EP1030914A2 - Oligonucleotides antisens contre la tenascine pour le traitement du vitiligo - Google Patents

Oligonucleotides antisens contre la tenascine pour le traitement du vitiligo

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Publication number
EP1030914A2
EP1030914A2 EP98954464A EP98954464A EP1030914A2 EP 1030914 A2 EP1030914 A2 EP 1030914A2 EP 98954464 A EP98954464 A EP 98954464A EP 98954464 A EP98954464 A EP 98954464A EP 1030914 A2 EP1030914 A2 EP 1030914A2
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Prior art keywords
seq
oligonucleotide
modified
alkyl
bridge
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English (en)
Inventor
Anuschirwan Peyman
Eugen Uhlmann
Caroline Weiser
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Sanofi Aventis Deutschland GmbH
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Aventis Pharma Deutschland GmbH
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Publication of EP1030914A2 publication Critical patent/EP1030914A2/fr
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    • 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
    • A—HUMAN NECESSITIES
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    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the invention relates to specific, optionally modified oligonucleotides with a length of up to 18 nucleotides, preferably with a length of 7-15 nucleotides, which correspond to sections of tenascin-coding sequences and which can bind to these sequences, their preparation and their use, for example for specific inhibition of the expression of tenascin and for the manufacture of medicinal products which can be used for the treatment of vitiligo.
  • Vitiligo is understood to mean an acquired lack of melanocytes, which creates hypopigmented areas of the skin, which are usually sharply defined and often symmetrically arranged, form one or two spots or cover almost the entire skin.
  • the hair in hypopigmented areas is usually white and also appears white in wood light.
  • the affected skin areas are susceptible to sunburn. The cause of the disease is unknown.
  • vitiligo is considered to be a disease acquired over the course of a lifetime, there is occasionally a family cluster (autosomal dominant, with incomplete penetrance and variable expression). It can also follow unusual physical trauma, especially a skull injury.
  • the available therapies include photochemotherapy (PUVA), for example with methoxypsoralen, phenylalanine, or khellin, the transplantation of cultivated Melanocytes, epidermal grafting, and treatment with steroids or placenta extracts.
  • PUVA photochemotherapy
  • Treatment with pseudocatalase has recently been reported (Schallreuter et al., Dermatology 190 (1995) 223).
  • Small stoves can also be covered with cosmetic make-up or tanning solutions.
  • Tenascin (Crossin, J. Cell. Biol. 61 (1996) 592) is an extracellular matrix glycoprotein which consists of six identical subunits which are linked at the amino terminus via disulfide bridges.
  • the Tenascin subunits have a characteristic domain structure: a cysteine-rich sequence at the amino-terminal end is followed by three sequence sections, each made up of repeating units, from units homologous to EGF, from units homologous to fibronectin (type III) and from units homologous to fibrinogen.
  • tenascin isoforms There are several isoforms of the tenascin subunits (hereinafter referred to as tenascin isoforms) which differ in the number of repeating units which are homologous to fibronectin type III. These isoforms are formed by alternative splicing of the Tenascin pre-mRNA and subsequent translation of the different Spliceva anten (A. Leprini et al., Perspectives on Developmental Neurobiology 2 (1994) 117-123). A human tenascin cDNA was developed by A. Siri et al. (Nucl. Acids Res. 19 (1991) 525-531) (sequence in Table 1).
  • This cDNA is stored in gene databases under the accession number X56160 and can be obtained under this number, for example under EMBIJGenbank / DDBJ / NBRF-PIR.
  • This cDNA contains a sequence section which codes for 12 repeating units which are homologous to fibrinogen type III.
  • the cDNAs of the other isoforms of human tenascins are truncated in this sequence section and code for less than 12 of these repeating units.
  • the expression of tenascin is limited in space and time and is assigned importance during the development of an organism and in the case of pathological changes (Crossin, vide supra). Such pathological changes include vitiligo, tumors and inflammation.
  • Antisense oligonucleotides (E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990); S. Agrawal, TIBTECH 1996, 376) offer a possibility for regulating gene expression.
  • WO 94/21664 (L. Denner et al.) Describes antisense oligonucleotides against tenascin, which are used to inhibit the proliferation of smooth cell muscles.
  • the oligonucleotides described there have a length of at least 18 nucleotides.
  • An object of the present invention was to provide new oligonucleotides which have advantageous properties and which can be used for the complete and / or partial inhibition of the gene expression of tenascin.
  • oligonucleotides that are up to 18 nucleotides in length can effectively influence the expression of tenascin.
  • the present invention relates to oligonucleotides with 7-17 nucleotide units, which are optionally modified.
  • the oligonucleotide has a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotides.
  • the oligonucleotide corresponds to sections of tenascin-coding sequences (ie the oligonucleotide has a sequence which is complementary to the corresponding section of a tenascin-coding sequence) and the oligonucleotide specifically binds to this tenascin-coding sequence (nucleic acid), for example to the tenascin gene and / or tenascin mRNA and / or tenascin cDNA, the tenascin coding sequence preferably being of human origin (eg human tenascin gene, human tenascin mRNA, human tenascin cDNA).
  • the section of the Tenascin coding sequence to which the oligonucleotide corresponds or to which the oligonucleotide is complementary has preferably a length of 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 nucleotide units (this applies in particular to the determination of the length of a modified and / or chimeric oligonucleotide or of oligonucleotide analogs).
  • a particular embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide having a length of 7 to 15 nucleotides and possibly being modified and which physiologically acceptable salts of the oligonucleotide.
  • a particular embodiment of the invention relates to an oligonucleotide which is directed against one or more specific regions of a tenascin-coding sequence, for example the start of transformation, the 5'-untranslated region, the coding region and / or the 3'-non-coding region .
  • the oligonucleotide can also be directed against one or more regions of a tenascin-coding sequence, which e.g. encodes for certain domains of tenascin, for example against the cysteine-rich domain, against a domain homologous to EGF, against a domain homologous to fibronectin type III and / or against a domain homologous to fibrinogen.
  • One embodiment of the invention relates to an oligonucleotide which binds to a nucleic acid which codes for one of the isoforms human Tenascins or parts thereof and inhibits their expression, the oligonucleotide being able to bind to a region of the nucleic acid which a) a part of the 5 ' non-coding region and / or the translation start or b) the translation start and / or a part of the coding region or C) comprises part of the coding region and / or part of the 3 ' non-coding region.
  • the invention relates in particular to an oligonucleotide that a sequence section of the human cDNA according to SEQ ID NO. 1 (Table 1) corresponds.
  • the invention further relates to an oligonucleotide which corresponds to a sequence section of the cDNA which is stored in gene databases under the accession number X56160.
  • an oligonucleotide can have, for example, one of the following sequences or parts thereof:
  • SEQ ID NO. . 9 3'- AAAGGAACGGGAGCG -5 '
  • SEQ ID NO. 19 3'- CCCGGTACTGA -5 'and SEQ ID NO. 20: 3'- CCACAGAAAGAAC -5 '.
  • sequences SEQ ID NO. 2 to SEQ ID NO. 20 correspond to sections of the tenascin-encoding cDNA as shown in Table 1.
  • An oligonucleotide that is one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 is complementary to a corresponding portion of a tenascin-encoding nucleic acid, e.g. a human Tenascin cDNA and can bind to this nucleic acid.
  • Sequences SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 18 are examples of oligonucleotides that have a sequence that is directed against the translation start of the tenascin coding sequences.
  • the invention also relates to derivatives of an oligonucleotide, for example its salts, in particular its physiologically tolerable salts.
  • Physiologically acceptable salts are understood as meaning compounds which are readily soluble, soluble and slightly soluble in water, for example as defined in the "German Pharmacopoeia” (9th edition 1986, official edition, German Pharmacist Verlag Stuttgart), page 19.
  • a special embodiment of the invention relates to the sodium salt of the oligonucleotide according to the invention.
  • Derivatives are also modified oligonucleotides.
  • An oligonucleotide can, for example, be composed entirely or partially of the natural nucleotides adenosine phosphate, guanosine phosphate, inosine phosphate, cytidine phosphate, uridine phosphate and thymidine phosphate.
  • One embodiment of the invention relates to an oligonucleotide which is composed of the natural nucleosides adenosine, guanosine, inosine, cytidine, uridine and thymidine and in which the nucleosides are linked to one another via phosphoric diester internucleoside bridges (“phosphoric diester bridges”).
  • an oligonucleotide can optionally contain one or more modifications, for example chemical modifications.
  • An oligonucleotide can be several of the same and / or have various modifications. Modifications can be located at certain nucleoside positions (nucleobase and / or ⁇ -D-2'-deoxyribose unit) and / or certain internucleoside bridges.
  • the chemical modification of an oligonucleotide can mean, for example, a) the complete or partial replacement of the phosphoric diester bridges (internucleoside bridge) by modified phosphorus bridges, phosphorothioate, phosphorus dithioate, NR 1 R 1 ' phosphoramidate, boranophosphate, phosphate (C ⁇ -C 2 ⁇ ) -O-alkyl esters, phosphate - [(C 6 -C ⁇ 2 ) aryl- (C ⁇ -C 2 ⁇ ) -O-alkyl] esters, (Ci- C 8 ) alkyl phosphonate and / or (C 6 -C ⁇ 2 ) arylphosphonate bridges are examples of modified phosphobridges, wherein
  • R 1 and R 1 ' independently of one another for hydrogen, (-C 8 -C 8 ) alkyl, (C 6 -C 20 ) aryl,
  • Methoxyethyl particularly preferably for hydrogen, (-CC 4 ) alkyl and / or
  • R 1 and R 1 together with the nitrogen atom carrying them form a 5-6-membered heterocyclic ring, which is also a further heteroatom from the series
  • phosphoric diester bridges May contain O, S, N; and / or b) the complete or partial replacement of the 3'- and / or 5'-phosphoric diester internucleoside bridges (“phosphoric diester bridges”) by “dephospho” bridges (described, for example, in Uhlmann, E. and Peyman, A. in “Methods in Molecular Biology “, vol. 20,” Protocols for Oligonucleotides and Analogs ", S.
  • PNA polyamide nucleic acid
  • PHONA phosphomono acid ester nucleic acid
  • nucleoside bases are examples of modified sugar units; and / or e) the modification or the complete or partial replacement of the natural nucleoside bases by modified (nucleoside) bases (“nucleobases”), where 5- (hydroxymethyl) uracil, 5-aminouracil, pseudouracil, dihydrouracil, 5- (C ⁇ - C 6) -alkyl-uracil, 5- (C 2 -C 6) -alkenyl-uracil, 5- (C 2 - C 6) alkynyl-uracil, 5- (C ⁇ -C6) alkyl-cytosine, 5 - (C 2 -C 6 ) alkenyl cytosine, 5- (C 2 -C 6 ) alkynyl cytosine, 5-fluorouracil, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine , 7-deaza-7-substit
  • oligonucleotide eg antisense oligonucleotide, triple helix-forming oligonucleotide
  • affinity for the tenascin-coding target sequence, pharmacokinetics) of the oligonucleotide favorably influence and / or when hybridizing the oligonucleotide to the target sequence can attack it with binding and / or crosslinking or can mean, where poly-lysine, intercalators such as pyrene, acridine, phenazine, phenanthridine, fluorescent compounds such as fluorescein, cross-linkers such as psoralen, azidoproflavin, lipophilic molecules such as (-C 2 -C 20 ) alkyl, lipids such as 1 , 2-Di-hexadecyl-rac-glycerol, steroids such as cholesterol, testosterone, vitamins such as vitamin E, poly- or oligo-ethylene glycol, (C ⁇ 2 -C ⁇ 8
  • the oligonucleotide has one or more chemical modifications which are selected independently of one another from a) the complete or partial replacement of the phosphoric diester bridges by phosphorothioate and / or (C 1 -C 8 ) alkylphosphonate bridges, b) the complete one or partial replacement of the sugar phosphate backbone by PNA units and / or PHONA units, c) the complete or partial replacement of the ⁇ -D-2'-deoxyribose units by 2'-F-2'-deoxyribose, 2'-O- (-CC 6 ) alkyl ribose and / or 2 '- [O- (-C 6 ) alkyl-O- (-C 6 ) alkyl] -ribose, d) the complete or partial replacement of the natural nucleoside Bases by 5- (C 2 -C 6 ) alkynyl uracii and / or 5- (C 2 -C 6 )
  • the oligonucleotide has one or more chemical modifications which can be selected independently of one another from the series comprising a) the Complete or partial replacement of the phosphoric diester bridges (phosphodiester bridges) by phosphorothioate bridges, b) Complete or partial replacement of the ß-D-2'-deoxyhbose units by 2'-F-2'-deoxyhbose, 2'-O- (C ⁇ - C 6 ) alkyl-ribose and / or 2 '- [O- (-C-C 6 ) alkyl-O- (-C-C ⁇ ) alkyl] -ribos ⁇ , c) the conjugation with lipophilic molecules, for example (C ⁇ 2 -C 2 o) -alkyl, with lipids, for example 1, 2-di-hexadecyl-rac-glycerol, with (-C 2 -C 8 ) -alkyl phosphate die
  • an oligonucleotide which can have one or more modifications and which has one of the sequences SEQ ID NO. 2 - SEQ ID NO. 20 or one of the sequences SEQ ID NO. 2 to SEQ ID NO. 20 corresponds to or corresponds to the corresponding sequence sections of a tenascin coding sequence and can bind to this section of the tenascin coding sequence.
  • oligonucleotide is provided, in the sequence of which each nucleotide (base and / or sugar and / or internucleoside bridge) is modified.
  • the oligonucleotide is entirely composed of phosphorothioates constructed (consistently modified phosphothioate, all internucleoside bridges modified).
  • an oligonucleotide is provided which has one of the sequences SEQ ID NO. 2 - SEQ ID NO.
  • an oligonucleotide is provided in that only a part of the phosphodiester bridges is replaced by phosphothioate bridges.
  • the invention includes oligonucleotides that are only minimally (or partially) modified. The principle of minimally modified oligonucleotides is described in A. Peyman, E. Uhlmann, Biol. Chem. Hoppe-Seyler, 377 (1996) 67-70.
  • oligonucleotides preferably 1-3 terminal nucleotide units (or preferably the corresponding internucleoside bridges) at the 5 'and / or at the 3' end and, if appropriate, additionally selected internal pyrimidine positions or preferably the corresponding ones Internucleoside bridges which are located at the 3 ' and / or 5 ' end of the corresponding pyrimidine nucleoside are modified or replaced, preferably internucleoside bridges being replaced by phosphorothioate bridges.
  • minimally modified oligonucleotides have particularly advantageous properties, for example they show particular nuclease stability with minimal modification.
  • a particular embodiment of the invention relates to an oligonucleotide in which selected internucleoside bridges are replaced by modified internucleoside bridges, preferably by phosphorothioate bridges.
  • the invention relates to an oligonucleotide in which either a) only certain phosphodiester internucleoside bridges or b) all phosphodiester internucleoside bridges are modified.
  • the invention further relates to a terminal in the 1-5
  • Internucleoside bridges at the 5 ' and / or at the 3 ' end of the oligonucleotide are modified.
  • the invention also relates to an oligonucleotide in which the 3 ' and / or 5 ' ends of non-terminal nucleosides, the one
  • Internucleoside bridges are modified.
  • oligonucleotide that is one of the sequences selected from the series of sequences SEQ ID NO. 21 to SEQ ID NO. 39, wherein
  • sequences SEQ ID NO. 21 to SEQ ID NO. 39 correspond to the sequences SEQ ID NO. 2 - SEQ ID NO. 20, i.e. they can bind to the same regions of a tenascin-coding sequence, but in contrast to SEQ ID NO. 2-20 some of the phosphodiester bridges are replaced by modified phosphodiester bridges or dephosphobridges, preferably by phosphothioate bridges (identified by an "s" in the sequence).
  • a chimeric oligonucleotide is constructed from at least two different sequence segments, for example a DNA segment and a modified segment, e.g. a PNA section and / or a PHONA section. These different sections give the entire oligonucleotide special properties.
  • a special form of chimeric oligonucleotides is described, for example, in Matteucci and Wagner, Nature 384 SUPP (1996) 20-22.
  • a chimeric oligonucleotide can e.g.
  • core sequence which consists of about seven nucleotides and which can activate RNase H and
  • flanking sequences which increase the affinity, specificity and / or nuclease stability of the oligonucleotide.
  • the "core sequence” can have modified internucleoside bridges at certain positions, for example the "core sequence” can contain phosphorothioate and / or phosphodiester bridges.
  • Suitable flanking sequences are, for example, sequences in which the sugar phosphate backbone (replacement of one or more sugar phosphate units) ) and / or ß-D-2 ' - Deoxyribose units are replaced.
  • Suitable flanking sequences are, for example, PNAs and / or 2'-O-alkyl derivatives such as 2'-O-methyl and / or 2'-O-propyl and / or 2'-methoxyethoxy derivatives.
  • a particular embodiment of the invention relates to a chimeric oligonucleotide which has one of the sequences SEQ ID NO. 40 - SEQ ID NO. 58, where x independently of one another stands for an unmodified or a modified phosphodiester internucleoside bridge or a dephospho bridge, preferably for phosphorothioate and / or phosphorus diester, and y independently of one another for the replacement of a sugar phosphate unit or a ⁇ -D-2 ' deoxyribose unit, preferably represents 2'-O-methyl-, 2'-O-propyl- and / or 2'-methoxyethoxyribose or a PNA building block, where
  • SEQ ID NO. 56 3'- GyGyTxGxGxTxAxCxCyCyC -5 '
  • SEQ ID NO. 57 3'- CyCxCxGxGxTxAxCyTyGyA -5 '
  • SEQ ID NO. 58 3'- CyCyAxCxAxGxAxAxAxGyAyAyC -5 '.
  • sequences SEQ ID NO. 40 - SEQ ID NO. 58 correspond to the sequences SEQ ID NO. 2 to SEQ ID NO. 20, i.e. they bind to the corresponding sequence sections of a tenascin-coding sequence, although the modifications mentioned are included.
  • the invention relates to methods for producing the oligonucleotides.
  • the oligonucleotides described can be made using various known chemical methods, e.g. using the standard phosphoramidite chemistry using iodine or TED (tetraethythiuram disulfide) as an oxidizing agent. This method is e.g. in Eckstein, F. (1991) "Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford.
  • the oligonucleotides can also be prepared by methods that optionally contain one or more enzymatic steps.
  • the invention relates to the use of the oligonucleotides.
  • the oligonucleotides can be used for hybridization or binding to tenascin-encoding (single-stranded and / or double-stranded) nucleic acids, for example DNA (e.g. genes, cDNA) and / or RNA (e.g. pre-mRNA, mRNA).
  • DNA e.g. genes, cDNA
  • RNA e.g. pre-mRNA, mRNA
  • this relates to the use of the oligonucleotides for hybridization with or binding to nucleic acids which have the sequence SEQ ID NO.
  • nucleic acids which have parts of this sequence for example sequences which code for tenascin isoforms
  • nucleic acids whose sequence deviates slightly from these sequences which have, for example, one or more point mutations.
  • the invention further relates to the use of the oligonucleotides for modulation and for the total or partial inhibition of the expression of tenascin or different tenascin isoforms or of mutants thereof, for example for the total or partial inhibition of transcription and / or translation.
  • the invention relates, for example, to the use of the oligonucleotides as antisense oligonucleotides.
  • the oligonucleotides can be used as aids in molecular biology.
  • the invention further relates to the use of the oligonucleotides as pharmaceuticals and / or diagnostic agents or the use of the oligonucleotides for the production of pharmaceuticals and / or diagnostic agents.
  • the oligonucleotides can be used in medicaments which are used for the prevention and / or treatment of diseases which are associated with the expression or overexpression of tenascin. Since the expression of tenascin is usually, i.e. e.g. in healthy people is limited in space and time, a deviation from this normal spatial and temporal expression can be regarded as overexpression.
  • the oligonucleotides can be used in diagnostic methods. Such diagnostic methods can e.g. used for the diagnosis or early detection of diseases that will be associated with an abnormal expression (e.g. overexpression) of Tenascin.
  • the invention also relates to a test kit which contains one or more oligonucleotides according to the invention and optionally other components.
  • a test kit which contains one or more oligonucleotides according to the invention and optionally other components.
  • Such a test kit can be used, for example, in diagnostics and for prevention, for example of skin cancer.
  • the invention further relates to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment of diseases in which tenascin or an overexpression of tenacsin is causal or involved.
  • the invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of diseases in which there is a malfunction or disruption of the immigration or the presence or storage of melanocytes in epithelial cell layers, for example in the epithelial cell layer of the epidermis, the choroid of the eye or the substantia nigra, underlies or is involved and of Addison's disease, diabetes mellitus, pernicious anemia and / or thyroid dysfunction.
  • the invention relates in particular to the use of the oligonucleotides or of medicaments which contain these oligonucleotides for the treatment and / or prevention of vitiligo and other depigmentation diseases or depigmentation disorders (for example the skin, hair, eyes), for example albinism and / or for the treatment of psoriasis and / or for the treatment of cancer, e.g. for inhibitors of tumor growth and tumor metastasis, for example in the case of melanoma and / or for the treatment of inflammation, in particular as an anti-inflammatory agent and / or for the treatment and / or prophylaxis of cardiovascular diseases, for example restenosis.
  • vitiligo and other depigmentation diseases or depigmentation disorders for example the skin, hair, eyes
  • albinism and / or for the treatment of psoriasis and / or for the treatment of cancer e.g. for inhibitors of tumor growth and tumor metastasis, for example in the case
  • the invention relates to the use of the oligonucleotides for the treatment of vitiligo or for the production of medicaments which can be used for the treatment of vitiligo.
  • the invention also relates generally (i.e. also oligonucleotides with a length of greater than or equal to 18 nucleotides) to the use of oligonucleotides for the treatment of vitiligo or the production of medicaments which can be used for the treatment of vitiligo.
  • the invention further relates to the use for the treatment of vitiligo in combination with known therapeutic methods, for example in combination a) with photochemotherapy (PUVA), e.g. using methoxypsoralen, phenylalanine and / or khellin and / or b) with the transplantation of cultured melanocytes ("epidermal grafting") and / or c) with a steroid treatment and / or d) with a treatment with placenta extracts and / or e) treatment with pseudocataiase.
  • PUVA photochemotherapy
  • the invention further relates to methods for the production of pharmaceuticals (pharmaceutical preparations).
  • pharmaceuticals pharmaceutical preparations
  • One or more different oligonucleotides or their physiologically compatible ones are used for the production of pharmaceuticals Salts mixed, where appropriate further pharmaceutical carriers and / or additives can be added.
  • the invention further relates to pharmaceutical preparations (medicaments) which contain one or more different oligonucleotides and / or their physiologically tolerable salts and, if appropriate, pharmaceutical carriers and / or additives.
  • the oligonucleotide (s) and / or their physiologically tolerable salts can be administered to animals, preferably to mammals, in particular to humans, as a medicament on their own, in mixtures with one another or in the form of pharmaceutical preparations.
  • the drugs can allow topical, percutaneous, parenteral and / or enteral use.
  • the preferred form of application depends on the particular circumstances.
  • a topical application e.g. preferred in the form of ointments, lotions or tinctures, emulsions, suspensions.
  • the frequency of application also depends on the individual circumstances.
  • a topical composition can be applied to the depigmented skin area once or twice a day.
  • Medicaments or pharmaceutical preparations can contain as active ingredient an effective dose of at least one oligonucleotide and / or a mixture of several oligonucleotides and, if necessary, additional, pharmaceutically perfect carriers and / or additives.
  • a pharmaceutical preparation can contain about 0.1% (percent by weight) or less to about 90% (percent by weight) or more of the therapeutically active oligonucleotide or the pharmaceutically active oligonucleotides.
  • the pharmaceutically effective dose of the respective oligonucleotide or an oligonucleotide which is part of a mixture of different oligonucleotides can vary within wide limits and must be adapted to the individual circumstances in each individual case.
  • the preparation of the pharmaceutical preparations can be carried out in a manner known per se, e.g. B. described in Remingtons Pharmaceutical Sciences (1985), Mack Publ. Co., Easton, PA. be carried out, where appropriate pharmaceutically inert inorganic and / or organic carriers can be used.
  • suitable pharmaceutically inert inorganic and / or organic carriers can be used.
  • pills tablets, dragees and / or hard gelatin capsules e.g. Lactose, corn starch and / or derivatives thereof, talc, stearic acid and / or their salts can be used.
  • As carriers for soft gelatin capsules and / or suppositories e.g. Fats, waxes, semi-solid and / or liquid polyols, natural and / or hardened oils can be used.
  • ⁇ As carriers for the preparation of solutions and / or syrups z. B. water, sucrose, invert sugar, glucose and / or polyols can be used. As carriers for the production of injection solutions, e.g. Water, alcohols, glycerin, polyols and / or vegetable oils can be used. As a carrier for microcapsules, implants and / or rods, for example, copolymers, e.g. from glycolic acid and lactic acid can be used. In addition, liposome formulations which are known to the person skilled in the art (N.
  • the dermal application can also be carried out, for example, with the aid of ionophoretic methods and / or with the aid of electroporation, and lipofectins and / or other (nucleic acid or DNA) carrier systems, for example those used in gene therapy, can also be used Systems are particularly suitable with the aid of which oligonucleotides can be introduced into eukaryotic cells or the nuclei of eukaryotic cells with great efficiency.
  • a pharmaceutical preparation can also contain additives, such as fillers, extenders, explosives, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colors, flavors or Flavoring agents, thickeners, diluents, buffer substances, furthermore solvents and / or solubilizers and / or agents for achieving a depot effect, and salts for changing the osmotic pressure, coating agents and / or antioxidants. They can also contain two or more different oligonucleotides and / or their physiologically tolerable salts and, in addition to at least one oligonucleotide, one or more other therapeutically active substances.
  • additives such as fillers, extenders, explosives, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colors, flavors or Flavoring agents, thickeners, diluents, buffer substances, furthermore solvents
  • the oligonucleotide was synthesized on an automatic DNA synthesizer (Applied Biosystems Model 380B or 394) using standard phosphoramidite chemistry and oxidation with iodine (F. Eckstein, Ed “Oligonucleotides and Analogues, A Practical Approach", IRL Press, Oxford, 1991 ).
  • TETD tetraethylthiuram disulfide
  • the oligonucleotide was first purified by butanol precipitation (Sawadogo, Van Dyke, Nucl. Acids Res. 19 (1991) 674). The sodium salt was then obtained by precipitation from a 0.5 M NaCl solution with 2.5 parts by volume of ethanol.
  • the oligonucleotide was determined using the
  • oligonucleotide showed that it was in each case in a purity of greater than 90%.
  • the methods for analyzing oligonucleotides are e.g. in Schweiber and Engler "Analysis of oligonucleotides” (in “Antisense - from technology to therapy", a laboratory manual and textbook, Schlingensiepen et al. eds., Biol. Science, Vol. 6 (1997) p. 78-103) .
  • ODN1 (Sequence SEQ ID NO. 24): 3'- GsGsAGGTsGGTsACsCsCsC -5 '
  • ODN 1 from Example 1 can e.g. closely mixed with 1g Dermatop® (Hoechst Aktiengesellschaft, Frankfurt am Main, Germany) base cream and the mixture stored at temperatures ⁇ 10 ° C.
  • Dermatop® Hoechst Aktiengesellschaft, Frankfurt am Main, Germany
  • the cream from Example 2 can then be applied, for example, twice a day (in the morning and in the afternoon or in the evening) to a depigmented skin area of a Vitiligo patient.
  • Table 1 Sequence SEQ ID NO. 1 :
  • TTCACAGGCC TGGACTGTGG CCAGCACTCC TGCCCCAGTG ACTGCAACAA CTTAGGACAA 1860
  • CAACAAGCCA CAACCAAAAC CACACTCACA GGTCTGAGGC CGGGAACTGA ATATGGGATT 2940
  • CAAGGGCATC AAACCAAGCC CTTGAGGGCT GAGATTGTTA CAGAAGCCGA ACCGGAAGTT 4980
  • ATCACAGCCC AGGGGCAGTA CGAGCTCCGG GTGGACCTGC GGGACCATGG GGAGACAGCC 6360

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Abstract

L'invention concerne des oligonucléotides spécifiques éventuellement modifiés pouvant comporter jusqu'à 18 nucléotides, qui peuvent correspondre à des fragments de séquences codant la ténascine et se lier à ces séquences. L'invention concerne également leur production et leur utilisation, par exemple pour l'inhibition spécifique de l'expression de la ténascine et pour la production de médicaments pouvant être utilisés afin de traiter le vitiligo.
EP98954464A 1997-11-15 1998-10-29 Oligonucleotides antisens contre la tenascine pour le traitement du vitiligo Withdrawn EP1030914A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19750702A DE19750702A1 (de) 1997-11-15 1997-11-15 Antisense Oligonucleotide gegen Tenascin zur Behandlung von Vitiligo
DE19750702 1997-11-15
PCT/EP1998/006868 WO1999025819A2 (fr) 1997-11-15 1998-10-29 Oligonucleotides antisens contre la tenascine pour le traitement du vitiligo

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EP1030914A2 true EP1030914A2 (fr) 2000-08-30

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EP (1) EP1030914A2 (fr)
JP (1) JP2001523451A (fr)
AR (1) AR013760A1 (fr)
AU (1) AU1156699A (fr)
DE (1) DE19750702A1 (fr)
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WO2023131829A1 (fr) 2022-01-05 2023-07-13 Ramakrishna Reddy Isanaka Formulation pour le traitement de troubles pigmentaires

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DE10238298A1 (de) * 2002-08-21 2004-03-04 Beiersdorf Ag Verwendung von Antisense-Oligonucleotiden zur Behandlung von degenerativen Hauterscheinungen
DE10254214A1 (de) * 2002-11-20 2004-06-09 Beiersdorf Ag Oligoribonukleotide zur Behandlung von degenerativen Hauterscheinungen durch RNA-Interferenz
FR2849381B1 (fr) * 2002-12-30 2005-02-25 Jean Noel Thorel Composition cosmetique, solaire et bio-bronzante
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WO2023131829A1 (fr) 2022-01-05 2023-07-13 Ramakrishna Reddy Isanaka Formulation pour le traitement de troubles pigmentaires

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DE19750702A1 (de) 1999-05-27
AR013760A1 (es) 2001-01-10
WO1999025819A2 (fr) 1999-05-27
JP2001523451A (ja) 2001-11-27
WO1999025819A3 (fr) 1999-09-10
US6878547B1 (en) 2005-04-12
AU1156699A (en) 1999-06-07

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