MXPA99000372A - Products analogues of proteins cationi - Google Patents

Abstract

The present invention relates to the life of circulation and / or in vivo absorption of the therapeutic, cationic proteins, which include but are not limited to basic proteins such as NT-3 and BDNF, can be increased by the generation of analogous products which they have a lower isoelectric point and, preferably, also a charge of the lower protein relative to the native sequence protein.

Description

ANALOGUE PROTEIN PRODUCTS CATIQNICAS FIELD OF THE INVENTION This invention relates generally to therapeutically active cationic protein polypeptide analog products, including, but not limited to, neurotrophic factor analogues known as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF). . More specifically, the invention relates to positively charged polypeptides in which modifications have been made to the native sequence, such that the analogous products have lower isoelectric points and, concomitantly, longer circulation times and / or improved absorption afterwards. of parenteral administration. The invention also relates to materials and methods for the recombinant production of such polypeptide analog products, to antibodies thereof, and to pharmaceutical compositions containing the analogous products that can be used for the treatment of various diseases and disorders, REF .: 29077 BACKGROUND OF THE INVENTION After administration of a therapeutic protein by a parenteral means, such as by subcutaneous, intravenous or intramuscular injection, the pharmacokinetic properties such as bioavailability, circulation time and rate of elimination can vary widely from protein to protein. Although there are active efforts in many laboratories to develop alternative routes of administration for protein products, little is known about the factors that govern the pharmacokinetic behavior of protein therapeutic products after such parenteral administration. It has been known that an increase in the molecular weight of a protein can result in a preferential uptake by the lymphatic system before the blood capillaries; Supersaxo et al., Phamaceutical Res., Volume 7, page 167 et esq. (1990). The molecular size of the therapeutic protein also plays a key role in insulin uptake, where the dissociation of a zinc-induced hexamer to a monomeric form has been shown to be the speed limitation step in the absorbance of insulin; Kang et al., Diabetes Care, Volume 14, pages 942-948 (1991). The clinical analysis in diabetic patients of the monomeric insulin analogue products, in which the exome association site has been eliminated, has demonstrated a faster uptake, leading to significant improvements in glucose control in diabetic patients; see Brange et al., Nature, Volume 333, page 679 et seq. (1988).

BRIEF DESCRIPTION OF THE INVENTION To assess the impact of the isoelectric point (pl) on the pharmacokinetic behavior of proteins, certain NT-3 and BDNF analog products have been produced, in particular, which have a relatively lower pH, yet retain the structure and biological activity of the protein in its "native" state (ie, the protein of the naturally occurring amino acid sequence, as well as the met "1" version thereof, both of which are referred to herein as "uncultivated type" ") From these studies, it has now been discovered that protein analog products designed to possess a lower pl and / or lower charge under physiological conditions than the non-cultured type molecule, can also exhibit in vivo circulation times. longer (ie, "lifespan") and improved absorption after administration by injection Although the invention is illustrated in this description with particular reference In addition to the NT-3 and human BDNF, this has broader applicability to any cationic protein, and particularly basic proteins which in their native sequence have a pl greater than about 7.0. It should be noted that the terms "protein" and "polypeptide" are used interchangeably throughout this description to imply the same thing. Established brieflyThe present invention is analogous products substitution, insertion and deletion of therapeutic proteins, cationic, and / or chemically modified versions of such therapeutic proteins, that are characterized by a lower pl while also exhibiting in longer circulation and / or higher absorption relative to unmodified (ie, native sequence) proteins. The analogous proteins of this invention are typically human therapeutic proteins which are usually, but not necessarily, basic proteins. Preferably, these proteins also have a lower charge under physiological conditions compared to the basic protein, unmodified. The present invention also concerns materials and methods for the recombinant production of such analogues (as a preferred practical method), as well as antibodies raised against analogs protein products, and to pharmaceutical compositions containing the analogues as agents biologically active for use in the treatment of diseases and physical disorders.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows the amino acid sequence for human NT-3 of the non-cultured type (ie, "native" sequence) which has been recombinantly produced in bacterial E cells. cal i and expressed with a methionine residue present at the N-terminus, ie r-metHuNT-3 (SEQ ID NO: 1). In the Figure, amino acid numbering begins with the first residue after the initial methionine (met "1), since the naturally occurring protein is normally expressed in mammalian cells without the methionine residue. of Sequences in the present account the Met residue as +1.) FIGURE 2 shows the nucleic acid sequence (SEQ ID NO: 2, and FIGURE 2A) and the amino acid sequence (SEQ ID NO: 2, and FIG. NO: 3, and Figure 2B) of a r-metHuNT-3 polypeptide analog product of the present invention, specifically, NT-3 (i-? I9) R61A, K64D (with the amino acid numbering in the starting Figure with the first residue after the initial methionine.) FIGURE 3 shows the nucleic acid sequence (SEQ ID NO: 4, and Figure 3A) and the amino acid sequence (SEQ ID NO: 5) and Figure 3B) of another r-metHuNT-3 polypeptide analogue product according to the present invention, specific n, NT-3 (? -? i7) R61A, K64D (the numbering of amino acids in the Figure starts again with the first residue after the initial methionine). FIGURE 4 shows the calibration curves of the ELISA (standard) assay for the NT-3 of the non-cultured type (ie, having the ID of SEQ.NO.:1) and for the analogous products of the NT-3. of the IDs. FROM THE SEC. NOS: 3 and 5. The optical density is marked against the concentration of the sample in nanograms per milliliter (ng / ml). The cross-reactivity for the NT-3 analog products in the ELISA assay is approximately ten percent. Serum samples from pharmacokinetic studies were analyzed by the ELISA, and concentrations of NT-3 of the non-cultured type and of the NT-3 analog products were determined by calibration against the appropriate standard curve. FIGURE 5 depicts a high performance, size exclusion liquid chromatogram (SEC-HPLC) in which the NT-3 of the non-cultured type is compared to the NT-3 analog products of Figures 2 and 3. The units of absorbance ("AU") are marked on the vertical axis, and the elution time to peak (in minutes) is shown on the horizontal axis. As shown, the analogous products are coeluted with the molecule of the non-cultured type, demonstrating that the non-covalent dimer structure of NT-3 of the non-cultured type has not been broken in any of the analogs. No aggregated protein was detected in any of these preparations. The Figure represents: () NT-3 of the non-cultivated type; () NT-3 (1-u7) R61A, K64D; and (•) FIGURE 6 depicts a high performance liquid cation exchange chromatogram (CEX-HPLC) in which the NT-3 of the non-cultured type is compared to the NT-3 analog products of Figures 2 and 3. The "AU" (vertical axis) indicates the absorbance units. The peak elution time is shown on the horizontal axis. In this figure the analogous products are eluted earlier than the NT-3 of the non-cultured type, which is consistent with the lower isoelectric points of the analogues. The figure represents () NT-3 of the non-cultivated type; () NT-3 (? - u7, R61A, K64D; and (.) NT-3 a-119) R61A, K64D. FIGURE 7 depicts a silver-stained SDS polyacrylamide gel electrophoresis chromatogram (SDS-PAGE) in which the NT-3 of the non-cultured type (see Figure 1) is compared to the NT-3 analog products of the Figures 2 and 3. All three samples were run as a single band with approximately the same molecular weight of the monomeric form. None of the samples is observed to contain significant amounts of higher molecular weight oligomers or lower molecular weight fragments. Lane 1: NT-3 (1-? 17) R61A, K64 D, 2.5 μg; Lane 2: NT-3a-u9) R61A, K64D, 2.5 μg; Track 3; NT-3 of the non-cultivated type, 2.5 μg; Lane 4: NT-3 of the non-cultivated type, 12.5 μg; Clue 5: molecular weight markers. FIGURE 8 shows the serum concentration profiles (in nanograms per milliliter) versus time (in hours?) For the ID proteins. FROM THE SEC. US: 1, 3 and 5 after intravenous (IV) administration to test rats. The dose level was 1 milligram per kilogram (mg / kg) of body weight. The concentration profiles are biphasic. The initial distribution phase was followed by a slower elimination phase. Each point on the graph represents an average of three animals. FIGURE 9 shows the serum concentration curves (in nanograms per milliliter) versus time (in hours) in rats for the ID proteins. FROM THE SEC. NOS: 1, 3 and 5 after subcutaneous administration (SC) of one mg / kg, with each point on the graph representing again an average of three animals. The absorption phase is characterized by an increase in serum concentration to a peak. NT-3 of the non-cultivated type (SEQ ID NO: 1) showed the fastest decrease after reaching the maximum concentration.

DETAILED DESCRIPTION OF THE INVENTION The principles of this invention have a broad applicability to any cationic protein for which a reduction in the pl and, optionally the loading of the prctein will result in an increase in the therapeutically relevant biological properties such as circulation time and / or absorption after parenteral administration. By way of illustration, such proteins include, but are not limited to, basic proteins such as NT-3, BDNF, growth factor and macrophage differentiation, and various known isoforms thereof that have essentially the same ability to increase platelet production. sanguineous in vi and ex vi vo (collectively referred to herein as "MGDF"), and keratinocyte growth factor (KGF). Detailed descriptions of these factors, their biological properties and methods for their preparation and analysis are given in the patent literature: NT-3 in the published PCT application WO 91/03569; BDNF in U.S. Patent Nos. 5,180,820 No. 5,229,500, No. 5,438,121 and No. 4,453,361 and in published PCT application WO 91/03568; MGDF in published PCT applications WO 95/26745, WO 95/21919, and WO 95/21920; and KGF in the published PCT application WO 90/08771. In essence, the aim of this invention is to make one or more modifications to the primary structure of the protein of the non-cultured type which retains the protein structure and the biological activity of the protein, but which also results in a lower isoelectric point and , preferably, a lower charge at a physiological pH. The particular manner in which these modifications are made is not critical, and any procedure which makes the aforementioned changes can be used to achieve the increases described in the properties. By way of illustration only, appropriate modifications can be made through the use of site-directed mutagenesis involving the addition of acidic residues to the sequence by insertion and / or replacement mutations, and / or removal of basic residues by mutations. of suppression and / or replacement. Alternatively, the chemical groups or portions can also be added to the selected sites (ie, at the amino acid residues) in the protein chain of the non-cultured type molecule to realize the same final purpose (i.e., the reduction of the pl and the load with the conservation of the structure and the biological activity). A specific example is the succinylation of the selected residues in the protein chain. The nucleic acids which encode the analogous products of the protein according to this invention (ie, wherein one or more amino acids are designed to differ from the non-cultured type polypeptide) can be produced using site-directed mutagenesis or amplification of PCR in which the polymer (s) have the mutations of the desired points. For a detailed description of suitable mutagenesis techniques, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and / or Ausubel et al., Editors, Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994). Chemical synthesis can also be used using the methods described by Engels et al., In Angew. Chem. Intl. Ed., Volume 28, pages 716-734 (1989), for preparing such nucleic acids. The DNA molecules can be used to express the polypeptides analogous products of the invention by recombinant methods familiar to those skilled in the art, including but not limited to the methods described in the patents or patent applications mentioned above for NT-3 , BDNF, KGF and MGDF. By way of illustration, a nucleic acid sequence encoding an analogous product polypeptide of this invention is inserted into a biologically functional vector, appropriate (eg, circular plasmid or viral DNA) for expression in a suitable host cell the vector includes regulatory sequences for the expression of the nucleic acid sequence, inserted and selected to be functional in the particular host cells, employed ( that is, the vector is compatible with the machinery of the host cell, such that amplification and / or expression of the gene can occur). The polypeptide can be amplified / expressed in prokaryotic host cells, yeast, insects (baculovirus systems) and / or eukaryotic cells. The selection of the host cell will depend at least in part on whether the polypeptide expression product should be glycosylated. If glycosylation is desired, then yeast, insect or mammalian host cells are preferred for use. Typically, the vectors will contain a 5 'flanking sequence (also referred to as a "promoter") and other regulatory elements, as well as enhancer (s), an element of origin or application, a transcriptional termination element, an intron sequence, complete containing a donor and acceptor binding site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. The 5 'flanking sequence may be the innate 5' flanking sequence of the gene of the non-cultivated type, or it may be homologous (ie, of the same species and / or strain as the host cell), heterologous (i.e., of a species). different from the species of the host cell or strain), hybrid (i.e., a combination of 5 'flanking sequences from more than one source), or synthetic. The source of the 5 'flanking sequence can be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, with the proviso that the 5' flanking sequence is -functional in, and can be activated by, the the host cell. The origin of the replication element is typically a part of the commercially acquired prokaryotic expression vectors, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain number of copies may, in some cases, be important for optimal expression of the polypeptide. If the selection vector does not contain an origin of the replication site, one can be chemically synthesized based on a known sequence and then ligated into the vector. The transcription termination element is typically located 3 'to the end of the polypeptide coding sequence and serves to terminate transcription to the polypeptide. Usually, the transcription termination element in prokaryotic cells is a fragment rich in G-C followed by a poly-T sequence. While the element is easily cloned from a library or even commercially purchased as part of a vector, it can also be easily synthesized using methods for the synthesis of nucleic acids such as those referred to above. A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Selection marker genes, typical encode proteins that (a) confer resistance to antibiotics or other toxins, for example ampicillin, tetracycline or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell complement; or (c) provide clinical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistant gene, the ampicillin resistant gene, and the tetracycline resistant gene. The ribosome binding element, commonly called the Shine-Dalgarno sequence (for prokaryotic organisms) or the Kozak sequence (for eukaryotic organisms), is necessary for the initiation of translation for mRNA. The element is typically located 3 'to the promoter and 5' to the coding sequence of the polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (ie, having a high content of A-G). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods set forth above and can be used in a prokaryotic vector. In those cases where it is desirable that the polypeptide be secreted from the host cell, a signal sequence can be used to direct the polypeptide out of the host cell where it is synthesized. Typically, the signal sequence is located in the coding region of the nucleic acid sequence, or directly at the 5 'end of the coding region. Many signal sequences have been identified, and any one of them that is functional in the selected host cell can be used herein. Consequently, the signal sequence can be homologous or heterologous to the polypeptide. Additionally, the signal sequence can be synthesized chemically using methods such as those referred to above. The host cells can be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as yeast, insect or vertebrate cells). The host cells, when cultured under suitable nutrient conditions, can synthesize the peptide, which can be subsequently collected by isolating the culture medium (if the host cell secretes it in the medium) or directly from the host cells that host it. produces (if not secret). After the collection the polypeptide can be purified using methods such as molecular tint chromatography, affinity chromatography and the like. In general, if the polypeptide is expressed in E. col i, this will contain a methionine residue at the N-terminus in its recovered form (ie, met "1), unless it is expressed in an E. coli strain in which the methionine is enzymatically split by the host .

Suitable cells or cell lines can also be mammalian cells, such as Chinese hamster ovary (CHO) cells or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, selection and production and purification of the product are well known in the art. Other suitable mammalian lines are the COS-1 and COS-7 monkey cell lines, and the CV-1 cell line. Additional, exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Also suitable are normal diploid cells, cell strains derived from the culture of primary tissue, as well as primary explantations. The candidate cells may be genotypically deficient in the selection gene, or they may contain a selection gene that acts dominantly. Still other suitable mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, cell lines from Balb-c or NIH mice, BHK hamster or Hak. The insertion (also referred to as "transformation" or "transfection") of the vector in the selected host cell can be performed using calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method. The selected method will be in part a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are discussed, for example, in Sambrook et al., Supra. The host cells containing the vector can be cultured using normal means well known to the skilled artisan. The media will usually contain all the nutrients necessary for the growth and survival of the cells. The suitable means for the culture of the E cells. col i are, for example, Caldo Luria (LB) and / or Caito Terrific (TB). Suitable media for the culture of eukaryotic cells are RPMI 1640, MEM, DMEM, all of which can be supplemented with serum and / or growth factors as required by the particular cell line being cultured. A suitable medium for insect culture is Grace medium supplemented with levadurolate, lactalbumin hydrolyzate, and / or fetal bovine serum as necessary. Typically, an antibiotic or other compound useful for the selective cultivation of transformed cells is added as an adjunct to the medium.

The compound to be used will be dictated by the appropriate marker element present in the plasmid with which the host cell is transformed. For example, where the selectable marker element is resistant to kanamycin, the compound added to the culture medium will be kanamycin. The amount of the polypeptide produced in the host cell can be assessed using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and / or activity assays such as gel exchange assays. DNA binding. If the polypeptide has been designed to be secreted from the host cells, most of the polypeptide will probably be found in the cell culture medium. However, if the polypeptide is not secreted, it will be present in the cytoplasm (for eucaryotic, bacterial, Gram-positive and insect host cells) or in the periplasm (for host cells of Gram-negative bacteria.) For the intracellular polypeptide , typically the host cells are first disrupted mechanically or osmotically to release the cytoplasmic contents in a buffered solution.The polypeptide is then isolated from this solution.The purification of the polypeptide from the solution can be carried out subsequently using a variety of techniques. has been synthesized such that it contains a residue such as Hexahistidine or another small peptide in either its carboxyl or amino terminus, it can be modified in a one-step procedure by passing the solution through an affinity column where the column matrix has a high affinity for the residue or for the polypeptide directly (ie, a monoclonal antibody). For example, polyhistidine binds with great affinity and specificity to nickel, so a nickel affinity column (such as Qiagen nickel columns) can be used for purification. (See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & amp;; Sons, New York, 1994). Where, on the other hand, the polypeptide has no residues or antibodies are available, other well-known purification methods can be used. Such methods include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, negative gel electrophoresis in combination with gel elution and preparative isoelectric focusing (machine / "Isoprime" technique, Hoefer Scientific). In some cases, two or more of these techniques can be combined to achieve increased purity. If it is anticipated that the polypeptide will be found primarily in the periplasmic space of the bacterium or the cytoplasm of eukaryotic cells, the contents of the periplasm or cytoplasm, including the inclusion bodies (eg, Gram-negative bacteria) if the polypeptide has formed such complexes can be extracted from the host cell using any normal technique known to the skilled artisan. For example, the host cells can be lysed to release the contents of the periplasm by the use of a French press, homogenization, and / or treatment with high frequency sound waves. The homogenized product can then be centrifuged. In addition to the preparation of the polypeptide analog products of this invention by recombinant DNA techniques, the polypeptides can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art, including that exposed. by Merrifield et al. in J. Am. Chem. Soc., Volume 85, page 2149 (1964), by Houghten et al., in Proc. Nati Acad. Scie, USA, Volume 82, page 5132 (1985) and by Stewart and Young in Solid Phase Peptids Synthesis, Pierce Chem. Co. Rockford, IL (1984). Chemically synthesized polypeptides can be oxidized using the methods set forth in those references to form disulfide bridges. The pl and loading of the protein analog products resulting from any of the aforementioned methods can be measured using standard techniques, such as those described further below in conjunction with the specific embodiments. Chemically modified polypeptide compositions (ie, "derivatives") wherein the polypeptide is linked to a polymer in order to modify the properties are included within the scope of the present invention. The polymer is typically soluble in water, so that the protein to which it binds does not precipitate in an aqueous environment, such as a physiological environment. The polymer can have an individual reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. A preferred reactive aldehyde is propionaldehyde of polyethylene glycol, which is stable in water, or monoalkoxy or aryloxy derivatives of 1 to 10 carbon atoms thereof (see U.S. Patent No. 5,252,714). The polymer can be branched or unbranched. Preferably, for the therapeutic use of the preparation of the final product, the polymer will be pharmaceutically acceptable. The water soluble polymer, or mixture thereof if desired, can be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose or other polymers based on carbohydrates, polyethylene glycol poly ( N-vinylpyrrolidone), propylene glycol homopolymers, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (for example, glycerol) and polyvinyl alcohol. In general, the polypeptide analog products of this invention will be useful for the same purposes for which proteins of the non-cultured type from which they are derived are known to be useful. For example, NT-3 is currently under clinical study for the treatment of peripheral neuropathies (including diabetics), while BDNF is under clinical study for the treatment of amyotrophic lateral sclerosis (ALS). KGF is known to be active as a tissue growth and repair factor, and is currently in clinical development for the treatment of mucositis induced by chemotherapy or radiation. MGDF (in the form of a derivative treated with PEG) is in clinical development for the stimulation of platelet production as an adjunct to thrombositopenia induced by chemotherapy. However, it is expected that the analogous products of this invention offer advantages over the unmodified forms from the standpoint of increased therapeutic lifetime and absorbability. For therapeutic purposes, the analogous product polypeptides of this invention will typically be formulated in suitable pharmaceutical compositions adapted for therapeutic delivery, which constitutes a further aspect of this invention. Such pharmaceutical compositions will typically comprise a therapeutically active amount of an analogous polypeptide, alone or together with one or more excipients, carriers or other ingredients normal to a pharmaceutical composition. The carrier material can be water for injection, preferably supplemented with other common materials in the solutions for administration to mammals. Typically, the analogous product polypeptide will be administered in the form of a composition comprising a purified form of the polypeptide (which may be chemically modified) in conjunction with one or more physiologically acceptable carriers, excipients or diluents. The buffered, neutral saline solution or the saline solution mixed with serum albumin are exemplaryly suitable carriers. Other carriers, diluents and excipients can be included as desired. The pharmaceutical compositions of this invention can be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 18th edition, AR Gennaro, ed., Mack Publishing Company, 1990) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors and are preferably inert in the doses and concentrations employed, and include buffers such as phosphate, citrate, acetate, succinate or other salts of organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspargin, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt formation counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). Any composition of this invention that is proposed to be used for in vi ve administration must be sterile. Sterilization is easily carried out by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using these methods can be conducted either before, or after, lyophilization and reconstitution. The composition for parenteral administration will ordinarily be stored in the lyophilized form or in solution. The amount of polypeptide that will be effective in the treatment of a particular disorder or condition will depend on the nature of the polypeptide and the disorder or condition, as well as the age and general health of the patient, and can be determined by clinical, normal procedures. Where possible, it will be desirable to determine the dose response curve of the first in vivo pharmaceutical composition, such as in bioassay systems, and then in animal model systems, useful in vivo prior to human analysis. In general, suitable in vivo amounts can be developed based on an understanding of the therapeutically effective doses known for the protein of the non-cultured type on which the analogues are based. The expert practitioner, considering the therapeutic context, type of disorder under treatment, etc., will be able to find out the proper dosage without undue effort. Introduction methods for administration purposes include intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous. In addition, the invention also encompasses pharmaceutical compositions comprising the polypeptide analog products administered by means of liposomes, microparticles or microcapsules, which may be particularly useful for achieving sustained release. Various delivery devices may be necessary in the case of some of the polypeptide analog products, such as those of NT-3, BDNF and other neurotrophic factors proposed for the treatment of neurological conditions associated with the brain and other areas of the nervous system. central. Such devices may include osmotic implants and pumps for intrathecal and intracranial delivery, for example. The analogous products of this invention can also be used according to normal procedures to generate antibodies that are useful for medically related purposes, such as for monitoring the blood levels of the corresponding analogue product in a subject undergoing therapeutic treatment. . Various methods known in the art can be employed for the production of polyclonal antibodies that recognize epitopes of the polypeptides. For the production of antibodies, several host animals can be immunized by injection with an analogous polypeptide, or fragment or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants can be used to increase the immune response, depending on the host species, including but not limited to, Freund mineral gels such as aluminum hydroxide (alum), surfactants such as lysolecithin, pluronic polyols, polyanions, polypeptides, oil emulsions, keyhole limpet emocianins, dinitrophenol, and potentially useful human adjuvants such as Bacille Calmette-Guerin and Coryne acteriujp pa rvum. For the preparation of the monoclonal antibodies directed towards the analogous polypeptides, any technique which provides for the production of antibody molecules by the continuous cell lines in culture can be used. For example, the hybridoma technique originally developed by Kohler and Milstein and described in Nature, Volume 256, pages 495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique described by Kozbor and collaborators in Immunology Today, volume 4, page 72 (1983) and the EBV-hybridoma technique for producing monoclonal antibodies described by Colé et al., in "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pages 77- 96 (1985), all are useful for the preparation of monoclonal antibodies. In addition, a molecular clone of an antibody can be prepared for an epitope or epitopes of the polypeptide with known techniques. In particular, the recombinant DNA methodology can be used to construct nucleic acid sequences encoding a moclonal antibody molecule or an antigen binding region thereof.; see, for example, Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). The antibodies are useful for diagnostic purposes both in vivo and in vitro, particularly in forms labeled to detect the presence of the polypeptides in a sample of fluid or tissue.

DESCRIPTION OF THE PREFERRED MODALITIES The invention is described in further detail with reference to the following materials, methods, procedures and test results. The amino acid residues of the proteins are identified in the conventional manner using the three letter designations, established (for example, "met" for methionine, "val" for valine, etc.) or in some cases the individual letter designations, established (for example, "M" for methionine, "R" for arginine, etc.) throughout the text.

Materials and methods: Preparation of NT-3 analog products. The amino acid residues for substitution in the native sequence of human NT-3 were selected with the aid of conservation of the core structure and biological / therapeutic activity (see Holland et al., J. Mol. Biol. Volume 239, pages 385-400, 1994, Ibanez et al., In Cell, Volume 69, pages 329-341, 1992 and also in EMBO Journal, Volume 12, pages 2281-2293, 1993). The actual substitutions that were made in the native sequence of human NT-3 are reflected in the sequences shown in Figures 2 and 3, respectively. In an analogue, shown in Figure 2, the following two substitutions were made: the arginine at position 61 (arg6?) Was replaced by alanine (ala), and the lysine at position 64 (lys? I) was replaced with aspartic acid (asp) This analogous products was designated "NT-3 (? - ?? 9) R61A, K64D". A second analogue, shown in Figure 3, had those same two amino acid substitutions and, in addition, truncated at residue 117 (thereby suppressing argua and thrug). this analogous product was designated "NT-3 (? - u7) R61A, K64D". To create these analogues, the mutations were introduced into the human NT-3 sequence by means of the Polymerase Chain Reaction Technology, Standard (PCR). For NT-3 (1-u9) R61A, K64D, oligonucleotides chemically synthesized in pairs were used to create fragments of the NT-3 gene comprising the frontal portion to the site of the codon mutations corresponding to the portions of the gene. and 64 and the posterior portion of the mutant codon gene at the end. A second PCT was carried out by combining the front and back portions to create the full-length nucleic acid molecule encoding the two mutations at positions 61 and 64, respectively. For NT-3 (? -117) R61A, K64D, the above procedure was repeated, except that the posterior portion omitted the codons for arginine and threonine at positions 118 and 119.

Expression in E col i. An encoding of the DNA sequence for a methionine residue at the 5 'end was included to express the analogous products in E. coli and a stop codon was placed at the 3' end in each case. In addition, the cut sites for the Xbal and HindIII restriction enzymes were placed at the 5 'and 3' ends of the gene, respectively, and a synthetic ribosome binding site was placed in an appropriate substance upstream of the initiating methionine. . The resulting synthetic gene fragments, flanked by the Xbal and HindIII restriction sites at the 5 'and 3' ends, respectively, contained a ribosome binding site, the ATG start codon (encoding methionine), the sequence of analog coding, and a stop codon. The fragments were digested with the restriction endonucleases Ndel and BamHI and then ligated into the vector pAMG12. The expression plasmid pAMG12 can be derived from the plasmid pCFM1656 (acquisition record of ATCC No. 69576, deposited on February 24, 1994) by making a series of base changes directed to the site by PCR overlaying the oligomutagenesis and the substitutions of DNA sequences. Starting with the Bglll site (base pair of plasmid No. 180) immediately 5 'to the replication promoter of the PcopB plasmid and proceeding to the plasmid replication genes, the changes of the base pairs (bp) are as follows: bp DAMG12 no. bp in PCFM1656 bp changed to. in PAMG12 # 204 T / AC / G # 428 A / TG / C # 509 G / AC / T # 617 insert two bp G / C # 679 G / CT / A # 980 T / AC / G # 994 G / CA / T # 1004 A / TC / G # 1007 C / GT / A # 1028 A / TT / A # 1047 C / GT / A # 1178 G / CT / A # 1466 G / CT / A # 2028 G / C deletion of bp # 2187 C / GT / A # 2480 A / TT / A # 2499-2502 AGTG GTCA TCAC CAGT # 2642 TCCGAGC deletion of bp AGGCTCG # 3435 G / C A / T # 3446 G / C A / T # 3643 A / T T / A In addition, the DNA sequence between the restriction sites AatlI (position # 4364 in pCFM1656) and Sac I I (position # 4585 in pCFM1656) is substituted with the following DNA sequence: [ext roe sticks j bear of Aat l I] 'CGTAACGTAT - «- ATGGTCTCCCCATGCGAGAGTAGGGAACT < - ?: CAGGCATCAA- 3 'GCACGCATTGCATACGTACCAGAGGGGTAC .- ?: TCTCATCCCTRGACGGTCCGTAGTT- -TAAAACGAAAGGCTCAGTCGAAAGACTCGGCCTTTCG ^ - AT TT CGTTCCGAGTCAGCTTTCGGACCCGGAAAGCAAAATAGACAACAAACAGCCAC - G ^ TAGGCGGCCCRCGCCTAAACTTGCAACGCTTCGTTGCC- -ACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGG- -TGCGAGAGGACTCATCC -CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG- -GGCCTCCCACCGCCCGTCCTGCGGGCGG ATTTGACGGTCCGTAGT TAATTCGTCTTC- -CCATCCTGACGGATGGCCTTTRRGCGTI CTACAAACTCTTTTGTTRATTTTTCTAAAT- -CGGTAGGACTGCCRACCGGAAAAACGCAAAGATGT??? ^? GAGAAAACAAATAAAAAGATTTA- AatII -ACATTCAAATATGGACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAAATTC- -TGTAAGTTTATACCTGCAGAGTATTAAAAATrrTTTAAGTAAACTGTTTACGATTTTAAG- -TTGATTAATATTCTCAATTGTGAGCGCTCACAATT ^ ATCGATTTGATTCTAGATTTGAGT- -AACTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGCTAAACTAAGATCTAAACTCA- -TTTAACTTTTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGCTCACTAGT- -AAATTGAAAATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTCGAGTGATCA- SacII -GTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAG AAGAAGAAGAAG- - C AGCTGGACGTCCC ATGGTACCTTCGAATG AGCTCCT A - GCGCCTTTCTTCTTCTTCTTC - -AAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCGGCCACCGCTGAGCAATAACTAGCATAA- - TTCTTTCGGGCTTTCCTTCGACTC AACCG ACGACGGTGGCGACTCGTTATTGATCGT ATT - -CCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTGTTGCTGAAAGGAGGAACCGCTCTT- -GGGGAACCCCSGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCCTTGGCGAGAA- -CACGCTCTTCACGC 3 '-GTGCGAGAAGTG 5' [sticky end of SacII] The product of the ligation was transformed into competent host cells of the E strain. col i FM15. The resulting colonies were selected for the production of recombinant protein from those colonies that produce the correct sized protein were verified by the DNA sequence. The correct strain was innoculated for fermentation by transferring a small amount to the Luria Broth (10 g / 1 of Tripticase-Peptone, 10 g / 1 of Yeast Extract, and 5 g / 1 of sodium chloride) and incubation at 30 ° C for sixteen hours with agitation at 250 rpm. The culture was transferred to a sterile medium that had been sterilized in place in a fermenter, then the cell mass was increased using continuous feeds of glucose and organic nitrogen, before being induced with lactose. After induction, the fermentation was stopped, the cells were harvested by centrifugation, the supernatant was removed and the remaining cell paste was frozen.

Purification of proteins. The cells of the paste were broken by homogenization with high pressure and the inclusion bodies were recovered by centrifugation. The inclusion bodies were solubilized in guanidine-HCl, then diluted in urea. After resting for several days, the solution was adjusted to pH 3, diluted with water, centrifuged and eluted in series through columns of cation exchange chromatography and hydrophobic interaction. The peak fractions were combined and sterile filtered.

Isoelectric point and protein loading. The isoelectric points of human NT-3 of the non-cultured type (t-metHuNT-3, SEQ ID NO: 1), and of the analogous products thereof (ie, SEQ ID NO. NOS: 3 and 5) were calculated using the "GCG" protein analysis / DNA sequence Logical Equipment Package available from Genetics Computer Groups, Incs. Madison Wisconsin. The loading of the molecule at a physiological pH (assumed to be pH 7.4) was estimated using the same logic equipment. The pl of the first analogous product T-3 (i-u9, R61A, K64D (SEQ ID NO: 3), was calculated to be approximately 0.9 pH units below that of NT-3 of the non-cultivated type (8.5, compared to 9.4) The loading at a physiological pH (7.4) for this analogous product represented a reduction of approximately 2.5 pH units from that of NT-3 of the non-cultivated type (ie from approximately +7 to + 4.5) The pl for the other analogue, NT-3 (? -7) R61A, K64D (SEQ ID NO: 5) was calculated to be approximately 8.2, which is approximately 1.2 pH units below that the pl of the NT-3 of the non-cultivated type (9.4) In addition, the loading at a physiological pH for this analogous product was decreased by approximately 3.5 pH units, to approximately +3.5, relative to the NT-3 of the type not cultivated (+7).

ELISA assay. The elisa assay was conducted in 96-well plates coated with a monoclonal antibody produced against human NT-3. A polyclonal rabbit antibody conjugated to a horseradish peroxidase was used as the secondary antibody. Serum samples, calibration standards and quality control samples were diluted with the phosphate buffer to a 50% serum matrix before assay.

The sample size was 100 microliters (μl) per well. Each sample was analyzed in duplicate. The limits of quantification were 0.65, 4.00 and 4.05 ng / ml of serum per NT-3 of the non-cultivated type, NT-3 (i-n9) R61A, K64D and NT-3a-? I7. R61A, K64D, respectively.

Size Exclusion Chromatography (SEC-HPLC). Size exclusion chromatography in each sample was performed using a Waters 600 system in conjunction with a G2000SWXL column (TosoHaas). The samples were eluted at a flow rate of 0.7 ml per minute (ml / min.) In a buffer consisting of 100 mM sodium phosphate, 0.5 M NaCl, pH 6.09. The peaks were detected at a wavelength of 230 nanometers (nm).

Chromatography of Cationic Exchange (CEX-HPLC). The CEX-HPLC was performed using a Waters 625 system with a Resource? Column. (Pharmacia, Uppsala, Switzerland). The samples were eluted at a flow rate of one milliliter per minute using a 0-1 M sodium chloride gradient in 20 mM Tris HCl, pH 8.5. The peaks were detected using a wavelength of 220 nm.

SDS-PAGE Silver Tinted. The proteins were diluted with 2% SDS, mixed with sample buffer, and heated for five minutes in boiling water. The separation was conducted according to the manufacturer's instructions using pre-molded TRIS-Tricine gradient gels, 10-20%, from ISS (Integrated Separations Systems, Natick, MA). The dyeing with silver was done according to the procedure of Blum et al. In Electrophoresis, Volume 8, pages 93-99 (1987).

Mytogenic Bioassay with 3T3trkC Cells. The biological activity of r-metHuNT-3 as a reference standard is determined by means of a mitogenic bioassay of cells using 3T3trkC cells. These cells were created by transferring 3T3 cells (ATCC), which normally does not express the trkC receptor on their surface, with the plasmid pcDNAl / neo (Invitrogen, San Diego, CA) modified to contain the DNA sequence for the receptor protein of human trkC. See Shelton et al., Journal of Neuroscience, Volume 15, page 477-491 (1995) for the sequence of the trkC gene, and Valenzuela et al., Neuron, Volume 10 pages 963-974 (1993) for an illustrative transfection procedure. The transfected cells are maintained at 37 ± 2 ° C, in a high humidity incubator under an atmosphere containing 5.5 ± 1.0% C02 and in a Minimum Essential Medium of Dulbecco with fetal bovine serum and Sulfate G-418. The cells were distributed in 96-well plates for each assay. After approximately twenty-four hours incubation time under the same conditions, the maintenance medium was replaced with RPMI 1640 and the samples cultured, NT-3 (? -? 19) R61A, K64D or NT-3U-U7) R61A, K64D in a dose of 1 milligram per kilogram of body weight (mg / kg) each. The test material was administered either (1) intravenously (IV) as a first dose, then subcutaneously (SC) twenty-four hours after the first dose, or (2) in the reverse manner. Serial blood samples were collected before dosing and at 1, 5, 15 and 30 minutes, and 1, 2, 4 and 8 hours after an IV dose. After subcutaneous dosing, samples were collected at 10 and 30 minutes and at 1, 2, 4, and 8 hours. Blood serum concentrations of NT-3 were determined using an ELISA assay (see above). The antibodies used in the assays were specific for the quantification of NT-3 of the cultured type. Although not fully optimized antibodies exhibited sufficient cross-reactivity with NT-3 (? -u9) R61A, K64D and NT-3 (? -u7) R61A, K64D to allow quantification of these analogous products as well. The standard curves were prepared for protein face (see Figure 4).Test Results: Characterization of the NT-3 analog products. It was observed that both analogs, NT-3 (? - u9) R61A, K64D and NT-3 (? -? I7) R61A, K64D, retain the activity of NT-3 of the non-cultured type (r-metHuNT-3) in the PC-12 in vitro bioassay (Table 1). In fact, the bioactivity of the analogous products as evaluated in this assay appeared to be somewhat greater than that of the NT-3 of the non-cultured type, indicating perhaps an increased affinity for the trkC receptor (NT-3).

Table 1. In Vitro Bioactivity of NT-3 Proteins Concentration Concentration Percentage of Product Exposed Measure NT-3 (mg / ml) analogous activity (mg / ml) expected (%) NT-3 of type no 0.32 0.23 72 cultivated NT-3 (i.n7) 0.36 1.18 328 R61A.K64D NT-3 (i - ?? 9) 0.25 1.31 524 R61A.K64D Both analogous products were eluted as non-covalent dimers in size exclusion HPLC, as well as for NT-3 of the non-cultured type (see Figure 5). As expected, there was no significant difference in the molecular weights of the three proteins. Significant protein aggregation was not detected for any analogue. The results of the cation exchange HPLC (see Figure 6) were consistent with a reduction in pl for both analogs. The change in protein loading probably explains the slight changes in the SDS-PAGE (Figure 7), compared to NT-3 of the non-cultivated type. Both of the analogous products retain the biological activity and the non-covalent dimer structure of the NT-3 of the non-cultivated type.

Pharmacokinetic behavior. The concentration curves in the serum after intravenous administration were observed to be biphasic (see Figure 8). There was a significant difference in the initial distribution phase between the different types of NT-3.

The lifetimes (aT? / 2) were 3.3, 5.4 and 7.6 minutes for NT-3 of the non-cultivated type, NT-3 (? -? I9) R61A, K64D and NT-3 < ? - ?? 7) R61A, K64D, respectively (see Table 2, later). The decrease observed in the elimination after intravenous administration could be due only to the slower distribution of the analogous NT-3 products. The most pronounced decrease in concentration during this phase was observed with NT-3 of the non-cultivated type. The lifetimes of the terminal phase (ßT? / 2) were similar for the three types and ranged from 0.9 to 1.0 hours, suggesting that the elimination mechanism for these types could be the same. The areas under the concentration-time curves (t-AUCinf) for the NT-3 (? - u9) R61A, K64D and NT-3,? -. 117) R61A, K64D were approximately 1.2 to 2 times that of the NT -3 of the non-cultivated type.

Table 2. Pharmacokinetic Parameters Obtained in Rats with a Given Single IV Dosage of 1 mg / kg of NT-3 Proteins 1 Area under the concentration curve in the serum time from time zero to infinity. The calculation of the area is through the trapezoidal method. 2 The elimination speed is calculated by: Dose + AUC. 3 Period of life of the distribution phase (aT? 2). 4 Period of life of the terminal phase (ßT? / 2).

After subcutaneous injection the concentration in the serum increased rapidly for NT-3 of the non-cultured type (see Figure 9). The time for the maximum concentration (TMAX) was approximately 0.17 hours (see Table 3, below). A slower absorption profile was observed for the specifically modified types, TMAX 'S for NT-3 (i-u9) R61A, K64D and NT-3 (? - u7) R61A, K64D were 0.67 and 1.33 hours, respectively . The maximum concentrations for NT-3 (? -? I9) R61A, K64D and NT-3 (1-? 17) R61A, K64D were 6 to 10 times higher than for NT-3 of the non-cultivated type, which suggests that the analogous products manifested a higher degree of absorption of the injection site after administration. In addition, the degree of absorption (bioavailability) is more typically determined from the ratio of the areas under the serum curves for the routes of subcutaneous and intravenous administration, Table 3. Bioavailability for NT-3 of the non-cultured type, NT-3 (? -? i9) R61A, K64D and NT-3 (1-u7) R61A, K64D were 2.2, 28.5 and 43.22%, respectively. Terminal life times for analogous products appear to be longer than those of the non-cultivated type.

Table 3. Pharmacokinetic Parameters Obtained in Rats with an Individual SC Dose of 1 mg / kg of NT-3 Proteins 1 Area under the curve of concentration in the serum time from time zero to infinity. The calculation of the area is through the trapezoidal method. 2 The bioavailability (F) is calculated by: (t-AUCsc + t-AUCiv) x 100%, where both areas were obtained from the same animal. 3 CMAX is the maximum concentration. 4 TMAX is the time for maximum concentation. 5 Period of life of the terminal phase (ßT? / 2) The results of these studies show that by increasing the pl of NT-3, one can decrease the rate of elimination after intravenous administration, at least initially, and can also increase the degree of absorption after subcutaneous administration. These results also show that the loading of the protein plays a significant role in the determination of pharmacokinetic behavior. In the case of a basic protein such as NT-3, as well as other cationic proteins, the decrease in the isoelectric point (or the charge at a physiological pH) can lead to a significant improvement in the absorption and bioavailability of the molecule after of subcutaneous administration. From this knowledge, and the description provided herein, it is possible to design new molecules of improved therapeutic value.

It should be noted that the analogous products illustrated in the above description are proposed to be exemplary only, and that additional analogue products of NT-3, as well as other proteins, can be created in view of the present disclosure to achieve points. lower isoelectric cells with longer circulation times and / or higher absorption. In a variation, for example, the analogous proteins, particular to the IDs. FROM THE SEC. NOS: 3 and 5 can be produced by expressing them in a mammalian cell or by secretion in a bacterial strain such that a product "without met" is obtained (ie, the methionine residue in the N-terminus is removed, to give as a result, the polypeptides of SEQ ID NOS: 6 and 7, respectively, as will also be true in the case of the BDNF analog products described below.

BDNF analog products Using the procedures described above for NT-3, the following BDNF analog products were prepared and purified from E. coli and the isoelectric point (pl) and the charge at a physiological pH for BDNF "of the non-cultivated type" (ie, a naturally occurring sequence; see U.S. Patent No. 5,180,820, Figure 5) (ID.

THE SEC. NO: 8) are also displayed for comparison purposes. The positions for substitution are numbered starting with the first residue after the methionine in the mature form of the protein as expressed in E. col i. (That is, the initial methionine residue is not counted).

BDNF, K65D, K73D, K95A, R97A The substitutions were lysine for aspartic acid. at positions 65 and 73, lysine for methionine at position 95 and arginine for alanine at position 97. (SEQ ID NO: 9). pl calculated: 8.46 charge: +4.0 BDNF, P60E, K65D, K73D, K95A Substitutions were proline for glutamic acid at position 60, lysine for arpartic acid at positions 65 and 73 and lysine for alanine at position 95. (SEQ ID NO: 10) ). pl calculated: 8.45 charge: +4.0 BDNF of the uncultivated type pl calculated: 10.23 charge: +9.5 Results of the Biological Test and Characterization 1) In Vitro Bioactivity: Myxogenic Bioassay with PC12 / trkC Cells. In this assay, the biological activity of BDNF of the non-cultured type and the BDNF analog products are determined quantitatively by measuring the incorporation of (MTS) into PC-12 cells (ATCC) that have been transformed to express the trkB receptor ( the high affinity receptor for BDNF). The transformed cells were maintained at 37 ± 2 ° C, in a high humidity incubator under an atmosphere containing 7.5 ± 1% carbon dioxide, in a Dulbecco Minimum Essential Medium containing bovine serum and equine fetal serum and 1% L-glutamine. The cells were distributed in well plates for the assay, and incubation was continued. After approximately forty-eight hours, the maintenance medium was replaced with RPMI 1640 Medium, the test samples were added and the incubation was continued under the same conditions for another 48 hours. The cells were then stained with MTS, incubated under the same conditions for another five hours, and the optical density for each well was read with a microplate reader at 490 nm. The results for the bioactivity of the two BDNF analog products against BDNF of the non-cultured type are given in Table 4, below.

Table 4. In Vitro Bioactivity of BDNF Proteins 2) In vivo Biological Analysis and Results. The biological properties of the analogous products were evaluated in vivo in male Sprague-Dawley rats at a dose of 3.0 milligrams per kilogram of body weight for BDNF, K65D, K73D, K95A, R97A and 1.7 milligrams per kilogram body weight for the BDNF, P60E, K65D, K73D, K95A. The test materials were administered intravenously and subcutaneously, followed by collection of blood samples and measurement of blood serum concentrations (see the same procedure described above) for NT-3 and analogous NT-3 products. ). The pharmacokinetic behavior is given below with respect to each form of administration.

Table 5. Pharmacokinetic Properties in Rats, Intravenous (IV) Individual Dose of BDNF Proteins Table 6. Pharmacokinetic Properties in Rats, Individual Subcutaneous (SC) Dose of BDNF Proteins These results show that the introduction of mutations in BDNF of the non-cultured type has a measurable effect on the pharmacokinetic properties. In particular, greater bioavailability is achieved in vi ve for the two analogous BDNF products compared to BDNF of the non-cultured type as reflected by the increased values for t-UAC (inf) after intravenous administration and the increase in t-AUC (inf) and F% after subcutaneous administration. The invention is defined in the appended claims (ix) DESCRIPTION OF THE SEQUENCE: I D. FROM THE SEC. NO: 1: Met Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser Leu Trp Val Thr Asp Lys Ser Ser Ala He Asp He Arg 20 25 30 Gly His Gln Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro 40 45 Val Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val 50 55 60 Lys Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser Gln Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Cal Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Wing 100 105 110 Leu Ser Arg Lys He Gly Arg Thr 115 120 INFORMATION FOR THE ID. FROM THE SEC. NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 360 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE. CDNA (xi) DESCRIPTION OF THE SEQUENCE: ID. FROM THE SEC, NO: 2 ATGTACGCTG AACACAAATC TCACCGTGGT GAATACTCTG TTTGCGACTC TGAATCTCTG 60 TGGGTTACCG ACAAATCTTC TGCTATCGAC ATCCGTGGTC ACCAGGTTAC CGTTCTGGGT 120 GAAATCAAAA CCGGTAACTC TCCGGTTAAA CAGTACTTCT ACGAAACCCG TTGCAAAGAA 180 GCTGCACCGG TTGACAACGG TTGCCGTGGT ATCGACGACA AACACTGGAA CTCTCAGTGC 240 AAAACCTCTC AGACCTACGT TCGTGCTCTG ACCTCTGAAA ACAACAAGCT TGTTGGTTGG 300 CGTTGGATTC GTATCGACAC CTCTTGCGTT TGCGCTCTGT CTCGTAAAAT CGGTCGTACC 360 (2) INFORMATION FOR THE ID. FROM THE SEC. NO: 3 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: ID. FROM THE SEC. NO: 3: Met Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Ser Leu Trp Val Thr Asp Lys Be Ser Wing Asp He Arg 20 25 30 Gly His Gln Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro 35 40 45 Val Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Wing Wing Pro Val 50 55 60 Asp Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser Gln Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Val Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Wing 100 105 110 Leu Ser Arg Lys He Gly Arg Thr 115 120 2) INFORMATION FOR THE ID. FROM THE SEC. NO: 4 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE. CDNA (xi) DESCRIPTION OF THE SEQUENCE: ID. FROM THE SEC. NO: 4: ATGTACGCTG AACACAAATC TCACCGTGGT GAATACTCTG TTTGCGACTC TGAATCTCTG 60 TGGGTTACCG ACAAATCTTC TGCTATCGAC ATCCGTGGTC ACCAGGTTAC CGTTCTGGGT 120 GAAATCAAAA CCGGTAACTC TCCGGTTAAA CAGTACTTCT ACGAAACCCG TTGCAAAGAA 180 GCTGCACCGG TTGACAACGG TTGCCGTGGT ATCGACGACA AACACTGGAA CTCTCAGTGC 240 AAAACCTCTC AGACCTACGT TCGTGCTCTG ACCTCTGAAA ACAACAAGCT TGTTGGTTGG 300 CGTTGGATTC GTATCGACAC CTCTTGCGTT TGCGCTCTGT CTCGTAAAAT CGGT 354 INFORMATION FOR THE ID. FROM THE SEC. NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 118 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE, protein (xi) DESCRIPTION OF THE SEQUENCE: ID. FROM SEC NO: 5: Met Ala Glu His Lys Ser His Arg Gly Glu Ser Val Cys Asp 1 5 10 15 Ser Glu Be Leu Trp Val Thr Asp Lys Ser Be Wing He Asp He Arg 20 25 30 Gly His Gln Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro 35 40 45 Val Lys Gln Phe Glu Thr Arg Cys Lys Glu Wing Arg Pro Val 50 55 60 Lys Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gln Cys 65 70 75 80 Lys Thr Ser Gln Thr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys 85 90 95 Leu Val Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Wing 100 105 110 Leu Ser Arg Lys He Gly 115 NFORMATION FOR THE ID. FROM THE SEC. NO: 6 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: unknown ii) TYPE OF MOLECULE, protein (xi) DESCRIPTION OF THE SEQUENCE: ID. FROM THE SEC. NO: 6: Ala Glu His Lys Ser His Arg Gly Glu Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val Thr Asp Lys Ser Ser Ala He Asp He Arg Gly 25 30 His Gln Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro Val 35 40 45 Lys Gln Phe Glu Thr Arg Cys Lys Glu Ala Ala Pro Val Asp 50 55 60 Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu 85 90 95 Val Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Ala Leu 100 105 110 Ser Arg Lys He Gly Arg Thr 115 (2) INFORMATION FOR THE ID. FROM THE SEC. NO: 7 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 117 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE, protein x i) DESC R I PC OF THE S ECUENC IA: I D. FROM THE S EC NO: 7: Ala Glu His Lys Ser His Arg Gly Glu Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val Thr Asp Lys Ser Be Ala He Asp He Arg Gly 20 25 30 His Gln Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro Val 35 40 45 Lys Gln Phe Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Asp 50 55 60 Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Ala Leu 100 105 110 Ser Arg Lys He Gly 115 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

Claims (48)

1. A polypeptide analog product of a cationic polypeptide, characterized in that the analog product has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues or by chemical modification of one or more amino acid residues in the native sequence, such that the isoelectric point is lower and the life of circulation and / or absorption in vi are increased for the analogous product relative to those same properties in the cationic polypeptide, unmodified.

2. A polypeptide according to claim 1, also characterized by a lower charge under physiological conditions compared to the cationic polypeptide, unmodified.

3. A polypeptide according to claim 1, characterized in that it is an analogous product of a cationic protein selected from the group consisting of neurotrophic factor-3 (NT-3), brain-derived neurotrophic factor (BDNF), growth factor and differentiation of macrophages and keratinocyte growth factor (KGF).

4. A polypeptide according to claim 3, characterized in that it is an analogous product of NT-3.

5. A polypeptide according to claim 4, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO: 3

6. A polypeptide according to claim 4, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO: 5

7. A polypeptide according to claim 4, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO 6

8. A polypeptide according to claim 4, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO: 7

9. A polypeptide according to claim 3, characterized in that it is an analogous product of BDNF.

10. A polypeptide according to claim 9, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO: 9

11. A polypeptide according to claim 9, characterized in that it has the amino acid sequence of the ID. FROM THE SEC. NO: 10

12. A DNA molecule that encodes a polypeptide analog product of a cationic polypeptide wherein the analog product has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues, or by a chemical modification of one or more amino acid residues in the native sequence, such that the isoelectric point is lower and the life of circulation and / or absorption m vi is increased for the analogous product relative to those same properties in the cationic polypeptide, unmodified.

13. A DNA molecule according to claim 12, characterized in that it encodes a product analogous to a cationic protein selected from the group consisting of NT-3, BDNF, MGDF and KGF.

14. A DNA molecule according to claim 13, characterized in that it encodes the polypeptide analog product of NT-3.

15. A DNA molecule according to claim 14, characterized in that it encodes the polypeptide of the ID. FROM THE SEC. NO: 3

16. A DNA molecule according to claim 14, characterized in that it encodes the polypeptide of the ID. FROM THE SEC. NO: 5

17. A DNA molecule according to claim 15, characterized in that it has the nucleic acid sequence of the ID. FROM THE SEC. NO: 2

18. A DNA molecule according to claim 16, characterized in that it has the nucleic acid sequence of the ID. FROM THE SEC. NO: 4

19. A DNA molecule according to claim 13, characterized in that it encodes an analogous product of BDNF polypeptide.

20. A DNA molecule according to claim 19, characterized in that it encodes the polypeptide of the ID. FROM THE SEC. NO: 9

21. A DNA molecule according to claim 19, characterized in that it encodes the polypeptide of the ID. FROM THE SEC. NO: 10

22. A biologically functional expression vector characterized in that it includes a DNA molecule according to claim 12 operably linked to the regulatory expression sequences.

23. A prokaryotic or eukaryotic host cell transformed or transfected with an expression vector according to claim 22 in a manner that allows the host cell to express the polypeptide encoded by the DNA molecule.

24. A bacterial host cell, transformed or transfected according to claim 23.

25. A host cell of E. col i, transformed or transfected according to claim 24.

26. A mammalian host cell, transformed or transfected according to claim 23.

27. A CHO cell, transformed or transfected according to claim 26.

28. A COS cell, transformed or transfected according to claim 26.

29. A process for the production of a polypeptide analog product of a cationic polypeptide, wherein the analog product has an amino acid sequence that differs from the native sequence of the original polypeptide by one or more amino acid residues, such that the isoelectric point is lower and the life of circulation and / or absorption in vi is increased for the analogous product relative to those same properties in the unmodified polypeptide, the method is characterized in that it comprises culturing under conditions of suitable nutrients a prokaryotic or eukaryotic host cell transformed or transfected with an expression vector comprising a DNA molecule encoding the polypeptide in a manner that allows the host cell to express the polypeptide, and optionally isolating the polypeptide product from the expression.

30. A method according to claim 29, characterized in that the polypeptide is an analogous product of NT-3.

31. A method according to claim 30, characterized in that the DNA molecule has been prepared by site-directed mutagenesis.

32. A method according to claim 30, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO: 3

33. A method according to claim 30, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO: 5

34. A method according to claim 30, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO 6.

35. A method according to claim 30, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO: 7

36. A method according to claim 29, characterized in that the polypeptide is an analogous product of BDNF.

37. A method according to claim 36, characterized in that the DNA molecule has been prepared by site-directed mutagenesis.

38. A method according to claim 36, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO: 9

39. A method according to claim 36, characterized in that the analogous product has the amino acid sequence of the ID. FROM THE SEC. NO: 10

40. A method according to claim 29, characterized in that the host cell is bacterial.

41. A method according to claim 40, characterized in that the bacterial host cell is E. col i.

42. A polypeptide product of expression in a eukaryotic or prokaryotic host cell of a DNA molecule according to claim 12.

43. An antibody against a polypeptide according to claim 1.

44. An antibody according to claim 43, characterized in that it is polyclonal.

45. An antibody according to claim 43, characterized in that it is monoclonal.

46. A PEG-treated derivative of a polypeptide according to claim 1.

47. A pharmaceutical composition characterized in that it comprises a therapeutically effective amount of a polypeptide according to claim 1 and a pharmaceutically acceptable carrier or diluent.

48. A method for the treatment of peripheral neuropathies, characterized in that it comprises administering to a patient having the disorder a therapeutically effective amount of a polypeptide according to claims 4 or 9.