Classifications

• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • 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
  • A61K31/12—Ketones
• • 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
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/42—Oxazoles
  • A61K31/423—Oxazoles condensed with carbocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • 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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • 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
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
  • A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
  • C12Y301/04053—3',5'-Cyclic-AMP phosphodiesterase (3.1.4.53)

Definitions

• the present invention relates to compositions and methods that combine the use of at least two agents that inhibit phosphodiesterases in the family defined as Family 4 (PDE4) and, more specifically, to combination therapies that include compounds such as roflumilast and a second active agent that inhibits a PDE4 of the B sub-family (i.e., a PDE4B such as PDE4B2).
• PDE4B Family 4
• the compositions and methods will attenuate an unwanted up-regulation of a PDE4B (e.g., PDE4B2) and may thereby improve treatment with the first agent (e.g., roflumilast).
• the second agent may improve the efficacy of the first agent, decrease the effective dose of the first agent, ameliorate the tolerance to the first agent that would otherwise develop (e.g., in patients with COPD exacerbation), reduce unwanted side effects caused by the first agent, or otherwise improve treatment regimes including administration of a PDE4 inhibitor.
• Phosphodiesterases are important enzymes because they hydrolyze and thereby inactivate the second messengers cAMP and cGMP (adenosine 3 '5 '-cyclic monophosphate and guanosine 3'5'-cyclic monophosphate, respectively).
• cAMP and cGMP adenosine 3 '5 '-cyclic monophosphate and guanosine 3'5'-cyclic monophosphate, respectively.
• PDEs also play a vital role in intracellular localization of cyclic nucleotide signaling and integrate those pathways with other signaling pathways (for review, see Halpin, Intl. J. of COPD 3(4):543-561, 2008).
• PDE4-PDE11 a super family of enzymes containing at least eleven families
• PDE4-PDE11 a super family of enzymes containing at least eleven families
• at least 21 genes have been identified to date that encode PDEs, and many studies have focused on the physiochemical and regulatory properties of the encoded proteins (see, e.g., Conti and Jin, Prog. Nucleic Acid Res. Mol. Biol. 63:1-38; Soderling and Beavo, Curr. Opin. Cell Biol. 12: 174-179, 2000; and Francis et al. , Prog. Nucleic Acid Res. Mol. Biol. 65: 1-52, 2001).
• Some of the 11 families include more than one member, with each member of the sub-family being identified by a capital letter after the Arabic number identifying the family (e.g., PDE4A, PDE4B, PDE4C, and PDE4D). Even further, most of the genes encoding PDEs have more than one promoter and the coding sequence is alternatively spliced.
• the splice variants are designated by a further Arabic numeral following the sub-family designation. For example, PDE4D3 is a PDE within family 4, sub-family D, and is designated as the third splice variant. There are at least 100 different PDE open reading frames (Halpin, supra, referencing Conti and Beavo, Annu. Rev. Biochem. 76:481-511, 2007).
• Phosphodiesterase 4B plays a key role in regulating inflammation.
• the present invention features pharmaceutical compositions including two active agents; a first agent that inhibits a phosphodiesterase in Family 4 (PDE4; e.g., a compound conforming to Formula I, such as roflumilast) and a second active agent that inhibits the expression or activity of a PDE4 in Family B (PDE4B; e.g., PDE4B2).
• PDE4 phosphodiesterase in Family 4
• PDE4B Family B
• the second active agent can inhibit the expression or activity of the PDE4B (e.g., PDE4B2) directly or indirectly.
• a direct inhibitor may inhibit the expression of the gene encoding PDE4B or directly bind to or otherwise directly interact with a PDE4B (e.g.
• compositions include a second active agent that indirectly inhibits the expression or activity of a PDE4B (e.g., PDE4B2), that agent may inhibit the expression of ⁇ , ⁇ ⁇ , p50, NFKB p65, or PKA-Cp or the activity of the expressed proteins (e.g., one could inhibit the formation of a complex including p50 and p65 (e.g., the ⁇ - ⁇ 50- ⁇ 65 complex or the ⁇ 50- ⁇ 65- ⁇ - ⁇ complex shown in Fig. 1).
• the first and second agents can be, independently, chemical compounds or biological compounds (e.g., a nucleic acid, peptide nucleic acid (PNA), or polypeptide).
• the first agent is a chemical compound and the second agent is a nucleic acid; in another embodiment, both the first and second agents are chemical compounds.
• the present compositions can include a compound of Formula I (e.g., roflumilast) as the first agent and an alkenyldiarylmethane (ADAM) compound (e.g., ADAM5 or ADAM6), a derivative thereof, or salt thereof that inhibits the activity of a PDE4B (e.g., PDE4B2).
• ADAM alkenyldiarylmethane
• the second active agent can be a steroid (e.g., a glucocorticoid such as dexamethasone, Cortisol, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA) and aldosterone).
• the second active agent is dexamethasone, curcumin, or HIF- ⁇ inhibitor.
• the pharmaceutical compositions can be formulated in various ways for administration to a patient or for use in the preparation of a medicament (e.g., a medicament for inflammation, such as an inflammatory lung disease, or for any other disease, disorder, or condition described herein as amenable to treatment).
• a medicament for inflammation such as an inflammatory lung disease, or for any other disease, disorder, or condition described herein as amenable to treatment.
• the present compositions can be formulated as particles for administration by inhalation, as a cream, gel, or ointment for topical administration, as a tablet or capsule for oral administration, or as a solution or suspension for injection (e.g., intravenous, intramuscular, intraperitoneal, or subcutaneous injection).
• the pharmaceutical compositions can include less of the first agent than would otherwise be required to achieve a certain outcome if the first agent were administered in the absence of the second agent.
• the first agent is a compound conforming to Formula I
• the amount of that compound administered in the presence of a second agent to achieve a given clinical result can be less than the amount of that compound administered in the absence of the second to achieve substantially the same result.
• the first agent is a compound of Formula I (e.g. , roflumilast) that is present in a unit dosage form in the amount of about 500 meg.
• the term "about” means ⁇ 10% of a referenced value (e.g.
• about 500 meg of roflumilast is 450-550 meg of roflumilast) or a value that includes an inherent variation of error for the device or the method being employed to determine the value, whichever is greater. While we may describe the methods herein as methods of treatment, any such descriptions can be equally well presented as "uses" and the invention can be claimed as the use of a composition described herein in, for example, the preparation of a medicament or in the preparation of a medicament for treating a disease, disorder or condition described herein (in accordance with varying patent practices throughout the world).
• the first and second active agents can be nucleic acid molecules that selectively inhibit the expression of a PDE4 (e.g. , a PDE in the 4 A or 4B subfamilies) or, in the case of the second agent, selectively inhibit a PDE4B (e.g. , PDE4B2).
• a PDE4 e.g. , a PDE in the 4 A or 4B subfamilies
• a PDE4B e.g. , PDE4B2
• kits including the compositions described herein.
• the first and second agents can be combined or contained separately, together with instructions for use.
• the invention features a kit comprising roflumilast, a second active agent that inhibits the expression or activity of a PDE4B (e.g., PDE4B2), instructions for use, and, optionally, one or more of a diluent, delivery device or dressing for use in administering the first or second active agent to a patient.
• a PDE4B e.g., PDE4B2
• the invention features an isolated cell that stably overexpresses a PDE4B (e.g. , PDE4B2).
• the cell may be a primary cell or may be immoralized and can be one of an established cell line.
• the cell can be a human cell. These cells can be used, among other things, in screening assays to identify inhibitors of PDE4/PDE4B/PDE4B2-mediated
• the PDE4/PDE4B/PDE4B2 gene product can be expressed from an expression vector such as a viral vector or plasmid or integrated into the cell's genome.
• the overexpression can be driven from a constitutively active or inducible promoter (e.g. , a promoter activated by transcription factors present in a tissue affected by a disease described herein (e.g. , a lung tissue-specific promoter).
• the invention features methods of treating a patient who has a condition as described herein (e.g. , an inflammatory lung disease such as COPD).
• the methods can be carried out by administering to the patient a therapeutically effective amount of at least one first agent (e.g., a compound of Formula I, such as roflumilast, or rolipram or cilomilast) and at least one second agent that inhibits the expression or activity of a PDE4B (e.g., PDE4B2).
• the first and second agents can be combined in a single dosage form or maintained in two dosage forms that are administered concurrently or sequentially by the same or different routes of
• the first agent can be administered orally, and the second agent can be administered topically (e.g., as eardrops to the ear to treat otitis media) by inhalation (e.g., to directly access the lungs), or by injection (e.g., intravenously).
• both the first and second agents are administered by inhalation (e.g., as a dry powder formulation in which the active agents are associated with a nanoparticle such as a liposome).
• the invention encompasses dry powder formulations of the first and second agents as well as dry powder and other formulations (e.g., liquids for infusion) in which one or more of the active agents are associated with a nanoparticle, such as a liposome.
• the invention features methods of identifying an inhibitor of PDE4B (e.g., PDE4B2).
• PDE4B2 regulates the expression of pro-inflammatory chemokines, including chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-C motif) ligand 7 (CCL7), C-X-C motif chemokine 10 (CXCLIO), and C-X-C motif chemokine 11 (CXCLll) (with CCL5, CCL7, CXCLIO, and CXCLll being referred to as the Group A chemokines), at least in part in a manner that is independent from PDE4B2's well known enzymatic activity.
• the assay can be variously configured in cultured cells or in vitro and identifies potential PDE4B2 inhibitors by their ability to suppress ⁇ -CA-induced NF-KB promoter activity and Group A chemokine expression in cells transfected with wild type
• the methods can include the steps of providing cells (e.g., cells such as BEAS-2B cells) transfected with an NF- ⁇ reporter vector (e.g., a vector including a detectable tag or detection system such as a luciferase-based system).
• the cells can optionally express or overexpress (e.g., by way of transfection) ⁇ -CA and a wild type and/or mutant PDE4B2.
• NF- ⁇ activity can be measured and the cells can be exposed to a potential inhibitor (e.g., cells in culture can be exposed to an inhibitor for about 1-12 hours) before measuring the expression or activity of NF- ⁇ or a Group A chemokine.
• a potential inhibitor that suppresses the expression or activity of NF- ⁇ or one or more of the Group A chemokines can be tested further as an inhibitor of PDE4B2.
• Fig. 1 is a schematic representation of PDE4B2 induction by NTHi and roflumilast via p65 and PKA-Cp.
• Figs. 2A-2I show results indicating that roflumilast synergizes with NTHi to up-regulate PDE4B2 expression in vitro and in vivo.
• Figs. 2A-2F PDE4B mRNA expression was analyzed.
• Fig. 2A is a bar graph illustrating PDE4B mRNA expression in BEAS-2B cells pretreated with roflumilast (Rof; 0.1 ⁇ ) for 1 h followed by a 1.5-hour stimulation with NTHi (MOI of 25, 50, and 100). At each multiplicity of infection, PDE4B mRNA was increased in the Rof-treated, NTHi-stimulated cells relative to control.
• Fig. 2A-2I show results indicating that roflumilast synergizes with NTHi to up-regulate PDE4B2 expression in vitro and in vivo.
• Figs. 2A-2F PDE4B mRNA expression was analyzed.
• BEAS-2B cells were pretreated with Rof (0.01, 0.1, and 1 ⁇ ) for 1 h followed by a 1.5-hour stimulation with NTHi.
• Rof 0.01, 0.1, and 1 ⁇
• PDE4B mRNA was increased in the Rof-treated, NTHi-stimulated cells relative to control.
• Fig. 2C primary NHBE cells were pretreated with Rof (0.1 ⁇ ) for 1 hour followed by a 1.5- hour stimulation with NTHi, increasing PDE4B mRNA significantly relative to control cells.
• BEAS-2B cells were pretreated with the transcriptional inhibitor actinomycin D (ActD; 5 ng/ml) and Rof (0.1 ⁇ ) for 1 hour followed by a 1.5-hour stimulation with NTHi.
• mice were inoculated with Rof (5 mg/kg i.p.) for 2 hours, followed by intratracheal inoculation with NTHi (5 x 10 7 cfu per lung). After 5 hours, PDE4B mRNA expression in lung tissues was analyzed and found to be significantly elevated in Rof-treated, NTHi-stimulated cells.
• Rof 5 mg/kg i.p.
• NTHi 5 x 10 7 cfu per lung
• Fig. 3 is a bar graph illustrating that PDE4B siRNA markedly depleted PDE4B2 expression in BEAS-2B cells; see Example 2; PDE4B is required for NTHi-induced expression of
• Figs. 4A-4B show that increased expression of PDE4B2 enhances NTHi-induced expression of chemokines.
• Fig. 4A is a photograph of a Western blot showing PDE4B2 expression in mock-transfected cells and cells that stably express PDE4B2.
• Fig. 4B is a panel of bar graphs illustrating the increased expression of the cytokines CCL5, CCL7, CXCL10, and CXCL11 in the mock- and PDE4B2-transfected cells treated as indicated with NTHi and Rof. The cells were pretreated with Rof for 1 hour followed by a five-hour stimulation with NTHi before mRNA expression was analyzed.
• Fig. 5 is a photograph of a Western blot; protein expression was analyzed after a 48-hour transfection with siRNAs directed to PKA-Ca and PKA-Cp in BEAS-2B cells, ⁇ -actin was analyzed as a control.
• Fig. 6 is a cartoon illustrating a drug and an siRNA, either of which may serve as a first or second agent as described herein, associated with a lipid-based nanoparticle for administration to a patient.
• Figs. 7A-7H are a collection of data generated primarily from studies aimed at determining whether dexamethasone can suppress the induction of PDE4B by NTHi and Rof.
• Fig. 8 is a representation of the mRNA sequence of human PDE4B (SEQ ID NO:l).
• Fig. 9 is a representation of the nucleic acid sequence of a human PDE4B2 (SEQ ID NO:2).
• Figs. 10A andlOB are schematic representations of the domains present in PDEs in the eleven known PDE families (A; from Halpin, supra, and originally published by Conti and Beavo, Annu. Rev. Biochem. 76:481-511, 2007) and a comparison of the splice variants in PDE4 family (B).
• compositions of the present invention include formulations (e.g., pharmaceutical or physiologically acceptable formulations) that can include two or more active agents that are useful in the treatment of inflammation (e.g. , an inflammatory lung disease, such as COPD).
• a pharmaceutical composition/formulation is a non-naturally occurring composition including at least one active agent, and such compositions are considered physiologically acceptable in the sense that they are non-toxic at the dosages prescribed.
• compositions of the invention are not limited to those that achieve a desired outcome by way of any particular mechanism of action, the compositions can include a first agent that inhibits the expression or activity of a PDE4 family member and a second agent that inhibits the expression or activity of one or more of the PDEs in the "B family" (i.e., a PDE4B, such as PDE4B2).
• a PDE4B such as PDE4B2
• first agent and second agent are used to indicate that the two agents are different from one another.
• each of the first and second agents may inhibit more than one PDE4 family member and may inhibit one or more of the same PDE4 family members.
• both the first and second agents may inhibit a phosphodiesterase in family 4, sub-family B, and both the first and second agents may inhibit more than one of the splice variants within a sub-family.
• both the first and second agents may inhibit PDE4B1 and PDE4B2.
• the first and/or second agent inhibits a phosphodiesterase in family 4, sub-family B, to the exclusion of PDEs in other families or subfamilies.
• the first and/or second agent may inhibit one or more of the splice variants of PDE4B but not any of the splice variants of PDE4D.
• active and “pharmaceutically active” to refer to the ability of an agent to affect its target (e.g. to activate, inhibit, up-regulate, or down-regulate) in vivo.
• active the effect an agent has on its target must be sufficient to confer a clinical benefit to a specific patient or generally to a population of patients (recognizing that a response can vary from person-to-person and may not be effective in some individuals).
• inhibitor refers to the ability of an agent to reduce the expression or activity of a stated target (here, typically an enzyme in the PDE4 family). While the inhibition does not have to achieve a complete and total reduction in the target's expression or activity, the reduction must occur to such an extent that it confers a benefit to a specific patient or generally to a population of patients (recognizing that a response can vary from person-to-person and may not be effective in some individuals). In the present case, that benefit can be, for example, an improved reaction to treatment with a PDE4 inhibitor such as roflumilast, cilomilast, or another PDE4 inhibitor disclosed herein. As noted elsewhere herein, the inhibitor may exert its action on the target directly (e.g., by inhibiting the
• the first agent and the second agent can be, independently, a chemical compound (e.g., a carbon-based small molecule having a molecular mass less than about 1,000 g/mol; a chemical compound may be referred to herein as a "drug"), a nucleic acid (e.g., a nucleic acid that mediates RNAi, a microRNA, or an antisense oligonucleotide), or a polypeptide (e.g., an antibody).
• a chemical compound e.g., a carbon-based small molecule having a molecular mass less than about 1,000 g/mol; a chemical compound may be referred to herein as a "drug”
• a nucleic acid e.g., a nucleic acid that mediates RNAi, a microRNA, or an antisense oligonucleotide
• a polypeptide e.g., an antibody
• compositions and methods can include more than one type of first agent and more than one type of second agent.
• the first agent can be a PDE4 inhibitor.
• the first agent can be an inhibitor of one or more of PDE4 isoforms, including, PDE4A (e.g., PDE4A1, PDE4A5, PDE4A8, PDE4A10, PDE4A11, and PDE4A7), PDE4B (e.g., PDE4B1, PDE4B2, PDE4B3, and PDE4B4), PDE4C (e.g., PDE4C1), and PDE4D (e.g., PDE4D1, PDE4D2, PDE4D3, PDE4D4, PDE4D5, PDE4D6, PDE4D7, PDE4D8, and PDE4D9).
• PDE4A e.g., PDE4A1, PDE4A5, PDE4A8, PDE4A10, PDE4A11, and PDE4A7
• PDE4B e.g., PDE4B1, PDE4B2,
• one of the substituents Rl and R2 is hydrogen, l-6C-alkoxy, 3-7C-cycloalkoxy, 3-7C-cycloalkylmethoxy, benzyloxy or 1-4C- alkoxy that is completely or partially substituted by fluorine, and the other is l-4C-alkoxy that is completely or partially substituted by fluorine;
• R3 is phenyl, pyridyl, phenyl that is substituted by R31, R32 and R33 or pyridyl that is substituted by R34, R35, R36 and R37, where
• R31 is hydroxyl, halogen, cyano, carboxyl, trifluoromethyl, l-4C-alkyl, l-4C-alkoxy, 1- 4C-alkoxycarbonyl, l-4C-alkylcarbonyl, l-4C-alkylcarbonyloxy, amino, mono- or di-l-4C- alkylamino or l-4C-alkylcarbonylamino;
• R32 is hydrogen, hydroxyl, halogen, amino, trifluoromethyl, l-4C-alkyl or l-4C-alkoxy;
• R33 is hydrogen, halogen, l-4C-alkyl or l-4C-alkoxy
• R34 is hydroxyl; halogen, cyano, carboxyl, alkyl, l-4C-alkoxy, l-4C-alkoxycarbonyl or amino;
• R35 is hydrogen, halogen, amino or l-4C-alkyl
• R36 is hydrogen or halogen
• R37 is hydrogen or halogen
• Rl is l-4C-alkoxy that is completely or partially substituted by fluorine.
• Rl is methoxy which is completely or partially substituted by fluorine.
• Rl is difluoromethoxy
• R2 is 3-5C-cycloalkoxy or 3-5C-cycloalkylmethoxy.
• R2 is 3-5C-cycloalkylmethoxy.
• R2 is cyclopropylmethoxy.
• R3 is pyridyl, or pyridyl that is substituted by R34, R35, R36 and R37.
• R3 is pyridyl that is substituted by R34, R35, R36 and R37.
• R3 is 3,5-dichloropyrid-4-yl.
• Rl is l-4C-alkoxy that is completely or partially substituted by fluorine;
• R2 is 3-5C-cycloalkoxy or 3-5C-cycloalkylmethoxy; and
• R3 is pyridyl, or pyridyl that is substituted by R34, R35, R36 and R37.
• Rl is methoxy which is completely or partially substituted by fluorine;
• R2 is 3-5C-cycloalkylmethoxy; and
• R3 is pyridyl that is substituted by R34, R35, R36 and R37.
• R34 is halogen or l-4C-alkyl
• R35 is hydrogen, halogen
• R36 is hydrogen or halogen
• Rl is difluoromethoxy
• R2 is cyclopropylmethoxy
• R3 is 3,5-dichloropyrid-4-yl.
• l-6C-Alkoxy is a radical which, in addition to the oxygen atom, contains a straight-chain or branched alkyl radical having 1 to 6 carbon atoms.
• Alkyl radicals having 1 to 6 carbon atoms which may be mentioned in this context are, for example, the hexyl, isohexyl (2-methylpentyl), neohexyl (2,2-dimethylbutyl), pentyl, isopentyl (3-methylbutyl), neopentyl (2,2-dimethylpropyl), butyl, isobutyl, sec-butyl, tert-butyl, propyl, isopropyl, ethyl and methyl radicals.
• 3-7C-Cycloalkoxy is, for example, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy and cycloheptyloxy.
• 3-7C-Cycloalkylmethoxy is, for example, cyclopropylmethoxy, cyclobutylmethoxy, cyclopentylmethoxy, cyclohexylmethoxy and cycloheptylmethoxy.
• l-4C-Alkoxy which is completely or partially substituted by fluorine is, for example, the 1,2,2-trifluoroethoxy, the 2,2,3,3,3-pentafluoropropoxy, the perfluoroethoxy and in particular the 1,1,2,2-tetrafluoroethoxy, the trifluoromethoxy, the 2,2,2-trifluoroethoxy and the
• Halogen within the meaning of the present invention is bromine, chlorine and fluorine.
• 1-4C-Alkyl is a straight-chain or branched alkyl radical having 1 to 4 carbon atoms.
• l-4C-Alkoxy is a radical which, an addition to the oxygen atom, contains one of the abovementioned l-4C-alkyl radicals. Examples are the methoxy and the ethoxy radicals.
• l-4C-Alkoxycarbonyl is a carbonyl group to which one of the abovementioned 1-4C- alkoxy radicals is bonded. Examples are the methoxycarbonyl (CH 3 O-CO-) and the
• l-4C-Alkylcarbonyl is a carbonyl group to which one of the abovementioned 1-4C- alkyl radicals is bonded.
• An example is the acetyl radical (CH 3 CO-).
• 1-4C- Alkyl carbonyloxy radicals contain, in addition to the oxygen atom, one of the abovementioned l-4C-alkylcarbonyl radicals.
• An example is the acetoxy radical (CH 3 CO-O-).
• Mono- or di-l-4C-alkylamino radicals are, for example, the methylamino, the
• a l-4C-alkylcarbonylamino radical is, for example, the acetamido radical
• Exemplary phenyl radicals substituted by R31, R32 and R33 are the radicals 2- acetylphenyl, 2-aminophenyl, 2-bromophenyl, 2-chlorophenyl, 2,3-dichlorophenyl, 2,4- dichlorophenyl, 4-diethylamino-2-methylphenyl, 4-bromo-2-trifluoromethylphenyl, 2-carboxy-5- chlorophenyl, 3,5-dichloro-2-hydroxyphenyl, 2-bromo-4-carboxy-5-hydroxyphenyl, 2,6- dichlorophenyl, 2,5-dichlorophenyl, 2,4,6-trichlorophenyl, 2,4,6-trifluorophenyl, 2,6- dibromophenyl, 2-cyanophenyl, 4-cyano-2-fluorophenyl, 2-fluorophenyl, 2,4-difluorophenyl, 2,6-difluorophenyl
• Exemplary pyridyl radicals substituted by R34, R35, R36 and R37 are the radicals 3,5- dichloropyrid-4-yl, 2,6-diaminopyrid-3-yl, 4-aminopyrid-3-yl, 3-methylpyrid-2-yl, 4- methylpyrid-2-yl, 5-hydroxypyrid-2-yl, 4-chloropyrid-3-yl, 3-chloropyrid-2-yl, 3-chloropyrid-4- yl, 2-chloropyrid-3-yl, 2,3,5,6-tetrafluoropyrid-4-yl, 3,5-dichloro-2,6-difluoropyrid-4-yl, 3,5- dibromopyrid-2-yl, 3,5-dibromopyrid-4-yl, 3,5-dichloropyrid-3-yl, 3,5- dimethylpyrid-4
• Suitable salts of the compounds described herein are all acid addition salts, but in particular all salts with bases.
• the salts can be pharmacologically tolerable, inorganic or organic acids and bases customarily used in pharmacy.
• Pharmacologically intolerable salts which can be obtained, for example, as process products during the preparation of the compounds on an industrial scale, are converted into pharmacologically tolerable salts by processes known to one of ordinary skill in the art.
• Suitable salts include water-soluble and water-insoluble acid addition salts with acids, such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulphuric acid, acetic acid, citric acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)benzoic acid, butyric acid, sulphosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid, stearic acid, toluenesulphonic acid,
• acids such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulphuric acid, acetic acid, citric acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)benzoic acid, butyric acid, sulphosalicylic acid, maleic acid,
• the acids being employed in salt preparation in an equimolar quantitative ratio or one differing therefrom depending on whether a mono- or polybasic acid is concerned and depending on which salt is desired.
• Other suitable salts are salts with bases. Examples of basic salts are lithium, sodium, potassium, calcium, aluminum, magnesium, titanium, ammonium, meglumine, tromethamine, or guanidinium salts.
• the bases being employed in basic salt preparation can be present in an equimolar quantitative ratio or one differing therefrom.
• the first agent is roflumilast or a salt thereof:
• first agent is rolipram or a salt thereof:
• Chemical compounds useful in the present compositions and methods can be purchased or synthesized, isolated, or purified by methods known in the art.
• Other compounds that can be employed as the first agent include cilomilast (developed by GlaxoSmithKline; see Christensen et al., J. Med. Chem. 41:821-835, 1998); BAY 19-8004 (developed by Bayer PLC; see
• ONO-6126 developed by Ono Pharmaceuticals; see Furuie et al, Eur. Respir. J. 22(SuppI 45):395s, 2003
• CI- 1044 developed by Pfizer; see Ouagued et al, Pulm. Pharmacol. Ther. 18:49-54, 2005
• HT-0712 developed by Inflazyme/Helicon; see MacDonald et al, Neurorehabil. Neural Repair 21:486-496, 2007
• ibudilast developed by Merck-Frosst; see Huang et al, Life. Sci.
• YM-976 developed by Yamanouchi; see Aoki et al., J. Pharmacol. Exp. Ther. 295:255-260, 2000
• V-11294A developed by Napp; see Gale et al., Br. J. Clin. Pharmacol. 54:478-484, 2002
• piclamilast, RP-73401 developed by Rohne-Poulenc-Rorer; see Chen et al, Acta Pharmacol. Sin. 25:1171-1175, 2004
• atizoram, CP-80633 developed by Pfizer; see Wright et al., Can. J. Physiol. Pharmacol.
• AN2728 under development by Anacor Pharmaceuticals
• apremilast developed by Celgene
• diazepam developed by Hoffmann-La Roche 1963
• luteolin supplied from peanuts that also possesses IGF-1 properties
• mesembrenone an alkaloid from the herb Sceletium tortuosum
• the first agent can up-regulate the expression or increase the activity of one or more PDE4 isoforms, including PDE4B isoforms, either by itself or in concert with other factors in the biological environment.
• PDE4B isoform can be PDE4B1,
• PDE4B2, PDE4B3, or PDE4B4 and the factors in the biological environment can include one or more of cyclic adenosine monophosphate (cAMP) elevators, lipopolysaccharide (LPE), or bacteria.
• cAMP cyclic adenosine monophosphate
• LPE lipopolysaccharide
• the bacteria can be nontypeable Haemophilus influenzae (NTHi).
• the Second Agent Any of the first agents described herein can be formulated with, packaged with, or administered with one or more of a second agent that inhibits, directly or indirectly, the expression or activity of a PDE4B ⁇ e.g., PDE4B2).
• the second agent can inhibit one or more other PDE4B isoforms, either instead of PDE4B2 or in addition to PDE4B2.
• the second agent can inhibit one or more of the splice Variants of PDE4B but not any of the splice variants of PDE4D.
• the second agent can inhibit one or more of the genes and/or enzymes that can up- regulate expression of a PDE4B isoform (e.g., PDE4B2).
• the second agent may inhibit the expression of ⁇ kinase ⁇ ( ⁇ ), ⁇ , transcription factor nuclear factor ⁇ (NF B) or a subunit thereof (e.g., p50 or p65), or protein kinase A-CP (PKA-Cp) or the activity of the expressed proteins (e.g., one could inhibit the formation of a complex including p50 and p65 (e.g., the ⁇ - ⁇ 50- ⁇ 65 complex or the p50-p65-PKA-Cp complex shown in Fig. 1)
• the second agent can inhibit one or more of the cellular pathways that PDE4B (e.g., PDE4B2) may up-regulate.
• PDE4B e.g., PDE4B2
• the pathway can be functioning in an enzymatic activity-dependent manner or an enzymatic activity-independent manner.
• the second agent can be a glucocorticoid, for example, dexamethasone or a biologically active derivative thereof:
• glucocorticoids include Cortisol, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), aldosterone, budesonide, hydrocortisone, triamcinolone, and pharmaceutically acceptable salts thereof.
• the second agent can also be an HIF- ⁇ inhibitor.
• the second agent can be hypoxia inducible factor- la inhibitor, dimethyloxaloylglycine (DMOG), chrysin, chetomin, YC-1, dimethyl-bisphenol A, 2-methoxyestradiol, IOX2, BAY 87-2243, PX-478 2HCI, FG- 2261, KC7F2, cryptotanshinone, EF-24, FM19G11, or PX 12.
• the second agent can be 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) or a derivative or salt thereof:
• the second agent can also be curcumin (i.e. , (lE,6E)-l,7-Bis(4-hydroxy-3- methoxyphenyl)-l,6-heptadiene-3,5-dione) or a derivative or salt thereof:
• the second agent can be or include ADAM5 or ADAM6 (Cullen et al, Bioorg. Med. Chem. Lett. 18:1530-1533 2008) or a pharmaceutically active derivative or salt thereof:
• the second agent can also be or include compound A-33 developed from a lead 2- arylpyrimidine derivative as described by Naganuma et al (Bioorganic & Medicinal Chemistry Letters 19(12):3174-3176, 2009), including compound 33 or a pharmaceutically active derivative or salt thereof:
• Triazine derivatives are also known to be potent inhibitors of PDE4B and can be used as second agents in the various embodiments of the present invention (Hagen et al., Bioorg. Med. Chem. Lett. 24(16) :4031-4034, 2014). Structural studies known in the art have demonstrated that potent and selective PDE4B inhibitors can bind CR3 (Control Region 3, located on the carboxyl side of the catalytic domain) and thereby lock the enzyme in a closed conformation. PDE4B selectivity is believed to be due to a single amino acid polymorphism in CR3 that selects the helical registration of the domain when it closes over the active site.
• each of R4, R5, and R6 is hydrogen, halogen, cyano, carboxyl, akyl, aryl, or hetercycle and can be optionally substituted with one or more halogen, cyano, carboxyl, akyl (e.g., methyl, ethyl, propyl, isopropyl, cyclopropyl), aryl, or heterocycle.
• R4 can be methyl, ethyl, propyl, isopropyl, or cyclopropyl.
• R5 can be aryl that is optionally substituted with halogen (e.g., F or CI) or heterocycle (e.g., furan or thiofuran) that is optionally substituted with halogen (e.g. , F or CI).
• R6 can be aryl that is optionally substituted with carboxylic acid, alkyl carboxylic acid (e.g., CH 2 C0 2 H,
• heterocycle e.g., piperidinone, imidazolidinone, tetrazole
• cyano alkyl cyano (e.g., CH 2 CN)
• alkyl heterocycle e.g., sulfonamide, or aminosulfonamide, or a derivative thereof.
• R NO* iPDE4B 3 ⁇ 4 5,6 nWI or a pharmaceutically active derivative or salt thereof.
• the second agent is a nucleic acid
• it can be a nucleic acid that mediates RNAi (e.g., an siRNA or shRNA) by targeting and inhibiting the expression of PDE4B2 or another target described herein, such as ⁇ - ⁇ .
• RNAi e.g., an siRNA or shRNA
• Other useful nucleic acid-based inhibitors include rnicroRNAs and antisense oligonucleotides. Relevant sequences and methods for generating inhibitory RNA molecules are known in the art. For easy reference, the mRNA sequence of human PDE4B is shown in Fig. 8.
• the selected agent may do so selectively ⁇ i.e., the agent may inhibit the expression of only PDE4B2 to the exclusion of other PDE4B variants or other PDE4 family members) or non-selectively (i.e., the agent may inhibit PDE4B2 as well as other PDE4B variants or other PDE4 family members).
• the mRNA sequence of PKA-CP is available through, for example, the NCBI "GenBank" website (see, e.g., Accession No. NM-002731 and Taylor et ⁇ , ⁇ . Rev. Biochem., 59:971-1005, 1990).
• PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP.
• CR3 Control Region 3
• the mRNA sequence of PKA-Cp is available through, for example, the NCBI
• the second agent can be a siRNA or a fragment thereof.
• siRNAs useful as the second agent i.e., as an agent that inhibits, directly or indirectly, the expression or activity of PDE4B2 are commercially available from, for example, Santa Cruz Biotechnology, Inc. (current catalog # sc-41599) and GE HealthCare.
• siRNAs, shRNAs, microRNAs and antisense oligonucleotides, frequently 19-21 nucleotides in length can be synthesized according to methods known in the art and customized as desired based on the sequence of the target to be inhibited.
• the second agent is an inhibitory RNA
• all of the agents may have a single sequence or may be a pool of different sequences (e.g., a pool of 3-7 siRNAs that inhibit the expression of PDE4B2, PKA-Cp, or another target described herein).
• the second agent can be a polypeptides or a fragment thereof.
• Polypeptides useful as the first or second agent include antibodies that specifically bind a target as described herein and, where the target is PKA-Cp, the polypeptide can be TTYADFIASGRTGRRNAIHD (SEQ ID NO:3) or an active fragment or other variant thereof.
• an ⁇ inhibitor significantly inhibited the synergistic induction of PDE4B2 by NTHi and roflumilast, but did not affect the induction of PDE4B2 by roflumilast alone. Therefore, in patients where COPD is exacerbated by or associated with NTHi infection, treatment can be carried out with, for example, roflumilast and an ⁇ inhibitor.
• the first agent and the second agent can be formulated to be administrated simultaneously or separately.
• the first agent and/or the second agent can be formulated in the form of a pill, a capsule, a granule, a tablet, a pallet, a suspension, an injection, an infusion, a suppository, a continuous delivery system, a syrup, a tincture, an ointment, a cream, eye drops, ear drops, a flush, a lavage, a slow absorbing depot, a dressing, a lozenge, or any pharmaceutically acceptable application or as a nutritional supplement.
• the first agent and/or the second agent, as disclosed herein, can be administered by any route appropriate to the condition to be treated. Suitable routes can include oral, inhalation, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), rectal, nasal, topical, vaginal, and the like.
• the formulations can be tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
• the formulations can include aerosol or dry powder including small particles.
• the formulation can also be a suspension (e.g., the first and/or second agent particles suspended in the liquefied propellant) or a solution (e.g., the first and/or second agents dissolved in liquefied propelleant.
• the particles can be in sizes of about 10 ⁇ or less. Preferably, the particles can be in sizes less than 5 ⁇ (e.g., 2-3 ⁇ ).
• the formulations can be prepared according to conventional methods and may be delivered with other therapeutic agents.
• the formulation can further include one or more of HFA propellant, surfactant co- solvent and/or excipient.
• formulation can include aqueous and nonaqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.
• a sterile injectable preparation e.g., a sterile injectable aqueous or oleaginous suspension.
• the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butane-diol or prepared as a lyophilized powder).
• a non-toxic parenterally acceptable diluent or solvent e.g., a solution in 1,3-butane-diol or prepared as a lyophilized powder.
• kits of the invention can include any one or more of the compositions described herein, including pharmaceutical compositions containing the first and second agents described herein in a formulation that is ready to administer or can be administered after further manipulation (e.g., after dilution or resuspension).
• the first agent and the second agent are within separate containers and packaged together within the kit.
• the kit can include instructions for use (e.g., a written document, a disclosure of aveb address, or an audio or visual presentation).
• the kit can include paraphernalia useful in administering the compositions contained therein (e.g., a needle, syringe, tubing, sterilant, gloves, mask, nebulizer, dropper, gauze, tape, or dressing).
• the kit can include one or more inhalation devices, for examples, an atomizer, nebulizer, vaporizer, metered dose inhaler (MDI), dry powdered inhaler, or the like.
• MDI metered dose inhaler
• compositions of the invention can be employed in human and veterinary medicine as therapeutics, where they can be used, for example, for the treatment and prophylaxis of the following illnesses: acute and chronic (in particular inflammatory and allergen-induced) airway disorders of varying origin (bronchitis, allergic bronchitis, bronchial asthma); dermatoses (especially of proliferative, inflammatory and allergic type), such as psoriasis (vulgaris), toxic and allergic contact eczema, atopic eczema, seborrhoeic eczema, Lichen simplex, sunburn, pruritus in the anogenital area, alopecia areata, hypertrophic scars, discoid lupus erythematosus, follicular and widespread pyodermias, endogenous and exogenous ache, acne rosacea and other proliferative, inflammatory and allergic skin disorders; disorders which are based on
• osteoarthritis and other arthritic conditions and inflammation of the joints disorders of the immune system (AIDS), types of shock (septic shock, endotoxin shock, gram-negative sepsis, toxic shock syndrome and ARDS (adult respiratory distress syndrome)) and also generalized inflammations in the gastrointestinal region (Crohn's disease and ulcerative colitis); disorders which are based on allergic and/or chronic, immunological false reactions in the region of the upper airways (pharynx, nose) and the adjacent regions (paranasal sinuses, eyes), such as allergic rhinitis/sinusitis, chronic rhinitis/sinusitis, allergic conjunctivitis and also nasal polyps; otitis media and other ear and sinus infections; but also disorders of the heart which can be treated by PDE inhibitors, such as cardiac insufficiency, or disorders which can be treated on account of the tissue-relaxant action of the PDE inhibitors, such as colics of the kidneys and of the ureters in
• Inflammation of the eye and tissues surrounding and affecting the eye can also be treated. While the invention is not limited to treatment of conditions that arise due to any particular molecular mechanism, treatable conditions may be recognized as associated with misexpression (e.g., overexpression) of histamine, platelet- activating factor (PAF), arachidonic acid derivatives such as leukotrienes and prostaglandins, cytokines, such as any of IL-1 to IL-12, alpha-, beta-, or gamma-interferon, tumor necrosis factor (TNF), or oxygen free radicals and proteases.
• the condition to be treated may be one in which mucus is overproduced.
• Embodiments in which the first agent is Roflumilast Roflumilast is useful in the treatment of inflammatory conditions of the lungs ⁇ e.g., chronic obstructive pulmonary disease (COPD), COPD associated with chronic bronchitis, and COPD exacerbations).
• COPD chronic obstructive pulmonary disease
• NHi Haemophilus influenzae
• the present compositions and methods can include roflumilast to reduce the risk of COPD exacerbations in patients with severe COPD associated with chronic bronchitis, a history of exacerbations, and infection with NTHi. Treatment may be contraindicated in patients with moderate to severe liver impairment (Child-Pugh B or C).
• the combination therapies described herein may improve one or more of the adverse effects reported in connection with roflumilast treatment (e.g., gastrointestinal problems such as abdominal pain, diarrhea, nausea; related signs such as weight loss and loss of appetite;
• headaches back pain; influenza; dizziness; insomnia; anxiety, depression, suicidal thoughts or pther mood changes; rhinitis and sinusitis; and urinary tract infections).
• the currently recommended dosage of roflumilast for patients with COPD is one 500 meg tablet per day, with or without food. This dosage may be maintained in the present compositions and methods, increased, or lessened (e.g., to 450-499 meg/day; 400-450 meg/day; 300-400 meg/day; or less than 300 meg/day (e.g. , 100-300 meg/day)).
• topical application forms (such as ointments) for the treatment of dermatoses can contain the first and/or the second active agents in a concentration of, for example, 0.1-99%.
• the dose for administration by inhalation is customarily between 0.01 and 0.5 mg/kg.
• the customary dose in the case of systemic therapy is between 0.05 and 2 mg per day.
• compositions can be formulated together with one or more solvents, gel formers, ointment bases and other active compound excipients (e.g., antioxidants, dispersants, emulsifiers, preservatives, solubilizers or permeation promoters), or any combination thereof.
• active compound excipients e.g., antioxidants, dispersants, emulsifiers, preservatives, solubilizers or permeation promoters
• Suitable inactive ingredients include lactose
• Suitable pharmaceutical formulations include, for example, powders, emulsions, suspensions, sprays, oils, ointments, fatty ointments, creams, pastes, gels, tablets, pills, capsules, and solutions.
• first and second agents described herein can be formulated for direct delivery to a site of inflammation.
• the first and/or second agents can be formulated as porous particles for delivery by inhalation.
• the agents can be administered directly as a powder (preferably in micronized form) or by atomizing solutions or suspensions which contain them.
• the first and/or second agents can be formulated as an aerosol.
• the present agents can be delivered by the AERx Essence® pulmonary delivery device and AERx® system currently sold by Aradigm.
• One or both of the first and second agents described herein can be incorporated into a nanoparticle (e.g., a therapeutic nanoparticle), which may improve the delivery profile and target specific tissues, such as lung tissue.
• the nanoparticles may be in the form of micron-scale dry powders and nanoparticles as described in Sung et al. (Trends Biotechnol, 25(12):563-570, 2007) or Azarmi et al. (Advanced Drug Delivery Reviews, 60(8):863-876, 2008) and may be Upid-based (e.g., liposomes or micelles) or non-lipid-based.
• the first and second agents can also be associated with (e.g., covalently or non- covalently bound to) mesoporous silica nanoparticles (MSNs), poly propyleneimine (PPI), quantum dots, and polymers (e.g., polyethylene glycol).
• MSNs mesoporous silica nanoparticles
• PPI poly propyleneimine
• quantum dots e.g., polyethylene glycol
• polymers e.g., polyethylene glycol.
• delivery vehicles including the first and second agents described herein are within the scope of the present invention, and the methods of treatment described herein can employ administration of the first and/or second agents in association with any such delivery vehicle.
• Methods of preparing and administering nanoparticles are well known in the art (see, e.g., Garbuzenko et al, Cancer Biol. Med., ll(l):44-55, 2014).
• disease encompasses various maladies which may be more frequently described in the literature as being an illness, condition, disorder, infection, syndrome, or the like.
• compositions and methods can incorporate a third agent, such as a
• cytochrome P450 enzyme inducer e.g. , rifampicin, phenobarbital, carbamazepine, and phenytoin
• an inhibitor of CYP3A4 or dual inhibitors of CYP3A4 and CYP1A2 (e.g., erythromycin, ketoconazole, fluvoxamine, enoxacin, and cimetidine).
• CYP3A4 e.g., erythromycin, ketoconazole, fluvoxamine, enoxacin, and cimetidine.
• these agents are avoided in conjunction with roflumilast, as they are thought to increase the patient's systemic exposure to roflumilast and therefore result in increased adverse reactions.
• the risks associated with such concurrent use are diminished in the context of the present invention, where compositions and treatments include an inhibitor of PDE4B2.
• Actinomycin D and protease inhibitor cocktail were purchased from Sigma-Aldrich.
• Myristoylated PKA inhibitor and ⁇ inhibitor IV were purchased from EMD Millipore.
• Roflumilast was purchased from Santa Cruz Biotechnology.
• N 6 -Phenyl-cAMP (6-Phe-cAMP), 8-pCPT-2'-0-Me-cAMP and Rp-8-CPT-cAMPS were purchased from BioLog.
• Forskolin was purchased from Enzo Life Sciences.
• Acrylamide was purchased from Wako Chemicals USA.
• Recombinant p65 protein was purchased from Active Motif.
• Recombinant PKA-C protein was purchased from R&D systems.
• Antibodies for PKA-C (sc-904), p65 (sc-8008), ⁇ -actin (sc-8432), -Tubulin (sc- 69969), PDE4B (sc-25812) and TFIIB (sc-225) were purchased from Santa Cruz Biotechnology, and antibodies for PKA-Ca (#4782) and c-Rel (#4727) were purchased from Cell Signaling.
• bronchial epithelial BEAS-2B cells were maintained in RPMI medium (Life Technologies) (ATCC® CRL-9609TM)- Human primary bronchial epithelial NHBE (Lonza) cells were maintained in bronchial epithelial growth media (BEGM) supplemented with BEGM SingleQuots (Jono et al., J. Biol. Chem. 277(47):45547-45557, 2002).
• BEAS-2B cells stably expressing human PDE4B2 were obtained by plasmid transfection following Geneticin selection (300 ⁇ g/mL). Cells were cultured in a humidified atmosphere of 5% C0 2 at 37°C.
• TaqMan reverse transcription reagents (Life Technologies) were used as described previously.
• quantitative RT-PCR analysis PCR amplifications were performed by using SYBR Green Universal Master Mix (Life Technologies). In brief, reactions were performed in triplicate containing 2 x Universal Master Mix, 1 ⁇ , of template cDNA, and 500 nM primers in a final volume of 12.5 xL, and the reactions were analyzed in a 96-well optical reaction plate (USA Scientific). Reactions were amplified and quantified by using a StepOnePlus Real-Time PCR System and the manufacturer's corresponding software
• RNAs were obtained by using the comparative Ct method and were normalized using human cyclophilin or mouse glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) as an endogenous control.
• GPDH mouse glyceraldehyde-3 -phosphate dehydrogenase
• PCR amplifications were performed with PrimeSTAR Max polymerase (Takara) by following the manufacturer's instruction.
• the primer sequences used are listed in the following Table.
• Cyclophillin A CGGGTCCTGGCATCTTGT GCAGATGAAAAACTGGGAACCA
• PDE4B 1 ACCTTTCCTGGGCACAGCCAC GCAGCGTGCAGGCTGTTGTG
• Plasmids, transfections and luciferase assay Plasmids, transfections and luciferase assay.
• the expression plasmids, constitutively active forms of IKKa ( ⁇ -CA, S176E/S180E) and ⁇ ( ⁇ -CA, S177E/S181E) and a dominant negative form of ⁇ ( ⁇ -DN, S32A/S36A) were previously described (Shuto et al., Proc. Natl. Acad. Sci. USA, 98(15):8774-8779, 2001; Ishinaga et al, supra).
• Luciferase reporter construct of NF- ⁇ pGL4.32 was purchased from Promega.
• siRNA-mediated knockdown Human validated siRNA oligos were obtained from GE Healthcare (Negative Control, D001810-10; PDE4B, L007648-01; PKA-Ca, M004649-01; ⁇ , M004650-00; p65, L003533-00; c-Rel, L004768-00). Cells were transfected with 50 nM siRNA using DharmaFECT-4 (Thermo Scientific) and collected or treated 48 h later. For the co-transfection of siRNA with DNA, cells were transfected with 10 nM siRNA using Lipofectamine 3000 (Life Technologies).
• HEPES at pH7.4, 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, supplemented with 1 mM Na 3 V0 4 and PIC) and incubated on ice for 10 min (Schreiber et al, Nucleic Acids Res. 17(15):6419, 1989).
• the cells were lysed by adding NP40 (0.5%) and vortexing for 15 sec. then centrifuged at 3,000 x g for 5 min before the supernatants were removed (cytosol fraction).
• Precipitates were resuspended in Buffer B (20 mM HEPES at pH7.5, 5 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, supplemented with 1 mM Na 3 V0 4 and PIC) and incubated on ice for 10 min. before vortexing and centrifuging at 16,000 x g for 15 min to recover the supernatants (nuclear fraction). Western blot.
• lysis buffer 50 mM Tris-HCl at pH7.4, 1% NP40, 0.25% deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, supplemented with 1 mM Na 3 V0 4 and PIC.
• cell extracts were recovered with Buffer A as described above.
• Cell or tissue extracts were separated on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences). The membrane was blocked with a solution of TBS containing 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk.
• PVDF polyvinylidene difluoride
• the membrane was then incubated in a 1:1,000 - 1:2,000 dilution of a primary antibody in 5% BSA-TBS-T. After washing (x3) with TBS-T, the membrane was incubated with a 1:5,000 dilution of the corresponding secondary antibody in 2.5% non-fat dry milk-TBS-T. Respective proteins were visualized using Amersham ECL Prime Regent (GE Healthcare Life Sciences).
• PDE4 activity was estimated from the difference between total and roflumilast-resistant PDE activity.
• Phos-tag PAGE Recombinant p65 proteins or nuclear extracts recovered without EDTA/EGTA were separated by SDS-PAGE, with 6% gels containing 50 ⁇ Mn 2+ -Phos-tag Acrylamide and transferred to PVDF membrane according to the manufacture's instructions (Kinoshita et al, Mol. Cell. Proteomics 5(4):749-757, 2006.
• kinase assay buffer (20 mM HEPES at pH7.5, 1 M MgCl 2 , 1 mM DTT, • 10 mM ATP) and incubated at 30 °C for 0.5 h.
• the reaction was stopped by adding 4 x SDS Sample buffer (0.24 M Tris-HCl at pH 6.8, 40% Glycerol, 8% SDS, 20% 2-Mercaptoehanol, 0.04% Bromophenol blue).
• mice and animal experiments For NTHi-induced inflammation in C57BL/6J mice (7 weeks old), anaesthetized mice were intratracheally inoculated with NTHi at a concentration of 5 x 10 7 CFU per mouse and saline was inoculated as control. The inoculated mice were then sacrificed 5 h after NTHi inoculation. For inhibition studies, the mice were pretreated with roflumilast intraperitoneally 2 h before NTHi inoculation. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Georgia State
• Example 1 Roflumilast synergizes with NTHi to up-regulate PDE4B2 expression in vitro and in vivo. Because the expression of PDE4 isoforms is induced by PDE4 inhibitors (Campos-Toimil et al., Br. J. Pharmacol. 154(l):82-92, 2008; Dlaboga et al., Brain Res.,
• Airway epithelial cells are the important sources of pro-inflammatory mediators induced by bacterial pathogens (Hallstrand et al, Clin. Immunol, 161(1):1-15, 2014). Thus, we first determined if NTHi induces the expression of a number of key pro-inflammatory mediators that have been shown to be critical in the pathogenesis of COPD.
• NTHi significantly up-regulated the expression of CCL5, CCL7, CXCLIO, CXCL11, Interleukin-8 (IL-8/CXCL8), granulocyte -macrophage colony-stimulating factor (GM-CSF) and TNF-a in BEAS-2B cells.
• IL-8/CXCL8 Interleukin-8
• GM-CSF granulocyte -macrophage colony-stimulating factor
• roflumilast inhibited NTHi-induced expression of these pro-inflammatory mediators to various extents.
• Roflumilast markedly inhibited the induction of TNF-a and GM-CSF, but modestly suppressed the induction of CCL5, CCL7 and IL-8. In contrast, it exhibited almost no inhibitory effect on CXCL10 and CXCL11 induction even at higher concentration.
• PDE4B2 depletion further significantly inhibited the induction of CCL5, CCL7, CXCL10 and CXCL11 (Group A) that were modestly or minimally inhibited by roflumilast alone.
• PDE4B2 depletion only minimally affected the induction of IL-8, GM-CSF and TNF-a (Group B) that were modestly or markedly inhibited by roflumilast alone.
• PDE4D was also up-regulated by NTHi and roflumilast, and previous studies have suggested that PDE4D has a different and non-redundant role from PDE4B (Ariga et al.,
• PDE4B2 up-regulation in NTHi-induced expression of chemokines we developed cells stably overexpressing wild-type PDE4B2 (PDE4B2-stable cells).
• PDE4B2 expression in PDE4B2-stable cells was higher in mock-transfected (Mock) cells (Fig. 4A).
• NTHi-induced expression of CCL5, CCL7, CXCL10 and CXCL11 was significantly enhanced in PDE4B2-stable cells compared to Mock cells (Fig. 4B).
• roflumilast was unable to fully inhibit the NTHi-induced expression of these chemokines in PDE4B2-stable cells even at the highest concentration tested (10 ⁇ ).
• synergistically up-regulated by NTHi and roflumilast may contribute, at least in part, to the decreased efficacy of roflumilast in suppressing CCL5, CCL7, CXCL10 and CXCL11 induction in bronchial epithelial cells.
• up-regulated PDE4B2 induces certain pro-inflammatory mediators in a manner that cannot be overcome by high doses of roflumilast (up to 10 ⁇ ; a very high level of this drug as the IC 5 o has been estimated at less than 1 nM) (Bender and Beavo, supra).
• PDE4B2 may regulate the expression of these chemokines at least in part independently of its well-known enzymatic activity by, e.g., acting as an adaptor protein.
• PDE4B2-WT wild-type PDE4B2
• PDE4B2-D392A catalytically inactive form of PDE4B2
• PDE4B2-WT and PDE4B2-D392A were equally expressed in transfected BEAS-2B cells, and PDE4 activity was significantly lower in the cells transfected with the PDE4B2-D392A mutant than in cells transfected with PDE4B2-WT.
• PDE4B2-WT markedly enhanced a constitutively active form of the inhibitor of NF- ⁇ (IKB) kinase ⁇ ( ⁇ -CA)- induced NF- ⁇ promoter activity in a dose-dependent manner and also enhanced ⁇ -CA- induced Group A-chemokine expression.
• PDE4B2-D392A also enhanced ⁇ - CA-induced NF- ⁇ promoter activity and chemokine expression although to a lesser extent compared with PDE4B2-WT.
• roflumilast up to 10 ⁇ was unable to fully inhibit the ⁇ -CA-induced NF- ⁇ promoter activity and Group A-chemokine expression in the cells transfected with PDE4B2-D392A.
• PDE4B2 enhances the inflammatory response in both PDE enzymatic activity-dependent and -independent manners, which may contribute to the tolerance to roflumilast.
• PKA inhibitor significantly suppressed the synergistic induction of PDE4B2 by NTHi and roflumilast.
• This inhibitor the polypeptide TTYADFIASGRTGRRNAIHD (SEQ ID NO:3), can be readily synthesized and is also available from Santa Cruz Biotechnologies.
• PKI and active fragments or variants thereof e.g. , polypeptides that are at least 80% (e.g. , 85%, 90%, or 95%) identical thereto, can be used in the present compositions and methods to inhibit PKA-Cp.
• PKA is a tetrameric enzyme consisting of two catalytic (C) and two regulatory (R) subunits. Binding of cAMP to R subunits results in relief of R subunit inhibition of the
• CRE cAMP response element
• Example 4 ⁇ - ⁇ 65 but not c-Rel is required for synergistic induction of
• NTHi is known as a potent activator of ⁇ (also known as IKK2), leading to the activation of NF-KB-dependent inflammatory response (Shuto et al., Proc. Natl. Acad. Sci. USA, 98(15) :8774-8779, 2001; Oeckinghaus and Ghosh, Cold Spring Harb. Perspect. Biol, l(4):a000034, 2009; Chen et al., Biochem. Biophys. Res. Commun., 324(3):1087-1094, 2004), we examined the requirement of ⁇ -NF-KB signaling in the synergistic induction of PDE4B2. We first evaluated the role of ⁇ .
• ⁇ inhibitor significantly inhibited the synergistic induction of PDE4B2 by NTHi and roflumilast, but did not affect the induction of PDE4B2 by roflumilast alone.
• BEAS-2B cells were transfected with the constitutively active form of IKKa ( ⁇ ⁇ -CA) and ⁇ ( ⁇ -CA).
• IKKa ⁇ ⁇ -CA
• ⁇ -CA ⁇ -CA
• ⁇ prevents the activation and nuclear translocation of NF- ⁇ complexes including p65 and c-Rel, which have been previously known to be activated by PKA-Ca and ⁇ , respectively (Gerlo et al, Cell Mol. Life Sci. , 68(23) :3823-3841, 2011; Yu et al., J. Mol. Med. (Berl), 82(9):621-628, 2004; Zhong et al., Cell, 89(3);413-424, 1997; Zhong et al., Mol. Cell, 1(5):661-671, 1998).
• NTHi induces nuclear translocation of p65 and c-Rel.
• Example 5 PKA-CP phosphorylates p65.
• p65 is physically associated with ⁇ by performing co- immunoprecipitation experiments.
• p65 and ⁇ - ⁇ were physically associated with each other in BEAS-2B cells transfected with both p65 and ⁇ .
• their interaction was enhanced by co-treatment of roflumilast with NTHi.
• roflumilast did not affect the nuclear expression level of p65 or ⁇ in BEAS-2B cells.
• Phosphorylation at multiple residues of p65 has been shown to regulate various functions of p65, such as DNA binding and transcriptional activities (Huang et al, Cell Signal, 22(9):1282-1290, 2010; Chaturvedi et al, Oncogene,
• phosphate-affinity (Phos- tag) PAGE a novel phosphate-binding tag-based method that has been developed to specifically decrease the migration speed of phosphorylated proteins so that the phosphorylated protein can be separated from non-phosphorylated protein (Kinoshita et al, Mol. Cell. Proteomics, 5(4):749- 757, 2006).
• the p65 constructs with mutation of other serine residues (S468A, S529A and S536A) known to be phosphorylated by other kinases were also analyzed.
• the intensity ratio of PKA ⁇ -induced phosphorylation was decreased in S276A- and S536A- transfected cells compared to the wild-type p65 -transfected cells.
• PDE4B2 expression was examined in the cells transfected with these different p65 phosphorylation site mutants.
• Example 6 Dexamethasone suppresses PDE4B induction by NTHi and Rof and improves the efficacy of Rof in suppressing NTHi-induced inflammation in lung epithelial cells in vitro and in mouse lung.
• Rof 1 ⁇
• FSK 1 ⁇
• Iso 0.1 ⁇
• DEX 10 nM
• PDE4B has been shown to be up-regulated by various inflammatory stimuli, which plays a critical role in mediating inflammatory response (Gobejishvili et al., Am. J. Physiol. Gastrointest. Liver Physiol, 294(4):G718-G724, 2008; Gobejishvili et al., J.
• PDE4B2 expression plays a critical role in NTHi-induced expression of chemokines CCL5, CCL7, CXCL10 and CXCL11, which have previously been shown to be crucial in the pathogenesis of COPD exacerbation.
• chemokines are increased in induced sputum, bronchoalveolar lavage (BAL) fluid or peripheral airways in patients with COPD (Saetta et al. , Am. J. Resp & Crit. Care Med., 165(10):1404- 1409, 2002; Fujimoto et al., Eur. Respiratory J., 25(4):640-646, 2005; Costa et al, Chest,
• PDE4B2 regulates the expression of certain chemokines in both an enzymatic activity-dependent and -independent manner. This may lead to the reduced efficacy of roflumilast in suppressing inflammation under certain clinical conditions due to the synergistically up-regulated PDE4B2.
• the effect of roflumilast in suppressing NTHi-induced CCL5, CCL7, CXCL10 and CXCL11 is less than its effect in suppressing some other pro-inflammatory mediators such as GM-CSF and TNF-a in bronchial epithelial cells.
• PKA-Cct activates NF- ⁇ signaling via phosphorylating p65 at Ser276 in a cAMP-independent manner (Zhong et al, Cell, 89(3):413-424, 1997). PKA-C has been shown to be maintained in an inactive state through association with ⁇ complex. Signals that lead to ⁇ degradation result in PKA-Ca activation and subsequent phosphorylation of p65 at Ser276.
• PKA-Ca and ⁇ also appear to differentially modulate the expression of proinflammatory mediators.
• proinflammatory mediators we found that some of the NTHi-induced proinflammatory mediators were increased upon PKA-Ca knockdown, which is line with the antiinflammatory role of PKA-Ca previously reported in other cell types (Ollivier et al., J. Biol. Chem., 271(34) :20828-20835, 1996).
• the role of ⁇ - ⁇ in mediating the inflammatory response remains largely unclear.
• we found that the expression of pro- inflammatory mediators was reduced by ⁇ - ⁇ knockdown, indicating the pro-inflammatory role of PKA-C .
• TNF-a mediated nuclear translocation of p65 is enhanced by PKA depletion using siRNA in HeLa cells (King et al. , PloS ONE, 6(4):el8713, 2011). Forskolin impairs the nuclear translocation of p65 in Jurkat T-lymphocytes (Neumann et at, EMBO J., 14(9):1991-2004, 1995). There are also NF- ⁇ nuclear translocation-independent mechanisms.
• cAMP inhibits NF-KB-mediated transcription without preventing the nuclear translocation of NF- ⁇ complex.
• cAMP/PKA inhibits NF-KB-dependent transcriptional activity by phosphorylating CREB, which competes with p65 for limiting amounts of NF- ⁇ co-activator CREB-binding protein (CBP) ⁇ Parry and Mackman, J. Immunol., 159(11) :5450-5456, 1997).
• CBP co-activator CREB-binding protein
• NF- ⁇ p65 phosphorylation at Ser276 by PKA activates NF- ⁇ and contributes to the malignant phenotype of head and neck cancer (Arun et al, Clin. Cancer Res., 15(19) -.5974-5984, 2009). It has been also shown that PKA-Ca activates NF- ⁇ signaling via phosphorylating p65 at Ser276 in an ⁇ -dependent but cAMP- independent manner (Zhong et al, 1997, supra).
• a kinase-interacting protein 1 (AKIPl) in various cell types or conditions also appear to be important for the effect of PKA on NF-KB activity. It has been shown that in cells with low levels of AKIPl, PKA-activating agents inhibit NF-KB transcriptional activity. In contrast, in cells with high levels of AKIPl, the PKA activation increases p65 phosphorylation at Ser276 and synergizes with NF- ⁇ activation (Gao et al., J. Biol. Chem., 285(36):28097-28104, 2010).
• Combined administration of roflumilast with a PKA-Cp -selective inhibitor may help attenuate the unwanted up- regulation of PDE4B2, thereby representing a promising therapeutic strategy to improve the efficacy, decrease the effective dose and possibly ameliorate the tolerance of PDE4-inhibitors in patients with COPD exacerbation.

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