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WO1992021353A1 - Compositions et systemes d'apport destines a l'administration par voie transdermique d'oligomeres neutres - Google Patents

Compositions et systemes d'apport destines a l'administration par voie transdermique d'oligomeres neutres Download PDF

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Publication number
WO1992021353A1
WO1992021353A1 PCT/US1992/004370 US9204370W WO9221353A1 WO 1992021353 A1 WO1992021353 A1 WO 1992021353A1 US 9204370 W US9204370 W US 9204370W WO 9221353 A1 WO9221353 A1 WO 9221353A1
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WIPO (PCT)
Prior art keywords
oligomer
neutral
skin
agents
region
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Application number
PCT/US1992/004370
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English (en)
Inventor
Lyle J. Arnold, Jr.
Kameron Maxwell
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Genta Incorporated
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Publication date
Application filed by Genta Incorporated filed Critical Genta Incorporated
Priority to JP5500490A priority Critical patent/JPH06507911A/ja
Publication of WO1992021353A1 publication Critical patent/WO1992021353A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/55—Phosphorus compounds
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates

Definitions

  • oligodeoxyribonucleotides for the treatment of conditions caused by viruses such as Herpes viruses.
  • they In order for Oligomers to be useful as therapeutic agents, they must be able to gain access to their sites of action. Since most of these sites of action are within the body of the animal to be treated, is necessary that the Oligomers be able to enter the body tissues. Moreover, such Oligomers may need to be able to cross cell membranes and to enter individual cells. It is possible that access to the body be achieved by injection, but this is not a preferred route of access, especially in cases where repeated administration or self- administration is involved. More preferred routes are by either skin or mucous membrane absorption.
  • Mucous mem ⁇ brane absorption can occur within the nasal passages, oral cavity, lung, upper or lower gastrointestinal tract or vaginal tract.
  • Topical skin delivery is also a preferred route of administration.
  • Local delivery of these thera-plastic agents has the additional benefit of potentially concentrating these agents near the site of action for enhanced effectiveness of the applied dose. This latter mode of application can also greatly reduce the cost of treatment.
  • Oligomer compositions that can maximally penetrate skin and mucous membranes.
  • Typi ⁇ cal drugs administered transdermally are small molecules having molecular weights less than 400 daltons. Oligomers which are macromolecules having molecular weights far in excess of 400 daltons would not be considered likely can ⁇ didates for transdermal delivery a priori. Summary of the Invention
  • the present invention is directed to a method of delivery of an antisense Oligomer for therapeutic purposes by administration across skin or mucous membranes.
  • Said Oligomers may have therapeutic activity by preventing or interfering with expression of a specific nucleic acid target sequence or the protein product of that sequence.
  • this method comprises the application to skin or mucous membrane of a formulation which comprises a neutral Oligomer which is complementary to and which can bind to or interact with said nucleic acid target sequence.
  • These Oligomers may hybridize to a single- stranded target nucleic acid sequence or alternatively may interact with a double-stranded target sequence to form a triple-stranded complex. (See, e.g.. USSN 368,027 "Forma ⁇ tion of Triple Helix Complexes of Double Stranded DNA Using Nucleoside Oligomers," the disclosure of which is incorporated herein by reference) .
  • the present invention is based upon our surprising finding that formulations comprising these neutral Oligomers which have no ionic internucleo- sidyl linkages are able to penetrate skin or mucous mem ⁇ brane at a substantially higher rate than Oligomers which have ionic internucleosidyl linkages.
  • Oligomers having only one ionic internucleo ⁇ sidyl linkage and the remainder of the internucleosidyl linkages neutral internucleosidyl linkages penetrate skin at a substantially lower rate than the neutral Oligomers of the present invention.
  • the delivery of these neutral Oligomers across skin or mucous membranes can be used in the direct or systemic treatment of various conditions by preventing expression of a nucleic acid target sequence.
  • deliv ⁇ ery of these neutral Oligomers across mucous membranes of the nose, the lung or gastrointestinal tract is suitable for systemic treatment, as well as direct treatment of that tissue.
  • These neutral oligomers may be used in direct treatment of skin or vaginal tissue.
  • the direct delivery of these neutral Oligomers across skin or mucous membranes may be used to prevent expression of specific nucleic acid sequences which may comprise either foreign nucleic acids from, for example, a virus, or an endogenous nucleic acid sequence.
  • topical administration of these neutral Oligomers may be used against viral, fungal and bacterial infections of the skin and mucous membranes such as genital warts caused by the human papilloma virus and infections caused by Herpes viruses.
  • topi ⁇ cal administration of neutral Oligomers may be used to block mediators of inflammation.
  • transdermal delivery of these neutral Oligomers may be used to treat conditions in which improper immune or inflammatory responses have been implicated such as psoriasis, atopic dermatitis, eczema, rheumatoid arth ⁇ ritis, allergic rhinitis and the like.
  • transdermal delivery of these neutral oligomers may be used to treat certain cancers of the skin and mucous membranes such as melanoma, mycosis fungoides, and squamous cell carcinoma (including of the cervix) by blocking of the expression of certain cellular factors which are involved in their proliferation.
  • the delivery of these neutral Oligomers across mucous membranes may be used in systemic treatment of a number of conditions.
  • these neutral Oligomers may be administered by inhalation to nasal and lung mucous mem ⁇ branes.
  • the systemic delivery of these Oligomers may be used to prevent expression of specified nucleic acid tar ⁇ get sequences which may comprise either foreign nucleic acid from, for example a virus, or an endogenous nucleic acid sequence.
  • One proposed target is to regulate over- expression of renin in the kidney. Since these Oligomers are reported to be cleared by the body through the kidney (see Colvin, O.M., et al.. Drug Metabolism and Disposi ⁇ tion. Vol. 18, pages 815-818 (1990)), administration of Oligomers across mucous membranes should result in a therapeutically effective concentration of Oligomer in the kidney.
  • the present invention is directed to compositions and delivery systems for the direct or sys ⁇ temic administration of neutral Oligomers across skin or mucous membranes.
  • the composi- tions of the present invention comprise a neutral Oligomer in a vehicle which comprises a short chain aliphatic alco ⁇ hol, preferably ethanol.
  • the vehicle com ⁇ prises a flux enhancer which increases transdermal (or transmembrane) flux of said oligomer.
  • Suitable enhancers include compounds which disrupt the skin or mucous mem ⁇ brane barrier to the Oligomer and/or change the partition ⁇ ing behavior of the Oligomer to the skin or mucous mem ⁇ brane.
  • Suitable enhancers include decylmethylsufoxide, and cyclic ketones, lactones, anhydrides or esters such as those described in PCT application PCT/US86/02583 (Publi ⁇ cation No. WO 87/03473), and other enhancers known to those skilled in the art.
  • compositions for transdermal delivery may optionally include a retention enhancer which increases retention of the Oligomer in the skin, particularly in the viable tis ⁇ sue (rather than the stratum corneum) .
  • Suitable retention enhancers include cyclic ketones such as those described in PCT/U586/02583 (Publication No. WO87/03473) .
  • nucleoside includes a nucleosidyl unit and is used interchangeably therewith, and refers to a subunit of a nucleic acid which comprises a 5 carbon sugar and a nitrogen-containing base.
  • the 5 carbon sugar is ribose; in DNA, it is a 2'-deoxyribose.
  • the term also includes analogs of such subunits.
  • non-nucleoside monomeric unit refers to a mono- meric unit which does not significantly participate in hybridization of an Oligomer to a target sequence. Such monomeric units must not, for example, participate in any significant hyhdrogen bonding with a nucleoside, and would exclude monomeric units having as a component, one of the 5 nucleotide bases or analogs thereof.
  • a “nucleoside/non-nucleoside polymer” refers to a polymer comprised of nucleoside and non-nucleoside mono ⁇ meric units.
  • oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 6 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means.
  • Oligomer refers to a chain of oligonucleosides which have internucleosidyl linkages linking the nucleoside monomers and thus, includes oligonucleotides, nonionic oligonucleoside alkyl and aryl-phosphonate analogs, alkyl- and aryl-phosphono- thioates phosphorothioate analogs of oligonucleotides, phosphoramidate analogs of oligonucleotides, neutral phosphate ester oligonucleoside analogs, such as phospho- triesters and other oligonucleoside analogs and modified oligonucleosides, and also includes nucleoside/non- nucleoside polymers.
  • nucleoside/ non-nucleoside polymers wherein one or more of the phos ⁇ phorous group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a, morpho- lino linkage, a formacetal linkage, a sulfamate linkage or a carbamate linkage.
  • alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl-phospho ⁇ nate internucleosidyl linkage.
  • methylphosphonate Oligomer (or "MP-Oligo- er") refers to Oligomers having at least one methylphos ⁇ phonate internucleosidyl linkage.
  • neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e. linkages having no net positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or aryl-phospho ⁇ nate linkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyltriester linkages; and non-phos ⁇ phorus-containing internucleosidyl linkages, such as sul- famate, morpholino, formacetal and carbamate linkages.
  • a neutral Oligomer may comprise a conjugate between a oligonucleoside or nucleoside/non-nucleoside polymer and a second molecule which comprises a conjuga ⁇ tion partner.
  • conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, photo-cross- linking agents such as psoralen, and the like.
  • con ⁇ jugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharmacokinetic distribution of the Oligonucleoside.
  • the essential requirement is that the oligonucleoside or nucleoside/non-nucleoside polymer that the conjugate comprises be neutral.
  • neutral alkyl- or aryl- phosphonate oligo ⁇ mer refers to neutral oligomers having neutral inter ⁇ nucleosidyl linkages which comprise at least one alkyl- or aryl- phosphonate linkage.
  • neutral methylphosphonate oligomer refers to neutral oligomers having internucleosidyl linkages which comprise at least one methylphosphonate linkage.
  • p in, e.g., as in ApA represents a phosphate diester linkage
  • p_ in, e.g., as in CpG represents a ethylphosphate linkage.
  • the term “tandem oligonucleotide” or “tandem Oligo ⁇ mer” refers to an oligonucleotide or oligomer which is complementary to a sequence located either on the 5 • or 3• side of a target nucleic acid sequence and which is co-hybridized with a second oligomer which is complemen ⁇ tary to the target sequence.
  • Tandem oligomers may improve hybridization of these oligomers to the target by helping to make the target sequence more accessible to such oli ⁇ gomers, such as by decreasing the secondary structure of the target nucleic acid sequence.
  • one of a pair of tandem Oligomers may improve the hybrid stability of the second tandem Oligomer by promoting a helical struc ⁇ ture at either the 5'- or 3'-end of said second Oligomer and vice-versa.
  • short chain aliphatic alcohol refers to an alcohol having from about 2 to about 20 carbon atoms in which the aliphatic (alkyl) chain may be either straight chained or branch chained and includes primary, secondary and tertiary alcohols, glycols and polyols.
  • flux enhancer refers to a substance which is used to increase transdermal flux of a compound.
  • a flux enhancer is typically applied to skin or mucous mem ⁇ brane in combination with the compound to increase trans ⁇ dermal flux of the compound. Enhancers are believed to function by disrupting the skin or mucous membrane barrier or by changing the partitioning behavior of the drug in the skin or mucous membrane.
  • FIG. l depicts a plot of cumulative amount of Oligo- mer penetrating hairless mouse skin versus time for sev ⁇ eral concentrations formulations of 14-mer in vehicle (EtOH/H 2 0/DMS, 80:15:5).
  • FIG. 2 depicts a plot of cumulative amount of a 14-mer having one internal anionic internucleosidyl link- age ("14-mer-lA") penetrating hairless mouse skin versus time (vehicle was EtOH (DMS/95:5).
  • FIG. 3 depicts a plot of percentage of applied dose of Oligomer absorbed versus time for formulations having varying concentrations of 14-mer.
  • FIG. 4 depicts a plot of cumulative amounts of 14-mer penetrating human cadaver skin versus times for several formulations of 14-mer.
  • Preferred neutral Oligomers include neutral alkyl- and aryl- phosphonate Oligomers and neutral Oligomers com ⁇ prising triester or phosphoramidate internucleosidyl link ⁇ ages.
  • neutral methylphosphonate Oligomers are particularly preferred.
  • particularly preferred are neutral methylphosphonate Oligomers having only methyl ⁇ phosphonate internucleosidyl linkages.
  • Oligomers which comprise one or more tri ⁇ ester internucleoside linkages, especially neutral ethyl- triester internucleoside linkages. It is believed that once such Oligomers have entered the cell, the ester group is cleaved to give a negatively charged diester linkage which is better retained inside the cell.
  • such Oligomers may incorporate either one or more methylphosphonate internucleoside link ⁇ ages or one or more nucleosides having a 2'-O-methyl ribo- syl moiety.
  • the presence of methylphosphonate inter ⁇ nucleoside linkages at the ends of the Oligomer gives exonuclease resistance and the incorporation of methyl ⁇ phosphonate internucleoside linkages or nucleosides which comprise a 2•-O-methyl ribosyl moiety in the middle of the Oligomer increases resistance of the Oligomer to endonucleases.
  • neutral Oligomers having from about 6 to about 40 nucleosides, more preferably from about 12 to about 20 nucleosides.
  • neutral Oligomers which comprise more than 20 nucleosides may be used, where complementarity to a longer sequence is desired, it may be advantageous to employ shorter tandem neutral Oligomers to maximize solubility and penetration through the skin or mucous membranes while competing for the development of a secondary structure of the target nucleic acid, such as a mRNA.
  • the neutral Oligomers comprise alkyl- or aryl- phosphonate Oligomers
  • the use of nucleoside units having 2'-0-alkyl- and, in particular, 2-O-methyl-ribosyl moieties in these neutral Oligomers may advantageously improve hybridization of the Oligomer to its complementary target sequence.
  • these neutral Oligomers may comprise a conjugate between a polynucleo- side or nucleoside/non-nucleoside polymer and a conjuga ⁇ tion partner.
  • Suitable conjugation partners include intercalating agents, alkylating agents, binding sub ⁇ stances for cell surface receptors, lipophilic agents, photo-crosslinking agents such as psoralen, hydrolytic or nucleolytic agents, pro-chelates or DNA modifying agents.
  • conjugation partners may include groups such as photo-crosslinking groups like psoralen, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving a targeted portion of a nucleic acid such as hydrolytic or nucleolytic agents like o-phen- anthrolinecopper or EDTA-iron may be incorporated in the Oligomers.
  • groups such as photo-crosslinking groups like psoralen, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving a targeted portion of a nucleic acid such as hydrolytic or nucleolytic agents like o-phen- anthrolinecopper or EDTA-iron may be incorporated in the Oligomers.
  • Oligomers may be labelled by any of several well known methods.
  • Useful labels include radioisotopes.
  • Isotopic labels include 3 H, 35 5, 32 P, 125 I, Cobalt and 14 C.
  • Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, and labelling, second strand synthesis, and reverse transcription.
  • Conjugation partners may also be introduced into the Oligomer by the incorporation of modified nucleosides or nucleoside analogs through the use of enzymes or by chem ⁇ ical modification of the Oligomer, for example, by the use of nonnucleotide linker groups.
  • these Oligomers When used to prevent function or expression of a single or double stranded nucleic acid sequence, these Oligomers may be advantageously derivatized or modified to incorporated a nucleic acid modifying group which may be caused - o react with said nucleic acid and irreversibly modify its structure, thereby rendering it non-functional.
  • a nucleic acid modifying group which may be caused - o react with said nucleic acid and irreversibly modify its structure, thereby rendering it non-functional.
  • USSN 565,299 discloses psoralen-derivatized Oligomers.
  • a wide variety of DNA modifying groups may be used as conjugation partners to derivatize these Oligomers.
  • DNA modifying groups include groups which, after the deriva ⁇ tized Oligomer forms a complex with a single stranded or double stranded DNA segment, may be caused to form a covalent linkage, cross-link, alkylate, cleave, degrade, or otherwise inactivate or destroy the DNA segment or a target sequence portion thereof, and thereby irreversibly inhibit the function and/or expression of that DNA segment.
  • the location of the DNA modifying groups in the Oli ⁇ gomer may be varied and may depend on the particular DNA modifying group employed and the targeted double stranded DNA segment. Accordingly, the DNA modifying group may be positioned at the end of the Oligomer or intermediate the ends. A plurality of DNA modifying groups may be included.
  • the DNA modifying group is photoreactable (e.g., activated by a particular wave ⁇ length, or range of wavelengths of light) , so as to cause reaction and, thus, cross-linking between the Oligomer and the double stranded DNA.
  • DNA modifying groups which may cause cross-linking are the psoralens, such as an aminomethyl- trimethyl psoralen group (AMT) .
  • AMT aminomethyl- trimethyl psoralen group
  • the AMT is advantageously photoreactable, and thus must be activated by exposure to particular wavelength light before cross-linking is effec- tuated.
  • Other cross-linking groups which may or may not be photoreactable may be used to derivatize these Oligomers.
  • the DNA modifying groups may comprise an alkylating agent group which is covalently bonded to the DNA segment to render it inactive.
  • alkyl ⁇ ating agent groups are known in the chemical art sand include groups derived from alkyl halides, haloacetamides, and the like.
  • DNA modifying groups which may be caused to cleave the DNA segment include transition metal chelating com ⁇ plexes such as ethylene diamine tetraacetate (EDTA) or a neutral derivative thereof.
  • transition metal chelating com ⁇ plexes such as ethylene diamine tetraacetate (EDTA) or a neutral derivative thereof.
  • Other groups which may be used to effect cleaving include phenanthroline, porphyrin and the like.
  • iron may be advantage- ously tethered to the Oligomer to help generate the cleav ⁇ ing radicals.
  • EDTA is a preferred DNA cleaving group
  • other nitrogen containing materials such as azo compounds or nitrenes or other transition metal chelating complexes may be used.
  • Suitable formulations comprise about 0.0001% to about 2% by weight of neutral Oligomer.
  • these neutral Oligomer for ⁇ mulations comprises about 2% to about 100% of a short chain aliphatic alcohol.
  • Suitable alcohols include etha- nol, isopropyl alcohol, propylene glycol and glycerol.
  • formulations of neutral Oligomers com ⁇ prising ethanol have demonstrated advantageous transdermal flux.
  • these neutral Oligomer formulations may additionally comprise a flux enhancer.
  • Suitable flux enhancers include those known to those skilled in the art and include decylmethylsulfoxide, dimethylsulfoxide as well as cyclic ketones, lactones, anhydrides and esters such as those disclosed in PCT Application No. PCT/US86/02583 (Publication Number W087/ 03473) .
  • Some of these flux enhancers also increase reten ⁇ tion of the Oligomer and, thus, act to increase the con ⁇ centration of Oligomer within the skin itself.
  • a flux enhancer which not only maximizes transdermal flux, but increases Oligomer retention in the skin.
  • Certain cyclic ketone and lactone enhancers have been reported to increase local retention as well and, thus, comprise a preferred class of enhancers for topical administration of Oligomer formulations.
  • the present invention pro ⁇ vides methods of preventing or interfering with expression of a specific target nucleic acid sequence by the trans ⁇ dermal administration of a neutral Oligomer which is com ⁇ plementary to and which can bind to or interact with a specific target nucleic sequence.
  • neutral Oligomers can penetrate skin or mucous membrane tissue.
  • neu- tral Oligomers may be applied to skin or mucous membrane to treat a variety of conditions either locally in the skin or mucous membrane (such as lung, gastrointestinal tract or vaginal tissue) or systemically by blocking or interfering with the expression of a specific target nucleic acid sequence.
  • the sequence to be blocked may comprise a "foreign" target nucleic acid sequence (i.e., one from an outside source such as a pathogen) or one of endogenous origin.
  • these neutral Oligomers may be used to directly treat viral, bacterial, fungal and other infections of the skin or mucous membranes caused by patho ⁇ gens.
  • Such conditions include those caused by a Herpes virus, a human papilloma virus (such as genital warts) , or species of Pseudomonas, Staphylococcus or Helicobacter.
  • These neutral Oligomers may also be used to block mediators of inflammation.
  • mediators include cyto- kines, growth factors, cell adhesion molecules or their ligands and receptors thereof as well as key enzymes in pathways leading to inflammation.
  • These blocking actions include preventing the expression of cytokines (such as IL-1) , growth factors (such as TGF- ⁇ and EGF) , or cell adhesion molecules (such as ELAM and ICAM) ; or the recep ⁇ tors for cytokines (such as IL-1), growth factors, or cell adhesion molecules.
  • cytokines such as IL-1
  • growth factors such as TGF- ⁇ and EGF
  • cell adhesion molecules such as ELAM and ICAM
  • Key enzymes whose expression may be blocked include protein kinase C and phospholipase A or C.
  • Neutral Oligomers which block the expression of phospholipase A 2 (PLA 2 ) or the receptor for IL-1 may be useful to treat inflammation of joints
  • These neutral Oligomers may be used therapeutically to treat immune or inflammatory-response related diseases or conditions.
  • Neutral Oligomers which block the expres ⁇ sion of IL-1, Transforming Growth Factor ⁇ (TGF ⁇ ) , amphi- doctorsn, or IL-6 may be useful in treating psoriasis.
  • These neutral Oligomers may also be useful in the treat ⁇ ment of atopic dermatitis.
  • these Oligomers may be used to block overexpression of IgE, said to be mediated by mast cells releasing histamine, and to be useful in the treatment of atopic dermatitis, eczema and asthma.
  • Oligomers which block IL-4 expression may be useful in the treatment of allergic rhinitis.
  • these Oligomers may be used in therapy of certain skin-related cancers such as melanoma and squa ous cell carcinoma, by blocking the expression of certain factors which promote cell growth and/or adhesion and are believed to be involved in metastasis.
  • These fac ⁇ tors include Epidermal Growth Factor (EGF) , and certain cellular adhesion factors.
  • EGF Epidermal Growth Factor
  • These neutral Oligomers may be used to prevent expres ⁇ sion of mediators of cell adhesion or their receptors so there will be no focus for cell recruitment or cell adhe ⁇ sion at a point of injury.
  • mediators include ELAM, ICAM, and thrombospondin.
  • Oligomers may also be used to block expression of retinoic acid receptors.
  • Oligomers may be useful to treat septic shock by blocking the expression of the IL-1 receptor antagonist.
  • target nucleic acid sequences which comprise a region of the gene to be blocked that is required for its expression.
  • These target sequences may comprise a portion of the gene, or a portion of a mRNA transcript thereof which is "available", i.e.. is in a state where the complementary neutral Oligomer is able to hybridize to the target sequence.
  • these target sequences are preferably single stranded and rela- tively free of secondary structure and bound protein.
  • Suitable target sequences include sequences which are at or proximate to a 5'-terminal translational start, a 3*-terminal polyadenylation signal, a mRNA cap site or a splice junction.
  • expression of renin especially in the juxta- glomerular apparatus (JGA) , is blocked by administration of these neutral Oligomers to skin or mucous membranes.
  • a preferred target sequence for the neutral Oligo ⁇ mers of the present invention comprises a sequence within the renin gene or its mRNA transcript. Renin is an enzyme which catalyzes the cleavage of angiotensinogen to produce angiotensin I.
  • Angiotensin I is further cleaved by angio- tensin converting enzyme to angiotensin II, which is a potent extracellular messenger involved in vasoconstric- tion.
  • Angiotension II also stimulates the adrenal cortex to secrete aldosterone.
  • Neutral Oligomers which are com ⁇ plementary to targets in the renin gene or mRNA tran ⁇ scripts thereof may be preferably administered by inhala ⁇ tion to the mucous membrane of the lung, thereby passing into the bloodstream, and reaching the kidney where renin is produced.
  • the permeability of hairless mouse skin for 14-mer-IA was about 10-times less than for 14-mer; the total amount of 14-mer which penetrated after 24 hours was about 5.5 /g/cm 2 in compar ⁇ ison to about 0.6 Ltg/cm 2 of 14-mer-IA.
  • the stratum corneum is the primary barrier to absorp ⁇ tion through the skin for most drugs, including macromole- cules.
  • an experiment was performed wherein the stratum corneum was removed from the skin by tape stripping.
  • tape stripped skin has been reported to have similar permeability characteristics to mucous membrane and, thus, has been proposed as a model system for mucous membrane. Penetration of the 14-mer through normal (unstripped) hairless mouse skin and skin following removal of the stratum corneum was examined and the rates compared.
  • Table I gives the cumulative amount of neutral methyl ⁇ phosphonate 14-mer penetrating stripped skins from two vehicles: EtOH/decylmethyl sulfoxide (DMS) (95:5, v/v) and ethanol (EtOH) .
  • DMS EtOH/decylmethyl sulfoxide
  • EtOH ethanol
  • the amount of the neutral methyl ⁇ phosphonate 14-mer penetrating skin was increased drama- tically when the stratum corneum was removed by tape stripping. Removal of the stratum corneum by tape strip ⁇ ping resulted in absorption of the entire dose of 14-mer absorbed through the stripped skin from both vehicles tested by 24 hours.
  • the concentration of neutral methylphosphonate 14-mer in the vehicle was related to the amount of drug penetrating a unit area of skin.
  • the driving force is related to the concentration of the neutral methylphosphonate 14-mer in the vehicle.
  • Table I depicts the penetration of 14-mer through hairless mouse skin using several different vehicles and at several different concentrations of 14-mer.
  • the cumulative amount of 14-mer which absorbed from a test vehicle over time is depicted in Figure 1.
  • the percent of the applied dose absorbed from the test vehi ⁇ cles for those concentrations of 14-mer is shown in Fig ⁇ ure 3.
  • the 0.05 mg/mL and 0.5 mg/mL formulations were both solutions, while the saturated (3.8 mg/mL) formula ⁇ tion was a suspension.
  • the sparing solubility of the 14-mer in the vehicle may effect the rate at which it was absorbed.
  • the cumulative amount of neutral methylphosphonate 14-mer which penetrated after 24 hours from the EtOH/H 2 0/ DMS (80:15:5) vehicle saturated with drug was about 3.5 jug/cm 2 . This was less than the cumulative amount which penetrated per cm 2 after 24 hours from the EtOH/DMS (95:5) vehicle (saturated) (wherein 5.5 ⁇ g/cm 2 was found to pene- trate) . (See Table I.)
  • the flux using the EtOH/H 2 0/DMS vehicle may have been reduced because the solubility of 14-mer in that vehicle was higher than EtOH/DMS and, thus, there was less driving force to partition the 14-mer out of the vehicle into the skin.
  • the permeability of human skin to the 14-mer was studied using the EtOH/H 2 0/DMS (80:15:5) vehicle saturated with neutral methylphosphonate 14-mer.
  • the results of this experiment are depicted in Figure 4 along with the data from the same experiment performed with the EtOH/DMS (95:5) and EtOH vehicles. While the permeability of the human skin toward the 14-mer from the EtOH/H 2 0/DMS (80:15:5) vehicle was lower than that from the water-free vehicles, the permeability profile was very similar. Com- pared to the hairless mouse data, the differences in per ⁇ meability were minimal. As had been the case with all the vehicles tested with human skin, human skin was much less sensitive to changes in vehicle composition than was hair ⁇ less mouse skin.
  • Drug products for topical application to skin should maximize the amount of drug retained in the target area within the skin.
  • the basal cells of the dermis are the regions to which the drug should be deli ⁇ vered.
  • Permeability experiments may indicate whether drug is being delivered through the skin and, therefore, that drug is being delivered to the dermal layer. However, it is important to know how much drug is retained in the skin at various times during the permeation experiment. There ⁇ fore, the skins used in the permeability experiments were routinely collected to analyze the amount of drug retained after 24 hours of exposure to the drug-containing vehicle. There are several ways that have been reported to assess drug retention.
  • a second approach that can be used is the separation of skin into layers (stratum corneum, viable epidermis, and dermis) .
  • a number of techniques have been used to separate skin into its component layers. Most techniques suffer from one or more problems, such as leaching of the drug into water during the separation pro- cess. For instance, a heat treatment technique for sepa ⁇ ration of the epidermis and the dermis requires submersion of the skin into hot water for several minutes prior to separation of the layers. To avoid this problem, a micro ⁇ wave technique to separate the dermis and epidermis has been described (Kumar, S., et al. , Pharm. Res. .6:740-741 (1989)) .
  • the first type of experiments used to determine drug Oligomer retention were those in which the entire skin sample was examined for Oligomer.
  • the experimental tech- nique used to measure retention is described in Example 5.
  • Table II the amounts of 14-mer recovered from skin samples following the 24 hour permeability experiments are listed.
  • the stratum corneum was removed from whole skin samples using microwave exposure for 5-8 seconds.
  • the amounts of 14-mer recovered from the samples is shown in Table IIB. Both hairless mouse and human skin were used; two samples of each were examined for total tissue reten ⁇ tion. Following removal of the stratum corneum from two additional skin samples, the 14-mer content was measured in the remaining viable epidermis and dermis of the sam ⁇ ples. Differences were observed between the hairless mouse and human skin data.
  • CAP A 10% acetic anhydride in acetonitrile.
  • CAP B ).625% N,N-dimethyl- aminopyridine in pyridine.
  • the 5•-dimethoxytrityi pro ⁇ tecting group is left on at the end of the synthesis to facilitate purification of the oligomers, as described below.
  • the crude, protected methylphosphonate oligomers are removed from the solid support by mixing with concentrated ammonium hydroxide for two hours at room temperature.
  • the solution is drained from the support using an Econo- ColumnTM (Bio-Rad, Richmond, CA) and the support is washed five times with 1:1 acetonitrile/water.
  • the eluted oligo- mer is evaporated to dryness under vacuum at room tempera ⁇ ture.
  • the protecting groups are removed from the bases with a solution of ethylenediamine/ethanol/aceto- nitrile/water (50:23.5:23.5:2.5) for 6 hours at room tem ⁇ perature.
  • the resulting solutions are then evaporated to dryness under a vacuum.
  • the 5*-dimethoxytrityl ("trityl”) containing oligo ⁇ mers are purified from non-tritylated failure sequences using a Sep-PakTM C 18 cartridge (Millipore/Waters Bedford, MA) as follows: The cartridge is washed with acetoni ⁇ trile, 50% acetonitrile in 100 mM, triethylammonium bicar- bonate (TEAB, pH 7.5) and 25 mM TEAB. Next, the crude methylphosphonate oligomer is dissolved in a small volume of 1:1 acetonitrile/water and then diluted with 25 mM TEAB to a final concentration of 5% acetonitrile.
  • TEAB triethylammonium bicar- bonate
  • This solu ⁇ tion is then passed through the cartridge.
  • the car- tridge is washed with 15-20% acetonitrile in 25 mM TEAB to eluate failure sequences from the cartridge.
  • the tri- tyl-on oligomer remaining bound to the cartridge is then detritylated by washing with 25 mM TEAB, 2% trifluoro- acetic acid, and 25 mM TEAB, in that order.
  • the trityl-selected oligomer is eluted from the cartridge with 50% acetonitrile/water and evaporated to dryness under vacuum at room temperature.
  • a linear gra ⁇ tower of 0-70% Buffer B is run over about 30-50 minutes at a flow rate of about 2.5 to 3.0 ml/minute. Fractions con ⁇ taining full length methylphosphonate oligomer are col- lected, evaporated under vacuum and resuspended in 50% acetonitrile/water.
  • the stratum corneum was removed from hairless mice for permeability experiments by using cellophane tape.
  • the tape was gently applied to the skin of a recently sacrificed animal and then pulled away from the body. This was repeated 12 to 15 times with fresh pieces of tape.
  • Human cadaver skin was obtained at autopsy through the Stanford University Medical Center.
  • the skin was excised using a dermatome from the thigh area of a 74 year old male within 24 hours post-mortem.
  • the thickness as measured with a Van Keuren light wave micrometer, ranged from 125 to 450 ⁇ m. The average thickness was 200 to 300 ⁇ m.
  • the skin was rinsed with phosphate buffered saline (pH 7.4), blotted dry and frozen for 6 months in triple- sealed bags evacuated of air. Prior to use, the skin was thawed and rinsed in PBS.
  • a diffusion console containing nine glass Franz dif ⁇ fusion cells was used in the permeability experiments.
  • the Franz cells were maintained at 37°c by thermostati ⁇ cally controlled water, which was circulated through a jacket surrounding the cell body.
  • Each skin was mounted and clamped between the cell body and the cell cap so that the epidermal side faced upward (vehicle side) .
  • the skins were then allowed to equilibrate for 1 hour in the diffu ⁇ sion cells prior to addition of the vehicle.
  • the exposed surface was 2.0 cm 2 .
  • the receptor was 0.01 M phosphate- buffered saline (pH 6.4) isotonic saline with 0.05% sodium azide added to prevent growth of microorganisms.
  • the Franz cells were closed to maximize drug concen ⁇ tration in the receptor phase.
  • the volume of the cells was 6.2 mL. the cells were stirred using a teflon-coated stir bar at 600 rpm.
  • the drug/vehicle mixtures were pipetted through the cell cap on to the skin [0.2 mL total vehicle added to 2.0 cm 2 (0.1 nK.cm 2 )].
  • a syringe needle was inserted through the side arm into the receptor solution and 300 ⁇ L was with ⁇ drawn. The volume removed was replaced by an equal volume of fresh saline. The solution effect was accounted for in the drug flux calculations.
  • the 14-mer (neutral methylphosphonate Oligomer having only methylphosphonate internucleosidyl linkages) and 14- mer-IA (methylphosphonate Oligomer having an internal ani- onic internucleosidyl linkage) were measured in the recep ⁇ tor solution by HPLC. These analyses were performed on a Waters 840 system consisting of two model 510 pumps, a model 481 UV detector, a model 710B WISP (sample proces ⁇ sor) , and a Digital computer model 350 microprocessor/ programmer. The column used to separate the 14-mer was a 3.9 mm x 15 cm 4 ⁇ m, Waters Nova-Pak C18. A gradient elu- tion was performed as follows for the 14-mer:
  • the homogenate was then centrifuged at 8,000 g for 15 minutes at room temperature. The supernatant was removed and analyzed directly by HPLC analysis (see below for conditions) . The chromatographic conditions were similar to those described above for the 14-mer with some minor changes noted below.
  • Vehicle containing oligomer and optionally enhancer 100 ⁇ L/cm 2 was placed on the surface of the skin (2 exposed surface cm 2 ) .
  • the highest retention of oligomer both in the viable tissues (dermal layer) and stratum corneum was observed from the ethanol/water/DMS vehicle.
  • the ratio of retained oligomer in stratum corneum to dermis was about 10-30 (Note: Since there was considerably more viable tissue than stratum corneum, the majority of oligomer retained was in the dermis) .
  • Tape stripping (to remove stratum corneum) of skin did not lead to a larger amount of 6-mer being retained in dermis as compared to retention in dermis using whole skin.
  • Table IV reports retention of 14-mer in dermis versus strateu corneum after treatment with 14-mer in various vehicle/enhancer combinations. Stratum corneum and dermis were separated by microwave treatment as described by Kumar et al. (Pharm. Res. 1:740-741 (1989)).
  • Total ⁇ g of 14-mer recovered from the homogenized skin sample corrected for loss of 14-mer during homogenization and centrifugation (see Example 4) ; the gm is the wet weight of the skin as measured prior to homogenization ⁇ M concentration of 14-mer in the skin were obtained from the molecular weight of the 14-mer and the assumed density of 1.0 for the skin sample (i.e.. 1.0 gm of skin is equal to 1.0 cc of skin) c
  • the HM skin used in this experiment was stripped to remove the stratum corneum d
  • concentration of 14-mer in this vehicle was 0.5 mg/mL compared to all the other experimental vehicles, which were saturated with excess solid 14-mer
  • n 2.
  • b Total ⁇ g of 14-mer recovered from the homogenized skin sample corrected for loss of 14-mer during homogenization and centrifugation (see Example 4) ; the gm is the wet weight of the skin as measured prior to homogenization
  • c ⁇ M concentration of 14-mer in the skin were obtained from the molecular weight of the 14-mer and the assumed density of 1.0 for the skin sample (i.e.. 1.0 gm of skin is equal to 1.0 cc of skin)
  • ⁇ he viable tissue is the tissue after the stratum corneum has been removed by microwave treatment (Kumar, et al.. Pharm. Res. .6:740-741 (1989)). It is a combination of the viable epidermis and the dermis.
  • SUBSTITUTESHEET The second value for the 6Mer came from a time study using a different skin donor, otherwise the data for the first three enhancers came from the same donor. The data for the last three enhancers came from the same experiment but from a different donor.
  • the values in this table are the means of three values from three separate cells, with the SD in parenthesis.
  • the 6 Mer time study had one cell for the 30 minute point and 2 cells for the other time points.

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Abstract

Des oligomères neutres peuvent être acheminés par voie transdermique et à travers des muqueuses à des fins thérapeutiques, de manière à bloquer l'expression d'une séquence d'acides nucléiques cible spécifique. Ces oligomères neutres sont utiles dans le traitement de divers états pathologiques, y compris ceux de la peau et des muqueuses, ainsi que des états affectant d'autres tissus.
PCT/US1992/004370 1991-05-31 1992-05-22 Compositions et systemes d'apport destines a l'administration par voie transdermique d'oligomeres neutres WO1992021353A1 (fr)

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EP0644765A1 (fr) * 1992-05-22 1995-03-29 Genta Incorporated Traitement de l'hyperproliferation cellulaire par inhibition de l'interleukine-1
WO1995031459A1 (fr) * 1994-05-11 1995-11-23 Antivirals Inc. Banque combinatoire de sous-unites morpholino et procede
EP0684764A1 (fr) * 1993-02-19 1995-12-06 Genta Incorporated Traitement de la calvitie associee aux androgenes au moyen d'oligomeres antisens
US6083978A (en) * 1996-03-05 2000-07-04 Sterix Limited Compounds with a sulfamate group
EP1080103A1 (fr) * 1998-05-21 2001-03-07 Isis Pharmaceuticals, Inc. Compositions et procedes pour l'administration non parenterale d'oligonucleotides
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US6727230B1 (en) 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US6887906B1 (en) 1997-07-01 2005-05-03 Isispharmaceuticals, Inc. Compositions and methods for the delivery of oligonucleotides via the alimentary canal
US6921776B1 (en) 1996-02-16 2005-07-26 Sterix Limited Compound
US6949520B1 (en) 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
US7001890B1 (en) 1997-01-23 2006-02-21 Coley Pharmaceutical Gmbh Pharmaceutical compositions comprising a polynucleotide and optionally an antigen especially for vaccination
US7129269B2 (en) 1996-03-05 2006-10-31 Sterix Ltd. Compound
US7354909B2 (en) 2001-08-14 2008-04-08 The United States Of America As Represented By Secretary Of The Department Of Health And Human Services Method for rapid generation of mature dendritic cells
US7521063B2 (en) 2000-01-14 2009-04-21 The United States Of America As Represented By The Department Of Health And Human Services Multiple CPG oligodeoxynucleotides and their use to induce an immune response
US7524828B2 (en) 1994-07-15 2009-04-28 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7534772B2 (en) 2000-06-22 2009-05-19 University Of Iowa Research Foundation Methods for enhancing antibody-induced cell lysis and treating cancer
US7585847B2 (en) 2000-02-03 2009-09-08 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US7615227B2 (en) 2001-12-20 2009-11-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce angiogenesis

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AU3056889A (en) * 1988-01-21 1989-08-11 Yale University Selective inhibition of gene expression by photoactivatable oligonucleotides
US5399676A (en) * 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity

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US4689320A (en) * 1983-10-17 1987-08-25 Akira Kaji Method for inhibiting propagation of virus and anti-viral agent

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Nucleic Acids Research, Volume 15, Number 24, issued 1987, C. CAZENAVE et al., "Rate of Degradation of (alpha)- and (beta)-oligodeoxy-nucleotides in Xenopus Oocytes. Implications for Anti-Messenger Strategies", pages 10507-10521, entire document. *
See also references of EP0587749A4 *

Cited By (36)

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EP0644765A4 (fr) * 1992-05-22 1997-02-26 Genta Inc Traitement de l'hyperproliferation cellulaire par inhibition de l'interleukine-1.
EP0644765A1 (fr) * 1992-05-22 1995-03-29 Genta Incorporated Traitement de l'hyperproliferation cellulaire par inhibition de l'interleukine-1
EP0684764A1 (fr) * 1993-02-19 1995-12-06 Genta Incorporated Traitement de la calvitie associee aux androgenes au moyen d'oligomeres antisens
EP0684764A4 (fr) * 1993-02-19 1997-10-22 Genta Inc Traitement de la calvitie associee aux androgenes au moyen d'oligomeres antisens.
US6727230B1 (en) 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
WO1995031459A1 (fr) * 1994-05-11 1995-11-23 Antivirals Inc. Banque combinatoire de sous-unites morpholino et procede
US7524828B2 (en) 1994-07-15 2009-04-28 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US7223741B2 (en) 1994-07-15 2007-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6653292B1 (en) 1994-07-15 2003-11-25 University Of Iowa Research Foundation Method of treating cancer using immunostimulatory oligonucleotides
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US7888327B2 (en) 1994-07-15 2011-02-15 University Of Iowa Research Foundation Methods of using immunostimulatory nucleic acid molecules to treat allergic conditions
US7846965B2 (en) 1996-02-16 2010-12-07 Sterix Limited Compound
US6921776B1 (en) 1996-02-16 2005-07-26 Sterix Limited Compound
US7202272B2 (en) 1996-02-16 2007-04-10 Sterix, Ltd. Compound
US6083978A (en) * 1996-03-05 2000-07-04 Sterix Limited Compounds with a sulfamate group
US7129269B2 (en) 1996-03-05 2006-10-31 Sterix Ltd. Compound
US7001890B1 (en) 1997-01-23 2006-02-21 Coley Pharmaceutical Gmbh Pharmaceutical compositions comprising a polynucleotide and optionally an antigen especially for vaccination
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6821957B2 (en) 1997-05-20 2004-11-23 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
US8691785B2 (en) 1997-07-01 2014-04-08 Isis Pharmaceuticals, Inc. Compositions and methods for non-parenteral delivery of oligonucleotides
US6887906B1 (en) 1997-07-01 2005-05-03 Isispharmaceuticals, Inc. Compositions and methods for the delivery of oligonucleotides via the alimentary canal
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
EP1080103A4 (fr) * 1998-05-21 2003-07-02 Isis Pharmaceuticals Inc Compositions et procedes pour l'administration non parenterale d'oligonucleotides
EP1080103A1 (fr) * 1998-05-21 2001-03-07 Isis Pharmaceuticals, Inc. Compositions et procedes pour l'administration non parenterale d'oligonucleotides
US6977245B2 (en) 1999-04-12 2005-12-20 The United States Of America As Represented By The Department Of Health And Human Services Oligodeoxynucleotide and its use to induce an immune response
US7776344B2 (en) 1999-09-27 2010-08-17 University Of Iowa Research Foundation Methods related to immunostimulatory nucleic acid-induced interferon
US6949520B1 (en) 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
US7521063B2 (en) 2000-01-14 2009-04-21 The United States Of America As Represented By The Department Of Health And Human Services Multiple CPG oligodeoxynucleotides and their use to induce an immune response
US7585847B2 (en) 2000-02-03 2009-09-08 Coley Pharmaceutical Group, Inc. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US7534772B2 (en) 2000-06-22 2009-05-19 University Of Iowa Research Foundation Methods for enhancing antibody-induced cell lysis and treating cancer
US7354909B2 (en) 2001-08-14 2008-04-08 The United States Of America As Represented By Secretary Of The Department Of Health And Human Services Method for rapid generation of mature dendritic cells
US7615227B2 (en) 2001-12-20 2009-11-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce angiogenesis

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