Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6957—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a device or a kit, e.g. stents or microdevices
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

• IRMs immune response modifiers
• certain IRMs may be useful for treating viral diseases (e.g., human papilloma virus, hepatitis, herpes), neoplasias (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis), and T ⁇ 2-mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis), and are also useful as vaccine adjuvants.
• viral diseases e.g., human papilloma virus, hepatitis, herpes
• neoplasias e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis
• T ⁇ 2-mediated diseases e.g., asthma, allergic rhinitis, atopic dermatitis
• Many of the IRM compounds are small organic molecule imidazoquinoline amine derivatives (see, e.g., U.S. Pat. No. 4,689,338),
• IRMs have higher molecular weights, such as oligonucleotides, including CpGs (see, e.g., U.S. Pat. No. 6,194,388).
• immune response modifiers of the invention can be attached to macromolecular support materials and, importantly, that they retain biological activity even while they remain attached to such material.
• This ability to form biologically active IRM-support complexes allows for a tremendous range of useful applications where one may not wish to release all of the IRM compound to be effective.
• the IRMs here can be active while attached to, e.g., implantable medical devices, particles, beads, polymers, and other supports, substrates, and matrix materials.
• immune system dendritic cells can be removed from a patient and activated ex vivo in the presence of a desired antigen by being placed in contact with a material lined with attached IRMs (e.g., container walls, beads, mesh, etc.). The activated dendritic cells can then be conveniently returned to the patient for therapeutic use, leaving the IRM behind so as to avoid systemic exposure.
• IRMs e.g., container walls, beads, mesh, etc.
• the IRMs are still biologically active when attached to a support complex, but surprisingly, the cytokine induction profile of the IRM can be altered in potentially desirable ways by virtue of such attachment. It has been found that attachment of some IRMs actually modifies the cytokine induction profile in favor of interferon ⁇ , which maybe important for certain therapeutic uses.
• the IRM may be covalently or non-covalently bound, preferably covalently bound, to the macromolecular support material. Attachment of an IRM to a macromolecular support material provides for the localized biological activity of the IRM and typically prevents, or at least reduces the occurrence of, the systemic distribution of the IRM.
• the present invention provides an IRM-support complex having an IRM compound attached to a macromolecular support material.
• the IRM compound may be covalently attached to the macromolecular support material.
• macromolecular support material refers to organic materials, inorganic materials, and combinations thereof that are generally biologically inactive relative to the biology being targeted by the IRM.
• the macromolecular support material is typically of a size and chemical nature to prevent the engulfment or penetration of the macromolecular material into cells, although this is not a necessary limitation.
• the macromolecular support material preferably has an average largest dimension of at least 1 nanometer (nm).
• the macromolecular support material may be part of a gel, a foam, a sponge, a fiber, or a bead.
• an IRM-support complex that includes an immune response modifier is attached to a polymer.
• the immune response modifier is covalently attached to the polymer.
• the polymer is a bioadhesive polymer.
• the present invention provides a medical article coated with the IRM-support complex that includes a polymer.
• the present invention also provides a medical article (i.e., medical device such as an implantable device) including an IRM-support complex, wherein the IRM-support complex includes an immune response modifier attached to a macromolecular support material
• the medical article may be a stent, a shunt, an artificial valve, a suture, a surgical clip, a surgical staple, an indwelling catheter, a dental implant, an orthopedic implant, a surgical prosthetic, an implantable vascular access port, an artificial heart, a ventricular assist pump, a blood oxygenator, a blood filter, a hemodialysis unit, a hemoperfusion unit, a conduit tube within a heart lung machine, a tube within a dialysis apparatus, a tube within a plasmapheresis unit, an artificial pancreas, an artificial liver, an artificial lung, an intraocular lens, or a contact lens.
• the present invention provides a medical article having disposed thereon an IRM, with the pro
• the present invention also provides a stent, shunt, or valve having a surface with an immune response modifier attached thereto, hi some embodiments, the immune response modifier may be covalently attached to the surface of the stent, shunt, or valve.
• the present invention also provides a method of making an IRM-support complex by attaching an immune response modifier to a macromolecular support material.
• the immune response modifier may be covalently attached to the macromolecular support material.
• the present invention also provides a method of treating a viral infection in a subject by administering to the subject an IRM-support complex having an IRM compound attached to a macromolecular support material.
• the IRM-support complex may be administered orally, nasally, ocularly, vaginally, transcutaneously, or rectally.
• the present invention also provides a method of treating an atopic immune response in a subject by administering to the subject an IRM-support complex having an IRM compound attached to a macromolecular support material.
• the LRM-substrate may be administered orally, nasally, vaginally, ocularly, transcutaneously, or rectally.
• the present invention also provides a method of treating solid tumors in a subject by administering to the subject an IRM-support complex having an IRM compound attached to a macromolecular support material.
• the IRM-substrate may be administered orally, nasally, vaginally, ocularly, transcutaneously, or rectally.
• the present invention also provides a method of preventing restenosis in a subject by implanting into the subject a stent having a surface with an immune response modifier associated therewith (preferably, attached thereto, and more preferably, covalently attached thereto).
• the present invention also provides a method of modifying the cytokine induction profile of an IRM by attaching the IRM to a macromolecular support complex, i some embodiments, the cytokine induction profile may be modified in favor of inlerferon ⁇ induction.
• the present invention also provides a method of reducing systemic adsorption of an immune response modifier in a subject by administering to the subject an IRM-support complex, the IRM-support complex including the immune response modifier attached to a macromolecular support material.
• the present invention also provides an IRM-support complex
• the present invention also provides a method of activating dendritic cells by permitting the cells contact an IRM compound attached to a support complex.
• the present invention also provides a method of treating cervical dysplasia in a subject by applying to the cervix an IRM-support complex comprising an IRM compound attached to a macromolecular support material.
• the present invention also provides a method of treating bladder cancer in a subject by applying to the bladder an IRM support complex comprising an IRM compound attached to a macromolecular support material.
• the present invention also provides a method of making an IRM-support complex, wherein the method involves attaching an immune response modifier to a macromolecular support material. Attaching the immune response modifier can involve covalently attaching it to the macromolecular support material. The method can also involve modifying the IRM to include an alkoxysilane moiety. The IRM- modified alkoxysilane is then attached to a silicon-containing support material.
• the IRM compound may be an agonist of at least one TLR, preferably an agonist of TLR6, TLR7, or TLR8.
• the IRM may also in some cases be an agonist of TLR9.
• the IRM compound may be a small molecule immune response modifier (e.g., molecular weight of less than about 1000 daltons).
• the IRM compound may comprise a 2-aminopyridine fused to a five-membered nitrogen-containing heterocyclic ring, or a 4-aminopyrimidine fused to a five-membered nitrogen-containing heterocyclic ring.
• the IRM compound may be a purine, imidazoquinoline amide, benzimidazole, lH-imidazopyridine, adenine, or a derivative thereof.
• an IRM-support complex comprising "an” LRM compound can be interpreted to mean that the complex includes at least one IRM compound.
• Figure 1 A is a graphical representation of the LFN ⁇ /TNF ⁇ ratio produced by human peripheral blood mononuclear cells incubated overnight with IRMl or IRM2 bound to monomeric avidin beads or tertrameric avidin beads.
• Figure IB is a graphical representation of the IFN ⁇ and TNF ⁇ produced by human peripheral blood mononuclear cells incubated overnight with IRMl or IRM2 bound to monomeric avidin beads or tertrameric avidin beads.
• the present invention is directed to the attachment of cytokine inducing and/or suppressing immune response modifiers (IRMs) to macromolecular support materials to form IRM-support complexes.
• IRMs cytokine inducing and/or suppressing immune response modifiers
• the IRMs retain biological activity following such attachment to a macromolecular support material.
• IRM-support complexes allow for the localized delivery of an IRM to a desired location in the body of a subject and typically prevent, or at least reduce the occurrence of, the systemic distribution of the IRM.
• macromolecular support material is a macromolecular material that is itself generally biologically inactive relative to the biology being targeted by the IRM.
• this definition of macromolecular support materials excludes bacteria and viruses, for example.
• the macromolecular support material may be of a size and chemical nature to prevent the engulfment or penetration of the macromolecular material into cells, in which case the IRM-support complex retains an extracellular location.
• the macromolecular support material may be of a size and chemical nature to allow engulfment by cells.
• the macromolecular support material may be of a size and chemical nature to allow selective deposition in solid tumors on the basis of the tumor's increased vascular permeability.
• the macromolecular support material is in the form of a solid (i.e., a solid support such as particles, fibers, membranes, films), but can also be in the form of a polymeric gel, sponge, or foam, for example.
• a macromolecular support material can be made of a variety of materials, including substrates made of ceramic, glassy, metallic, or polymeric materials, or combinations of materials.
• substrate substrate
• support material support
• support may also be used herein to refer to a macromolecular support material.
• an IRM is attached to a macromolecular support material.
• the term "attached” includes both covalent bonding and non- covalent chemical association (e.g., ionic bonding and hydrogen bonding) of an immune response modifier with a macromolecular support material.
• Non-covalent association is preferably by specific, high affinity protein-ligand interaction, as opposed to nonspecific hydrogen bonding.
• the immune response modifiers are attached to a macromolecular support material by means of covalent bonding.
• the terms “coupled,” “conjugated,” “bonded,” or “immobilized” may also be used herein to represent “attached.”
• “attached” excludes mere coating of the macromolecular support material with an IRM.
• Such attachment of an IRM to a macromolecular support material can be used to reduce the occurrence of, or prevent, the systemic absorption of the IRM, and can minimize the systemic side effects sometimes observed with the systemic administration of an IRM. Also, such attachment of an IRM to a substrate can serve to limit or focus the effect of the IRM to a localized region for a desired duration, and if the support material can be removed, the IRM can then be easily removed at will along with it. This provides very important control over where and how long the IRM is applied.
• One or more IRMs can be attached to a solid support or other macromolecular support material. Also, one IRM can be attached to multiple solid supports or other macromolecular support materials.
• the substrate having the IRM attached thereto can be used in a variety of medical applications, which can be therapeutic, prophylactic (e.g., as a vaccine adjuvant), or diagnostic.
• treating a condition or a subject includes therapeutic, prophylactic, and diagnostic treatments.
• the IRM would preferentially activate precursor plasmocytoid dendritic cells (pDC cells) of the blood to stimulate LNF ⁇ formation, for a localized antiproliferative effect, with reduced systemic distribution of the IRM.
• pDC cells precursor plasmocytoid dendritic cells
• An IRM can be attached to a macromolecular support complex and used in wound dressings, wound packing materials, wound sealants, sutures, and surgical clips to promote healing and/or reduce the occurrence of scarring.
• An IRM-support complex can be applied to the vagina or uterus to treat vaginal infections, such as, e.g., herpes or papilloma virus.
• vaginal infections such as, e.g., herpes or papilloma virus.
• one or more IRMs can be attached to (as opposed to blended or dissolved in) a macromolecular support material, such as a material incorporated into a gel or foam, for application within the vagina or uterus.
• An IRM-support complex can be applied to the nasal cavity.
• one or more IRMs can be attached to a macromolecular support material, such as a gel, foam, or spray for application to the nasal passages and/or sinuses.
• An IRM-support complex can be applied to the eye to treat, e.g., viral infections, such as herpes.
• one or more IRMs can be attached to a macromolecular support material for inclusion in an ophthalmic preparation for application to the eye and/or to ophthalmic devices, such as intraocular lenses and contact lenses.
• An IRM can also be attached to a macromolecular support material, such as a polymer, for the formation of a compound depot in the body of a subject to promote the long term, localized effect of the IRM.
• a macromolecular support material such as a polymer
• an IRM can be attached to a macromolecular support material, such as an oligomer, a polymer, a bead, a tissue culture flask, a tissue culture plate, a microtiter plate, or a column, for use in, e.g., ex- vivo treatment of immune cells, experiment testing, or a diagnostic assay in which an IRM is a component.
• a macromolecular support material such as an oligomer, a polymer, a bead, a tissue culture flask, a tissue culture plate, a microtiter plate, or a column
• a diagnostic assay in which an IRM is a component.
• use of an LRM-support complex can enhance cellular contact with an IRM, can facilitate the removal of an IRM from a diagnostic assay, can allow for the concentrated delivery of an IRM, and can assist in the conservation of IRM reagents.
• Immune response modifiers include compounds act on the immune system by inducing and/or suppressing cytokine biosynthesis.
• IRMs possess potent immunostimulating activity including, but not limited to, antiviral and antitumor activity, and can also down-regulate other aspects of the immune response, for example shifting the immune response away from a T H 2 immune response, which is useful for treating a wide range of T H 2 mediated diseases.
• IRMs can also be used to modulate humoral immunity by stimulating antibody production by B cells. Further, various IRMs have been shown to be useful as vaccine adjuvants (see, e.g., U.S. Pat. Nos. 6,083,505 and 6,406,705, and International Publication No. WO 02/24225).
• IL-6, IL-8, IL-10, IL-12, MIP-1, and/or MCP-1 can also inhibit production and secretion of certain T H 2 cytokines, such as IL-4 and IL-5.
• Some LRMs are said to suppress IL-1 and TNF (see, e.g., International Publication No. WO 00/09506).
• preferred IRMs are so-called small molecule IRMs, which are relatively small organic compounds (e.g., molecular weight under about 1000 daltons, preferably under about 500 daltons, as opposed to large biologic protein, peptides, and the like).
• IRMs are known to be agonists of at least one Toll-like receptor (TLR). IRMs that are agonists for TLRs selected from 6, 7, 8, and 9 may be particularly useful for certain applications. Some small molecule IRMs are agonists of TLRs such as 6, 7, and 8, while oligonucleotide IRM compounds are agonists of TLR9, and perhaps others. Thus, in some embodiments, the IRM that is attached to a macromolecular support material may be a compound identified as an agonist of one or more TLRs.
• IRM compounds that activate a strong cytotoxic lymphocyte (CTL) response may be particularly desirable as vaccine adjuvants, especially for therapeutic viral and/or cancer vaccines because a therapeutic effect in these settings is dependent on the activation of cellular immunity.
• CTL cytotoxic lymphocyte
• IRM compounds that are TLR 8 agonists may be particularly desirable for use with therapeutic cancer vaccines because antigen presenting cells that express TLR8 have been shown to produce IL-12 upon stimulation tlirough TLR8.
• IRM compounds that are TLR 7 agonists and/or TLR 9 agonists may be particularly desirable for use with prophylactic vaccines because the type I interferon induced by stimulation through these TLRs is believed to contribute to the formation of neutralizing T ⁇ l-like humoral and cellular responses.
• LRM compounds that are both TLR 7 and TLR 8 agonists may be particularly desirable for use with therapeutic viral vaccines and/or cancer vaccines because TLR7 stimulation is believed to induce the production of type I UN and activation of innate cells such as macrophages and NK cells, and TLR8 stimulation is believed to activate antigen presenting cells to initiate cellular adaptive immunity as described above. These cell types are able to mediate viral clearance and/or therapeutic growth inhibitory effects against neoplasms.
• one way to determine if an IRM compound is considered to be an agonist for a particular TLR is if it activates an NFkB/luciferase reporter construct through that TLR from the target species more than about 1.5 fold, and usually at least about 2 fold, in TLR transfected host cells such as, e.g., HEK293 or Namalwa cells relative to control transfectants.
• TLR transfected host cells such as, e.g., HEK293 or Namalwa cells relative to control transfectants.
• Preferred LRM compounds include a 2-aminopyridine fused to a five-membered nitrogen-containing heterocyclic ring.
• IRMs are small organic molecules (e.g., molecular weight under about 1000 Daltons, preferably under about 500 Daltons, as opposed to large biologic protein, peptides, and the like) such as those disclosed in, for example, U.S. Pat. Nos.
• classes of small molecule IRM compounds include, but are not limited to, compounds having a 2-aminopyridine fused to a five-membered nitrogen- containing heterocyclic ring.
• Such compounds include, for example, imidazoquinoline amines including, but not limited to, substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ether substituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, and 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amines; tetrahydroimidazoquinoline amines including, but not limited to, amide substituted tetraliydroimidazoquinoline amines, sul
• IRM compounds comprise a 2-aminopyridine fused to a five- membered nitrogen-containing heterocyclic ring.
• IRMs said to induce interferon (among other things), include purine derivatives (such as those described in U.S. Pat. Nos. 6,376,501, and 6,028,076), imidazoquinoline amide derivatives (such as those described in U.S. Pat. No. 6,069,149), lH-imidazopyridine derivatives (such as those described in apanese Patent Application No. 9-255926), benzimidazole derivatives
• Examples of small molecule IRMs that comprise a 4-aminopyrimidine fused to a five-membered nitrogen-containing heterocyclic ring include adenine derivatives
• LRM compounds examples include 2-propyl[l,3]thiazolo[4,5- c]quinolin-4-amine, which is considered predominantly a TLR 8 agonist (and not a substantial TLR 7 agonist), 4-amino- ⁇ , ⁇ -dimethyl-lH-imidazo[4,5-c]quinoline-l- ethanol, which is considered predominantly a TLR 7 agonist (and not a substantial TLR 8 agonist), and 4-amino-2-(ethoxymethyl)- ⁇ 4 ⁇ -dimethyl-6,7,8,9-tetrahydro-lH- imidazo[4,5-c]quinoline-l -ethanol, which is a TLR 7 and TLR 8 agonist.
• 4-amino- 04a - dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol has beneficial characteristics, including that it has a much lower CNS effect when delivered systemically compared to imiquimod.
• IRM compounds include, e.g., N-[4-(4-amino- 2-butyl-lH-imidazo[4,5-c][l,5]naphthyridin-l-yl)butyl]-N'-cyclohexylurea, 2-methyl- 1 -(2-methylpropyl)- lH-imidazo [4,5-c] [ 1 ,5]naphthyridin-4-amine, 1 -(2-methylpropyl)- lH-imidazo [4, 5 -c] [ 1 , 5 ]naphthyridin-4-amine, N- ⁇ 2- [4-amino-2-(ethoxymethyl)- ⁇ H ⁇ imidazo [4,5-c] quinolin- 1 -yl] -1,1 -dimethylethyl ⁇ methanesulfonamide, N- [4-(4-amino- 2-ethyl-lH-imid
• IRMs include large biological molecules such as oligonucleotide sequences.
• IRM oligonucleotide sequences contain cytosine-guanine dinucleotides (CpG) and are described, for example, in U.S. Pat. Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705.
• CpG-containing oligonucleotides can include synthetic immunomodulatory structural motifs such as those described, for example, in U.S. Pat. Nos. 6,426,334 and 6,476,000.
• Other IRM nucleotide sequences lack CpG and are described, for example, in International Patent Publication No. WO 00/75304.
• IRMs Various combinations of IRMs can be used if desired.
• LRMs such as imiquimod - a small molecule, imidazoquinoline IRM, marketed as ALDARA (3M Pharmaceuticals, St. Paul, MN) - have been shown to be useful for the therapeutic treatment of warts, as well as certain cancerous or pre-cancerous lesions (See, e.g., Geisse et al., J. Am. Acad. Dermatol, 47(3): 390-398 (2002); Shumack et al, Arch. Dermatol, 138: 1163-1171 (2002); U.S. Pat. No. 5,238,944 and U.S. Pat.
• Conditions that may be treated by administering an IRM-support complex of the present invention include, but are not limited to:
• viral diseases such as, for example, diseases resulting from infection by an adenoviras, a herpesvirus (e.g., ⁇ SV-I, ⁇ SV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picomavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), apapovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus
• infectious diseases such as chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, or parasitic diseases including but not limited to malaria, pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection; and
• neoplastic diseases such as intraepithelial neoplasias, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, renal cell carcinoma, leukemias including but not limited to myelogeous leukemia, chronic lymphocytic leukemia, multiple myeloma, non- Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-cell lymphoma, and hairy cell leukemia, and other cancers;
• leukemias including but not limited to myelogeous leukemia, chronic lymphocytic leukemia, multiple myeloma, non- Hodgkin's lymphoma, cutaneous T-cell lymphoma, B-cell lymphoma, and hairy cell leukemia, and other cancers;
• T R -mediated atopic diseases, such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen's syndrome;
• atopic diseases such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen's syndrome;
• an IRM-support complex of the present invention may be useful as a vaccine adjuvant for use in conjunction with any material that raises either humoral and/or cell mediated immune response, such as, for example, live viral, bacterial, or parasitic immunogens; inactivated viral, tumor-derived, protozoal, organism-derived, fungal, or bacterial immunogens, toxoids, toxins; self-antigens; polysaccharides; proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines; autologous vaccines; recombinant proteins; glycoproteins; peptides; and the like, for use in connection with, for example, BCG, cholera, plague, typhoid, hepatitis A, hepatitis B, hepatitis C, influenza A, influenza B, parainfluenza, polio, rabies, measles, mumps, rubella, yellow fever, tetanus, diphtheria, hemophilus influenza b
• Certain IRM-support complexes of the present invention may be particularly helpful in individuals having compromised immune function.
• certain complexes may be used for treating the opportunistic infections and tumors that occur after suppression of cell mediated immunity in, for example, transplant patients, cancer patients and HIV patients.
• a macromolecular support material can be porous or nonporous, depending on preferred final use.
• a macromolecular support material can be continuous or non-continuous depending on ultimate desired usage.
• a macromolecular support material can be made of a variety of materials, which may be organic, inorganic, or combinations thereof, including substrates made of ceramic, glassy, metallic, oligomeric or polymeric materials, or combinations of materials.
• silicon-based materials e.g., silica-based materials can be used.
• the term "metal" includes metalloids such as silicon.
• a macromolecular support material can be flexible or inflexible depending on ultimate desired usage.
• a copolymer is used to refer to a polymer prepared from two or more monomers, and includes terpolymers, tetrapolymers, etc.
• Exemplary synthetic polymers include, but are not limited to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose prop
• Fluoropolymeric materials can also be used. Examples of such materials are disclosed in U.S. Pat. Nos. 6,630,047; 6,451,925; and 6,096,428.
• Exemplary natural polymers include, but are not limited to: alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), zein, and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. Copolymers and mixtures ofany of these polymers could be used if desired.
• polymeric hydrogel materials can be constructed from poly(vinyl alcohol) precursors as disclosed in U.S. Pat. Nos. 4,528,325 and 4,618,649 or from poly(methyl methacrylate).
• Poly(methyl methacrylate) is commercially available and is often used in ophthalmic devices such as intraocular lenses, contact lenses, and the like.
• a suitable hydrogel can be natural, synthetic, or a combination thereof.
• the hydrogel can be thermally responsive to a designed temperature such as, for example, a hydrogel as described in U.S. Patent Application Serial Number 10/626261, filed July 24, 2003.
• the thermally responsive hydrogels can be harden when they are warmed up to body temperature, can be further harden upon
• Preferred bioadhesive polymers include crosslinked polymers of acrylic acid. Suitable examples include acrylic acid polymers crosslinked with allyl sucrose or allyl pentaerythriol such as those polymers designated as carbomers. Suitable carbomers include, for example, those available as CARBOPOL 97 IP NF Polymer and
• CARBOPOL 974P NF Polymer available from Noveon, Inc., Cleveland, OH.
• crosslinked acrylic acid polymers include those crosslinked with divinyl glycol such as those designated as polycarbophils.
• Suitable polycarbophils include, for example, those available as NOVEON AA-1 USP Polycarbophil, available from Noveon, Inc., Cleveland, OH.
• Bioadhesive organic polymers are preferred for certain applications of IRMs.
• the IRM is to be used for treating cervical dysplasia or bladder cancer, a bioadhesive polymer is desired.
• the adhesive qualities of the formulation would allow the IRM to be in contact with the biological tissue allowing for greater contact time for cytokine induction.
• the IRM is not simply dissolved or blended into a formulation from which it is to be released, but is attached to the support material by a sufficiently strong bond (which sometimes may require a covalent bond) so that under the circumstances of intended use the IRM is biologically active during use while it is attached to the support.
• the IRM is covalently attached to the support material.
• Gels, creams, films, salves, coatings, sticks, colloids, pastes, and foams incorporating IRM-support complexes can be applied to a variety of bodily surfaces, and among the many uses may include, e.g., wound dressings and wound packing materials. These sorts of solid, semi-solid, or viscous preparations can serve to promote the retention of the IRM compounds on the bodily surface and also to prevent the systemic adsorption of the IRM.
• Bodily surfaces can include, but are not limited to, gastrointestinal tract, vagina, uterus, bladder, oral cavity, nasal passages, periodontal surfaces, rectum, ocular surfaces, or surfaces of the ear.
• Particles can be the substrate to which an JJRM is attached.
• particles can be in the form of beads, including, but not limited to carbohydrate beads and latex beads, such as those commercially available from many suppliers, including, for example Biorad and Pierce.
• oxide-containing particles e.g., silica particles
• the particles can also be in the form of microparticles, such as microspheres, microcapsules, etc. Nanoparticles, such as quantum dots can be used as well.
• Ceramic supports, glass supports, and metallic supports are all known in the art and are commercially available or can be prepared by a variety of known techniques.
• Woven and nonwoven webs are useful as substrates having either regular or irregular physical configurations of surfaces from which the IRMs can extend. Fibrous webs are particularly desired because such webs provide large surface areas, with nonwoven fibrous webs being preferred due to ease of manufacture, low material cost, and allowance for variation in fiber texture and fiber density.
• a wide variety of fiber diameters e.g., 0.05 micrometer ( ⁇ m) to 50 micrometers, can be used. Web thickness can be varied widely to fit the application, e.g., 0.2 micrometer to 10 centimeters (cm) thick or more.
• Fibrous webs can be prepared by methods known in the art, or by modifications of methods known in the art.
• Nonwoven webs can be prepared by melt-blowing as is known to those skilled in the art. hi general, a molten polymeric material is extruded in such a way as to produce a stream of melt blown polymer microfibers. The fibers are collected on a collection screen, with the microfibers forming a web.
• the nonwoven webs can also optionally include a permeable support fabric laminated to one or both sides of the web, or can additionally contain reinforcing fibers.
• Exemplary materials useful to prepare nonwoven fibrous webs include polymers and copolymers of monomers that form fibrous webs.
• Suitable polymers include polyalkylenes such as polyethylene and polypropylene, polyvinyl chloride, polyamides such as the various nylons, polystyrenes, polyarylsulfones, poly( vinyl alcohol), polybutylene, poly(ethylene vinyl acetate), polyacrylates such as polymethyl methacrylate, polycarbonate, cellulosics such as cellulose acetate butyrate, polyesters such as poly(ethylene terephthalate), polyimides, and polyurethanes such as polyether polyurethanes, and combinations thereof.
• Nonwoven webs can also be prepared from combinations of co-extruded polymers such as polyesters and polyalkylenes. Copolymers of the monomers that provide the above-described polymers are also included within the scope of the present invention. Nonwoven webs can also be combined webs, which are an intimate blend of fine fibers and crimped staple fibers.
• Suitable substrates for attachment of LRMs can also include microporous membranes, fibers, hollow fibers, or tubes, all of which are l ⁇ iown in the art.
• the same materials useful for preparing webs are also suitable for preparing fibers and membranes.
• An exemplary microporous membrane is one made from thermoplastic polymeric material using a thermally induced phase separation technique that involves melt blending a thermoplastic polymer with immiscible liquid at a temperature sufficient to form a homogeneous mixture, forming an article from the solution into a desired shape, cooling the shaped article so as to induce phase separation of the liquid and the polymer and to ultimately solidify the polymer, and removing at least a substantial portion of the liquid leaving a microporous polymer matrix.
• This method and the preferred compositions used in the method are described in detail in U.S. Pat. Nos. 4,957,943; 4,539,256; and 4,726,989.
• polymeric supports can also be hydrophobic polyolefin membranes prepared by thermally induced phase separation techniques, but also having a hydrophilic polymeric shell interlocked about such hydrophobic membrane surfaces.
• the support materials having and an IRM associated therewith can include a combination of materials.
• they can include a combination of inorganic and organic materials or a combination of different organic polymers. This can occur by layering the materials, for example.
• One or more of the materials can be associated (e.g., attached) to the particulate support material on the outermost surface such that an
• IRM is masked or hidden from a body's immune system until it reaches its targeted site of action.
• polymers of lactic acid and glycolic acid in the form of particles having one or more IRMs attached thereto can have a coating of a polyalkylene oxide polymer (e.g., polyethylene glycol) thereon (see, e.g., Gref et al, Colloids and Surfaces B: Biointerfaces 18, 301-313, 2000).
• the polyalkylene oxide can function to mask the IRM from the body's immune system until it reaches its targeted site of action.
• balloon expandable stents examples include the MULTILL K stent by Guidant Corp., the Coronary Stent S670 by Medtronic AVE, the NLR stent by Boston Scientific Corp., the CROSS FLEX stent by
• the vessels in which the stent of the present invention can be deployed include, but are not limited to, natural body vessels such as ducts, arteries, trachea, veins, intestines, bile ducts, ureters and the esophagus.
• cancerous blockages inside body passageways e.g., esophagus, bile ducts, trachea, intestine, vasculature and urethra, among others
• stents which operate to hold open passageways that have been blocked by the cancerous growth or tumors.
• stents do not prevent the ingrowth of the cancerous material througli the interstices of the stent.
• Systemic therapies that have been investigated for the prevention of restenosis include agents directed at treatment of endothelial loss, anti-platelet agents (e.g., aspirin), vasodilators (e.g., calcium channel blockers), antithrombotics (e.g., heparin), anti-inflammatory agents (e.g., steroids), agents that prevent vascular smooth muscle cell (VSMC) proliferation (e.g., colchicine), and promoters of re-endothelialization (e.g., vascular endothelial growth factor).
• anti-platelet agents e.g., aspirin
• vasodilators e.g., calcium channel blockers
• antithrombotics e.g., heparin
• anti-inflammatory agents e.g., steroids
• agents that prevent vascular smooth muscle cell (VSMC) proliferation e.g., colchicine
• promoters of re-endothelialization e.g., vascular endothelial
• Cytokines are mediators of both the acute and chronic inflammatory response. Many cytokines such as tumor necrosis factor alpha, interferon-alpha, interferon-gamma, interleukins 1, 4, 13, and monocyte chemoattractant protein- 1, and nitric oxide have been identified to play a role in the inflammatory response after vascular tissue injury. While the exact role that these and other cytokines play is not clear, there is evidence to suggest alterations in the fibroproliferative responses seen in oral, dermal and vascular tissues can be accomplished by altering the local cytokine profile in the tissue. For example interferon-alpha and gamma have been shown to reduce collagen synthesis by dermal fibroblasts and hypertrophic scar fibroblasts.
• Immune response modifiers include small molecules that trigger the production of cytokines from antigen presenting cells through, for example, toll-like receptor (TLR) pathways such as, for example, TLR 7 and/or 8.
• TLR toll-like receptor
• Some IRMs can direct the innate immune response to produce cytokines, such as IL-12 and interferon-gamma, that stimulate a THI or cell-mediated response. This T H I cytokine response can also lead to the reduction in cytokines implicated in the T H 2 response.
• medical device and “medical article” are used interchangeably and refer generally to any device that has surfaces that can, in the ordinary course of their use and operation, contact bodily tissue, organs or fluids such as blood.
• medical devices include, without limitation, stents, stent grafts, anastomotic connectors, leads, needles, guide wires, catheters, sensors, surgical instruments, angioplasty balloons, wound drains, shunts, tubing, urethral inserts, pellets, implants, pumps, vascular grafts, valves, pacemakers, and the like.
• a medical device can be an extracorporeal device, such as a device used during surgery, which includes, for example, a blood oxygenator, blood pump, blood sensor, or tubing used to carry blood, and the like, which contact blood which is then returned to the subject.
• a medical device can likewise be an implantable device such as a vascular graft, stent, stent graft, anastomotic connector, electrical stimulation lead, heart valve, orthopedic device, catheter, shunt, sensor, replacement device for nucleus pulposus, cochlear or middle ear implant, intraocular lens, and the like.
• Implantable devices include transcutaneous devices such as drug injection ports and the like.
• the medical devices include a shunt, an artificial valve, a suture, a surgical clip, a surgical staple, an indwelling catheter, a dental implant (with the proviso that the dental implant is not a periochip inserted into the periodontal cavity), an orthopedic implant, a surgical prosthetic, an implantable vascular access port, an artificial heart, a ventricular assist pump, a blood oxygenator, a blood filter, a hemodialysis unit, a hemoperfusion unit, a conduit tube within a heart lung machine, a tube within a dialysis apparatus, a tube within a plasmapheresis unit, an artificial pancreas, an artificial liver, an artificial lung, an intraocular lens, or a contact lens.
• the affinity constant between avidin and biotin is remarkably high (the dissociation constant, Kd, is approximately 10 "15 M, see, Green, N., Biochem J, 89, 599, 1963) and is not significantly lessened when biotin is coupled to a wide variety of biomolecules. Numerous chemistries have been identified for coupling biomolecules to biotin with minimal or negligible loss in the activity or other desired characteristics of the biomolecule. A review of the biotin-avidin technology can be found in Applications of Avidin-Biotin Technology to Affinity-Based Separation, Bayer, et al., J. of
• the avidin-biotin bond is stable in serum and in the circulation (R. D. Wei, D. H. Kou, S. L. Hoo, Experientia, Vol. 27, pp. 366-368, 1970). Once formed, the avidin-biotin complex is unaffected by most extremes of pH, organic solvents and denaturing conditions. Separation of streptavidin from biotin requires conditions, such as 8M guanidine, pH 1.5, or autoclaving at 121°C for 10 minutes.
• IRMs may be biotinylated using any known methodologies.
• IRMs may be biotinylated chemically, using activated biotin analogues, such as N- hydroxysuccinimidobiotin (NHS-biotin), which is commercially available from Pierce Chemical Company, Rockford, IL, and requires the presence of a free primary amino group on the IRM.
• NHS-biotin N- hydroxysuccinimidobiotin
• Immune response modifiers may be covalently bonded to a macromolecular support material by any of the methods l ⁇ iown in the art.
• U.S. Pat. Nos. 4,722,906, 4,979,959, 4,973,493, and 5,263,992 relate to devices having biocompatible agents covalently bound via a photoreactive group and a chemical linking moiety to the biomaterial surface.
• U.S. Pat. Nos. 5,258,041 and 5,217,492 relate to the attachment of biomolecules to a surface through the use of long chain chemical spacers.
• 5,002,582 and 5,263,992 relate to the preparation and use of polymeric surfaces, wherein polymeric agents providing desirable properties are covalently bound via a photoreactive moiety to the surface.
• Others have used photochemistry to modify the surfaces of biomedical devices, e.g., to coat vascular grafts. (See, e.g., Kito, H. et al., ASAIO Journal 39:M506-M511, 1993; and Clapper, D. L., et al., Trans. Soc. Biomat.
• PVA polyvinyl alcohol
• IRMs could be attached to macromolecular supports in a similar fashion to the methods described in U.S. Pat. Nos. 5,200,471, 5,344,701, 5,486,358, 5,510,421, and 5,907,016. These patents disclose macromolecular supports having biologically active agents covalently bound via the reaction of a nucleophilic-functional group on the biologically active agent with an azlactone functional group on the macromolecular support.
• the IRM can be attached to a macromolecular support material using a linking group.
• the linking group includes a reactive group capable of reacting with a reactive group on the substrate to form a covalent bond.
• Suitable reactive groups include those discussed in Hermanson, G. (1996), Bioconjugate Techniques, Academic Press, Chapter 2 "The Chemistry of Reactive Functional Groups", 137-166.
• the linking group may react with a primary amine (e.g., an N-hydroxysuccinimidyl ester or an N-hydroxysulfosuccinimidyl ester); it may react with a sulfhydryl group (e.g., a maleimide or an iodoacetyl), or it may be a photoreactive group (e.g.
• the linking group may also be an alkoxysilyl group (e.g., a triethyoxysilyl group) that can be covalently coupled to an IRM.
• the alkoxysilyl group can then be covalently coupled to a silicon-containing support material such as silica, which may be in the form of particles.
• the substrate includes a chemically active group accessible for covalent coupling to the linking group.
• a chemically active group accessible for covalent coupling to the linking group includes groups that may be used directly for covalent coupling to the linking group or groups that may be modified to be available for covalent coupling to the linking group.
• suitable chemically active groups include, but are not limited to, primary amines and sulfhydryl groups.
• attachment may occur by reacting an immune response modifier with a crosslinker and then reacting the resulting intermediate with a substrate.
• crosslinkers suitable for preparing bioconjugates are known and many are commercially available. See for example, Hermanson, G. (1996) Bioconjugate Techniques, Academic Press.
• Attachment also may occur, for example, according to the method shown in Reaction Scheme I in which the substrate is linked to the IRM moiety through R ⁇ .
• a compound of Formula III is reacted with a heterobifunctional crosslinker of Formula IV to provide a compound of II.
• RA and R B each contain a functional group that is selected to react with the other.
• R A contains a primary amine
• R B contains an amine-reactive functional group such as an N- hydroxysulfosuccinimidyl ester.
• R A and R B may be selected so that they react to provide the desired linker group in the conjugate.
• the reaction generally can be carried out by combining a solution of the compound of Formula III in a suitable solvent such as N,N-dimethylformamide with a solution of the heterobifunctional cross-linker of Formula IV in a suitable solvent such as N,N-dimethylformamide.
• the reaction may be run at ambient temperature.
• the product of Formula II may then be isolated using conventional techniques.
• step (2) of Reaction Scheme I a compound of Formula II that contains reactive group Z A is reacted with the substrate to provide the JJRM-couples substrate of Formula I.
• the reaction can be carried out by combining a solution of the compound of Formula II in a suitable solvent such as dimethyl sulfoxide with the substrate.
• a suitable solvent such as dimethyl sulfoxide
• the reaction may be run at ambient temperature or at a reduced temperature (approximately 4°C). If Z is a photoreactive group such as a phenyl azide then the reaction mixture will be exposed to long wave UV light for a length of time adequate to effect cross-linking (e.g., 10-20 minutes).
• the average number of immune response modifier moieties per substrate surface area may be controlled by adjusting the amount of compound of Formula II used in the reaction.
• a compound of Formula II may be synthesized without using a heterobifunctional crosslinker. So long as the compound of Formula II contains the reactive group ZA, it may be reacted with the substrate using the method of step (2) above to provide an IRM-coupled substrate.
• the R groups can be hydrogen or organic groups that can optionally include various substitutions. They can include alkyl groups, alkenyl groups, including haloalkyl groups, aryl groups, heteroaryl groups, heterocyclyl groups, and the like.
• preferred R groups include hydrogen, alkyl groups having 1 to 4 carbon atoms (i.e., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, and cyclopropylmethyl), and alkoxyalkyl groups (e.g., methoxyethyl and ethoxymethyl).
• R 3 and R are independently hydrogen or methyl or R 3 and R 4 join together to form a benzene ring, a pyridine ring, a 6-membered saturated ring or a 6-membered saturated ring containing a nitrogen atom.
• R 3 and R are independently hydrogen or methyl or R 3 and R 4 join together to form a benzene ring, a pyridine ring, a 6-membered saturated ring or a 6-membered saturated ring containing a nitrogen atom.
• haloalkyl is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like.
• aryl as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl.
• heteroaryl includes aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N).
• Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, and so on.
• Heterocyclyl includes non-aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N) and includes all of the fully saturated and partially unsaturated derivatives of the above mentioned heteroaryl groups.
• exemplary heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, isothiazolidinyl, and imidazolidinyl.
• the aryl, heteroaryl, and heterocyclyl groups can be unsubstituted or substituted by one or more substituents independently selected from the group consisting of alkyl, alkoxy, methylenedioxy, ethylenedioxy, alkylthio, haloalkyl, haloalkoxy, haloalkylthio, halogen, nitro, hydroxy, mercapto, cyano, carboxy, formyl, aryl, aryloxy, arylthio, arylalkoxy, arylalkylthio, heteroaryl, heteroaryloxy, heteroarylthio, heteroarylalkoxy, heteroarylalkylthio, amino, alkylamino, dialkylamino, heterocyclyl, heterocycloalkyl, alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, haloalkylcarbonyl, haloalkoxycarbonyl, alkylthiocarbonyl
• the IRM is attached to the substrate through a linking group at the N 1 nitrogen of the imidazole ring.
• the linking can occur at different positions on the ring system. Examples of which are shown below for imidazoquinoline amines, imidazonaphthyridine amines and imidazopyridine amines respectively.
• the attachment is effected using the method of Reaction Scheme I starting with an IRM containing reactive group R A at the desired attachment point.
• the IRM-support complexes of the present invention can be incorporated into a wide variety of formulations, including, for example gels, creams, dispersions, or solutions.
• formulations can include solvents (e.g., propylene glycol, sorbitol, polyethylene glycol, hexylene glycol, dipropylene glycol), oils (e.g., mineral oil, vegetable oils, fatty acid triglycerides, isopropyl mysristate, isopropyl palmitate, and isostearic acid), emulsifiers (polysorbate 60, sorbitan monostearate, polyglyceryl-4 oleate, polyoxyethylene(4) lauryl ether, poloxamers, and sorbitan trioleate), preservatives (e.g., methylparaben and propyl paraben), neutralizers (e.g., sodium hydroxide), etc.
• solvents e.g., propylene glycol, sorbito
• formulations are disclosed in U.S. Pat. No. 6,245,776 and U.S. Patent Publication No. 2003/0199538. If the formulation is a gel, for example, it can be a preformed gel or formed at the application site.
• An amount of an LRM-support complex effective for a given therapeutic or prophylactic application is an amount sufficient to achieve the intended therapeutic or prophylactic application.
• the precise amount of IRM-support complex used will vary according to factors known in the art including but not limited to the physical and chemical nature of the IRM compound, the nature of the macromolecular support material, the intended dosing regimen, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the IRM compound, and the species to which the formulation is being administered. Accordingly it is not practical to set forth generally the amount that constitutes an amount of IRM support complex effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
• the dosing regimen may depend at least in part on many factors known in the art including but not limited to the physical and chemical nature of the IRM compound, the nature of the macromolecular support material, the amount of IRM being administered, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the IRM-support complex, and the species to which the formulation is being administered. Accordingly it is not practical to set forth generally the dosing regimen effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
• JJRM compounds used in the examples are shown in Table 1.
• the oil was purified by column chromatography on silica gel (eluting with 97.5:2.5 chloroform:methanol) to provide 12.4 g of N-[2-(7-benzyloxy-2- ethoxymethyl- lH-imidazo[4,5-c]quinolin- 1 -yl)- 1 , 1 -dimethylethyljmethanesulfonamide as a beige solid.
• the crude product was purified by column chromatography on silica gel (eluting sequentially with 95:5 and 92.5:7.5 dichloromethane:methanol) to provide 8.5 g of tert-butyl ⁇ 6-[2-ethoxymethyl-l-(2- methanesulfonylamino-2-methylpropyl)- lH-imidazo [4,5-c] quinolin- 1 - yloxy]hexyl ⁇ carbamate as a white solid.
• Example 1 IRMl and IRM2, each containing a biotin moiety, were coupled to ULTRALINK immobilized monomeric avidin beads (item number 53146, PIERCE
• Phosphate buffered saline 1.4 mL was added, the suspension was mixed by vortexing, the beads were allowed to settle, and then supernatant (1.4 mL) was removed. The wash procedure was repeated 5 times and then the beads were resuspended to 1 mL to provide a solids concentration of 50%.
• PBS represents the PBS buffer control
• BdlMono represents the monomeric avidin bead control (50%) slurry)
• Bd2Tetra represents the tetrameric avidin bead control (50% slurry)
• IRMl represents 0.7 ⁇ M unbound IRMl
• IRM2 represents 0.7 ⁇ M unbound 1RM2
• IRMlMono represents LRMl bound on Bdl (0.7 ⁇ M IRM in 50% slurry)
• IRM2Mono represents 1RM2 bound on Bdl (0.7 ⁇ M IRM in 50% slurry)
• IRMl Terra represents IRMl bound on Bd2 (0.7 ⁇ M IRM in 50% slurry);
• IRM2Tetra represents IRM2 bound on Bd2 (0.7 ⁇ M IRM in 50% slurry).
• the dispersion was centrifuged to remove the solvents.
• Example 3 Preparation of IRM Grafted Nanoparticles A dispersion of SiO 2 particles (0.49 g of 2327, 20 nm ammonium stabilized colloidal silica sol, 41 % solids; Nalco, Naperville, IL) was placed in a 5 mL vial. The dispersion was diluted with 0.2 g of deionized water and 0.5 g of DMSO.
• Example 5 Preparation of IRM Grafted Nanoparticles The procedure of Example 3 was repeated except that the amount of N-[3-(4- amino-2-ethoxymethyl-6,7-dimethyl-lH-imidazo[4,5-c]pyridin-l-yl)propyl]-N'-[3- (triethoxysilyl)propyl]urea was reduced from 33 mg to 8.5 mg.
• Example 6 Preparation of IRM Grafted Nanoparticles The procedure of Example 3 was repeated except that the amount of PEG triethoxysilane was increased from 12.4 mg to 31.0 mg.
• Example 7 Preparation of LRM Grafted Nanoparticles The procedure of Example 2 was repeated except that 34 mg of 7V-[4-(4-amino- 2-propyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-N'-[3-(triethoxysilyl)propyl]urea was used in lieu of 7V-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-lH-imidazo[4,5-c]pyridin- l-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea.
• Example 8 Preparation of IRM Grafted Nanoparticles The procedure of Example 3 was repeated except that 34 mg of N-[4-(4-amino- 2-propyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-iV'-[3-(triethoxysilyl)propyl]urea was used in lieu of N-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-lH-imidazo[4,5-c]pyridin- l-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea.
• Example 9 Preparation of IRM Grafted Nanoparticles The procedure of Example 8 was repeated except that the amount of N-[4-(4- amino-2-propyl- lH-imidazo [4,5-c] quinolin- 1 -yfjbutyl] -N '- [3 - (triethoxysilyl)propyl]urea was reduced from 34 mg to 17 mg.
• Example 8 The procedure of Example 8 was repeated except that the amount of N-[4-(4- amino-2-propyl- 1 H-imidazo [4,5-c] quinolin- 1 -yl)butyl] -N'- [3 - (triethoxysilyl)propyl]urea was reduced from 34 mg to 8.5 mg.
• the laminated sample was placed under a photoreactor consisting of 6 germicidal lamps (G15T8 bulb, 15W, available from General Electric Company, Nela Park, Cleveland, OH).
• the laminated sample was placed 7.6 - 10 cm away from the bulbs with fluoropolymer film facing the UV lamps.
• the sample was subjected to UV irradiation for 10 min.
• the treated fluoropolymer film was removed and immersed in THF for 2 hours.
• the film was removed from the THF and subjected to further washing with THF.
• the film was dried under a stream of N 2 gas.
• the grafted film was sampled and analyzed by ESCA:
• Example 13 Preparation of an IRM Grafted Fluoropolymer Film Glass microscope slides (5.1 cm by 7.6 cm) were cleaned with acetone and distilled water. One surface of the glass substrate was coated with a methanol solution containing 10 wt-% of 3-aminopropyl triethoxysilane and 5 wt-% of n-phenyl propyl triethoxy silane (both are available form GELEST, INC. 11 Steel Rd.
• the treated fluoropolymer film was removed and immersed in methanol for 2 hours to remove any residual primers. The film was removed from the methanol and subjected to further washing with methanol.
• the resulting triethoxyoxysilane grafted FEP film was then coated with a solution of 7V-[4-(4-amino-2-propyl- lH-imidazo [4,5 -c] quinolin- 1 -yl)butyl] -N'- [3 - (triethoxysilyl)propyl]urea in DMSO (1 wt-%>) and subsequently subjected to heat treatment in an oven at 50 °C overnight.
• Example 14 Preparation of an LRM Self Assembled Monolayer A glass microscope slide was cleaned with H 2 O 2 /concentrated sulfuric acid in a 1:3 mixture and subsequently washed with distilled water. The cleaned glass slide was immersed in a solution of 7Y-[3-(4-amino-2-ethoxymethyl-6,7-dimethyl-lH- imidazo[4,5-c]pyridin-l-yl)propyl]-N'-[3-(triethoxysilyl)propyl]urea in DMSO (1 wt- %) for 30 min.
• Example 15 IRMs were covalently coupled to gold particles to form nanometer-sized IRM- gold conjugates through a two-step reaction: the gold surface was functionalized with carbonate by reacting with thiol carbonate; the carbonate functional group was then coupled to the primary amine group of an IRM catalyzed by a carbodiimide.
• the final mixture was shaken at 3 ⁇ z at room temperature for another 12 hours (hr) followed by centrifugation at 14,000 revolutions per minute (rpm) for 30 minutes (min). After removing the supernatant, the precipitant was washed with 0.5 mL of PBS buffer twice before being redispersed in 1 millilter (mL) of PBS.
• a Field Emission SEM micrograph showed that, the modified particles were well separated and distributed. The infrared spectrum showed that there was a significant increase at the -NH- signal, indicating the successful coupling of IRM to the colloidal gold.
• Example 16 Similarly, a gold conjugate was also made with 10 nm colloidal gold (catalog number: 154011, ICN Biomedicals).
• Example 17 LRM-gold particles was also made from avidin-biotin or anti biotin-biotin coupling: reacting the commercially available gold-strepavidin (Amersham
• Example 18 An IRM conjugate of ferritin, a metaloprotein containing 4000 to 5000 Fe ions, was synthesized through direct coupling between the carboxyl group of [(4- amino-l-isobutyl-lH-imidazo[4,5-c]quinolin-2-yl)methoxy]acetic acid and the primary amine of ferritin catalyzed by l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
• EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
• the average ratio of [(4-amino-l- isobutyl-lH-imidazo[4,5-c]quinolin-2-yl)methoxy]acetic acid to ferritin in the conjugate was determined to be 0.6 (M/M), based on the UV spectrum measurement of [(4-amino-l-isobutyl-lH-imidaz[4,5-c]quinolin-2-yl)methoxy]acetic acid in the initial solution and the eluted solution.
• the recovery rate of ferritin was 95% after passing through the column.
• the eluted fraction was verified by HPLC, which showed a single peak. No significant lost of iron was observed during the modification.
• the conjugate showed biological activities in a test with RAW cells.
• Example 19 An IRM was covalently immobilized onto functionalized superparamagnetic particles using a modified protocol based on the manufacturer's suggested protocol.
• the mixture was centrifuged at 3000G for 10 min. The supernatant was removed and the IRM concentration was determined by UV absorption at 247 nm.
• the beads were washed with 7 mL of methanol 3 times and 7 ml of Dulbecco's PBS 3 times.
• the IRM content in the modified beads was calculated by subtracting the amount of IRM found in the supernatant and washes from the amount of IRM that was initially combined with the beads.
• An IRM was covalently immobilized onto nanosized superparamagnetic particles using the following procedure.
• An IRM was covalently attached to core shell superparamagnetic particles using the following procedure.
• a portion (1 mL) of water-based silica coated superparamagnetic particles 50 mg, SiMAG-1, Chemicell Gmbh, Berlin, Germany
• a water based dispersion of core shell magnetic particles with dimensions in the range of 100 nm was diluted with 5mL de-ionized water and 15 mL 2-propanol.
• IRMs can be covalently attached to a bioadhieve polymer as follows:: IRMs can be covalently attached to bioadhesive crosslinked polymers of acrylic acid through amide or ester formation. An IRM containing a pendant amine or hydroxyl group is reacted with a free carboxylic acid group on the polymer to form an amide or an ester respectively. IRM compounds containing pendant amine or hydroxyl groups and methods of making them are known. See, for example, U.S. Pat. Nos.
• IRM-bioadhesive polymer complex and other suitable complexes can be incorporated into a gel, cream, or solution formulation.
• Such formulations can include solvents, oils, emulsifiers, etc. Examples of suitable formulations are disclosed inU.S. Pat. No. 6,245,776 and U.S. Patent Publication No. 2003/0199538.
• Example 23 An IRM was covalently immobilized in a hydrogel using the following procedure. Preparation of Component A
• Polyethylene glycol disuccinimidyl succinate (PEG-SS2, prepared according to the method of Example 1 in U.S. Pat. No. 5,583,114) was dissolved in sterile water at 300 mg/mL.
• Component A (1 mL) and Component B (0.5 mL) were combined.
• a transparent hydrogel formed in about 10 seconds.
• Collagen fibers (available from Caliochem) were combined with a solution of 1- aminobutyl)-2-propyl-lH-imidazo[4,5-c]quinolin-4-amine in tetrahydrofuran (5 g of 1 wt-%)). 1,3-Dicyclohexylcarbodiimide (50 mg) was added and the mixture was placed in a in an ultrasonic bath at 45°C for 24 hours. The collagen fibers were removed from the solution and thoroughly washed with tetrahydrofuran. A sample was analyzed by
• Example 26 Preparation of IRM Grafted Poly(ethylene terephthlate) Film Pieces of poly(ethylene terephthlate) film (available from 3M Company, St. Paul, MN) were combined with a solution of l-(4-aminobutyl)-2-propyl-lH- imidazo[4,5-c]quinolin-4-amine in tetrahydrofuran (2 g of 2.5 wt-%). The mixture was placed in a in an ultrasonic bath at 45°C for 24 hours. The pieces of film were removed from the solution and thoroughly washed with tetrahydrofuran. Electron Spectroscopy of Chemical Analysis (ESCA), 52° with respect to the sample surface, of a sample showed the existence of nitrogen at 6-7% of the total composition. Analysis of an untreated PET film did not show nitrogen.
• ESA Electron Spectroscopy of Chemical Analysis
• Example 27 Preparation of IRM Grafted Acrylate Beads
• Oxirane functionalized acrylic beads (160 mg, average 150 ⁇ m in diameter, from Sigma Chemical, Cat. No. O-76280) were suspended in PBS (2 mL) and incubated for 30 minutes.
• the pH of the mixture was brought up to 9 by the addition of IN sodium hydroxide.
• the reaction mixture was shaken (3 Hz) at room temperature for 72 hours. The reaction mixture was then centrifuged at 3000 G for 5 min.

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