WO1994018835A1 - Treatment of androgen-associated baldness using antisense oligomers - Google Patents
Treatment of androgen-associated baldness using antisense oligomers Download PDFInfo
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- WO1994018835A1 WO1994018835A1 PCT/US1994/001748 US9401748W WO9418835A1 WO 1994018835 A1 WO1994018835 A1 WO 1994018835A1 US 9401748 W US9401748 W US 9401748W WO 9418835 A1 WO9418835 A1 WO 9418835A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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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
- A61K8/60—Sugars; Derivatives thereof
- A61K8/606—Nucleosides; Nucleotides; Nucleic acids
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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- 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
- C12N15/1137—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 against enzymes
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- 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
- C12N15/1138—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 against receptors or cell surface proteins
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- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/99—Oxidoreductases acting on the CH-CH group of donors (1.3) with other acceptors (1.3.99)
- C12Y103/99005—3-Oxo-5alpha-steroid 4-dehydrogenase (acceptor) (1.3.99.5), i.e. steroid-5alpha-reductase
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- C12N2310/10—Type of nucleic acid
- C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
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- 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
- Androgens are steroid hormones found circulating at varying levels in both men and women. They are essential in sex differentiation, development, and reproductive function. However, androgens can also play a role in undesirable physiological conditions, including different types of baldness.
- MPB male pattern baldness
- Hair follicles initially appear in utero. No new follicles are created after birth, and it is believed that none are lost in adult life. However, in MPB, hair follicles do become progressively smaller (miniaturized) . Hair follicles exhibit cyclic activity. Each period of active growth of hair (anagen) alternates with a resting period (telogen) , separated by a relatively short transi ⁇ tion phase (catagen) . Hair growth on the human scalp is a mosaic of follicular activity with each follicle at a stage independent of its neighbors. At any one time, between 4-24% (average 13%) of follicles are in telogen and ⁇ 1% in catagen.
- anagen may occupy three years or more; however, the percentage of follicles in telogen increases with age, resulting in a gradual thinning.
- MPB the ratio of telogen to anagen is increased still further.
- the hairs in affected areas become steadily shorter and finer, and ultimately may be reduced to the short ( ⁇ 2cm) , fine, unpigmented hair known as vellus hair.
- the endocrine system does not directly initiate or curtail the activity of the hair follicle, androgens do accelerate or retard the normal cyclic activity of hair growth described above.
- Testosterone is the major circulating androgen. Because circulating T is largely bound to sex hormone binding globulin (SHBG) , the availability of T depends not only on its total concentration, but also on the level of SHBG. While plasma T levels in MPB appear to be normal, SHBG levels tend to be low. This implies that bald males may have higher levels of free testosterone. This implication is borne out by the demonstration that bald males have high T concentrations in their saliva.
- SHBG sex hormone binding globulin
- DHT 5-alpha-dihydrotestosterone
- balding men have increased 5-alpha-reductase activity in the hair follicles and skin of the frontal scalp, it has been suggested that this enzyme may be involved in development of MPB.
- Two genes have been reported, each of which codes for a distinct 5-alpha- reductase enzyme (Genbank locus-HUM5AR and HUMSRDA) .
- the effects of androgens in MPB are mediated by the binding of an androgen (primarily DHT) to the androgen receptor (AR) .
- Androgens bind specifically to the AR, which is either situated in the nucleus or transferred to it from the cytoplasm.
- the AR belongs to a subfamily of steroid/thyroid hormone/retinoic acid receptors, whose activity is controlled by the tight and specific binding of the cognate ligand.
- Evidence for the involvement of the .AR in MPB includes the demonstration that androgenic alopecia (a type of pattern baldness in women) can be alleviated by treatment with antiandrogens.
- antiandrogens such as spironolactone, cyproterone acetate, flutamide and cimetidine, bind to the AR and competitively inhibit DHT binding.
- sebaceous glands of bald scalps were found to have greater binding affinity and capacity for androgens than those in hairy scalps.
- Minoxidil is a potent vasodilator which has been used as a treatment for hypertension.
- a noted side effect of this treatment was the growth of hair on parts of the body. This led to the testing of topical minoxidil on balding areas of the scalp. The result in some cases was an apparent decrease in vellus hairs with a concomitant increase in terminal hairs. Many of the subjects studied reported that their rate of hair loss decreased. However, not all subjects responded to treatment with minoxidil.
- minoxidil will not help the majority of balding men, although it does help a specific population of minimally balding young men.
- the reason for the effectiveness of minoxidil is not known. It might be due to an increase in blood flow caused by the vasodilating effect of the drug.
- the longterm effects of minoxidil treatment are not known.
- Other treatments are directed at reducing the production of DHT from testosterone, thereby preventing its cytosol-nuclear binding and/or translocation.
- Topical or intralesional progesterone can also be used to reduce the production of DHT from T. Since progesterone is similar in structure to testosterone, it competes with testosterone for 5-alpha-reductase, the enzyme that converts testosterone to DHT.
- the present invention is directed to methods of treating androgen-associated hair loss, particularly hair loss in men, more particularly to methods of decreasing the progression of male pattern baldness and also to pharmaceutical compositions useful for these methods. These methods and pharmaceutical compositions are parti ⁇ cularly suited to the treatment of hair loss associated with increased levels of protein-bound DHT in scalp. According to one aspect, Oligomers complementary to a target sequence in genes which result in increased amounts of androgen receptor bound-5- ⁇ -dihydrotestosterone in scalp tissue are used to down-regulate genes or their transcription products.
- the topography of male pattern baldness has to do with both the number of androgen receptor (“AR”) molecules of the follicular cells and the activity of 5-alpha- reductase (“5-C.-RE”) in different areas of the scalp.
- AR androgen receptor
- 5-alpha- reductase 5-alpha- reductase
- the androgen receptor may be involved in other types of hair loss aside from MPB.
- androgenic alopecia a type of hair loss in women, has been shown to respond to treatment with antiandrogens.
- the methods and pharmaceutical compositions of the present invention may be useful in the treatment of other types of androgen-associated hair loss.
- these methods and compositions may be useful in treating other conditions where localized (as opposed to systemic) down-regulation of the AR or 5- ⁇ -RE is desirable.
- the present invention is also directed to methods of decreasing levels of protein-bound 5-alpha-dihydro- testosterone in a localized and tissue-specific manner without significantly interfering with testosterone metabolism in other tissues or systemically by exposing the cells of the tissue to be treated with an Oligomer or Oligomers which inhibit or alter expression of the AR or 5- ⁇ -RE.
- Oligomers include those which interact with a target sequence selected from a gene coding for the AR or 5- ⁇ -RE or a sequence immediately upstream from the transcription site of the gene or their transcription products.
- the present invention is directed to a method of treating androgen-associated hair loss by decreasing levels of 5-alpha-dihydrotestosterone which are present in follicles and bound to protein, and according to a preferred aspect decreasing levels of DHT bound to the androgen receptor in scalp tissue without significantly interfering with testosterone synthesis and/or metabolism in other tissues.
- This method comprises exposing scalp cells to an amount of an Oligomer or Oligomers sufficient to provide a decrease in the rate of hair loss, preferably by a cosmetically significant amount.
- the Oligomer or Oligomers interact with a gene coding for the AR or 5- ⁇ -RE or a sequence immediately upstream from the transcription start site of the gene or their transcription products and thereby inhibit or alter expression of the AR or 5- ⁇ -RE.
- Suitable Oligomers for use in the methods and pharma ⁇ ceutical compositions of the present invention include (a) an antisense Oligomer having a sequence complementary to
- SUBSTITUTE SHEET (RULE 26; a sequence of RNA transcribed from a target gene present in the cells; (b) an antisense Oligomer having a nucleo ⁇ side sequence complementary to a single stranded DNA target sequence; (c) an antisense Oligomer having a nucleoside sequence complementary to a single RNA or DNA strand contained within a duplex (d) a Third Strand Oligomer having a sequence complementary to a selected double stranded nucleic acid sequence of a target gene present in the cells; and (e) a Triplex Oligomer Pair which is complementary to a single-stranded nucleic acid sequence of a target gene or its transcription product or to a single-stranded sequence contained within a duplex.
- the target gene is advantageously selected from the group consisting of those genes encoding 5-alpha-reductase and the androgen receptor. According to a preferred aspect, the Oligomer is applied topically to the scalp tissue.
- the present invention is directed to a method of treating androgen- associated hair loss which comprises exposing scalp to an amount of an Oligomer which decreases the rate of hair loss wherein said Oligomer is selected from an antisense Oligomer having a sequence complementary to that of RNA transcribed from a gene for androgen receptor or an antisense Oligomer having a sequence complementary to a sequence of RNA transcribed from a gene for 5-alpha- reductase.
- the Oligomer is a neutral Oligomer.
- Neutral Oligomers such as methylphos- phonate Oligomers are cleared rapidly through the kidneys .
- methylphosphonate Oligomers which are rapidly cleared from the plasma and are excreted in the urine.
- the Oligomers used according to the present invention preferably comprise Oligomers which have a neutral back- bone.
- Neutral Oligomers are preferred, in part, due to their advantageous uptake through the skin when applied topically.
- these Oligomers are substantially neutral. More preferably, neutral Oligomers are used.
- Particularly preferred are substantially neutral methyl- phosphonate Oligomers. According to an especially pre ⁇ ferred aspect, neutral methylphosphonate Oligomers are employed.
- purine or “purine base” includes not only the naturally occurring adenine and guanine bases, but also modifications of those bases such as bases sub ⁇ stituted at the 8-position, or guanine analogs modified at the 6-position or the analog of adenine, 2-amino purine, as well as analogs of purines having Sarbon replacing nitrogen at the 9-position such as the 9-deaza purine derivatives and other purine analogs.
- 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 term includes not only those nucleosidyl units having A, G, C, T and U as their bases, but also analogs and modified forms of the naturally-occurring bases, including the pyrimidine-5- donor/acceptor bases such as pseudoisocytosine and pseudouracil and other modified bases (such as 8- substituted purines) .
- the 5-carbon sugar is ribose; in DNA, it is 2' -deoxyribose.
- nucleoside also includes other analogs of such subunits, including those which have modified sugars such as 2'-0-alkyl ribose.
- R is hydrogen or an alkyl or aryl group.
- Suitable alkyl or aryl groups include those which do not sterically hinder the phosphonate linkage or interact with each other.
- the phosphonate group may exist in either an "R” or an "S” configuration.
- Phosphonate groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
- phosphodiester or diester refers to
- phosphodiester groups may be used as inter ⁇ nucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
- non-nucleoside monomeric unit refers to a mono- meric unit wherein the base, the sugar and/or the phos ⁇ phorus backbone has been replaced by other chemical moieties.
- a “nucleoside/non-nucleoside polymer” refers to a polymer comprised of nucleoside and non-nucleoside monomeric units.
- oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 4 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.
- the term "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 or phosphorodithioate analogs of oligonucleotides, phosphoramidate analogs of oligo- nucleotides, neutral phosphate ester oligonucleoside analogs, such as phosphotriesters and other oligonucleo ⁇ side analogs and modified oligonucleosides, and also includes nucleoside/non-nucleoside polymers.
- nucleoside/non-nucleoside polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a sulfamate linkage, or a carbamate linkage. It also includes nucleoside/non-nucleoside polymers wherein both the sugar and the phosphorous moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs.
- nucleoside/non- nucleoside polymers wherein the base, the sugar, and the phosphate backbone of a nucleoside are either replaced by a non-nucleoside moiety or wherein a non-nucleoside moiety is inserted into the nucleoside/non-nucleoside polymer.
- said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
- alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl-phos ⁇ phonate internucleosidyl linkage.
- Suitable alkyl- or aryl- phosphonate groups include alkyl- or aryl- groups which do not sterically hinder the phosphonate linkage or interact with each other.
- Preferred alkyl groups include lower alkyl groups having from about 1 to about 6 carbon atoms.
- Suitable aryl groups have at least one ring having a conjugated pi electron system and include carbocyclic aryl and heterocyclic aryl groups, which may be optionally substituted and preferably having up to about 10 carbon atoms.
- the term " ethylphosphonate Oligomer” (or "MP- Oligomer”) refers to Oligomers having at least one methylphosphonate internucleosidyl linkage.
- neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e., linkages having no positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or aryl- phos ⁇ phonate linkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyltriester linkages; and non-phosphorus-containing internucleosidyl linkages, such as sulfamate, morpholino, formacetal, thioformacetal, and carbamate linkages.
- a neutral Oligomer may comprise a conjugate between an oligonucleoside or nucleoside/non-nucleoside polymer and a second molecule which comprises a conjugation partner.
- conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, nucleic acid modifying groups including photo- cross-linking agents such as psoralen and groups capable of cleaving a targeted portion of a nucleic acid, and the like.
- conjugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharma- cokinetic distribution of the Oligomer.
- the essential requirement is that the oligonucleoside or nucleoside/non- nucleoside polymer that the Oligomer conjugate comprises be substantially neutral and capable of hybridizing to its complementary target sequence.
- substantially neutral in referring to an Oligomer refers to those Oligomers in which at least about 80 percent of the internucleosidyl linkages between the nucleoside monomers are nonionic linkages.
- neutral alkyl- or aryl- phosphonate Oligomer refers to neutral Oligomers having neutral internucleosidyl 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.
- tandem oligonucleotide or “tandem Oligomer” 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 comple ⁇ mentary 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 Oligomers, such as by decreasing the secondary structure of the target nucleic acid sequence.
- one member of a pair of tandem Oligomers may improve the hybrid stability of the second tandem Oligomer to the target nucleic acid sequence by promoting a helical structure 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 membrane in combination with the compound to increase transdermal 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.
- Triplex Oligomer Pair refers to first and second Oligomers which are optionally covalently linked at one or more sites and which are complementary to and are capable of hydrogen bonding to a segment of a single
- SUBSTITUTE SHEET (RULE 25) stranded target nucleic acid, such as RNA or DNA, and, thus, together with the single stranded target nucleic acid, are capable of forming a triple helix structure therewith.
- the term "Third Strand Oligomer” refers to Oligomers which are capable of hybridizing to a segment of a double stranded nucleic acid, such as a DNA duplex, an RNA duplex or a DNA-RNA duplex, and forming a triple helix structure therewith.
- triplex Oligomer Pair or first and second Oligomers
- Third Strand Oligomer refers to Oligomers having base sequences which are capable of forming or recognizing hydrogen bonds (and base pairing or hybridizing) with the base sequence of the nucleic acid to form a triple helix structure.
- substantially complementary refers to Oligomers, including Triplex Oligomer Pairs or Third Strand Oligomers which may lack a complement for each nucleoside in the target sequence, have sufficient binding affinity for the target sequence to form a stable duplex or triple helix complex, as the case may be, and thereby specifically recognize the target sequence and selectively inhibit or down-regulate its expression.
- triplet or "triad” refers to a hydrogen bonded complex of the bases of three nucleosides between a base (if single stranded) or bases (if double stranded) of a target sequence, a base of a Second Strand and a Third Strand (if a single stranded target sequence) or a base of a Third Strand (if a double-stranded target) .
- Figure 1 depicts thermal denaturation profiles for double stranded and triple-stranded complexes formed between Oligomer 2 and a target sequence.
- Figure 2 depicts clearance from plasma of a tritium- labelled tetramer in a mouse model.
- Figure 3 depicts clearance from plasma of a tritium labelled dodecamer in a mouse model.
- DHT 5- alpha-dehydrotestosterone
- Such down-regulation may be effected by use of an Oligomer which may bind to a protein's active site to modulate its function or Oligomers such as antisense Oligomers, Third Strand Oligomers and Triplex Oligomer pairs. Suitable nucleoside sequences for these Oligomers may be determined from the sequences of target genes. Preferred sequences of the target region are described herein.
- the Oligomer selected may be any of a number of types, including those having a charged or uncharged backbone.
- Preferred Oligomers include alkyl- and aryl- phosphonate Oligomers, especially preferred are methylphosphonate Oligomers.
- Other preferred Oligomers include phosphorothioate Oligomers, morpholino analogs, formacetal analogs and peptide nucleic acid (“PNA”) analogs.
- the Oligomers each comprise from about 4 to about 40 nucleosides, more preferably, from about ⁇ to 30 nucleosides. Especially preferred are Oligomers of about 8 to about 20 nucleosides.
- tandem Oligomers are employed.
- Preferred tandem Oligomers include those which comprise a total of about 20 to about 40 nucleosides.
- Oligomers having the selected internucleoside linkages may be conveniently prepared according to synthetic techniques known to those skilled in the art. For example, commercial machines, reagents and protocols are available for the synthesis of Oligomers having phosphodiester and certain other phosphorus-containing internucleoside linkages. See also Gait, M.J., Oligonucleotide Synthesis: A Practical Approach (IRL Press, 1984) ; Cohen, Jack S., Oligodeoxynucleotides Antisense Inhibitors of Gene Expression, (CRC Press, Boca Raton, FL, 1989) ; and Oligonucleotides and Analogues: A Practical Approach, (F. Eckstein, 1991) . Preparation of Oligomers having certain non-phosphorus-containing internucleoside linkages is described in United States Patent No. 5,142,047, the disclosure of which is incorporated herein by reference.
- chirally pure Oligomers are used according to the present invention.
- Oligomers comprising at least one chirally pure internucleosidyl linkage may be used and may be preferred.
- Such Oligomers may be prepared using methods such as those described in Lesnikowski et al . , Nucleic Acids Research 18 (8) .2109-2115 (1990) and Stec et al., Nucleic Acids Research 19 (21) :5883-5888 (1991) .
- Oligomers which are nucleoside/ non-nucleoside polymers. Suitable Oligomers also include chimeric oligonucleotides which are composite RNA, DNA analogues (Inoue et al. , FEBS Lett.
- Suitable Oligomers include Oligomers having chimeric backbones.
- Such chimeric backbone Oligomers include Oligomers having mixed phosphate backbones including nucleoside sequences which are capable of activating RNaseH and nucleoside sequences which do not activate RNaseH, and thus allow site directed cleavage of an RNA molecule. See U.S. Patent No. 5,149,797 which is incorporated herein by reference.
- Chimeric backbone Oligomers also include Oligomers having a mixture of internucleosidyl linkages which may or may not include phosphorus atoms, such as morpholinyl linkages, formacetal linkages, peptide nucleic acid (PNA) linkages and the like.
- PNA peptide nucleic acid
- Oligomers having a neutral backbone for example, methylphosphonate Oligomers with cleaving or cross-linking moieties attached, may prove advantageous in certain circumstances; such Oligomers may have a longer half-life in vivo since the neutral structure reduces the rate of nuclease digestion while the cleaving or cross-linking moiety may promote inactivation of target polynucleotide sequences.
- these antisense Oligomers have a sequence which is complementary to a portion of the RNA transcribed from the selected target gene.
- duplexes between the antisense Oligomer and the RNA transcribed from the target gene.
- the duplexes so formed may inhibit translation, processing or transport of an mRNA sequence or may lead to digestion by the enzyme RNaseH.
- Single stranded Oligomers may also bind to a duplex DNA target such that a duplex is formed with one of the two DNA strands, and the second DNA of the target strand is displaced from the duplex.
- Preferred is the formation of a duplex by the Oligomer with the coding strand of the DNA duplex target ("invading duplex") .
- the invading duplex so formed may inhibit transcription.
- down regulation of 5-alpha reductase or the androgen receptor may be accomplished by triple helix formation using a Third Strand Oligomer or a Triplex Oligomer Pair having sequences selected such that the Oligomer (s) are complementary to and form a triple helix complex with a target sequence of double stranded or single stranded nucleic acid and thereby interfere with or prevent expression of the targeted nucleic acid sequence.
- Triple strand formation can occur in one of several ways.
- a single stranded Oligomer may form a triple strand with duplex DNA or RNA; two separate or connected Oligomers may form a triple strand with single stranded DNA or RNA; two separate or connected Oligomers may bind to one of the duplex DNA or RNA strands and displace the other such that it is not involved in triple strand formation.
- Oligomers including Third Strand Oligomers and Triplex Oligomer Pairs
- the Oligomer employed will have a sequence that is complementary to the sequence of the target nucleic acid.
- absolute complementarity may not be required; in general, any Oligomer having sufficient complementarity to form a stable duplex (or triple helix complex as the case may be) with the target nucleic acid is considered to be suitable. Since stable duplex formation depends on the sequence and length of the hybridizing Oligomer and the degree of complementarity between the antisense Oligomer and the target sequence, the system can tolerate less fidelity (complementarity) when longer Oligomers are used.
- Oligomers which form triple helix complexes.
- Oligomers of about 8 to about 40 nucleosidyl units in length which have sufficient complementarity to form a duplex or triple helix structure having a melting temperature of greater than about 40°C under physiological conditions are particularly suitable for use according to the methods of the present invention.
- the concentration of Oligomer used may vary, depending upon a number of factors, including the extent of hair loss condition to be treated, the type and the specificity of the particular antisense Oligomer, Triplex Oligomer Pair, or Third Strand Oligomer selected. It is believed that significant inhibition as demonstrated by a cosmetically significant decrease in progression of hair loss may be obtained at concentrations in about the 10 ⁇ M range; however, under other conditions, higher or lower concentrations of Oligomer may be preferred.
- Oligomers are to be administered transdermally, preferred are neutral Oligomers.
- these Oligomers may comprise a conjugate between a polynucleoside or nucleoside/non-nucleoside polymer and a conjugation partner.
- Suitable conjugation partners include inter ⁇ calating agents such as acridine, alkylating agents, binding substances for cell surface receptors, lipophilic agents, photo-crosslinking agents such as psoralen, other cross-linking agents, pro-chelates, or nucleic acid modifying agents, including groups capable of cleaving a targeted portion of a nucleic acid such as hydrolytic or nucleolytic agents like o-phenanthroline copper or EDTA- iron, all of which may be incorporated in the Oligomers.
- 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 chemical modification of the Oligomer, for example, by the use of non-nucleotide 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 incorporate a nucleic acid modifying group which may be caused to react with said target nucleic acid and irreversibly modify its structure, thereby rendering it non-functional .
- Conjugates may be introduced to alter the pharmacodynamics or toxicity of the oligonucleotides in the body.
- a cleavable moiety may be attached according to patent 4,588,525, such cleavable moiety being particularly useful with topical application of the conjugate.
- nucleic acid modifying groups may be used as conjugation partners to derivatize these Oligomers.
- Nucleic acid modifying groups include groups which, after the derivatized Oligomer forms a complex with a single stranded or double stranded nucleic acid segment, may be caused to cross-link, alkylate, cleave, degrade, or otherwise inactivate or destroy the target nucleic acid segment or a target sequence portion thereof, and thereby irreversibly inhibit the function and/or expression of that nucleic acid segment.
- the location of the nucleic acid modifying groups in the Oligomer may be varied and may depend on the particular nucleic acid modifying group employed and the targeted nucleic acid segment. Accordingly, the nucleic acid modifying group may be positioned at the end of the Oligomer or intermediate between the ends. A plurality of nucleic acid modifying groups may be included.
- the nucleic acid modifying group is photoreactable (e.g., activated by a particular wavelength, or range of wavelengths of light) , so as to cause reaction and, thus, cross-linking between the
- nucleic acid modifying groups which may cause cross-linking are the psoralens, such as an aminomethyltrimethyl psoralen group (AMT) .
- AMT aminomethyltrimethyl psoralen group
- the AMT is advantageously photoreactable, and thus must be activated by exposure to particular wavelength light before cross ⁇ linking is effectuated.
- Other cross-linking groups which may or may not be photoreactable may be used to derivatize these Oligomers.
- the nucleic acid modifying groups may comprise an alkylating agent group which is covalently bonded to the nucleic acid segment to render the target inactive.
- alkylating agent groups are known in the chemical arts and include groups derived from alkyl halides, haloacetamides and the like.
- Polynucleotide modifying groups which may be caused to cleave the polynucleotide segment include moieties which generate radicals, as well as moieties which promote cleavage through nucleophilic attack. Transition metal chelating complexes, such as ethylenediaminetetraacetate (EDTA) or a neutral derivative thereof, can be used to generate radicals.
- EDTA ethylenediaminetetraacetate
- cleavage agents include nucleophilic agents and hydrolytic agents that promote the addition of water at the phosphorus internucleotide linkages. Such agents include amines, substituted guanidinium groups, imidazole groups and the like.
- Preferred neutral Oligomers include neutral alkyl- and aryl-phosphonate Oligomers and neutral Oligomers comprising morpholino or phosphoramidate linkages. Especially preferred are neutral methylphosphonate Oligomers. In view of their demonstrated ability to penetrate skin, including tape stripped skin, (which has had the stratum corneum removed and which has been reported as a model for mucous membrane) , particularly preferred are neutral methylphosphonate Oligomers having only methylphosphonate internucleosidyl linkages.
- Oligomers which may be neutral until they enter cells and once inside are converted to charged species through chemical or biological processes.
- Such charged oligonucleotides may contain other moieties that stabilize the oligonucleotides to nuclease degradation.
- Substituents such as 2' -O-methylribose groups, various base modifications, and analogs of the phosphorous backbone, such as phosphorothioates, can increase resistance to nucleases.
- the presence of methylphosphonate or other neutral internucleoside linkages in the Oligomer give exonuclease resistance.
- 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 instead to employ shorter neutral tandem Oligomers which total more than 20 nucleosides in order to maximize solubility and penetration through the skin while competing for the development of a secondary structure of the target nucleic acid, such as an mRNA.
- These tandem Oligomers may also increase specificity of binding to the target sequence.
- the neutral Oligomers comprise alkyl- or aryl- phosphonate Oligomers
- nucleoside monomeric units having modified ribosyl moieties may advantageously improve hybridization of the Oligomer to its complementary target sequence.
- Suitable formulations comprise about 0.0001% to about 10% by weight of neutral Oligomer.
- neutral Oligomer formulations which comprise about 2% to about 100% of a short chain aliphatic alcohol.
- Suitable alcohols include ethanol, isopropyl alcohol, propylene glycol and glycerol .
- formulations of neutral Oligomers comprising 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, di ethylsulfoxide as well as cyclic ketones, lactones, anhydrides and esters such as those described in PCT Application No. PCT/US86/02583 (Publication Number W087/ 03473) . Some of these flux enhancers also increase retention of the Oligomer and, thus, act to increase the concentration of Oligomer within the skin itself.
- the present invention includes liposomal delivery of the Oligomers.
- Various methods and types of liposomal vesicles for drug delivery have been described. See, e.g. , Remington's Pharmaceutical Sciences (1990) .
- the Oligomers may be encapsulated by a liposome. Such liposome complexes advantageously may act to enhance delivery of Oligomer.
- the present invention is directed to methods of preventing or reducing hair loss using Oligomers which interfere with the expression of the enzyme 5-alpha-reductase, or with the expression of the androgen receptor itself.
- Oligomers include antisense Oligomers, Third Strand Oligomers and Triplex Oligomer Pairs.
- a method of decreasing hair loss by preventing or interfering with the expression of the human androgen receptor or the 5-alpha-reductase enzyme by administration of an Oligomer which is complementary to a target sequence on the DNA or an mRNA transcribed therefrom which codes for the androgen receptor or 5-alpha-reductase or to a sequence immediately upstream from the transcription start site for the mRNA.
- the Oligomer administered may be either an antisense Oligomer, a Third Strand Oligomer, or a Triplex Oligomer Pair.
- the antisense Oligomer is complementary to a sequence of RNA transcribed from a target gene, to a single-stranded DNA target sequence, or to a single RNA or DNA strand contained within a duplex.
- the Third Strand Oligomer has a base sequence selected so that it is capable of hydrogen bonding with a sequence of a double stranded nucleic acid and forming a triple helix complex therewith.
- the first and second Oligomers of the Triplex Oligomer Pair have sequences selected such that they are complementary to and capable of hydrogen bonding with a targeted single-stranded nucleic acid sequence of a target gene or its transcription product or to a single strand of a duplex and together with the single stranded nucleic acid form a triple helix complex.
- the target gene is selected from the group consisting of those genes encoding the androgen receptor or the enzyme 5-alpha-reductase and is considered to include a target sequence immediately upstream from the transcription start site of that .gene.
- the target sequence would include sequences from -500 to +20 (relative to the transcription start site) of the androgen receptor gene or 5-alpha reductase gene. More perferably suitable sequences would include target sequences in the area from -100 to +20 (relative to the transcription start site) of the androgen receptor gene or 5-alpha reductase gene.
- Oligomers of appropriate length preferably from about 8 to 40 nucleosides, more preferably from about 12 to about 20 nucleosides, are selected so as to be adjacent to or cover these sites when hybridized to the target, in part or in whole.
- Preferred sites when the target sequence is mRNA include, in both the androgen receptor and 5-alpha-reductase genes, the 5' untranslated region, the translation initiation region including regions slightly downstream of the AUG start codon (preferably up to about 20 nucleotides downstream from the AUG initiation codon), splice acceptors, splice donors, and the 3' untranslated region.
- the preferred target sites would include the sequence ranging from 18-50, with reference to the nucleotide positions of the human androgen receptor gene (Genbank locus:HT-TMARB) .
- a preferred target of this sequence range would include the sequence:
- TTC CCC CAC TCT CTC TC corresponding to the nucleotide positions 28-44 of the androgen receptor gene (Genbank locus:HUMARB) .
- a second preferred target of this sequence ranging would include the sequence:
- CTC TCT CTC ACC TC corresponding to the nucleotide positions 36-50 of the androgen receptor gene.
- a preferred triplex target site would include the sequence range from 109-126 of exon 4 of the human androgen receptor gene (Genbank locus :HUMARC4) .
- a preferred target of this sequence range would include the sequence: [3] UCU CUC UUC CUU CCC, 0 corresponding to the nucleotide positions 109-123 of exon 4 of the human androgen receptor gene.
- Oligomers of the appropriate length preferably from about 8 to 40 nucleosides and more preferably from about 12 to about 25 nucleosides especially from about 12 to about 20 nucleosides, are selected so as to have sequences which hybridize to sites immediately adjacent to these sites or hybridize with and cover these sites, in part or wholly, as defined by the nucleotide positions included above for 5-alpha-reductase and the androgen receptor.
- the sequence of the Oligomers is the reverse complement of the sequence of the targeted region so as to be able to hybridize to the targeted region.
- the Oligomers are selected to form sequence-specific hydrogen bonding interactions with the double stranded nucleic acid target .
- the first and second Oligomers are selected so as to form sequence specific hydrogen bonding interactions with a single stranded nucleic acid, and together form a triple helix structure.
- Oligoribonucleotides may be synthesized using the following procedures:
- the oligoribonucleotides were synthesized using 5'-0- dimethoxytrityl-2' -0-t_ert-butyldimethylsilyl-3 ' -O-N,N- diisopropyl-/S-cyanoethylphosphoramidite nucleosides (purchased from either Millipore or Pennisula Labora- tories) .
- the syntheses were done on a 1 ⁇ mole scale with a Milligen 8750 automated DNA synthesizer using standard Milligen phosphoramidite procedures with the exception that the coupling times were extended to 12 minutes to allow adequate time for the more sterically hindered 2'-0- tert-butyldimethylsilyl RNA monomers to react.
- the oligonucleotides were handled under sterile, RNase-free conditions. Water was sterilized by overnight treatment with 0.5% diethyl- pyrocarbonate followed by autoclaving. All glassware was baked for at least 4 hours at 300°C. The oligonucleotides were deprotected and cleaved from the support by first treating the support bound oligomer with 3/1 ammonium hydroxide/ethanol for 15 hours at 55°C. The supernatant, which contained the oligo- nucleotide, was then decanted and evaporated to dryness.
- the resultant residue was then treated with 0.6 mL of 1 M tetrabutylammonium fluoride in tetrahydrofuran (which contained 5% or less water) for 24 hours at room tempera ⁇ ture.
- the reaction was quenched by the addition of 0.6 mL of aqueous 2 M triethylammonium acetate, pH 7.
- Desalting of the reaction mixture was accomplished by passing the solution through a Bio-Rad 10DG column using sterile water. The desalted oligonucleotide was then dried.
- oligoribonucleotides Purification of the oligoribonucleotides was carried out by polyacrylamide gel electrophoresis (PAGE) con ⁇ taining 15% 19/1 polyacrylamide/bis-acrylamide and 7 M urea using standard procedures (See Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual, pages 184-185 (Cold Spring Harbor 1982)). The gels were 20 cm wide by 40 cm long and 6 mm in width. The oligoribonucleotides
- the melting temperatures (Tm) at which half of each complex had dissociated to single strands was 45.8°C and 2.3°C (2:1 mole ratio MP:RNA) for Oligomer 1 and Oligomer 2, respectively (see Table I) .
- the entire melting curve for MP Oligomer 2 and its target at 2:1 and 1:1 ratios is shown in Figure 1.
- Figure 1 depicts a thermodenaturation profiles for double-stranded and triple-stranded complexes formed between Oligomer 2 and a target sequence.
- Oligomer 1 (Androgen Rec. #1 44 . 1°C (1 : 1) target: 5' gag-aga-gag-tgg-ggg-aa) 45 , . 8 ° C (2 : 1)
- Oligomer 2 (Androgen Rec. #2 41 . 1 ° C ( 1 : 1 ) target: 5' gag-gtg-gag-aga-gag) 42 . 3 ° C ( 1 : 2 )
- methylphosphonate oligomers from mouse serum was measured with two oligomers: 3 H-tetramer (1689-3) 3 H-(dT) 4 and a 12-mer (2054-2) 3 H-C2-(TC) 6 (where C2 referred to a 2-carbon non-nucleotide linker with a primary amine) .
- mice (Jackson Laboratory) 9-10 weeks old were injected in the tail vein with 27 nmol (3 x 10 5 dpm) of oligomer in 200 ⁇ l phosphate buffered saline.
- mice were mildly anesthetized with metofane (methoxyflurane) during the procedure, and each mouse was bled no more than 7 or
- Figures 2 and 3 depict plots of the clearance from plasma of the 4-mer ( Figure 2) and the 12-mer ( Figure 3) .
- Hairless mice male, HRS/J strain, 8 to 10 weeks old, 20 to 25 g
- HRS/J strain 8 to 10 weeks old, 20 to 25 g
- hairless mice male, HRS/J strain, 8 to 10 weeks old, 20 to 25 g
- HRS/J strain 8 to 10 weeks old, 20 to 25 g
- skins were rinsed with physiological saline and used within one hour.
- 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 7.4) 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 onto 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. Permeability results are tabulated in Table II.
- the 14-mer (neutral methylphosphonate Oligomer of 14 nucleosides having only methylphosphonate internucleosidyl linkages) and 14-mer-IA (methylphosphonate Oligomer of 14 nucleosides having an internal anionic internucleosidyl linkage) were measured in the receptor solution by HPLC.
- the column used to separate the 14-mer was a 3.9 mm x 15 cm 4 ⁇ m, Waters Nova-Pak C18. A gradient elution was performed as follows for the 14-mer:
- HPLC conditions were altered somewhat for measure ⁇ ment of the 14-mer-IA. Again, a gradient elution profile was used as described below.
- the homogenate was then centrifuged at 8,000 g for 15 minutes at room temperature. The supernatant was removed and analyzed directlyby HPLC analysis (see below for conditions) .
- the chromatographic conditions were similar to those described above for the 14-mer in permeability experiments with some minor changes noted below.
- Human skin which had been dermatomed to a thickness of about 5-200 ⁇ m was used.
- the skin was mounted in a closed glass Franz diffusion cell (as described in Exam ⁇ ple 5) .
- Vehicle containing oligomer and, in some instances, enhancer 100 ⁇ L/cm 2 was placed on the surface of the skin (2 cm 2 exposed surface) .
- the amount of oligomer diffusing through and remain- ing in the skin was measured by HPLC. (See Example 5) .
- the cumulative amount was greater in hairless mouse skin, but was generally within an order of magnitude.
- 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 V reports retention of 14-mer in dermis versus stratum corneum after treatment with 14-mer in various vehicle/enhancer combinations.
- Stratum corneum and dermis were separated before analysis by microwave treatment as described by Kumar et al. (Phar . Res. 6.:740-741 (1989)) .
- 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 6) ; 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) d
- the viable tissue is the tissue after the stratum corneum has been removed by microwave treatment (Kumar, e_t al. , Pharm. Res. 6_:740-741 (1989)). It is a combination of the viable epidermis and the dermis.
- the values in this table are the means of three values 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.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP94909689A EP0684764A4 (en) | 1993-02-19 | 1994-02-18 | Treatment of androgen-associated baldness using antisense oligomers. |
CA002156512A CA2156512C (en) | 1993-02-19 | 1994-02-18 | Treatment of androgen-associated baldness using antisense oligomers |
JP6519151A JPH08506961A (en) | 1993-02-19 | 1994-02-18 | Treatment of androgen-related baldness using antisense oligomers |
AU62437/94A AU692148B2 (en) | 1993-02-19 | 1994-02-18 | Treatment of androgen-associated baldness using antisense oligomers |
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US1954393A | 1993-02-19 | 1993-02-19 | |
US08/019,543 | 1993-02-19 |
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WO1994018835A1 true WO1994018835A1 (en) | 1994-09-01 |
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PCT/US1994/001748 WO1994018835A1 (en) | 1993-02-19 | 1994-02-18 | Treatment of androgen-associated baldness using antisense oligomers |
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EP (1) | EP0684764A4 (en) |
JP (1) | JPH08506961A (en) |
AU (1) | AU692148B2 (en) |
CA (1) | CA2156512C (en) |
IL (1) | IL108712A0 (en) |
WO (1) | WO1994018835A1 (en) |
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FR2739553A1 (en) * | 1995-10-06 | 1997-04-11 | Oreal | USE OF BRADYKININE ANTAGONISTS TO STIMULATE OR INDUCE HAIR GROWTH AND / OR STOP THE HAIR LOSS |
WO1997038728A1 (en) * | 1996-04-15 | 1997-10-23 | Dyad Pharmaceutical Corporation | Combination therapy for androgenic alopecia with antisense oligonucleotides and minoxidil |
EP1080226A1 (en) * | 1998-05-21 | 2001-03-07 | Isis Pharmaceuticals, Inc. | Compositions and methods for topical delivery of oligonucleotides |
EP1080103A1 (en) * | 1998-05-21 | 2001-03-07 | Isis Pharmaceuticals, Inc. | Compositions and methods for non-parenteral delivery of oligonucleotides |
WO2001079504A1 (en) * | 2000-04-13 | 2001-10-25 | Millennium Pharmaceuticals, Inc. | 23155 NOVEL PROTEIN HUMAN 5-α REDUCTASES AND USES THEREFOR |
EP1175488A1 (en) * | 2000-03-08 | 2002-01-30 | IRA Istituto Ricerche Applicate S.R.L. | Cosmetic or pharmaceutical composition useful in inhibiting or delaying human alopecia by means of topical application of the composition |
US6489163B1 (en) * | 1996-05-08 | 2002-12-03 | Board Of Regents, The University Of Texas System | Ribozyme mediated inactivation of the androgen receptor |
US6887906B1 (en) | 1997-07-01 | 2005-05-03 | Isispharmaceuticals, Inc. | Compositions and methods for the delivery of oligonucleotides via the alimentary canal |
WO2005042741A2 (en) * | 2003-10-21 | 2005-05-12 | Dyad Pharmaceutical Corporation | METHODS AND COMPOSITIONS FOR TREATING 5alpha-REDUCTASE TYPE 1 AND TYPE 2 DEPENDENT CONDITIONS |
EP1598421A2 (en) | 2004-05-19 | 2005-11-23 | Beiersdorf AG | Oligoribonucleotides for influencing hair growth |
US8318689B2 (en) | 2001-11-09 | 2012-11-27 | Centre National De La Recherche Scientifique | SiRNA-based cancer treatment |
US9175291B2 (en) | 2012-10-11 | 2015-11-03 | Isis Pharmaceuticals Inc. | Modulation of androgen receptor expression |
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JP6768253B2 (en) * | 2015-10-09 | 2020-10-14 | 井上 肇 | How to get information about androgenetic alopecia in the subject |
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CA2110040A1 (en) * | 1991-05-31 | 1992-12-10 | Lyle J. Arnold, Jr. | Compositions and delivery systems for transdermal administration of neutral oligomers |
AU684774B2 (en) * | 1992-12-08 | 1998-01-08 | Genta Incorporated | Formation of triple helix complexes using a novel motif |
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- 1994-02-18 EP EP94909689A patent/EP0684764A4/en not_active Ceased
- 1994-02-18 CA CA002156512A patent/CA2156512C/en not_active Expired - Fee Related
- 1994-02-18 JP JP6519151A patent/JPH08506961A/en active Pending
- 1994-02-18 AU AU62437/94A patent/AU692148B2/en not_active Ceased
- 1994-02-18 WO PCT/US1994/001748 patent/WO1994018835A1/en not_active Application Discontinuation
- 1994-02-20 IL IL10871294A patent/IL108712A0/en unknown
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Also Published As
Publication number | Publication date |
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IL108712A0 (en) | 1994-05-30 |
CA2156512A1 (en) | 1994-09-01 |
CA2156512C (en) | 2002-11-19 |
EP0684764A1 (en) | 1995-12-06 |
AU6243794A (en) | 1994-09-14 |
JPH08506961A (en) | 1996-07-30 |
AU692148B2 (en) | 1998-06-04 |
EP0684764A4 (en) | 1997-10-22 |
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