Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/51—Lyases (4)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/01—Hydro-lyases (4.2.1)
  • C12Y402/01011—Phosphopyruvate hydratase (4.2.1.11), i.e. enolase

Definitions

• the present invention relates to methods and pharmaceutical compositions for the treatment of autoimmune diseases, especially rheumatoid arthritis.
• RA Rheumatoid arthritis
• RA aetiology of RA.
• Several causes have been proposed including: 1) environmental factors (tobacco, microbial pathogens), 2) genetic factors (susceptibility alleles HLA-DRB 1 *0401 and *0404 in Caucasian population, allele T 1858 of PTPN22), 3) hormonal factors 4) immunological factors. Both innate and adaptative immunity are involved in the onset and the maintenance of the disease respectively. Many cells (Antigen presenting cells, T cells, T regulatory cells and B cells) are involved in the pathogenesis of RA.
• This multicellular system plays a pivotal role in the different phases of the disease with direct cell interactions and cytokinic communication (TNF-alpha, IL-lbeta, IL-1 Ra, IL-4, IL-10, IL-17, RANK-L).
• TNF-alpha IL-alpha
• IL-1 Ra IL-1 Ra
• IL-4 IL-10
• IL-17 RANK-L
• alpha-Enolase is a multifunctional protein expressed in various tissues such as liver, thymus, kidney and also in synovial tissue. It is a key glycolytic enzyme which converts 2- phospho-glycerate into phosphoenolpyruvate. In addition to this property, alpha-Enolase exerts several biological functions related to its cellular localization. Its ability to serve as a plasminogen receptor on the surface of a variety of cells (epithelial, endothelial and hematopoietic cells) suggests that it can play a role in the intravascular and pericellular fibrinolysis.
• MBP-1 Myc binding protein 1
• MBP-1 an alternative translation initiation product of the alpha- Enolase RNA, may be found in the nucleus, where it acts as a transcriptional repressor of the c-myc proto-oncogene, with subsequent regulation of cell growth and differentiation.
• ENO is a target of antibodies in a wide spectrum of infectious and autoimmune diseases ENO such as ulcerative colitis, Crohn's disease, primary sclerosing cholangitis, systemic lupus erythematosus, autoimmune hepatitis, Belief s disease.
• Antibodies against ENO have been found in 0 to 6%> of healthy controls. Nevertheless, to date, the presence of autoantibodies against citrullinated ENO remains exclusive of RA. It has been proposed that the pathogenic role of ENO antibodies may be explained by a molecular mimicry process.
• An ENO immunodominant peptide was identified and referred as to EP1. Antibodies to this epitope were observed in 37-62% of sera obtained from patients with RA.
• This immunodominant peptide showed 82% homology with a peptide of ENO from Porphyromonas gingivali (referred as to por-EPl).
• the levels of antibodies to this immunodominant peptide correlated highly with the levels of antibodies to the bacterial peptide.
• antibodies to the human peptide cross-react with citrullinated recombinant Porphyromonas gingivalis enolase.
• the present invention relates to an a-enolase polypeptide for use in the prophylactic treatment of an autoimmune disease in a subject in need thereof.
• the inventors have evaluated the clinical and immunological effects of recombinant non-citrullinated ⁇ -enolase in the well-known collagen induced athritis model.
• Different doses (10 - 100 ⁇ g) of ⁇ -enolase were indeed intraperitoneally injected to 6 week-old DBA/1 mice one day prior to collagen II arthritis induction.
• Both clinical (weight, global and joint scores, tarsal thickness) and biological (anticollagen II and anti-a-enolase [home-made ELISA] antibodies, cytokines [IL-lb, IL-2, TNF-a, IL-6, IL-4, IL-10, IL-17, IFNg]) levels were assessed during a 72 days follow-up period.
• the present invention relates to an a-enolase polypeptide for use in the prophylactic treatment of an autoimmune disease in a subject in need thereof.
• the prophylactic administration of ⁇ -enolase polypeptide of the invention should serve to prevent or attenuate said autoimmune disease in said subject.
• subject preferably human
• at high risk for an autoimmune disease are prophylactically treated with the ⁇ -enolase polypeptide of the invention.
• examples of such subjects include, but are not limited to, humans with a family history of autoimmune disease.
• said autoimmune disease result in part of the presence of autoantibodies against citrullinated a-enolase.
• said automimmne disease is rheumatoid arthritis.
• a-enolase or "ENO” has its general meaning in the art and refers to the glycolytic enzyme which converts 2-phospho-glycerate into phosphoenolpyruvate.
• the term includes naturally occurring ⁇ -enolase and function conservative variants and modified forms thereof.
• the ⁇ -enolase can be from any source, but typically is a mammalian (e.g., human and non-human primate) a-enolase, and more particularly a human a-enolase.
• the sequence of a- enolase protein and nucleic acids for encoding such a protein are well known to those of skill in the art.
• An exemplary sequence of human ⁇ -enolase is provide as set forth in SEQ ID NO: l and an exemplary sequence of murine ⁇ -enolase is provide as set forth in SEQ ID NO:2 [ Figure 1].
• any ⁇ -enolase protein or gene sequence variant may be used as long as it has the properties of an a-enolase.
• “Function conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70 % to 99 % as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
• a “function-conservative variant” also includes a polypeptide which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75 %, most preferably at least 85%, and even more preferably at least 90 %, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
• a-enolase polypeptide refers to any polypeptide that comprises the immunodominant fragment of a-enolase EP1 (or function conservative variant thereof) consisting of the amino acid sequence ranging from position 5 to position 21 in SEQ ID NO: l or ranging from position 6 to position 21 in SEQ ID NO:2 ( Figure 1). Accordingly, the term encompasses ⁇ -enolase itself or fragments thereof comprising the immunodominant fragment of ⁇ -enolase EP1.
• a function conservative variant of the immunodominant fragment EP1 may be represented by the peptide ranging from position 5 to position 21 in SEQ ID NO:3 (por-EPl).
• polypeptides of the invention are recombinant polypeptides that are not citrullinated.
• ⁇ -enolase polypeptides of the invention can be conformationally constrained to retain their immunogenicity properties.
• cyclization is well known in the art and generally involves the introduction of a disulfide bound between two cysteine residues.
• the cycle is formed through a side chain to side chain ring involving a monosulfide or disulfide bridge between pairs of cysteines, penicillamines, homocysteines, combinations of the foregoing, or other pairs of amino acids in which the side chains are linked with either one or two sulfur atoms.
• Methods for the synthesis of disulfide cyclic polypeptide are well known in the art and are described for example in US3,929,758, US4,216,141; and US4, 102,877.
• Polypeptides of the invention may thus comprise cysteine residues at terminal ends to allow the cyclisation of the polypeptides.
• the a-enolase polypeptide is selected from the group consisting of CKIHAREIFDSRGNPTVEC (SEQ ID NO:4), CIHAREIFDSRGNPTVEC (SEQ ID NO :5) or CKIIGREILDSRGNPTVEC (SEQ ID NO :6) that are cyclised by a disulfide bound between the two cysteine residues.
• a-enolase polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
• modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
• the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
• a strategy for improving drug viability is the utilization of water-soluble polymers.
• Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
• water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
• PEG Polyethylene glycol
• Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
• PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
• copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
• PEGylation techniques for the effective modification of drugs.
• drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
• the PEG chains typically 2000 daltons or less
• Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
• the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
• These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
• the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
• increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomerular filtration (e.g., less than 45 kDa).
• linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
• a specific trigger typically enzyme activity in the targeted tissue.
• this type of tissue activated drug delivery is particularly useful where delivery to a specific site of bio distribution is required and the therapeutic agent is released at or near the site of pathology.
• Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes (see e.g., technologies of established by VectraMed, Plainsboro, N.J.).
• Such linkers may be used in modifying the a-enolase polypeptides described herein for therapeutic delivery.
• a-enolase polypeptides may be produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art.
• ⁇ -enolase polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques.
• Various automatic synthesizers are commercially available and can be used in accordance with known protocols, a-enolase polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc.
• the purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art.
• recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.
• a variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
• microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,
• Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.
• Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below.
• Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art.
• Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
• vectors comprising polynucleotide molecules for encoding the a- enolase-derived proteins.
• Methods of preparing such vectors as well as producing host cells transformed with such vectors are well known to those skilled in the art.
• the polynucleotide molecules used in such an endeavor may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host.
• the expression vectors include DNA encoding the given protein being operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes.
• suitable transcriptional or translational regulatory sequences such as those derived from a mammalian, microbial, viral, or insect genes.
• regulatory sequences include transcriptional promoters, operators, or enhancers, mR A ribosomal binding sites, and appropriate sequences which control transcription and translation.
• expression vector expression construct
• expression cassette any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
• a suitable expression vector for expression of the peptides or polypeptides of the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Methods for the construction of mammalian expression vectors are disclosed, for example, in EP-A-0367566; and WO 91/18982.
• 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 oligonucleotide, siR A, shR A or ribozyme 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 rous 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 such as a retrovirus.
• retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
• adenovirus adeno-associated virus
• SV40-type viruses polyoma viruses
• Epstein-Barr viruses Epstein-Barr viruses
• papilloma viruses herpes virus
• vaccinia virus
• Non-cytopathic viruses include retroviruses (e.g., lentivirus), 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.
• viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
• AAV adeno-associated virus
• 12 different AAV serotypes AAVl to 12
• Recombinant AAV are derived from the dependent parvovirus AAV2.
• the adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
• the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
• wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
• the adeno- associated virus can also function in an extrachromosomal fashion.
• Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. 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. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript.
• 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. It may also be administered into the epidermis or a mucosal surface using a gene-gun.
• 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 micro encap sulation.
• promoters/promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells.
• the nucleic acid being expressed is under transcriptional control of a promoter.
• a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the protein of interest (i.e., a-enolase, a variant and the like).
• a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence.
• under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. Any promoter that will drive the expression of the nucleic acid may be used.
• the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
• a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
• a promoter might include either a human or viral promoter.
• Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta]-actin, rat insulin promoter, the phosphoglycerol kinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest.
• CMV human cytomegalovirus
• Another regulatory element that is used in protein expression is an enhancer. These are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Where an expression construct employs a cDNA insert, one will typically desire to include a polyadenylation signal sequence to effect proper polyadenylation of the gene transcript. Any polyadenylation signal sequence recognized by cells of the selected transgenic animal species is suitable for the practice of the invention, such as human or bovine growth hormone and SV40 polyadenylation signals.
• Another aspect of the invention relates to a nucleic acid molecule encoding for an a- enolase polypeptide according to the invention for use in the prophylactic treatment of an autoimmune disease as defined above.
• said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector as above described.
• a further object of the invention relates to a vector comprising a nucleic acid encoding for an a-enolase polypeptide for use in the prophylactic treatment of an autoimmune disease as defined above.
• a further object of the invention relates to a host cell comprising a nucleic acid encoding for an a-enolase polypeptide (or a vector comprising a nucleic acid thereof) for the prophylactic treatment of an autoimmune disease as defined above.
• the ⁇ -enolase polypeptides (or nucleic acid encoding for a ⁇ -enolase polypeptide or a vector comprising a nucleic acid encoding for a ⁇ -enolase polypeptide) of the invention are administered in a therapeutically effect amount in said subject.
• a “therapeutically effective amount” is meant a sufficient amount of a-enolase polypeptides (or nucleic acid encoding for a ⁇ -enolase polypeptide or a vector comprising a nucleic acid encoding for a ⁇ -enolase polypeptide) to preserve the vascular endothelial cell barrier integrity at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• 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 or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
• a further object of the invention relates to pharmaceutical compositions comprising a a-enolase polypeptide (or nucleic acid encoding for a a-enolase polypeptide or a vector comprising a nucleic acid encoding for a ⁇ -enolase polypeptide) for use in the prophylactic treatment of an autoimmune disease.
• the ⁇ -enolase polypeptide (or nucleic acid encoding for a a-enolase polypeptide or a vector comprising a nucleic acid encoding for a ⁇ -enolase polypeptide) may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, 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.
• the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganis
• Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the a-enolase polypeptide (or nucleic acid encoding for a a-enolase polypeptide or a vector comprising a nucleic acid encoding for a ⁇ -enolase polypeptide) can be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the patient being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual patient.
• parenteral administration such as intravenous or intramuscular injection
• other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
• FIGURES are a diagrammatic representation of FIGURES.
• Figure 1 Aminoacid sequence of native alpha-enolase from human, mouse and Porphyromonas Gingivalis Figure 2: Clinical effects of prophylactic a-enolase injection in murine collagen- induced arthritis.
• FIG. 3 Clinical effects of prophylactic por-EPl injection in murine collagen- induced arthritis: Prophylactic injection (IP) of cyclic P. Gingivalis peptide (ckiigreildsrgnptvec), derivated from enolase, allows to decrease significantly arthritis severity in CIA. We can observe a dose-effect, better results were obtained with a dose of l0 ⁇ g of peptide (means+/-SEM are indicated).
• Figure 4 prophylactic enolase injection induces both anticollagen II antibodies titer decrease and anti-enolase antibodies titer increase in murine collagen induced arthritis: (A) anti-CII Ab and (B) anti-ENO Ab titers.
• CIA is a well-established experimental animal model close to human RA, with which it shares many clinical, immunological and histopatho logical features (Courtenay, J.S., et al, Immunisation against heterologous type II collagen induces arthritis in mice. Nature, 1980. 283(5748): p. 666-8.].
• CIA can be induced in genetically (H-2 q or H-2 r ) susceptible strains (DBA/1, B10.Q, B10.RIII) of mice by immunization with native heterologous collagen II (CII), a known component of cartilage. Both humoral and cellular immunity are implicated in this model, because anti-CII antibodies and Cll-specific Thl cells are necessary for the development of arthritis.
• T cell deficient mice do not develop disease.
• the resulting disease is a chronic proliferative synovitis with important cartilage destruction, bone erosion leading to joint deformations.
• CIA is an appropriate model, which has been widely used to evaluate approved RA therapies (etanercept, anakinra, abatacept, toclizumab) and compounds that were discontinued during phase II or III clinical trials.
• the inventors have evaluated the clinical and immunological effects of recombinant non-citrullinated a-enolase in the well-known collagen induced athritis model.
• Different doses (10 - 100 ⁇ g) of ⁇ -enolase were indeed intraperitoneally injected to 6 week- old DBA/1 mice one day prior to collagen II arthritis induction.
• Both clinical (weight, global and joint scores, tarsal thickness) and biological (anticollagen II and anti-a-enolase [homemade ELISA] antibodies, cytokines [IL-lb, IL-2, TNF-a, IL-6, IL-4, IL-10, IL-17, IFNg]) levels were assessed during a 72 days follow-up period.

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