WO2011064395A2 - Inhibiteurs et antagonistes des canaux calcium dans le traitement de l'asthme - Google Patents

Inhibiteurs et antagonistes des canaux calcium dans le traitement de l'asthme Download PDF

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WO2011064395A2
WO2011064395A2 PCT/EP2010/068546 EP2010068546W WO2011064395A2 WO 2011064395 A2 WO2011064395 A2 WO 2011064395A2 EP 2010068546 W EP2010068546 W EP 2010068546W WO 2011064395 A2 WO2011064395 A2 WO 2011064395A2
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seq
cavl
cells
sequence
gene expression
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WO2011064395A3 (fr
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Lucette Pelletier
Jean-Charles Guery
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Inserm
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • the invention relates to the use of specific antagonists of the Cavl .2 and/or Cavl .3 subunits of voltage-dependent L-type calcium (Cavl) channels, preferably expressed in Th2 lymphocytes, for use in preventing and/or treating inflammatory diseases in a subject in need thereof.
  • the present invention also relates to an inhibitor of CACNA1C and/or CACNA1D gene expression for use in preventing and/or treating such inflammatory diseases.
  • the inflammatory diseases are allergic diseases. More preferably, the inflammatory diseases are respiratory allergic diseases.
  • Respiratory diseases such as allergic rhinitis and asthma are widespread conditions with complex and multifactorial etiologies.
  • the severity of the conditions vary widely between individuals, and within individuals, dependent on factors such as genetics, environmental conditions, and cumulative respiratory pathology associated with duration and severity of disease. Both diseases are a result of immune system hyper-responsiveness to innocuous environmental antigens, with asthma typically including an atopic (allergic) component.
  • asthma the pathology manifests as inflammation, mucus overproduction, and reversible airway obstruction which may result in scarring and remodelling of the airways.
  • Mild asthma is relatively well controlled with current therapeutic interventions including beta-agonists and low dose inhaled corticosteroids or cromolyn.
  • moderate and severe asthma are less well controlled, and require daily treatment with more than one long-term control medication to achieve consistent control of asthma symptoms and normal lung function.
  • doses of inhaled corticosteroids are increased relative to those given to mild asthmatics, and/or supplemented with long acting beta-agonists or leukotriene inhibitors.
  • beta-agonists can decrease dependence on corticosteroids, they are not as effective for total asthma control as corticosteroids (e.g., reduction of episodes, emergency room visits). With severe asthma, doses of inhaled corticosteroids are increased, and supplemented with both beta-agonists and oral corticosteroids. Severe asthmatics often suffer from chronic symptoms, including night time symptoms; limitations on activities; and the need for emergency room visits. Additionally, chronic corticosteroid therapy at any level has a number of unwanted side effects. Allergic rhinitis is an inflammation of the nasal passages, and is typically associated with watery nasal discharge, sneezing, congestion and itching of the nose and eyes. It is frequently caused by exposure to irritants, particularly allergens.
  • Subjects suffering from allergic rhinitis mostly suffer from seasonal symptoms due to exposure to allergens, such as pollen, that are produced during the natural plant growth season(s). A smaller proportion of sufferers have chronic allergies due to allergens that are produced throughout the year such as house dust mites or animal dander.
  • Treatments are available for the treatment of allergic rhinitis including oral and nasal antihistamines, and decongestants. Antihistamines are utilized to block itching and sneezing and many of these drugs are associated with side effects such as sedation and performance impairment at high doses. Decongestants frequently cause insomnia, tremor, tachycardia, and hypertension. Nasal formulations, when taken improperly or terminated rapidly, can cause rebound congestion.
  • Anticholinergics and leukotriene receptor antagonists have substantially fewer side effects, but they also have limited efficacy. Similarly, prescription medications are not free of side effects. Nasal corticosteroids can be used for prophylaxis or suppression of symptoms; however, compliance is variable due to side effects including poor taste and nasal irritation and bleeding. Allergen immunotherapy is expensive and time consuming and carries a low risk of anaphylaxis.
  • Respiratory allergic diseases are currently considered as inflammatory disorders involving the conducting airways and characterized by infiltration of the airway wall with inflammatory cells driven mostly by Th2 lymphocytes, mast cells and eosinophils.
  • Th2 lymphocytes by producing inflammatory cytokines as IL-4, IL-5, IL-9 and IL-13, are thought to play a central role in both the initiation and maintenance of the disease.
  • cytokines such as IL-13, IL-4, IL-5, cytokine receptors such as IL-4R alpha, transcription factors such as STAT-6, GATA-3, or NFkB, and chemokines receptor such as CCR3.
  • Th2 lymphocytes express voltage-dependent L-type calcium (Ca v l) channels, said Ca v l channels being essential for TCR-induced calcium signalling in Th2 lymphocytes and for production of IL-4, IL-5 and IL-13 cytokines.
  • Ca v l channels being essential for TCR-induced calcium signalling in Th2 lymphocytes and for production of IL-4, IL-5 and IL-13 cytokines.
  • the inventors aim to provide a new therapy for treating inflammatory diseases, and particularly allergic diseases, preferably respiratory allergic diseases such as asthma, by targeting said Ca v l channels in Th2 lymphocytes.
  • the present invention relates to a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Ca v l) channels, preferably expressed in Th2 lymphocytes, or to an inhibitor of CACNA1C and/or CACNAID gene expression for use as a medicament.
  • a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Ca v l) channels preferably expressed in Th2 lymphocytes, or to an inhibitor of CACNA1C and/or CACNAID gene expression for use as a medicament.
  • the present invention also relates to a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Ca v l) channels, preferably expressed in Th2 lymphocytes, for use in preventing and/or treating inflammatory diseases, and particularly allergic diseases, preferably respiratory allergic diseases such as asthma and allergic rhinitis.
  • the present invention also relates to an inhibitor of CACNA1C and/or CACNAID gene expression for use in preventing and/or treating inflammatory diseases, and particularly allergic diseases, preferably respiratory allergic diseases such as asthma and allergic rhinitis.
  • the present invention also relates to specific antisense oligonucleotides that inhibit CACNA1C and/or CACNAID gene expression.
  • DHPRs dihydropyridine receptors
  • nicardipine was tested the effect of nicardipine in a model of experimental allergic asthma in mice and observed that nicardipine altered TCR-driven calcium response in Th2 cells as well as the release of IL-4, IL-5 and IL-13 cytokines, which are implicated in the pathogenesis of asthma, thereby impeding the development of Th2 mediated airway inflammation and reducing the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2 cytokines (20).
  • nicardipine might be potentially useful in the treatment of asthma.
  • the concentrations of nicardipine used for preventing or treating asthma in this model were 30 times higher than those used in the treatment of experimental cardiovascular pathologies for inhibiting calcium signalling in excitable cells.
  • the inventors then investigated about the specific molecular target of DHP antagonists in T-cells.
  • the DHP are known to target voltage-dependent L-type calcium (Ca v l) channels.
  • the Ca v channels are hetero-oligomeric protein complexes comprising a main pore-forming CCi-subunit in association with auxiliary ⁇ , ⁇ 3 ⁇ 4_ ⁇ subunits and optionally ⁇ -subunit.
  • the ⁇ 3 ⁇ 4 subunit is composed of four membrane- spanning domains (TIV), and each domain consists of six transmembrane segments (S1-S6). Beside the cytoplasmic N- and C-termini, joining the domains are intracellular regions comprising the loops linking domain I-II, domain II-III and domain III- IV.
  • the human ⁇ 3 ⁇ 4 subunit is encoded by 10 genes, of which 4 genes CACNA1S, CACNA1C, CACNA1D and CACNA1F respectively encode the L-type calcium (Ca v l) channels referred to as Cavl. l, Cav 1.2, Cav 1.3 and Cav 1.4 channels (Jurkat-Rott et al, The impact of splice isoforms on voltage-gated calcium channel CCi subunits, J Physiol 554.3, pp 609-619, 2003).
  • the inventors aim to provide a new therapy for preventing and/or treating inflammatory diseases, particularly respiratory allergic diseases such as asthma by using a specific antagonist of at least one of Ca v 1.2 and Ca v 1.3 subunits expressed in Th2 lymphocytes, or by using an inhibitor of CACNA1C and/or CACNA1D gene expression.
  • the CACNA1C and CACNA1D genes are expressed in the Th2 lymphocytes, thereby giving the Ca v 1.2 and Ca v 1.3 subunits.
  • Ca v l means voltage-dependent L-type calcium channels.
  • Cavl.2 subunit and “Cav 1.3 subunit” have their general meanings in the art.
  • the Cavl.2 and Cavl.3 subunits are respectively encoded by the CACNA1C and CACNA1D genes. These subunits may include naturally occurring Ca v 1.2 or Ca v 1.3 subunits and variants and modified forms thereof. They can be from any source, but typically are mammalian (e.g., human and non-human primate) Ca v 1.2 and Ca v 1.3 subunits, particularly human Cavl.2 and Cavl.3 subunits.
  • Cavl.2 subunit expressed in the Th2 lymphocytes and “Cavl.3 subunit expressed in the Th2 lymphocytes” respectively refer to the isoforms of the Cavl.2 and Cavl.3 subunits which are expressed in the Th2 lymphocytes.
  • inflammatory diseases refer to any diseases marked by the generation, activation and recruitment of inflammatory cells into the tissues. These cells may belong to the innate (eosinophils, mast cells, basophils) or adaptative (T-cells especially Th2-cells) immune system.
  • allergic diseases also referred to as atopic diseases
  • allergens any disorder of the immune system resulting from an inappropriate immune response towards normally harmless environmental compounds called allergens. Such a response depends on the nature of the allergen, environmental and genetic factors. For example, some genetic diseases such as the Netherton syndrome are constantly associated with allergic manifestations. Allergy is one form of hypersensitivity that is called type I (or immediate) hypersensitivity. It is characterized by excessive activation of innate immune cells, including eosinophils, mast cells and basophils, as well as by the production of IgE specific antibodies.
  • allergic reactions include eczema, urticaria, allergic rhinitis (perennial and seasonal), asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees.
  • allergic diseases refer to eczema, urticaria, allergic rhinitis, asthma, food allergies, and reactions to the venom of stinging insects.
  • respiratory allergic diseases refer to any obstructive diseases of the airways including:
  • asthma includes bronchial, allergic, intrinsic, extrinsic, exercise- induced, drug-induced (including aspirin and NSAID-induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness;
  • COPD chronic obstructive pulmonary disease
  • bronchitis including eosinophilic bronchitis
  • cystic fibrosis cystic fibrosis
  • rhinitis acute and chronic rhinitis including rhinitis medicamentosa, and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever);
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA), tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., FXR) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • proteins e.g., FXR
  • an “inhibitor of gene expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene. Consequently an “inhibitor of CACNA1C and/or CACNA1D gene expression” refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the CACNA1C and/or CACNA1D genes. Said inhibitor of CACNA1C and/or CACNA1D gene expression may be identified as described in example 1 (particularly "Transfection experiment” and "Immunoblotting").
  • Nucleic acids are polymers of nucleotides, wherein a nucleotide comprises a base linked to a sugar which sugars are in turn linked one to another by an interceding at least bivalent molecule, such as phosphoric acid.
  • the sugar is either 2'- deoxyribose (DNA) or ribose (RNA).
  • An "oligonucleotide” generally refers to a nucleic acid molecule having less than 30 nucleotides.
  • oligodeoxynucleotide or “ODN” means a monocatenary DNA molecule having less than 30 nucleotides; thus an ODN corresponds to an oligonucleotide of which the sugar is deoxyribose.
  • Antisense poly- or oligonucleotides refer to synthetic poly- or oligonucleotides that mimic the complementary nature of the naturally occurring nucleic acids after which they are designed. They may contain modified bases, sugars, or linking molecules; such modified structures can impart an increased stability to the antisense poly- or oligonucleotides.
  • An example of an antisense oligonucleotide is an antisense molecule composition that has a phosphorothioate backbone.
  • an antisense ODN is an antisense DNA molecule which has a phosphorothioate or morpholino backbone.
  • a "polypeptide” is a polymer of amino acid residues joined by peptide bonds, and a peptide generally refers to amino acid polymers of 12 or less residues. Peptide bonds can be produced naturally as directed by the nucleic acid template or synthetically by methods well known in the art.
  • a "protein” is a macromolecule comprising one or more polypeptide chains. A protein may further comprise substituent groups attached to the side groups of the amino acids not involved in formation of the peptide bonds. Typically, proteins formed by eukaryotic cell expression also contain carbohydrates.
  • receptor antagonist is meant a natural or synthetic compound that has a biological effect opposite to that of a receptor agonist.
  • the term is used indifferently to denote a "true” antagonist and an inverse agonist of a receptor.
  • a "true” receptor antagonist is a compound which binds the receptor and blocks the biological activation of the receptor, and thereby the action of the receptor agonist, for example, by competing with the agonist for said receptor.
  • An inverse agonist is a compound which binds to the same receptor as the agonist but exerts the opposite effect. Inverse agonists have the ability to decrease the constitutive level of receptor activation in the absence of an agonist.
  • Cavl.2 or Cavl.3 subunit antagonist includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the Cavl.2 or Cavl.3 subunit in the patient.
  • Said biological activity of Cavl.2 or Cavl.3 subunit is the capacity of allowing the entry of calcium in the cells expressing said Cavl.2 or Cavl.3 subunit.
  • said biological activity of Cavl.2 or Cavl.3 subunit expressed in Th2 lymphocytes is the capacity of allowing the entry of calcium in the Th2 lymphocytes.
  • Such Cavl.2 or Cavl.3 subunit antagonists include any agent that can block the Cavl.2 or Cavl.3 subunit activation or any of the downstream biological effects of the Cavl.2 or Cavl.3 subunit activation in the Th2 lymphocytes.
  • Cavl.2 or Cavl.3 subunit antagonists are specific (ie selective) for the Cavl.2 or Cavl.3 subunit, and preferably specific for the Cavl.2 or Cavl.3 subunit expressed in Th2 lymphocytes, as compared with the other Cavl subunits, such as the Cavl.l and Cavl.4 subunits.
  • the affinity of the antagonist for the Cavl.2 or Cavl.3 subunit is at least 10-fold, preferably 25-fold, more preferably 100-fold, still preferably 500-fold higher than the affinity for the other Cavl subunits (Cavl.l and Cavl.4).
  • the affinity of an antagonist for the Cavl.2 or Cavl.3 subunit may be quantified by measuring the activity of the Cavl.2 or Cavl.3 subunit in the presence a range of concentrations of said antagonist in order to establish a dose-response curve. From that dose response curve, an IC 50 value may be deduced which represents the concentration of antagonist necessary to inhibit 50% of the response to an agonist in defined concentration.
  • IC 50 value may be readily determined by the one skilled in the art by fitting the dose- response plots with a dose-response equation as described by De Lean et al. (1979). IC 50 values can be converted into affinity constant (Ki) using the assumptions of Cheng and Prusoff (Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (150) of an enzymatic reaction, Biochem Pharmacol. 1973 Dec 1;22(23):3099-108).
  • a specific or selective Cavl.2 subunit antagonist is a compound for which at least one of the ratios (i) Ki Cavl. l : Kj Cavl.2, and (ii) IC 50 Cavl.l: IC 50 Cavl.2, and at least one of the ratios (i') Ki Cavl.4 : K ; Cavl.2 and ( ⁇ ') IC 50 Cavl.4: IC 50 Cavl.2, are above 10: 1, preferably 25 : 1 , more preferably 100: 1, still preferably 1000: 1.
  • a specific or selective Cavl.3 subunit antagonist is a compound for which at least one of the ratios (i) Ki Cavl. l : Kj Cavl.3, and (ii) IC 50 Cavl.l: IC 50 Cavl.3, and at least one of the ratios (i') K ; Cavl.4 : K ; Cavl.3 and ( ⁇ ') IC 50 Cavl.4: IC 50 Cavl.3, are above 10: 1, preferably 25: 1, more preferably 100: 1, still preferably 1000: 1.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • antibody refers to a protein capable of specifically binding an antigen, typically and preferably by binding an epitope or antigenic determinant or said antigen.
  • antibody also includes recombinant proteins comprising the binding domains, as well as variants and fragments of antibodies. Examples of fragments of antibodies include Fv, Fab, Fab', F(ab')2, dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed from antibody fragments.
  • treating refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition.
  • preventing refers to preventing one or more symptoms of such disorder or condition.
  • the term "subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human.
  • a “therapeutically effective amount” as used herein is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject.
  • a “therapeutically effective amount of the active agent” to a subject is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the subject.
  • the present invention aims the use of an inhibitor of CACNAIC and/or CACNAID gene expression or the use of a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Cavl) channels as a medicament.
  • the present invention also aims the use of an inhibitor of CACNAIC and/or CACNAID gene expression in preventing and/or treating inflammatory diseases, and particularly allergic diseases, preferably respiratory allergic diseases such as asthma and allergic rhinitis.
  • the inhibitor is an inhibitor of CACNAIC gene expression.
  • the present invention also aims the use of a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Cavl) channels in preventing and/or treating these inflammatory diseases.
  • the antagonist is a specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits of voltage-dependent L-type calcium (Cavl) channels expressed in Th2 lymphocytes.
  • Ca v 1.2 and Ca v 1.3 subunits are indeed expressed in murine and human Th2 lymphocytes, thanks to their corresponding genes CACNAIC and CACNAID. Moreover, specific isoforms of both Cavl.2 and Cavl.3 subunits are expressed in murine and human Th2 lymphocytes.
  • the inhibitors of gene expression include, but are not limited to, antisense oligonucleotides, siRNAs, shRNAs, ribozymes and DNAzymes.
  • the antagonists include, but are not limited to, small organic molecules, antibodies and ap tamers.
  • One aspect of the invention relates to the use of an inhibitor of CACNA1C and/or CACNA1D gene expression.
  • Inhibitors of CACNA1C and/or CACNA1D gene expression for use in the present invention may be based on antisense oligonucleotides constructs.
  • Antisense oligonucleotides including antisense RNA and antisense oligodeoxynucleotides molecules, the antisense RNA or DNA molecules being modified or not, would act to directly block the translation of Cavl.2 or Cavl.3 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Cavl.2 or Cavl.3 proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Cavl.2 or Cavl.3 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion or inhalation.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131).
  • the inhibitors of CACNA1C and/or CACNA1D gene expression for use in the present invention are antisense oligodeoxynucleotides (ODNs).
  • ODNs are synthetic polymers, whose monomers are deoxynucleotides like those in DNA, and wherein their sequence (3' ⁇ 5') is antisense (ie complementary to the sequence of a molecule of mRNA).
  • Antisense ODNs act by blocking the synthesis of the Cavl.2 or Cavl.3 protein. This is achieved by the binding of the ODN to the Cavl.2 or Cavl.3 mRNA, as described in Example 1.
  • antisense ODNs are particularly useful as therapeutics for pulmonary inflammation, airway hyperresponsiveness, and/or respiratory allergic diseases like asthma.
  • Lung provides an ideal tissue for aerosolized antisense ODNs for several reasons: the lung can be targeted non-invasively and specifically, it has a large absorption surface, and it is lined with surfactant that may facilitate distribution and uptake of antisense ODNs. Delivery of antisense ODNs to the lung by aerosol results in excellent distribution throughout the lung in both mice and primates. Moreover, antisense ODNs have relatively predictable toxicities and pharmacokinetics based on backbone and nucleotide chemistry. Pulmonary administration of antisense ODNs results in minimal systemic exposure, potentially increasing the safety of such compounds as compared to other classes of drugs.
  • Some antisense ODNs of this mRNA have been synthesized and shown as particularly successful in the treatment of asthma on a mouse model, by significantly inhibiting CACNA1C gene expression (see examples 1 and 3).
  • preferred inhibitors of CACNA1C gene expression are the antisense oligonucleotides, preferably antisense ODNs, that comprise at least 15 nucleotides complementary to (i) at least one of the sequences SEQ ID NO : 6, SEQ ID NO:48 and SEQ ID NO:49, or to (ii) at least one sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO:6 or SEQ ID NO:48 or SEQ ID NO:49.
  • antisense oligonucleotides preferably antisense ODNs, that comprise at least 15 nucleotides complementary to (i) the exon lb of the sequence SEQ ID NO : 6, ie SEQ ID NO: 7, or to (ii) at least one sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO : 7.
  • antisense oligonucleotides preferably the antisense ODNs, that have the following sequence:
  • antisense oligonucleotides preferably antisense ODNs, comprising at least one of the sequences SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:76 and SEQ ID NO: 77.
  • nucleic acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about 15 nucleotides, usually at least about 20 nucleotides.
  • FASTA FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG) , Madison, Wis.
  • GCG Genetics Computer Group
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1.
  • sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266: 131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • CACNA1D gene expression product here mRNA, present in human Th2 cells
  • This partial sequence corresponds to SEQ ID NO : 8. Therefore, preferred inhibitors of CACNA1D gene expression are the antisense oligonucleotides, preferably antisense ODNs, that comprise at least 15 nucleotides complementary to (i) the sequence SEQ ID NO : 8 or to (ii) at least one sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO:8.
  • antisense oligonucleotides preferably antisense ODNs, that have the following sequence:
  • antisense oligonucleotides preferably antisense ODNs, comprising at least one of the sequences SEQ ID NO:3 to SEQ ID NO:5.
  • the present invention relates to an antisense oligonucleotide chosen from the group consisting of: antisense oligonucleotides that comprise at least 15 nucleotides complementary to at least one of the sequences SEQ ID NO : 6, SEQ ID NO: 48 and SEQ ID NO:49, or to at least one sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO:6 or SEQ ID NO:48 or SEQ ID NO:49; and - antisense oligonucleotides that comprise at least 15 nucleotides complementary to the sequence SEQ ID NO : 8 or to at least one sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO: 8.
  • the antisense oligonucleotide is selected from antisense oligodeoxynucleotides of sequence chosen from SEQ ID NO: l to SEQ ID NO:5 or SEQ ID NO:73 to SEQ ID NO:77, antisense oligodeoxynucleotides comprising at least one of the sequences SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, and antisense oligodeoxynucleotides of sequence having at least 90% or 95% or more identity to the sequence SEQ ID NO: l or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO: 73 or SEQ ID NO: 74 or SEQ ID NO: 75 or SEQ ID NO:
  • Small inhibitory RNAs can also function as inhibitors of CACNA1C and/or CACNA1D gene expression for use in the present invention.
  • CACNA1C and/or CACNA1D gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CACNA1C and/or CACNA1D gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g.
  • Ribozymes and DNAzymes can also function as inhibitors of CACNA1C and/or CACNA1D gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • DNAzymes are enzymatic DNA molecules with catalytic action. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Cavl.2 or Cavl.3 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of CACNA1C and/or CACNA1D gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • ODNs, siRNAs, shRNAs, ribozymes and DNAzymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense ODN, siRNA, shRNA, ribozyme or DNAzyme nucleic acid to the cells and preferably cells expressing Cavl.2 or Cavl.3.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense ODNs, siRNAs, shRNAs, ribozymes and DNAzymes nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus vaccini
  • Non-cytopathic viruses include retroviruses (e.g., lenti virus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art (see e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • vectors according to the invention are viral vectors, like lentivirus vectors.
  • lentivirus may be pLentiLox 3.7 as described in Karnabi et al, 2009 (Silencing of Cavl.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA, Biochemical and Biophysical Research Communications 384 (2009) 409-414).
  • the inhibitors of CACNA1C and/or CACNA1D gene expression according to the invention can be identified according to different screening methods.
  • the method can consist in introducing the antisense oligonucleotide or siRNA into the cells by transfection with chemicals (as lipofectin or Hpofectamin) or by electroporation.
  • shRNA products can be encoded by adequate vectors allowing the generation of retroviral particles. In that case, cells to be tested would be transduced with retrovirus or lentiviral vectors (see Karnabi et al above).
  • the specific antagonist of at least one of the Cavl.2 and Cavl.3 subunits is a small organic molecule.
  • DHP are not considered as specific antagonists according to the present invention, because they bind to all the Cavl.l, Cavl.2, Cavl.3 and Cavl.4 subunits.
  • the specific antagonist of the Cavl.2 or Cavl.3 subunit may consist in an antibody (the term including antibody fragment) that can block the Cavl.2 or Cavl.3 subunit activation.
  • Antibodies directed against of the Cavl.2 or Cavl.3 subunit can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against the Cavl.2 or Cavl.3 subunit can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495-7); the human B-cell hybridoma technique (Cote RJ, Morrissey DM, Houghton AN, Beattie EJ Jr, Oettgen HF, Old LJ. Generation of human monoclonal antibodies reactive with cellular antigens. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2026-30); and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp.
  • Cavl.2 or Cavl.3 subunit antagonists useful in practicing the present invention also include anti-Cavl.2 or anti-Cavl.3 antibody fragments including but not limited to F(ab') 2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Cavl.2 or Cavl.3.
  • Humanized anti-Cavl.2 or anti-Cavl.3 antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the Cavl.2 or Cavl.3 subunit antagonist is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990 (Tuerk C. and Gold L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas P, Cohen B, lessen T, Grishina I, McCoy J, Brent R. (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature, 380, 548-50). Then after raising aptamers directed against the Cavl.2 or Cavl.3 subunit as above described, the skilled man in the art can easily select those blocking Cavl.2 or Cavl.3 subunit activation.
  • compositions Another object of the invention relates to a method for treating and/or preventing an inflammatory disease, particularly an allergic disease, and preferably a respiratory allergic disease comprising administering to a subject in need thereof an inhibitor of CACNAIC and/or CACNAID gene expression or a specific antagonist of at least one of Cavl.2 and Cavl.3 subunits, preferably expressed in Th2 lymphocytes.
  • Another object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of CACNAIC and/or CACNAID gene expression or a specific antagonist of at least one of Cavl.2 and Cavl.3 subunits, preferably expressed in Th2 lymphocytes, in a physiologically acceptable vehicle.
  • Cavl.2 or Cavl.3 subunits antagonists or inhibitors of CACNAIC or CACNAID gene expression may be administered in the form of a pharmaceutical composition which comprises a physiologically acceptable vehicle, as defined below.
  • physiologically acceptable vehicle it is meant an inert vehicle, ie compatible with the envisaged application.
  • said antagonist or inhibitor is administered in a therapeutically effective amount.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions may contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • Cavl.2 or Cavl.3 subunits antagonists or inhibitors of CACNA1C or CACNA1D gene expression may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • the active principle alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • Cavl.2 or Cavl.3 subunits antagonists or inhibitors of CACNA1C or CACNA1D gene expression are adapted for intranasal administration ; thus preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for an intranasal administration.
  • Compositions and devices for delivery to the lung and nose are well known.
  • the inhibitors and antagonists according to the invention can be soluble in aqueous solution and may be delivered using standard nebulizer devices. They can also be delivered using other devices such as dry powder inhalers or metered dose inhalers which can provide improved patient convenience as compared to nebulizer devices, resulting in greater patient compliance.
  • FIGURES The invention will further be illustrated in view of the following figures and examples.
  • FIG. 1 Ca v 1.2 and Ca v 1.3 are up regulated in Th2- but not in Thl-cells.
  • cDNAs from Th2-, Thl-cells and brain used as a positive control were tested for expression of auxiliary Ca v l beta subunits and ⁇ actin (right panel).
  • FIG. 2 Ca v 1.2 and Ca v 1.3 expression is reduced by transfection with specific antisense oligodeoxynucleotides.
  • Th2-cells treated with lipofectin (lipo), or transfected with Ca v 1.2 (1.2), Ca v 1.3 (1.3) sense (S) or antisense (AS) were analyzed 48 h after transfection by western blotting with anti-Ca v 1.2 or Ca v 1.3 monoclonal antibodies. Actin was used as a loading control. The intensity of bands was quantified and normalized to the level of actin.
  • the histograms in (C) represent the mean + ISD from six experiments where lyzates from cells treated as in (B) were immuno-blotted with anti-Ca v 1.2 (white bars) or anti-Ca v 1.3 (black bars) antibodies; * p ⁇ 0.02 when compared to Th2-cells transfected with control ONs.
  • Figure 3 Ca v lAS reduce the TCR-dependent Ca 2+ response and cytokine production by
  • Th2-cells transfected with Ca v 1.2 sense (S), Ca v 1.2 antisense (AS) or a mixture of Ca v 1.2 and Ca v 1.3 AS ODNs were loaded with Fura-2/AM. Intracellular Ca 2+ levels were recorded for 2 min before stimulation with anti-TCR mAb (arrow) and for 7- 8 min after stimulation. At least 45 cells were recorded and [Ca 2+ ]; calculated for each cell were averaged at each time-point. Standard errors are not indicated, but they were less than 10% of the mean. One representative experiment out of 6 is shown.
  • FIG. 4 Th2-cells transfected with Ca v lAS lose their ability to induce asthma.
  • IL-4 concentration was reduced in the BALF of mice transferred with Th2AS (black bars) compared to mice injected with Th2 or Th2S (white bars). Data represent the mean + ISD from 5 mice and are representative of 5 experiments.
  • FIG. 5 Ca v lAS impair the development of asthma.
  • a and B BALB/c mice immunized with OVA in alum were exposed 15 days later to daily doses of intranasal OVA for 5 days.
  • CD4 + T-cells were purified from the lungs.
  • An intracellular staining with the monoclonal anti- Ca v 1.2 antibody was performed (A).
  • cDNAs were prepared and analyzed for the expression of Ca v 1.2 (white bars) and Ca v 1.3 (grey bars) by real time PCR (B). Data were normalized to levels of the housekeeping gene HPRT and represent the mean + SD from three independent experiments. Results obtained with cDNAs from Th2-cells obtained after three rounds of stimulation were shown for comparison.
  • Ca v lAS suppress airway hyper-reactivity in an experimental model of active asthma.
  • BALB/c mice were immunized with OVA in alum and were exposed 15 days later to daily doses of intranasal OVA mixed with Ca v 1.2 plus Ca v 1.3 sense (Ca v lS) or antisense (Ca v lAS) ODNs for 5 days.
  • Mice treated with Ca v lAS black circles displayed no or only mild airway hyper-reactivity upon exposure to increasing concentrations of intranasal methacholine, unlike mice exposed to either OVA alone (Ct, open squares) or mice treated with Ca v lS (open circles); * p ⁇ 0.01, 5 mice per group.
  • One representative experiment out of three is shown. The dashed line represents the results obtained with mice primed with OVA in alum and given intranasal PBS.
  • Th2- but not Thl-cells express Ca v 1.2 and anti-Ca v 1.3 proteins. Lyzates from differentiated Thl and Th2-cells were resolved by SDS-PAGE and then immunoblotted (50 ⁇ g protein/lane) with anti-Ca v 1.2 or anti-Ca v 1.3 polyclonal antibodies. The anti-Ca v 1.2 and anti-Ca v 1.3 antibodies were pre-incubated (Th2-pep) or not with the antigenic peptide before use. The figure shows one representative experiment out of five performed.
  • FIG. 8 Ca v lAS reduce dihydropyridine receptor expression in Th2-cells.
  • Th2-cells collected after three rounds of stimulation were treated with lipofectin alone, or were transfected with Ca v 1.2 (1.2) or Ca v 1.3 (1.3) sense (S) or antisense (AS).
  • Cells were stained 24 h later with ST-BODIPY-labelled DHP.
  • the fluorescence intensity was quantified by using the Image J software and the intensity/per cell was averaged. Results represent the mean of fluorescence intensity/per cell obtained from five independent experiments.
  • FIG. 9 Ca v lAS do not alter the proliferation of Th2-cells
  • Th2-cells were transfected with a mixture of Ca v 1.2 and Ca v 1.3 antisense (Th2AS) or sense (Th2S). The cells were stained 48h later with carboxyfluorescein succinimidyl ester (CFSE) and stimulated on plates coated with anti-TCR mAb in the presence of anti-CD28 mAb. The fluorescence was then analyzed by FACS 48h later and showed that Th2AS and Th2S proliferated equally well. The histogram shows the percentage of dividing cells in Th2, Th2S and Th2AS. Results are expressed as mean + SD of 4 cultures. Figure 10.
  • CFSE carboxyfluorescein succinimidyl ester
  • Ca v lAS modify neither the calcium response nor IL-4 production induced by stimulation with thapsigargin or PMA/ionomycin.
  • A) Th2-cells were either treated with lipofectin only (lipo) or transfected with Ca v 1.2 (1.2) or Ca v 1.3 (1.3) sense (S) or antisense (AS) ODNs and were loaded with Fura2-AM. The [Ca 2+ ]; was recorded before stimulation (Unst.) and after stimulation with thapsigargin (Thapsi). [Ca 2+ ]; was recorded in 60-70 cells for each condition. The peak value for [Ca ]i was determined for each cell and the mean value calculated for each sample.
  • the histograms represent the mean + 1SD of the mean peak values of [Ca 2+ ]; from six independent experiments.
  • Th2-cells transfected with Ca v lAS can localize into the lungs of BALB/c mice upon adoptive transfer.
  • 24 h after transfection of Th2-cells with a mixture of Ca v 1.2 and Ca v 1.3 sense (Th2S) or antisense (Th2AS) cells were injected intravenously in BALB/c mice that were given intranasal OVA for 7 days.
  • the lungs were collected, digested and CD4 + T- cells were purified and labelled with anti-clonotypic KJ1.26 antibodies.
  • the same numbers of KJ1.26 + cells were recovered from mice injected with Th2, Th2S or Th2AS.
  • One representative experiment out of the three performed is shown.
  • FIG. 12 Ca v 1.2 transcripts are expressed in CD4+CRTH2+ cells.
  • cDNA from CRTh2+ cells were amplified with the primers listed in Example 2 : 94°C for 1 min. then 40 cycles (90°C for 45 sec, 55°C for 40 sec, 72°C for 1 min.). In some experiments neuroblastoma was used as a positive control.
  • a) indicates the location of primers used in PCR 1 to 5 relative to the numbers of exons and to the expected structure of the channel (Tiwari et al. PNAS 2006).
  • the rectangle area schematizes the cell membrane.
  • FIG. 13 Ca v 1.2 channels are preferentially expressed in Th2 cells as compared to Thl cells.
  • cDNAs were prepared from 5 samples of CRTH2+ (Th2) and CRTH2- cells differentiated along the Thl pathway.
  • Real time PCR was developed to measure the amount of transcripts encoding Ca v 12 and the housekeeping gene GAPDH in Th2 and Thl cells. Results were normalized relative to the expression of GAPDH which is stable in the two cell subsets. * p ⁇ 0.01 compared to Thl cells.
  • Nicardipine inhibits the entry of Ca + and the production of IL-4 in Th2 cells upon TCR stimulation.
  • Nicardipine ( ⁇ ) reduced the production of IL-4 by CRTH2+ cells upon stimulation through the TCR as assessed by ELISA 24h after stimulation with plate-bound anti-CD3 and soluble anti-CD28mAb, with or without the drug.
  • FIG. 15 Effect of Ca v lAS on Ca v 1.2 expression in transfected CRTH2+ cells.
  • CRTH2+ cells were transfected with putative Ca v 1.2 specific antisense oligodeoxynucleotides (AS, AS1 to AS5 cf table 2) or control oligonucleotides and tested 48h later for Ca v 1.2 expression by staining with anti-Ca v 1.2 mAb. Fluorescence was then quantified. Results are expressed as the mean + 1SD of 10 to 20 cells.
  • AS5 induced the most important inhibtion of Ca v 1.2 expression. The numbers in brackets represent the percentages of inhibition.
  • FIG. 6 Ca v lAS reduce the production of IL-5 and IL-13 by CRTH2 + cells.
  • CRTH2+ cells were transfected with Ca v lAS or control oligonucleotides and stimulated 48h later with plate-bound anti-CD3 and soluble anti-CD28niAb for 24h. Cytokine concentrations were measured by ELISA. The numbers in brackets represent the percentages of inhibition.
  • mice 7-10 week old female BALB/c mice (Janvier Ets, Le Genest St.Isle, France) and DO11.10 BALB/c mice, which express a transgenic T-cell receptor (TCR) specific for the 323-339 ovalbumin (OVA) peptide, were cared for in our animal facility.
  • TCR transgenic T-cell receptor
  • OVA ovalbumin
  • CD4+ T cells were isolated from spleens of DO11.10 BALB/c mice by negative selection with the Dynal® Mouse CD4 Negative Isolation Kit (Invitrogen Carlsbad, CA). CD4+ T cells were cultured in RPMI 1640 supplemented with 10% foetal calf serum (ATGC, noisysy Le Grand, France), 1% pyruvate, 1% non-essential amino acids, 2 mM glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 50 ⁇ beta-mercaptoethanol.
  • CD4+ T-cells (10 6 / well) were differentiated along the Th2 pathway by weekly stimulation with 2.5 x 10 6 irradiated BALB/c spleen cells/well, 0.3 ⁇ OVA 323-339 peptide (Neosystem, France), 10 ng/ml IL-4 and 10 ⁇ g/ml anti- IFNgamma (XMG1.2) antibody (both purchased from BD Pharmingen, San Diego, CA).
  • Thl- cell differentiation was achieved by weekly stimulation of 10 6 CD4+ T cells with APCs (5 x 10 6 /well), 5 ng/ml IL-12 (BD Pharmingen) and 10 ⁇ g/ml anti-IL-4 antibody (11B.11, BD Pharmingen).
  • Cytokine concentration was measured by ELISA (Savignac et al, 2001) in the supernatants of T cells (2.5xl0 4 cells/well) stimulated with plate-bound anti-TCR mAb (H57- 597, BD Pharmingen, 1 ⁇ g/ml) for 24 h.
  • Cavl. l 5'-tgtggtatgtcgtcacttcctcc-3' (SEQ ID NO: 9) and 5'-cgtcaatgatgctgccgatg-3' (SEQ ID NO: 10);
  • Cavl.2 5'-caagccctcacaaaggaatgc-3' (SEQ ID NO: 11) and 5'-aaagttgcccctgctgtgtcactc-3' (SEQ ID NO: 12);
  • beta2 5'-ataaccacagagagggagccaca-3' (SEQ ID NO: 19) and 5'-tatacatccctgttccactcgcca-3' (SEQ ID NO: 20);
  • beta3 5'-tccctggacttcagaaccagcag-3' (SEQ ID NO: 21) and 5'-ttgtggtcatgctccgagtcctg-3' (SEQ ID NO: 22);
  • beta4 5'-tgagacacagcaaccattctacagaga-3' (SEQ ID NO: 23) and 5'- atgtcgggagtcatggctgcatc-3' (SEQ ID NO: 24);
  • beta actin 5'-tggaatcctgtggcatccatgaaac-3' (SEQ ID NO: 25) and 5'- taaaacgcagctcagtaacagtccg-3' (SEQ ID NO: 26).
  • PCR conditions were 94°C for 45 sec, followed by 55°C (Cavl and ⁇ subunits) or 60°C ( ⁇ - actin) for 45 sec, and 72°C for 1 min over 21 cycles (beta-actin) or 40 cycles (Cavl and ⁇ subunits).
  • Cavl.2 5'agacgcgggtggttttttta-3' (SEQ ID NO: 27) and 5'-gaagacgctgttccggttacc-3' (SEQ ID NO: 28);
  • Cavl.3 5'-tgaggccaaaagtaaccccgagg-3' (SEQ ID NO: 29) and 5'-cgccatcatagccgaaaggacaat-3' (SEQ ID NO: 30);
  • GATA-3 5'-gccatgggttagagaggcag-3' (SEQ ID NO: 31) and 5'- ttggagactcctcacgcatgt-3' (SEQ ID NO: 32);
  • T-bet 5'-cggtaccagagcggcaagt-3' (SEQ ID NO: 33) and 5'- cagctgacccaagaggaatca-3' (SEQ ID NO: 34); Orail: 5'-gcttcgccatggtagcgat-3' (SEQ ID NO: 35); and 5'-gctgatcatgagggcaaac-3' (SEQ ID NO: 36);
  • Stiml 5'-gccacagcttggcctgg-3' (SEQ ID NO: 37) and 5'-gctccatcaggctgtgg-3' (SEQ ID NO: 38);
  • Kvl.3 5'-ccagcacctctcctcttcag-3' (SEQ ID NO: 39) and 5'- agggcatacacagaccaagg-3' (SEQ ID NO: 40);
  • KCa3.1 5'-tgccatttgcgtactgagag-3' (SEQ ID NO: 41) and 5'-gacaaaggaggaaggcagtg-3' (SEQ ID NO: 42);
  • HPRT 5'-ctggtgaaaggacctctcg-3' (SEQ ID NO: 43) and 5'- tgaagtactcattatagtcaagggca-3' (SEQ ID NO: 44).
  • Quantification of target gene expression was calculated by normalising the values relative to the expression of HPRT.
  • Thl or Th2-cells specific for the OVA 323-339 peptide were transfected with Cavl.2 or Cavl.3 sense, mismatched or antisense ODNs by using Lipofectin® (Invitrogen).
  • the sequences of the ODNs are given in Table E2.
  • FACS analysis showed that transfection with FITC-conjugated control or antisense ODNs labelled 60-70% of cells (data not shown).
  • Table E2 Oligodeoxynucleotides used in transfection experiments :
  • L01776 and AJ4377291.1 refer to the murine Cavl.2 and Cavl.3a sequences respectively.
  • Membranes were saturated with TBS/Tween containing 1% milk and 1% BSA and incubated overnight with anti-Cavl.2 and anti-Cavl.3 polyclonal (Alomone Labs, Jerusalem, Israel) or monoclonal antibodies (UC Davis NINDS/NIMH NeuroMab facility, Davis CA). Blots were developed with the appropriate HRP-conjugated secondary antibody and Amersham Biosciences Enhanced Chemiluminescence system (GE Healthcare, Little Chalfont, UK).
  • Membranes were then washed and incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 0.1 M 2-Mecaptoethanol) at 60°C for 30 min to be reprobed with anti-actin antibodies as a loading control.
  • stripping buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 0.1 M 2-Mecaptoethanol) at 60°C for 30 min to be reprobed with anti-actin antibodies as a loading control.
  • Thl and Th2 transfer experiments Single cell intracellular Ca 2+ measurements were done as previously described (20, 23) in cells loaded with 5 ⁇ Fura2-AM before being stimulated with anti-TCR plus anti-CD28 antibodies, thapsigargin or ionomycin. Thl and Th2 transfer experiments. Thl and Th2-cells transfected as described above were injected intravenously into BALB/c recipient mice (5 x 10 6 cells per mouse) and the mice were then given intranasal OVA protein (50 ⁇ g in PBS) once per day for 7 days. Broncho- alveolar lavage fluid (BALF) was collected 24 hours after the final OVA administration. In vivo administration of Cavl-specific antisense ODN.
  • BALF Broncho- alveolar lavage fluid
  • mice primed intraperitoneally with 100 ⁇ g OVA protein in 2 mg alum (Sigma) were challenged 15 days later with a mixture of intranasal OVA (50 ⁇ g in PBS) and control or Cavl.2 and Cavl.3 antisense ODN (200 ⁇ g corresponding to 15 nmoles/ day) in PBS for 5 days. 24h after the final OVA administration, the mice were anaesthetized and BALF was collected by cannulation of the trachea and lavage with PBS.
  • BALF Airway inflammation and stimulation assays.
  • BALF was centrifuged at 300 g. Cell pellets were suspended in PBS and the number of cells was determined by trypan blue exclusion. Cells were then centrifuged onto slides and stained with May-Grunwald Giemsa to identify inflammatory cells. Lung samples were fixed in formalin overnight, stored in 70% ethanol, and 5 ⁇ -thick sections were stained with hematoxylin and eosin.
  • CD4+ T cells 25 x 10 4 / well) were purified from peribronchial lymph nodes and stimulated for 24 h with OVA (100 ⁇ g/ml) and irradiated BALB/c spleen cells as a source of APCs (2 x 10 6 / well).
  • Airway hyper-responsiveness It was assessed 24 h after the final OVA inhalation by using a whole body plethysmograph (EMKA Technologies, France). Baseline-enhanced pause (Penh) values were recorded for 3 min before the mice were exposed to intranasal methacholine as previously described (Gomes et al, 2007). The Penh values measured during 20 min sequence were averaged and expressed as the fold increase over baseline values.
  • Th2- but not Thl-cells express Ca v 1.2 and Ca v 1.3 channels.
  • Multiple splice variants may encode Ca v 1.2 channels with some exons that characterize the cardiac form such as the exon la instead of the exon lb, and the use of exons 34a, 41a and 45 that were excluded in other forms (25).
  • the sequences of Ca v 1.2 cloned from Th2-cells did not express cardiac specific exons and differed from the Genbank accession L01776 sequence (cloned from the BALB/c brain) only by the use of the exon 8 instead of the exon 8a (exons that are known to be exclusive) (25) and by the absence of exon 33.
  • the sequences described as encoding the four voltage sensors in classical Ca v l channels (26) were also evidenced in Th2-cell sequences.
  • Ca v l specific antisense oligodeoxynucleotides (Ca v lAS) to reduce translation of the mRNAs.
  • Ca v lAS Ca v l specific antisense oligodeoxynucleotides
  • Th2-cells transfected with Ca v lAS lose their ability to induce asthma.
  • Th2-cells OVA-specific transgenic Th2-cells were transfected with Ca v l AS (Th2AS) or Ca v lS (Th2S) and injected into BALB/c mice that were given intranasal OVA to induce airway inflammation. Similar numbers of transgenic T-cells, identified by the staining with anti- KJ1.26 antibodies were found in the lungs of mice after the transfer of either Th2AS or Th2S (see Figure 11) suggesting that Ca v lAS do not prevent Th2-cells to localize into the lungs.
  • Th2AS Ca v l AS
  • Th2S Ca v lS
  • mice injected with Th2AS were dramatically reduced in the bronchoalveolar lavage fluid (BALF) of mice injected with Th2AS when compared to the controls ( Figure 4A). This was consistent with the absence or minor lung inflammation in these animals when compared to the controls (data not shown).
  • the lack of lung inflammation in mice injected with Th2AS was accompanied by a decrease in the IL-4 concentration in the BALF ( Figure 4C) and a reduction in the number of lymphocytes in the lung-draining lymph nodes ( Figure 4D) when compared to controls.
  • CD4 + T cells from the draining lymph nodes produced less IL-4, IL-5 and IL-13 when stimulated with antigen-presenting cells (APCs) and OVA as compared to controls ( Figure 4E).
  • APCs antigen-presenting cells
  • OVA oxygen-presenting cells
  • Example 2 Human Th2-cells express Ca y l channels
  • CD4+ T- cells were then purified by negative selection by incubation with a cocktail of antibodies (anti-CD8, CD14, CD16, CD19, CD36, CD56, CDdwl23, CD235a) and magnetic beads coated with anti-mouse IgG (kit M-450 Dynabeads).
  • CD4+ (around 90% pure) contained from 1 to 5% of CRTH2+cells.
  • CRTH2 is a receptor for prostaglandin D2 shown to be mainly expressed by memory Th2-cells.
  • CRTH2+ cells were collected by positive selection by using the human CD294 (CRTH2) MicroBead kit (Miltenyi). They were around 70% pure and intracellular staining with anti-interleukin (IL)-4 and anti-interferon gamma (IFNg) showed that around 50-60% of these cells produced IL-4 upon stimulation with PMA (and ionomycin) versus around 3-5% in CD4+ or CRTH2- CD4+ T-cells.
  • IL interleukin
  • IFNg anti-interferon gamma
  • nu mbers correspond to the position i n N M001129827.1, the va ria nt 2 of huma n CACNAIC tra nscri pts
  • CD4 + T cells were purified from healthy blood donors by negative selection and stained with anti-CRTH2-PE (phycoerythrin) antibodies.
  • CRTH2 the receptor for prostaglandin D2 is considered as a cell surface marker for human memory Th2 + cells. This population represents 0.5 to 2% of CD4 + T cells. These cells were then purified on magnetic beads coated with anti- PE antibodies. CRTH2 + cells were around 80 to 85% pure. These cells were then cultured in the presence of beads coated with anti-CD3 and anti-CD28 mAbs plus IL-4, IL-2 and anti- IFNy antibodies for 14 to 28 days. At the end of the culture, intracellular cytokine staining reveals that around 50% of cells produced IL-4 and less than 5% of cells produced IFNy (data not shown).
  • CRTH2- cells were differentiated along the Thl pathway by setting them in the presence of beads coated with anti-CD3 and anti-CD28 mAbs plus IL-12, IL-2 and anti-IL-4 antibodies for 14 to 28 days. Intracellular cytokine staining reveals that more than 70% of cells produced IFNy and less than 3-5% synthetized IL-4 (not shown).
  • mRNAs were extracted from CRTH2+ and CRTH2- (Thl) CD4+ T cells, reverse-transcribed, and PCR were performed with primers specific for Ca v 1.2 encoding gene (table 1) and the housekeeping gene.
  • Table 1 Sequences of primers used for sequencing human Ca v 1.2 channel mRNA in CRTH2 + cells
  • NM-001129839 used as a reference is reported as the variant 13 of human Ca v 1.2 in databases
  • Nicardipine (5 or 10 ⁇ ), a dihydropyridine antagonist of dihydropyridine receptors alias Ca v l channels reduced the production of Th2 cytokines upon stimulation of human Th2 cells with coated anti-CD3niAb and soluble anti-CD28 mAb (Fig.14).
  • CRTH2 + cells were also transfected with different Ca v lAS (Table 2). These antisense were chosen from litterature and predictive databases.
  • the Ca v lAS reduced the expression from 10 to 70% depending upon the AS (Fig. 15).
  • AS6 the AS analogous to the Ca v lAS used for inhibiting mouse Ca v 1.2
  • Ca v 1.2 by 80% (not shown).
  • Preliminary results suggested that Ca v l AS, and especially AS5 and AS6 decreased the production of IL-5 and IL- 13 (Fig.l6).
  • Eosinophils also express these Ca v 1.2 channels
  • Human Th2 cells overexpress Ca v 1.2 and likely Ca v 1.3 channels.
  • the sequences of Ca v 1.2 channels are similar to neuronal isoforms of the channels.
  • the protein is detected in these Th2 cells.
  • Th2 cells express approximately 10 times more Ca v 1.2 channels than Thl cells.
  • Nicardipine reduces Th2 cytokine production by CRTH2+.
  • Preliminary results suggest that CavlAS and especially Cavl.5 and Cavl.6 AS decreased Cavl.2 expression and Th2 cytokine production. These latter results are recent and should be confirmed.
  • Dihydropyridine receptors are selective markers of Th2 cells and can be targeted to prevent Th2-dependent immunopathological disorders. J Immunol 2004;172:5206-5212.

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Abstract

Cette invention concerne un inhibiteur de l'expression du gène CACNAlC et/ou CACNAlD pour traiter et/ou prévenir les maladies inflammatoires, de préférence, les maladies allergiques, et mieux encore, les maladies respiratoires d'origine allergique. Cette invention concerne également un antagoniste spécifique constitué d'au moins un des sous-motifs Cavl.2 et Cavl.3 des canaux calcium de type L voltage-dépendants pour prévenir et/ou traiter ces maladies inflammatoires.
PCT/EP2010/068546 2009-11-30 2010-11-30 Inhibiteurs et antagonistes des canaux calcium dans le traitement de l'asthme WO2011064395A2 (fr)

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