucleotide and an enzymatic moiety that digests the target polynucleotide. Specificity of ribozyme inhibition is related to the length the antisense region and the degree of complementarity of the antisense region to the target region in the target polynucleotide.
• ribozyme inhibitors comprising antisense regions from 5 to about 50 nucleotides in length, including all nucleotide lengths in between, that are perfectly complementary, as well as antisense regions that include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target p 11 O ⁇ -encoding polynucleotide.
• Ribozymes useful in methods of the invention include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo, to the extent that the modifications do not alter the ability of the ribozyme to specifically hybridize to the target region or diminish enzymatic activity of the molecule. Because ribozymes are enzymatic, a single molecule is able to direct digestion of multiple target molecules thereby offering the advantage of being effective at lower concentrations than non-enzymatic antisense oligonucleotides.
• RNAi technology for inhibiting pi 1 O ⁇ expression.
• the invention provides double-stranded RNA (dsRNA) wherein one strand is complementary to a target region in a target pi 1 O ⁇ -encoding polynucleotide.
• dsRNA molecules of this type are less than 30 nucleotides in length and referred to in the art as short interfering RNA (siRNA).
• dsRNA molecules longer than 30 nucleotides in length and in certain aspects of the invention, these longer dsRNA molecules can be about 30 nucleotides in length up to 200 nucleotides in length and longer, and including all length dsRNA molecules in between.
• complementarity of one strand in the dsRNA molecule can be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi 1 O ⁇ -encoding polynucleotide.
• dsRNA molecules include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo.
• dsRNA double stranded
• siRNA short-interfering RNA
• Circular RNA lasso inhibitors are highly structured molecules that are inherently more resistant to degradation and therefore do not, in general, include or require modified internucleotide linkage or modified nucleotides.
• the circular lasso structure includes a region that is capable of hybridizing to a target region in a target polynucleotide, the hybridizing region in the lasso being of a length typical for other RNA inhibiting technologies.
• the hybridizing region in the lasso may be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target pi lO ⁇ -encoding polynucleotide.
• RNA lassos are circular and form tight topological linkage with the target region, inhibitors of this type are generally not displaced by helicase action unlike typical antisense oligonucleotides, and therefore can be utilized as dosages lower than typical antisense oligonucleotides. Preparation and use of RNA lassos is described in U.S.
• the inhibitors of the invention may be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.
• a carrier molecule such as a linear polymer (e.g., polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide.
• carriers for use in the pharmaceutical compositions of the invention include carbohydrate-based polymers, such as trehalose, mannitol, xylitol, sucrose, lactose, sorbitol, dextrans, such as cyclodextran, cellulose, and cellulose derivatives. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Other carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337.
• Still other useful carrier polymers known in the art include monomethoxy-polyethylene glycol, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.
• Derivatization with bifunctional agents is useful for cross-linking a compound of the invention to a support matrix or to a carrier.
• One such carrier is polyethylene glycol (PEG).
• the PEG group may be of any convenient molecular weight and may be straight chain or branched.
• the average molecular weight of the PEG can range from about 2 kDa to about 100 kDa, in another aspect from about 5 kDa to about 50 kDa, and in a further aspect from about 5 kDa to about 10 kDa.
• the PEG groups will generally be attached to the compounds of the invention via acylation, reductive alkylation, Michael addition, thiol alkylation or other chemoselective conjugation/ligation methods through a reactive group on the PEG moiety (e.g., an aldehyde, amino, ester, thiol, haloacetyl, maleimido or hydrazino group) to a reactive group on the target inhibitor compound (e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimido or hydrazino group).
• a reactive group on the PEG moiety e.g., an aldehyde, amino, ester, thiol, haloacetyl, maleimido or hydrazino group
• target inhibitor compound e.g., an aldehyde, amino, ester, thiol, ⁇ -haloacetyl, maleimid
• Cross-linking agents can include, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
• Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
• reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos.
• compositions of the invention may also include compounds derivatized to include one or more antibody Fc regions.
• Fc regions of antibodies comprise monomeric polypeptides that may be in dimeric or multimeric forms linked by disulfide bonds or by non-covalent association.
• the number of intermolecular disulfide bonds between monomeric subunits of Fc molecules can be from one to four depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2) of antibody from which the Fc region is derived.
• the term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms of Fc molecules, with the Fc region being a wild type structure or a derivatized structure.
• the pharmaceutical compositions of the invention may also include the salvage receptor binding domain of an Fc molecule as described in WO 96/32478, as well as other Fc molecules described in WO 97/34631.
• Such derivatized moieties preferably improve one or more characteristics of the inhibitor compounds of the invention, including for example, biological activity, solubility, absorption, biological half life, and the like.
• derivatized moieties result in compounds that have the same, or essentially the same, characteristics and/or properties of the compound that is not derivatized.
• the moieties may alternatively eliminate or attenuate any undesirable side effect of the compounds and the like.
• Compounds that compete with an inhibitor compound described herein for binding to PI3K ⁇ are also contemplated for use in the invention. Methods of identifying compounds which competitively bind with PI3K ⁇ , with respect to the compounds specifically provided herein, are well known in the art.
• inhibitor as used herein embraces compounds disclosed, compounds that compete with disclosed compounds for PI3K ⁇ binding, and in each case, conjugates and derivatives thereof.
• Methods include administration of an inhibitor to an individual in need, by itself, or in combination as described herein, and in each case optionally including one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants/flavoring, carriers, excipients, buffers, stabilizers, solubilizers, other materials well known in the art and combinations thereof.
• any pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents known in the art that serve as pharmaceutical vehicles, excipients, or media may be used.
• exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma, methyl- and propylhydroxybenzoate, talc, alginates, carbohydrates, especially mannitol, ⁇ -lactose, anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextrans, gum acacia, and starch.
• compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present inhibitor compounds. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
• Pharmaceutically acceptable fillers can include, for example, lactose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, calcium sulfate, dextrose, mannitol, and/or sucrose.
• Inorganic salts including calcium triphosphate, magnesium carbonate, and sodium chloride may also be used as fillers in the pharmaceutical compositions.
• Amino acids may be used, such as use in a buffer formulation of the pharmaceutical compositions.
• Disintegrants may be included in solid dosage formulations of the inhibitors. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge, corn starch, potato starch, and bentonite may all be used as disintegrants in the pharmaceutical compositions. Other disintegrants include insoluble cationic exchange resins.
• Powdered gums including powdered gums such as agar, Karaya or tragacanth may be used as disintegrants and as binders. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.
• crystalline cellulose examples include crystalline cellulose, cellulose derivatives such as methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC), acacia, corn starch, and/or gelatins
• Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
• An antifriction agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process.
• Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, talc, and waxes.
• Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. Suitable glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of the therapeutic into the aqueous environment, a surfactant might be added as a wetting agent. Natural or synthetic surfactants may be used.
• Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents such as benzalkonium chloride and benzethonium chloride may be used.
• Nonionic detergents that can be used in the pharmaceutical formulations include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the pharmaceutical compositions of the invention either alone or as a mixture in different ratios.
• Controlled release formulation may be desirable.
• the inhibitors of the invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums.
• Slowly degenerating matrices may also be incorporated into the pharmaceutical formulations, e.g., alginates, polysaccharides.
• Another form of controlled release is a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push the inhibitor compound out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. Colorants and flavoring agents may also be included in the pharmaceutical compositions.
• the inhibitors of the invention may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
• the therapeutic agent can also be administered in a film coated tablet.
• Nonenteric materials for use in coating the pharmaceutical compositions include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy- methyl cellulose, povidone and polyethylene glycols.
• Enteric materials for use in coating the pharmaceutical compositions include esters of phthalic acid. A mix of materials might be used to provide the optimum film coating.
• Film coating manufacturing may be carried out in a pan coater, in a fluidized bed, or by compression coating.
• Compositions can be administered in solid, semi-solid, liquid or gaseous form, or may be in dried powder, such as lyophilized form.
• the pharmaceutical compositions can be packaged in forms convenient for delivery, including, for example, capsules, sachets, cachets, gelatins, papers, tablets, capsules, ointments, granules, solutions, inhalants, aerosols, suppositories, pellets, pills, troches,' lozenges or other forms known in the art.
• the type of packaging will generally depend on the desired route of administration.
• Implantable sustained release formulations are also contemplated, as are transdermal formulations.
• Such pharmaceutical compositions may be for administration for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intratracheal, intrathecal, intraocular, retrobulbar, intrapuhnonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in the cornea); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea.
• intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intratracheal, intrathecal, intraocular, retrobulbar, intrapuhnonary (e.g., aerosolized drugs) or subcutaneous injection including depot administration for long term release e.g., embedded under the splenic capsule, brain, or in
• the treatment may consist of a single dose or a plurality of doses over a period of time.
• the methods of the invention involve administering effective amounts of an inhibitor of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers, as described above.
• a chosen route of administration may dictate the physical form of the compound being delivered.
• the invention provides methods for oral administration of a pharmaceutical composition of the invention. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042) at Chapter 89.
• Solid dosage forms include tablets, capsules, pills, troches or lozenges, and cachets or pellets.
• liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
• Liposomal encapsulation may include liposomes that are derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
• the formulation will include a compound of the invention and inert ingredients which protect against degradation in the stomach and which pe ⁇ nit release of the biologically active material in the intestine.
• the inhibitors can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
• the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
• the capsules could be prepared by compression.
• pulmonary delivery of the present inhibitors in accordance with the invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
• Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
• Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn H nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.
• each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and or carriers useful in therapy.
• the inhibitors of the invention are most advantageously prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns), for example, 0.5 to 5 ⁇ m, for most effective delivery to the distal lung.
• Formulations suitable for use with a nebulizer will typically comprise the inventive compound dissolved in water at a concentration range of about 0.1 to 100 mg of inhibitor per mL of solution, 1 to 50 mg of inhibitor per mL of solution, or 5 to 25 mg of inhibitor per mL of solution.
• the formulation may also include a buffer.
• the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the inhibitor caused by atomization of the solution in forming the aerosol.
• Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive inhibitors suspended in a propellant with the aid of a surfactant.
• the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1 ,2-tetrafluoroethane, or combinations thereof.
• Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
• Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent or diluent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
• a bulking agent or diluent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
• Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the inhibitor to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
• Formulations for nasal delivery may include dextran or cyclodextran
• Toxicity and therapeutic efficacy of the PI3K ⁇ selective compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Additionally, this information can be determined in cell cultures or experimental animals additionally treated with other therapies such as radiation, chemotherapeutic agents, and antiangiogenic agents.
• the pharmaceutical compositions are generally provided in doses ranging from 1 pg compound/kg body weight to 1000 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, and 1 to 20 mg/kg, given in daily doses or in equivalent doses at longer or shorter intervals, e.g., every other day, twice weekly, weekly, or twice or three times daily.
• the inhibitor compositions may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product.
• Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient.
• the frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
• the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.
• a suitable dose may be calculated according to body weight, body surface area or organ size.
• FIG. 1 shows a count of inflammatory cell infiltration (total cells and differential cellular component) of BAL fluids isolated from OVA- sensitized/challenged mice treated with p 11 O ⁇ inhibitor IC87114 or control in BAL fluid.
• Macrophage Mac
• Lym lymphocyte
• Neu neutrophils
• Es eosinophils
• Figure 2 depicts total lung inflammation, defined as the average of the peribronchial and perivascular inflammation scores, in OVA-sensitized/challenged mice treated with IC87114 or control. Bars represent mean ⁇ SEM from 6 independent experiments. #, p ⁇ 0.05 vs. SAL+SAL; *, p ⁇ 0.05 vs. OVA+SAL.
• Figure 3 shows airway responsiveness to aerosolized methacholine measured in unrestrained, conscious OVA-sensitized/challenged mice treated with IC87114 or control, at 72 h after the last challenge. Readings of breathing parameters were taken for 3 min after each nebulization during which Penh values were determined. Data represent mean ⁇ SEM from 6 independent experiments. #, p ⁇ 0.05 vs. SAL+SAL; *, p ⁇ 0.05 vs. OVA+SAL.
• EXAMPLES The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof.
• EXAMPLE 1 SELECTIVE PI3K ⁇ INHIBITOR INHIBITS MAST CELL DEGRANULATION
• Mast cells are centrally important in allergic inflammation of the airways. Mast cell activation is mediated by antigen/IgE cross-linking of the Fc ⁇ RI receptor, complement components and cytokines activation, leading to a spontaneous release of preformed histamine and proteases from secretory granules. These activated cells then transcribe, translate and secrete proinflammatory cytokines and chemokines which lead to increased inflammation.
• Non-specific PI3-kinase inhibitors such as wortmannin have been shown to inhibit mast cell degranulation from rat basophilic leukemia cells (RBL- 2H3) (Kitani et al., Biochem Biophys Res Commun. 183:48-54, 1992).
• RBL- 2H3 rat basophilic leukemia cells
• PI3-kinase subunits in regulating mast cell degranulation remains unclear [Tkaczyk et al, JBiol Chem. 278:48474-84 (2003); Windmiller et al, JBiol Chem. 278:11874-8 (2003)].
• Bone marrow cells from mouse femur were cultured in mouse IL-3- containing RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies, Carlsbad, CA), 2 raM glutamine, and 50 ⁇ M 2-ME in humidified 95% air/5% CO 2 at 37°C [Saito, H., F. et al., J. Immunol. 138:3927-3934 (1987)].
• BMMC bone marrow-derived mast cells
• DNP antidinitrophenyl
• Tyrode buffer 112 mM NaCl, 2.7 mM KC1, 0.4 mM NaH 2 PO 4 , 1.6 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes [pH 7.5], 0.05% gelatin, 0.1% glucose), resuspended in Tyrode buffer to 1 x 10 cells/ml and incubated with pi 10-delta inhibitor or vehicle control (0.3% DMSO) for 30 minutes.
• DNP antidinitrophenyl
• DNP-HSA human serum albumin
• results of the ELISA indicate that mast cells pretreated with IC87114 (5 ⁇ M) reduce the amount of histamine in the cell supernatant by approximately 40% compared to vehicle control, and increase the amount of histamine detected in the cell pellet accordingly.
• This demonstrates that inhibition of pi lO ⁇ kinase reduces the amount of histamine released from mast cell storage granules which has utility as an effective therapy for decreasing histamine release from mast cell activation mediated through Fc ⁇ RI.
• BMMCs were sensitized by an overnight incubation with 0.5-1 ⁇ g/ml antidinitrophenyl (DNP) IgE mAb, washed once in BMMC medium in the absence of IL-3 and resuspended in the same medium at 1 x 10 7 cells/ml.
• DNP antidinitrophenyl
• cytokines include TNF- ⁇ , IL-6, IL-2, and granulocyte/monocyte-colony stimulating factor (GM-CSF). Results show that stimulation with 10 ng/ml antigen and addition of only 2 ⁇ M IC87114 to mast cells decreases the amount of TNF- ⁇ secreted by mast cells to undetectable levels.
• IL-6 100 ng/ml of antigen plus 3 ⁇ M inhibitor inhibits TNF- ⁇ production from activated mast cells by 85%.
• Assessment of IL-6 levels after addition of the p 11 O ⁇ kinase inhibitor demonstrates similar effects.
• Addition of IC87411 to activated mast cells (10 ng/ml antigen) showed a dose dependent reduction in IL-6 production, from 6000 pg/ml with no inhibitor, reduced to approximately 2000 pg/ml IL-6 in the presence of 50 ⁇ M inhibitor.
• Bone marrow mast cells isolated from pi lO ⁇ knockout mice were also assessed for IL-6 secretion levels after activation by antigen (100 ng/ml).
• pi lO ⁇ inhibitor up to 10 ⁇ M had no effect on levels of IL-6 in knockout cells, indicating that the effect of the inhibitor is through inhibition of the protein kinase pi lO ⁇ isoform.
• GM-CSF secretion by activated mast cells was also inhibited by approximately 45% after addition of the p 110 ⁇ inhibitor.
• Levels of the inflammatory cytokine IL-2 were also measured from antigen stimulated BMMC.
• BMMC activated with 10 ng/ml antigen showed a dose response inhibition of IL-2 secretion upon addition of p 110 ⁇ inhibitor, with levels decreased from control values of approximately 10 pg/ml to ⁇ 4 pg/ml IL-2 in the presence of 20 ⁇ M inhibitor, a 2.5 fold reduction in IL-2 secretion.
• Chemokines play a significant role in attracting cells to the site of inflammation thereby promoting the inflammatory response.
• Antigen activated BM mast cells were assessed for chemokine production in the presence and absence of pi lO ⁇ inhibitor using Multi-Analyte Profile Technology (Rules-Based Medicine, Inc. Austin, TX).
• pi lO ⁇ inhibitor (5 ⁇ M) on chemokine production from activated mast cells resulted in approximately 57% inhibition of eotaxin levels, approximately 37% inhibition of lymphotactin, approximately 60% inhibition of macrophage-derived chemokine (MDC) secretion, and approximately 35% inhibition in the levels of macrophage-inflammatory proteins MlP-l ⁇ and MlP-l ⁇ .
• MDC macrophage-derived chemokine
• Akt activation has previously been associated with secretion of cytokines from activated rat basophilic leukemia cells or cells isolated from tyrosine kinase knockout mice [Kitaura et al, J. Exp. Med. 192:729-39 (2000)]. Broad inhibition of class la PI3Ks in certain cell types, such as endothelium, using LY294002 has been shown to not only to reduce phosphorylation of Akt in response to TNF- ⁇ , but also in non-cytokine stimulated cells, as these lipid kinases are essential for both cell motility and survival. [See Madge et al, J. Biol. Chem., 275:15458-15465 (2000)]. Because Akt activation occurs through several different biochemical pathways, it was necessary to determine the role PI3K pi lO ⁇ played in triggering Akt phosphorylation in activated mast cells. To assess the effects of PI3K inhibitor on downstream signaling,
• BMMC were stimulated with a dose range of antigen and incubated with IC87114 as above with 0, 2 ⁇ M, 5 ⁇ M, 20 ⁇ M or 50 ⁇ M inhibitor.
• Cells were lysed as described above and phsophoserine or phosphotyrosine levels for several pi lO ⁇ downstream effectors were measured by Western blot.
• Antibodies used in the Western analysis include: Akt pS473 ; 3-phos ⁇ hoinositide-dependent kinase (PDKl) pS241 ; pERK; pJNK; Lyn pY396 and Bruton's tyrosine kinase (Btk) pY223.
• Antibodies were obtained from Cell Signaling Technology (Beverly, MA), Upstate Biotechnology (Lake Placid, NY), Zymed, and Santa Cruz Biotechnology.. Western blot analysis demonstrated that pi lO ⁇ inhibitor as low as 5 ⁇ M decreased Akt phosphorylation after 10 minutes of antigen stimulation and significantly inhibited phosphorylation after 30 minutes of antigen stimulation. The 50 ⁇ M inhibitor dose again significantly inhibited S243 Akt phosphorylation after 2 minutes of antigen stimulation, and completely abolished phosphorylation at 10 or 30 minutes of antigen stimulation.
• pi lO ⁇ selective inhibitor regulation of Akt which is involved in mast cell cytokine secretion, will provide an effective therapeutic method for modulating ongoing inflammation in an already developed mast cell disorder by reducing cellular infiltrate to the site of inflammation, or may act preventatively in disorders such as allergy or asthma to prevent the onset of an immune reaction.
• EXAMPLE 4 ALLERGEN-INDUCED AIRWAY INFLAMMATION LEADS TO INCREASED ACTIVITY OF PllO ⁇ IN LUNG TISSUE PI3K activity is stimulated after antigen challenge in a murine model of allergic asthma, and administration of wortmannin or LY294002, two broad- spectrum inhibitors of PI3Ks, attenuate inflammation and airway hyperresponsiveness (AHR) (Ezeamuzie et al., Am JRespir Crit Care Med 164:1633-39, 2001; Kwak et al., J. Clin. Invest 111 : 1083-92, 2003).
• AHR airway hyperresponsiveness
• pi lO ⁇ was reported to be essential for allergen-IgE-induced mast cell degranulation and vascular permeability (Ali et al., Nature 431:1007-11, 2004).
• OVA ovalbumin
• mice On days 21, 22, and 23 after the initial sensitization, the mice were challenged for 30 min with an aerosol of 3% (wt/vol) OVA in saline (or with saline as a control) using an ultrasonic nebulizer (NE-U12, Omron, Japan).
• IC87114 0.1 or 1 mg/kg body weight/day
• DMSO vehicle control
• IC87114 0.1 or 1 mg/kg body weight/day
• DMSO vehicle control
• PIP3 levels increased from approximately 15 pmol/ ml pre-challenge to approximately 75 pmol/ml at 1 hr, approximately 100 pmol/ml at 24 hr, and approximately 160 pmol/ml at 48 hrs and 72 hrs, indicating that class I PI3K activity increased approximately 4.6- , 6.1-, 9.5-, and 9.6-fold, respectively, after OVA inhalation, compared with the pre- challenge period. In contrast, no significant changes in PI3K activity were observed after saline inhalation. Activation of these kinases has been linked to phosphorylation of Ser- 473 of Akt, an event crucial for Akt enzymatic activation (Alessi et al., Curr.
• Akt levels were measured by Western blot. Protein extracts from lung tissue homogenates (30 ⁇ g/lane) were electrophoresed in polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA), transferred electrophoretically to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA), and incubated overnight at 4°C with anti phoshposerine antibodies as described above. Consistent with increased PI3K activity, levels of p-Akt protein in the lung tissues were also increased at 72 h after OVA inhalation, as detected by Western blot, compared with levels in the control animals that received saline inhalation. No significant changes in total Akt protein levels were observed.
• the level of phosphorylation was similar to saline control levels indicating that pi lO ⁇ contributed significantly to the overall class I PI3K activity in allergen-induced Akt activation. While PLP3 formation and Akt phosphorylation indicated that the PI3K family plays a role in allergy and asthma, administration of the pi lO ⁇ specific inhibitor described herein demonstrates that the important PI3K for allergy and asthma is pi lO ⁇ .
• mice were sacrificed with an overdose of pentobarbital- Na (100 mg/kg of body weight/administered intraperitoneally). Blood was drawn by puncture of the vena cava and centrifuged. Serum was shock frozen in liquid nitrogen and stored at -70°C for IgE measurements. BAL was performed as described (Kwak et al., supra). Briefly, the chest cavity was exposed to allow for expansion, after which the trachea was carefully intubated and the catheter secured with ligatures. Pre-warmed 0.9% NaCl solution was slowly infused into the lungs and withdrawn. Total BAL cells were counted using a hemocytometer.
• Differential cell counts were obtained from BAL cells spun down onto slides with a cytocentrifuge (Shannon Scientific Ltd., Cheshire, United Kingdom) and treated with Diff-Quik solution (Dade Diagnostics of Puerto Rico Inc. Aguada, Puerto Rico). Two independent, blinded investigators counted the cells using a microscope. Approximately 400 cells, in each of four different random locations were counted. The mean number from the two investigators was used to estimate the cell differentials. For cytokine and leukotriene measurements, supernatants of BAL were shock frozen in liquid nitrogen and stored at -70°C until use.
• OVA inhalation significantly increased the absolute numbers of eosinophils, lymphocytes, and neutrophils, as compared with saline control ( Figure 1).
• Intratracheal administration of IC87114 reduced the number of eosinophils, lymphocytes, and neutrophils detected in BAL fluids at 72 hours post challenge by 79.8%, 63.5%, and 80%, respectively, compared with mice treated with vehicle control.
• mice were sacrificed and the lungs and trachea were filled intratracheally with a fixative (0.8% formalin, 4% acetic acid) using a ligature around the trachea.
• Lungs were removed and lung tissues were fixed with 10% (vol/vol) neutral buffered formalin.
• the specimens were dehydrated and embedded in paraffin.
• 4 ⁇ m sections of fixed embedded tissues were cut on a Leica model 2165 rotary microtome (Leica Microsystems Nussloch GmbH,, Nussloch, Germany), placed on glass slides, deparaffinized, and stained with hematoxylin 2, eosin-Y (Richard- Allan Scientific, Kalamazoo, MI) and periodic acid-Schiff (PAS).
• OVA- exposed mice showed numerous inflammatory cells in the peribronchiolar zone and accumulation of mucus and cellular debris within the lumen of the bronchioles.
• IC87114-treated mice showed substantial attenuation in the eosinophil-rich leukocyte infiltration in the peribronchiolar regions and in the amount of debris present in the lumen.
• representative sections of each group were stained with periodic acid-Schiff (PAS) for detection of goblet cells.
• PAS periodic acid-Schiff
• EXAMPLE 6 EFFECTS OF PllO ⁇ INHIBITOR ON CYTOKINES AND CHEMOKINES IN ALLERGEN INDUCED AIRWAY INFLAMMATION Eosinophil accumulation and subsequent activation in bronchial tissues is known to play a critical role in the pathogenesis of allergic airway inflammation (Busse et al., NEnglJMed 344:35062, 2001; Humbles et al., Science 305:1776-79, 2004).
• Eosinophil transmigration into the airways is a multistep process that is orchestrated by Th2 cytokines (IL-4, IL-5, and IL-13), and coordinated by specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM- 1 and VLA-4 (10, 11).
• Th2 cytokines IL-4, IL-5, and IL-13
• specific chemokines such as eotaxin in combination with adhesion molecules such as VCAM- 1 and VLA-4 (10, 11).
• IL-13 is a potent inducer of eotaxin expression in airway epithelial cells (Tigani et al., Eur J Pharmacol 433:217-23, 2001) Given the essential role of Th2 cytokines in evoking allergic inflammatory responses, the concentrations of IL-4, IL-5, and IL-13 were measured from BAL fluid as well as in the lung tissue of OVA-challenged mice that received either pi 10 ⁇ inhibitor or vehicle control.
• IL-l ⁇ , TNF- ⁇ , IL-4, IL-5, IL-13, and RANTES were quantified in the supematants of BAL fluids by enzyme immunoassays according to the manufacturer's protocol (IL-l ⁇ , TNF- ⁇ , IL-4, and IL-5; Endogen, Inc., Woburn, MA; IL-13 and RANTES; R&D Systems, Inc., Minneapolis, MN).
• the lower detection limit for IL-l ⁇ , TNF ⁇ , IL-4, IL-5, IL-13, and RANTES in these assays was 3, 10, 5, 5, 1.5, and 2 pg/ml, respectively.
• BAL fluids were also increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation compared with the levels after saline inhalation, from approximately 100 pg/ml TNF ⁇ and 15 pg/ml IL-l ⁇ in control animals up to approximately 280 pg/ml TNF ⁇ and 30 pg/ml IL-1 ⁇ .
• IC87114 reduced the increased levels of these proinflammatory cytokines by more than 50%, down to approximately 140 pg/ml TNF ⁇ and 15 pg/ml IL-l ⁇ .
• One of the responses to these cytokines is the induction of leukocyte- endothelial adhesion molecules.
• ICAM-1 and VCAM-1 proteins in the lung tissue were increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation and these levels were substantially reduced by the administration of IC87114.
• Western blot analysis revealed that protein levels of the chemokines eotaxin and RANTES in the lung tissue were increased significantly (p ⁇ 0.05) at 72 h after OVA inhalation compared with the saline control.
• Administration of IC87114 reduced the increased levels of these chemokines by more than 50%.
• enzyme immunoassays revealed that increased levels of RANTES in BAL fluids at 72 10. h after OVA inhalation were also significantly (p ⁇ 0.05) reduced by IC87114 treatment. Effect ofIC87114 on serum IgE levels' and LTC4 release in BAL fluid IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE (Emson et 1., JExp Med 188:399-404, 1998). The biological 15 activities of IgE are mediated through high affinity IgE receptors (Fc ⁇ RI) on mast cells and basophils.
• Fc ⁇ RI high affinity IgE receptors
• TMBS 30 teframethylbenzidine substrate
• TMBS 30 teframethylbenzidine substrate
• the plates were read at 450 nm on a microplate reader (Molecular Dynamics, Sunnyvale, CA).
• Total serum IgE was measured by capture ELISA in a manner similar to the detection of OVA-specific IgE.
• a biotinylated rat anti-mouse IgE (PharMingen) was used to detect captured IgE in place of the biotinylated OVA.
• Substantial elevation in total IgE and OVA-specific IgE was observed in serum from OVA-challenged mice (approx. 12 ng/ml total IgE, approx.
• LTC 4 Levels of LTC 4 were quantified in the supematants of BAL fluids by enzyme immunoassay according to the manufacturer's protocol (Cayman Chemical Co., Ann Arbor, MI). The lower detection limit for LTC 4 in this assay was 10 pg/ml.
• the BAL fluid levels of the LTC 4 were 3.1-fold higher in the OVA- sensitized/challenged mice (approximately 40 pg/ml) than in the mice receiving saline only (approximately 13 pg/ml) (p ⁇ 0.05 compared to saline).
• IC87114 (0.1 and 1 mg/kg), significantly (p ⁇ 0.05) inhibited LTC 4 levels by 37 and 50%, respectively decreasing LTC 4 levels to approximately 26 pg ml and 21 pg/ml, respectively.
• the amounts of LTC 4 in the BAL fluid of OVA-sensitized/challenged mice treated with vehicle control were not significantly different from those of the saline control group.
• Start of an inspiration is determined by extrapolating from a straight line drawn from two levels of the rising inspiratory phase of the box pressure signal.
• Time of inspiration (TI) is defined as the time from the start of inspiration to the end of inspiration; time of expiration (TE) as the time from the end of inspiration to the start of the next inspiration.
• the maximum box pressure signal occurring during one breath in a negative or positive direction is defined as peak inspiratory pressure (PIP) or peak expiratory pressure (PEP), respectively. Recordings of every 10 breaths are extrapolated to define the respiratory rate in breaths per minute.
• the relaxation time (Tr) is defined as the time of pressure decay to 36% of the total expiratory pressure signal (area under the box pressure signal in expiration).
• Airway responsiveness was substantially increased in the OVA- challenged group in response to methacholine inhalation as compared with the saline- challenged group (Figure 3).
• Administration of IC87114 to OVA-sensitized mice prior to OVA challenge showed a significant (p ⁇ 0.05) attenuation in Penh measured at all methacholine levels tested suggesting a role for pi lO ⁇ in immune-mediated events leading to airway hyperresponsiveness in vivo.
• These results are contemplated to be associated with reduction in Th2 cytokine production, tissue eosinophilia, and mast cell activation, following pllO ⁇ inhibition.
• Allergic airway inflammation and AHR development involve multiple inflammatory cells and a wide array of mediators.
• EXAMPLE 8 EFFECTS OF PllO ⁇ INHIBITORS ON RAT MAST CELL DEGRANULATION Mast cells and basophils express Fc ⁇ RI, the high affinity receptor for
• IgE rat basophil leukemia cells
• RBL-2H3 rat basophil leukemia cells
• Cells were then plated in a 24 well plate at a concentration of 4 x 10 5 cells/ml, and cultured with 25 ⁇ l of 1 mCi/ml 3 H-labeled serotonin (0.5 ⁇ Ci/ml final) and 1 ⁇ g/ml anti-DNP IgE overnight at 37°C. Cell media was aspirated from the wells and cells washed twice by adding 500 ⁇ l of PBS to the well and inverting the plate onto a stack of paper towels!
• a final volume of 200 ⁇ l PBS was added and cells equilibrate ⁇ 2 min in a 37°C water bath, and 10 ⁇ l DNP- albumin was added to each well (10 ng/ml final) and incubated for the 10 to 30 min at 37°C.
• the reaction was stopped by transferring the buffer from each well into a liquid scintillation vial.
• Wells were washed two times using 500 ⁇ l of 1% Triton X- 100 in PBS incubated 10 min at room temperature, and the liquid transferred to the vial for measuring.
• pi lO ⁇ inhibitors were effective at reducing the levels of mast cell degranulation mediated by IgE crosslinking.
• Mast cell degranulation plays a significant role in mediating allergic reactions and other type I hypersensitivity responses in vivo.
• an animal model of dermal hypersensitivity was used. To sensitize, the shaved dorsal skin of Lewis rats was injected with either saline or anti-DNP monoclonal IgE (1.25 to 25 ng in 50 ⁇ l per site) intradermally.
• Ketotifen (10 mg/kg) was intraperitoneally injected 30 minutes before antigen challenge. Blood samples were drawn immediately after the wheal size measurements and plasma concentration of IC87114 was determined by determined liquid-liquid extraction by liquid chromatography/mass spectroscopy as described previously (Puri et al., Blood 103:3448-3456, 2004). Type I hypersensitivity responses showed a dose dependent response to IC87114, decreasing to approximately 70% of control at a dose of 20 mg/kg, to approximately 55% at a dose of 60 mg/kg. The positive control ketotifen (10 mg/kg) decreased sensitivity responses to approximately 35% of the control response. These results indicate that pi lO ⁇ plays a role in mediating type I hypersensitivity reactions in sensitized animals, indicating that administration of pi lO ⁇ inhibitors may reduce or prevent type I sensitivity reactions
• EXAMPLE 10 EFFECTS OF PllO ⁇ INHIBITOR ON HUMAN MAST CELL DEGRANULATION To determine the effect of pi lO ⁇ inhibitors on human mast cells, cells were isolated from human cord blood, differentiated to mast cell lineage and assayed for degranulation and histamine release in the presence of p 11 O ⁇ inhibitors .
• CD34 + human cord blood cells were isolated and differentiated using stem cell factor and IL-4 following the protocol set out in Hsieh et al. (JExp Med. 193:123-33, 2001), in the presence and absence of methylcellulose (Iida et al, Blood 97:1016-22, 2001). Cells were cultured for approximately 5 weeks and were harvested by centrifugation at 1000 rpm for 3 minutes.
• D-PBS Dulbecco's- phosphate buffered saline

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