MXPA97003362A - Conjugates of bdnf and nt-3 with a polymer solubleen a - Google Patents

Abstract

The present invention relates to BDNF and NT-3 derivatives of neurotrophic factors by binding these polypeptides to a water-soluble polymer, for example, polyethylene glyc

Description

CONJUGATES OF BDNF AND NT-3 WITH A SOLUBLE POLYMER IN WATER FIELD OF THE INVENTION The present invention relates to a novel class of BDNF and NT-3 derivatives wherein a molecule of BDNF and NT-3 is bound to a water soluble polymer, and to a method for preparing such derivatives. I BACKGROUND OF THE INVENTION Proteins for therapeutic use are currently available in suitable forms, in widely suitable amounts as a result of advances in recombinant DNA technologies. Chemical derivatives of such proteins can effectively block a proteolytic enzyme from physical contact with the structure of the protein itself, and thus prevent degradation. Additional advantages may include, under certain circumstances, increased stability and circulation time of the therapeutic protein and decrease in REF: 24658 -inmunogenicity. However, it should be noted that the effect of modifying a particular protein can not be predicted. A review article describing the modification of proteins and fusion proteins is Francis, Focus on Growth Factors 3: 4-10, published by Mediscript, Mountview Court, Friern Barnet Lane, London, England (1992). Polyethylene glycol ("PEG" or "peg") is such a chemical moiety that has been used in the preparation ("pegylation") of therapeutic, therapeutic products ("pegylated proteins"). For example, Adagen®, a pegylated adenosine deaminase formulation is approved to treat combined, severe immunodeficiency disease; pegylated superoxide dismutase has been used in clinical trials to treat head injury; pegylated alpha interferon has been tested in phase I clinical trials to treat hepatitis; Pegylated glucocerebrosidase and pegylated hemoglobin are reported to have been preclinically tested. For some proteins, it has been shown that the binding of polyethylene glycol protects them against proteolysis, Sada et al., J. Fermentation Bioengineering 71: 137-139 (1991). Methods for joining certain portions of polyethylene glycol are available. See U.S. Patent No. 4,179,337 (Davis et al.), And U.S. Patent No. 4,002,531 (Royer). For a review, see Abuchowski et al., In Other Water-soluble Polymers have been used to modify proteins, such as ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly 1, 3, 6-trioxane, ethylene / maleic anhydride copolymers, and polyane acids (either homopolymers or random copolymers). For polyethylene glycol, a variety of means have been used to attach the polyethylene glycol molecules to the protein. In general, the polyethylene glycol molecules are connected to the protein by means of a reactive group found in the protein. Amino groups, such as those in the plant residues or in the N-terminations, are suitable for such binding. For example, the Royer patent, supra, states that reductive alkylation was used for the attachment of polyethylene glycol molecules to an enzyme. European Patent Application No. 0 539 167, published on April 28, 1993, establishes that peptides and organic compounds with free amino group (s) are modified with a derivative of P-i idato or soluble organic polymers. in water, related. U.S. Patent No. 4,904,584 (Shaw) refers to the modification of Usin residues in proteins for the attachment of polyethylene glycol molecules by means of reactive amine groups. A specific therapeutic protein that has been chemically modified is the granulocyte colony stimulation factor, ie, G-CSF. See European patent publications Nos. EP 0 401 384, EP 0 473 268, and EP 0 335 423. Another example is pegylated IL-6, described in US Pat. No. 5, 264,209 (Mikayama et al.). also European Patent Publication No. 0 154 316, published September 11, 1985, reports reacting a lymphokine with a polyethylene glycol aldehyde. The pegylation (reaction with polyethylene glycol) of the protein molecules in general results from a mixture of chemically modified protein molecules. As an illustration, the protein molecules with five lysine residues and one free amino group in the N-termination reacted in the above methods, can result in a heterogeneous mixture, some having six polyethylene glycol portions, some five, some four, some three, some two, some one and some zero. Among the molecules with several, the polyethylene glycol moieties can not be joined in the same location in different molecules. The above methods typically require a binding portion between the protein and the polyethylene glycol molecule. The procedure described by Delgado et al. In "Coupling of PEG to Protein by Activation with Tresyl Chloride, Applications in Immunoaffinity Cell Partitioning," 'Separations Using Aqueous Phase Systems, Applications in Cell Biology and Biotechnology, Plenum Press, New York, NY (1989 ), on pages 211-213, involves the use of tresyl chloride and does not result in linking groups between the polyethylene glycol and protein portions. This method can be difficult to use to produce therapeutic products because the use of tresyl chloride can result in toxic byproducts.

Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) reports the modification of CD4 immunoadhesin with monomethoxypolyethylene glycol aldehyde ("MePEG glycol") by means of reductive alkylation. The authors report that the in vitro binding capacity of the modified CD4-IgG (to the gp 120 protein) decreased in the proportion correlated to the degree of MePEGylation. Brain-derived growth factor (BDNF) and neurotrophin-3 (NT-3) are known polypeptides that belong to a different class of neurotrophic factors called neurotrophins that include nerve growth factors (NGF). These factors promote the survival and maintenance of neuronal functions and are primary candidates for the therapeutic treatment of neurodegenerative diseases. Barde et al., Neuron 2: 1525-1534 (1989); Snider et al., Cell 77: 627-638 (1994). Methods for the identification and recombinant production of these factors have been described in the patent literature; see U.S. Patent No. 5,169,762 (Gray et al.) for NGF, U.S. Patent Nos. 5,180,820 (Barde et al.) and 5,229,500 (Barde et al.) for BDNF, and published PCT application No. WO 91/03569 for NT-3. BRIEF SUMMARY OF THE INVENTION Briefly stated, in one aspect, the present invention provides BDNF and NT-3 derivatives in which the polypeptide portion of BDNF and NT-3 binds to a water soluble polymer. More particularly, the present invention includes derivatives of BDNF and NT-3 wherein the polypeptides are reacted with portions of water-soluble, reactive (ie "activated") polymers to bind the polymer to the polypeptide. Such binding can be performed by reactions discussed herein, such as acylation or alkylation. Acylation or alkylation with polyethylene glycol or other water-soluble polymer can be carried out under conditions whereby the main product is mono- or polydivitized. The polydivitization generally involves the attachment of polyethylene glycol or other water-soluble polymer to the e-amino groups of the polypeptide residues of the Usin and may additionally involve the binding of the polymer to the N-terminus of the polypeptide. The monoderivatization preferentially involves the binding of the polymer to the a-amino group of the N-termination residue of a polypeptide portion of BDNF and NT-3, thereby providing for the selective binding of a portion of water-soluble polymer. in the N-termination of the polypeptide. This is provided for a substantially homogeneous preparation of polymer conjugated / BDNF or polymer / NT-3 molecules as well as (if used, polyethylene glycol) a preparation of BDNF or NT-3 molecules having the directly coupled polyethylene glycol moiety. to the BDNF or NT-3 portion. The BDNF or NT-3 derivatives of this invention are useful for the same purposes for which the trophic factors of BDNF or NT-3 are known to be useful, in particular, for the promotion of survival and maintenance of neurons in In vitro and in vivo, and as potential therapeutic agents for the treatment in humans of neurodegenerative diseases, such as Parkinson's, aminotrophic lateral sclerosis (ALS), Huntington's disease, retinal degeneration, peripheral neuropathies and Alzheimer's disease, among others. As shown in some of the subsequent additional examples, the derivatization according to this invention can also result in an increased capacity of the molecule to migrate through the brain tissue, thereby resulting in a greater ease of delivery to the brain. the therapeutic targets located within the brain. In another aspect, also further described in detail, below, this invention provides a method for the preparation of BDNF and NT-3 derivatives of the aforementioned variety, in which the water-soluble polymers, especially polyethylene glycol, join the group α-amino in the N-terminus of the polypeptide (BDNF or NT-3), to obtain a homogeneous population of derivatized molecules, ie, conjugates of these polypeptides with the polymer.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 shows an example of acylation of BDNF (or NT-3) using active N-hydroxysuccinimidyl (NHS) esters of monomethoxypolyethylene glycol aldehydes to result in a polypeglylated product. In the figure, k represents the number of MPEG molecules reacted with a BDNF or NT-3 molecule, n represents the degree of MPEG polymerization used in the reaction (where n = 2000 by MPEG having a molecular weight of 100 kDa and n = 40 per MPEG having a molecular weight of 2 kDa), and m represents the total number of primary amino groups per molecule of BDNF or NT-3. FIGURE 2 shows an example of non-specific reductive alkylation of BDNF (or NT-3) using active aldehydes of monomethoxy polyethylene glycol to result in a polypeglylated product. In this figure, k, m and n are the same as ned above. FIGURE 3 shows an example of reductive alkylation at the specific site of BDNF or NT-3 in the a-amino group of the N-terminus residue in the polypeptide, using active aldehyde monomethoxy polyethylene glycol to result in a substantially monopegylated product ( in the N-termination). FIGURE 4 shows the immunohistochemistry of colin acetyltransferase (ChAT) of axotomized facial motor neurons in an animal model in vivo for the loss of peripheral nerve function.

Yan et al., J. Neurosci. 14 (9): 5281-5291 (1994). The right facial nerves of adult female rats were transected and the animals were treated subcutaneously each day for seven days as follows: PBS (A), non-pegylated BDNF at 5 milligrams per kilogram of body weight, mg / kg (B), N-terminally pegylated BDNF at 0.3 mg / kg (C), or randomly pegylated BDNF at 0.3 mg / kg (D). In the rats treated with PBS, the axotomy resulted in a greater drop in ChAT immunoreactivity in the injured facial nuclei (facial nucleus of panel A on the right side). In contrast, treatment with both non-pegylated BDNF and PEGylated BDNF (panels B, C and D) attenuated the injury-induced decrease in ChAT immunoreactivity. The symbols are as follows: FN, facial nucleus; py, pirimidal tract; Sp5, nucleus of the trigeminal spinal tract. The scale bar (in panel D) represents 1 mm. FIGURE 5 shows a dose response curve following the treatment of the test animals with non-pegylated BDNF and pegylated BDNF in the same model of axotomized facial motor neurons as in FIGURE 4. The animals received a daily subcutaneous injection (sc) vehicle only (0), pegylated BDNF (D), pegylated BDNF at the N-terminus (•) or randomly pegylated BDNF (i) at the indicated doses for a period of seven days. The values were established as mean ± SEM (n = 4). The data were analyzed by ANOVA followed by the Dunnett t test. *, p < 0.05; **, p, 0.01, BDNF treatments against vehicle. FIGURE 6 is a bar graph showing the penetration of non-pegylated BDNF ("NAT-BDNF") or pegylated BDNF ("PEG-BDNF") into the brain of live rats after single injection into the center of the striatum law. Twenty hours later, the animals were perfused-fixed with 4% of formaldehyde. The brains were removed, sectioned and stained with an antibody specific for BDNF using the procedure described by Yan et al. In Soc. Neurosci. Abs. 20: 1306 (1994). The total penetration volume of BDNF in the brain tissue was quantified by integrating all sections of BDNF immunoreactive tissue. The values were established as the mean ± SEM (n = 4). The data were analyzed by the Student t test. *, p < 0.0001. FIGURE 7 is a bar graph showing penetration of non-pegylated BDNF and pegylated BDNF into the brain of living rats after continuous local administration for a period of seven days. The total penetration volume of BDNF was quantified as in FIGURE 6. The values were established as the mean ± SEM (n = 4). The data were analyzed by the Student t test. *, p <; 0.0001. FIGURE 8 shows the retrograde transport through brain tissue of non-pegylated BDNF (panels A and B) and pegylated BDNF (panels C and D) to dopaminergic neurons located internally after infusion in the striatum of rat brain live, using the same procedure described in FIGURE 7. Panels B and D are elongations of the square sections in panels A and C, respectively. The symbols in panel A have the following meaning: SNC, compact nigra substance; SNR, crosslinked nigra substance; and VTA, ventral tegmental area. The bar in panel B indicates 500 μm for panels A and C, and 200 μm for panels B and D.

DETAILED DESCRIPTION OF THE INVENTION Contemplated for use in the practice of this invention are BDNF or NT-3 of native or natural sequence (i.e. occurring naturally), as well as fragments, precursors and polypeptide molecules that represent one or more amino acid substitutions, deletions or additions derived from the natural sequence exhibiting biological properties similar to the molecules of the natural sequence, for example, chimeras, analogs and the like. Accordingly, unless specifically indicated otherwise, the terms "BDNF" and "NT-3" should be taken to mean these polypeptides in any of the foregoing forms. Methods for the preparation of BDNF or NT-3 are known, particularly by recombinant means, which is typically the most practical way to obtain large amounts. Useful methods are described in the scientific and patent literature, including those set forth in the aforementioned US patents Nos. 5,180,820 and 5,229,500, and in published PCT application WO 91/03569, all of which are incorporated herein by reference .

Particularly preferred for use in the practice of this invention are BDNF and NT-3 of the natural sequence as expressed recombinantly in prokaryotic and eukaryotic cells, including recombinant polypeptide products, expressed from human nucleotide sequences ( "r-HuBDNF" and "r-HuNT-3"), including those expressed in bacterial cells to include a methionine residue at the N-terminus (ie, "r-metBDNF" and "r-metNT-3") . Examples of such polypeptides are those having the sequences shown in IDEN. FROM THE SEC. NO: 1 ("r-HuBDNF"), IDEN. FROM THE SEC. NO: 2 ("r-metHuBDNF"), IDEN. FROM THE SEC. NO: 3 ("r-HuNT-3") and IDEN. FROM THE SEC. NO: 4 ("r-metHuNT-3"). PEGylation of BDNF or NT-3 can be carried out by any of the pegylation reactions known in the art. See, for example: Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 154 316; EP 0 401 384; and the other publications cited herein that refer to pegylation. Preferably, the pegylation is carried out by means of an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or a reactive, analogous water-soluble polymer). These preferred means for derivatization with polyethylene glycol are discussed in more detail below.

Acylation PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with a BDNF or NT-3 polypeptide. Virtually any reactive PEG molecule can be used to carry out the pegylation of these polypeptides. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS"). As used herein, "acylation" is contemplated to include without limitation the following types of linkages between BDNF or NT-3 and a water soluble polymer such as PEG: amide, carbamate, urethane, and the like; See Bioconjugate Chem. 5: 133-140 (1994). The reaction conditions can be selected from any of those known in the art or those developed subsequently but conditions such as temperature, solvent and pH that could inactivate the BDNF or NT-3 species to be modified should be avoided. The reaction conditions that are applied in general, will be described later. An exemplary reaction with an NHS ester or monomethoxy-PEG is depicted in FIGURE 6. PEGylation by acylation generally results in a polymethylated BDNF or NT-3 product, where the e-amino groups of Usin are pegylated by means of an acyl linkage group. Preferably, the connection link will be an amide. Also preferably, the resulting product will be substantially (e.g.,> 95%) mono-, di- or tripeglylated. Nevertheless, some species with higher degrees of PEGylation (up to the maximum number of lysine e-amino acid groups of BDNF or NT-3 plus an a-amino group at the amino terminus of BDNF or NT-3) will normally be formed in amounts depending on of the specific reaction conditions, used. If desired, pegylated, more purified species can be separated from the mixture containing unreacted species by normal purification techniques, including, but not limited to, dialysis, salt displacement, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis Alkylation Pegylation by alkylation generally involves reacting a PEG aldehyde derivative with a polypeptide such as BDNF or NT-3 in the presence of a reducing agent. PEGylation by alkylation can also result in poly-pegylated BDNF or NT-3. An exemplary reductive alkylation reaction to produce a polypeglylated product is shown in FIGURE 7. In addition, one can manipulate the reaction conditions as described herein to substantially favor pegylation only in the a-amino group of the N-termination of the BDNF or NT-3 polypeptide (ie, a monopegylated species). A reductive, exemplary alkylation reaction with BDNF or NT-3 to produce a monopegylated product is shown in FIGURE 8. In either case of monopegylation or polypeylation, the PEG groups are preferentially bound to the polypeptide via a -CH group -NH-. With particular reference to the -CH2- group, this type of link is referred to herein as an "alkyl" linkage. Derivatization by means of reductive alkylation to produce a monopegylated product takes advantage of or evaluates the differential reactivity of different types of primary amino groups (Usin versus N-terminus) available for derivatization in BDNF or NT-3. The reaction is carried out at a pH (see below) which allows one to take advantage of pKa differences between the e-amino groups of the Usino residues and those of the a-amino group of the N-terminus residue of the polypeptide. By such selective derivatization, the binding of a water soluble polymer containing a reactive group, such as an aldehyde, to a polypeptide is controlled. The conjugation with the polymer takes place predominantly at the N-terminus of the polypeptide and no significant modification of other reactive groups occurs, such as the amino side chain amino groups of Usin. In an important aspect, the present invention provides a substantially homogeneous preparation of monopolymer / BDNF or monopolymer / NT-3 conjugate molecules, ie BDNF or NT-3 to which substantially one polymer molecule has been bound (ie, >90%) in a single location only. More specifically, if polyethylene glycol is used, the present invention also provides pegylated BDNF or NT-3 possibly lacking antigenic linking groups and having the polyethylene glycol molecule directly coupled to the BDNF or NT-3 polypeptide. Thus, in a preferred aspect, the present invention relates to pegylated BDNF or NT-3 wherein the PEG group (s) are linked via acyl or alkyl linkages. As discussed above, such products can be monopegillated or polypeglylated (eg, containing 2-6, preferably 2-5, PEG groups). The PEG groups generally bind to the polypeptide on ay / oe amino in the polypeptide chain, but it is also contemplated that the PEG groups could be attached to any amino group of the polypeptide structure that is sufficiently reactive to become bound to a PEG group under suitable reaction conditions. The polymer molecules used in both the acylation and alkylation approaches can be selected from water-soluble polymers or a mixture thereof. The selected polymer must be soluble in water, so that the polypeptide to which it binds does not precipitate in an aqueous environment, such as a physiological environment.

The selected polymer must be modified to have an individual reactive group, such as an active ester for acylation or an aldehyde for alkylation, preferably, so that the degree of polymerization can be controlled as provided in the present method. A preferred reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or mono alkoxy or aryloxy derivatives of 1 to 10 carbon atoms thereof (see US 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 can be selected from the group consisting of, for example, polyethylene glycol (for example, monomethoxy polyethylene glycol), dextran, poly (N-vinyl pyrrolidone), propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (for example, glycerol) and polyvinyl alcohols. For the alkylation reactions, the selected polymer must have a reactive, individual ester group. For the present reduced alkylation, the selected polymer must have an individual reactive aldehyde group. In general, the water soluble polymer will not be selected from naturally occurring glycosyl residues since they are usually made more conveniently by mammalian recombinant expression systems. The polymer can be of any molecular weight, and can be branched or unbranched. A water soluble polymer, particularly preferred for use herein is polyethylene glycol. As used herein, polyethylene glycol means that it encompasses any of the PEG forms that have been used to derivatize other proteins, such as mono-, alkoxy- or aryloxy-polyethylene glycol of 1 to 10 carbon atoms. In general, the derivatization can be carried out under any suitable condition used to react a biologically active substance with an activated, water-soluble polymer molecule. Methods for preparing pegylated BDNF or NT-3 will generally comprise the steps of (a) reacting a BDNF or NT-3 polypeptide with polyethylene glycol (such as an ester derivative or PEG reactive aldehyde) under conditions whereby BDNF or NT-3 comes to join one or more PEG groups, and (b) obtain the reaction product. In general, the optimal reaction conditions for the acylation reactions will be determined on a case-by-case basis based on known parameters and the desired result. For example, the larger the PEG: protein ratio, the larger it will be. the percentage of polypeglylated product. In the method of this invention, reductive alkylation to produce a substantially homogeneous population of monopolymer / polypeptide conjugate molecules will generally comprise the steps of: (a) reacting a BDNF or NT-3 polypeptide with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to allow selective modification of the a-amino group at the amino terminus of the polypeptide and (b) obtain the reaction product. For a substantially homogeneous population of monopolymer / BDNF or monopolymer / NT-3 conjugate molecules, the reductive alkylation reaction conditions are those that allow selective binding of the water-soluble polymer portion to the N-terminus of the polypeptide . Such reaction conditions will in general provide different pKa between the lumps of e-amino from usina and the a-amino group at the N-terminus (the pKa which is the pH at which 50% of the amino groups are protonated and 50% do not) . The pH also affects the ratio of polymer to the polypeptide that is used. In general, if the pH is lower, a larger excess of polymer to polypeptide will be desired (ie, the less reactive is the a-amino group of the N-terminus, the polymer will be more necessary to achieve optimum conditions). If the pH is higher, the polymer: polypeptide ratio needed will not be larger (ie, more reactive groups are available, so that a few polymer molecules are needed). For purposes of the present invention, the pH will generally fall within the range of 3-9, preferably 3-6. Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer, a few numbers of polymer molecules that can be attached to the polypeptide. Similarly, the branching of the polymer should be taken into account when optimizing these parameters. In general, the higher the molecular weight (or more branches), the higher the polymer: polypeptide ratio. In general, for the pegylation reactions contemplated herein, the preferred average molecular weight for the polymer is from about 2 kDa to about 100 kDa (the term "about" indicates ± 1 kDa). A more preferred average molecular weight is from about 5 kDa to about 50 kDa, and especially from about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to BDNF or NT-3 will generally vary from 1: 1 to 100: 1, preferentially (for polyperacylation) 1: 1 to 20: 1 and (for monopegylation) 1: 1 to 5: 1. By using the conditions set forth above, the reductive alkylation according to this invention will provide selective linkages of the polymer to any BDNF or NT-3 polypeptide protein having an α-amino group at the amino terminus, and will provide a substantially homogeneous preparation. of monopolymer conjugate / polypeptide protein. The term "monopolymer / polypeptide conjugate" is used herein to mean a composition comprised of an individual polymer molecule bound to a BDNF or NT-3 molecule. The monopolymer / polypeptide conjugate will have a polymer molecule located at the N-terminus, but not on lysine side groups. The preparation will preferably be greater than 80% monopolymer / polypeptide conjugate, and more preferably greater than 90% monopolymer / polypeptide conjugate, with the rest of observable molecules that are unreacted (i.e., polypeptide that is lacking). of the polymer portion). The following examples provide a preparation that is at least about 90% monopolymer / polypeptide conjugate, and about 10% unreacted polypeptide. The monopolymer / polypeptide conjugate is biologically active. For the present reductive alkylation, the reducing agent must be stable in aqueous solution and preferably capable of reducing only the Schiff base formed in the initial process of reductive alkylation. Preferred reducing agents can be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane. A particularly preferred reducing agent is sodium cyanoborohydride. Other reaction parameters, such as solvent, reaction times, temperatures, etc., as well as the means of purification of products, can be determined on a case-by-case basis based on published information with reference to the derivatization of proteins with soluble polymers. in water (see the publications cited here). Exemplary details are shown in the Examples given below. One may prefer to prepare a mixture of polymer / polypeptide conjugate molecules by acylation and / or alkylation methods, and the advantage provided herein is that one can select the proportion of monopolymer / polypeptide conjugate to be included in the mixture. In this way, if desired, one can prepare a mixture of various polypeptides by various numbers of bound polymer molecules (ie, di-, tri- tetra- etc.) and combine with the monopolymer / polypeptide conjugate material prepared using the present methods, and in this way obtain a mixture with a predetermined proportion of monopolymer / polypeptide conjugates. As mentioned, the polymer / polypeptide conjugates according to this invention can be used for the same purposes for which BDNF or NT-3 is known to be useful. These polypeptides have previously been shown to be effective, for example, as trophic factors that promote the survival and maintenance of neurons in vitro, thus making possible the study and use of neurons in research. Such cells are normally very difficult to keep alive in a culture. These same biological activities support the use of such in vivo factors for the neurological study of animals and offer the potential as therapeutic agents for the treatment of neurodegenerative diseases that are associated with neuronal function losses. For therapeutic purposes, the polymer / polypeptide conjugates of this invention can be formulated into, and administered in, any biocompatible, sterile, pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. The amount of the BDNF / polymer or NT-3 / polymer conjugate that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by normal clinical techniques. Where possible, it is desirable to determine the dose response curve for the pharmaceutical compositions of the invention first in vitro, for example, as in the BDNF and NT-3 bioassay systems described in the literature and then in the model of useful animals before testing it in humans. Methods of administration will include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary and oral. In addition, it may be desirable to introduce the pharmaceutical compositions into the central nervous system by any suitable route, including intraventricular or intrathecal injection. In addition, it should be desirable to administer the pharmaceutical compositions locally to the area in need of treatment. This can be achieved, for example, by local infusion during surgery, by injection, by means of a catheter or by the use of an implant of a porous, non-porous, gelatinous, fibrous or membranous material.

DESCRIPTION OF THE SPECIFIC MODALITIES The preparation of the BDNF derivatives and Particular NT-3 according to this invention and its physical and biological properties are shown in the following text. These examples are presented to more fully illustrate the present invention and are not intended to be limiting. In these examples, human BDNF and NT-3 are used as they are recombinantly prepared in E. coli (ie, r-metHuBDNF and r-metHuNT-3), unless otherwise specified.

EXAMPLE 1 Preparation of conjugate of monoMPEG (6kDa) -BDNF with the binding site in the N-terminal α-amino group (s) To a stirred, cooled (4 ° C) solution of r-metHuBDNF (2.5 mg / ml) in 100 mM sodium phosphate, pH 4.0, containing 20 mM NaCNBH3, a two-fold molar excess of activated methoxypolyethylene glycol aldehyde was added. (MPEG), which has an average molecular weight of 6,000 daltons (ie, 6 kDa).

The degree of modification of the protein during the course of the reaction was monitored by size exclusion chromatography using a 6HR 10/30 superoxide column (Pharmacia) eluted at a flow rate of 0.4 ml / min with sodium phosphate 100 mM, pH 6.9, containing 0.5 M NaCl. After ten hours, analysis by size exclusion chromatography indicated that all of the polypeptide (which exists in the solution as a dimer) had been converted essentially to the two possible forms of the N-terminally pegylated derivative: MPEG conjugated to one or both of the N-terminations of the BDNF dimer. The reaction mixture was then diluted a total of five times with sterile water and applied to an ion exchange column of HiLoad 16/10 S Sepharose HP (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 7.5. The reaction mixture was loaded onto the column at a flow rate of 1 ml / min, and the unreacted MPEG aldehyde was eluted with three column volumes of the same buffer. A gradient of five hundred minutes, linear from 0% to 100% of 20 mM sodium phosphate, pH 7.5, containing 0.75 M NaCl, was used to elute the two forms of the N-terminally pegylated BDNF dimer. The fractions containing the MPEG-BDMF derivatives were pooled, concentrated, and sterile filtered.

EXAMPLE 2 Preparation of conjugate of monoMPEG (20 kDa) -BDNF with the binding site in the N-terminal α-amino group (s) The procedure of Example 1 was repeated, except when using a methoxypolyethylene glycol (MPEG) aldehyde of 20,000 daltons (20 kDa) and a pH of 5.0.

EXAMPLE 3 Preparation of polyMPEG (6 kda) -BDNF conjugate by reductive alkylation with MPEG aldehydes To a stirred, cooled (4 ° C) solution of r-metHuBDNF (10 mg / ml) in 100 mM BICINE, pH 8, containing 20 mM NaCNBH3, a four-fold molar excess of methoxypolyethylene glycol activated aldehyde (MPEG) was added. ), which has an average molecular weight of 6 kDa.

The degree of protein modification during the course of the reaction was monitored by size exclusion chromatography using a 6HR 10/30 superoxide column (Pharmacia) eluted at 0.4 ml / min with 100 mM sodium phosphate, pH 6.9 , which contains 0.5 M NaCl. After ten hours, analysis by size exclusion chromatography indicated that all the polypeptide had been modified with MPEG. The reaction mixture was then diluted five times with sterile water, the pH was adjusted to 7 (using phosphoric acid), and the mixture was applied to an ion exchange column of HiLoad 16/10 S Sepharose HP (Pharmacia) equilibrated with buffer of 20 mM sodium phosphate, pH 7.5. The reaction mixture was loaded onto the column at a flow rate of 1 ml / min, and the unreacted MPEG aldehyde was eluted with three column volumes of the same buffer. A gradient of five hundred minutes, linear from 0% to 100% of 20 mM sodium phosphate, pH 7.5, containing 0.75 M NaCl, was used to elute the MPEG-BDNF conjugate. Fractions containing MPEG-BDNF conjugates were pooled, concentrated, and sterile filtered.

EXAMPLE 4 Preparation of polyMPEG conjugate (6 kDa) - BDNF by reductive acylation with MPEG aldehydes The procedure of Example 3 was repeated, except that a six-fold molar excess of MPEG aldehyde was used.

EXAMPLE 5 Preparation of conjugates of polyMPEG (6kDa) -BDNF by acylation with activated MPEG derivatives To a stirred, cooled solution (4 ° C) of r-metHuBDNF (10 mg / ml) in buffer of BICINE 0.1 M, pH 8, a four-fold molar excess of carboxymethyl ester MPEG, having a molecular weight, was added. average of 6 kDa. The polymer was dissolved by gentle agitation and the reaction was continued at the same temperature. The degree of protein modification during the course of the reaction was monitored by size exclusion chromatography using a 6HR 10/30 superoxide column (Pharmacia) eluted at 0.4 ml / min with 100 mM sodium phosphate, pH 6.9 , which contains 0.5 M NaCl. After three hours, size exclusion chromatography indicated that all the BDNF dimer had been modified with MPEG. The reaction mixture was then diluted four times with sterile water and the pH of the mixture was adjusted to 7 (using 0.5M phosphoric acid). This solution was applied to an ion exchange column of HiLoad 16/10 S Sepharose HP (Pharmacia) equilibrated with 20 M sodium phosphate buffer, pH 7.5. The unreacted MPEG aldehyde was eluted with three column volumes of the same buffer. A gradient of five hundred minutes, linear from 0% to 100% of 20 mM sodium phosphate, pH 7.5, containing 0.75 M NaCl, was used to elute MPEG-BDNF conjugates. Fractions containing MPEG-BDNF conjugates were pooled, concentrated, and sterile filtered.

EXAMPLE 6 Preparation of conjugates of polyMPEG (6 kDa) -BDNF by acylation with activated MPEG derivatives The procedure of Example 5 was repeated, except that a mol to mol ratio of reactants is used.

EXAMPLE 7 Preparation of conjugates of polyMPEG (20 kDa) -BDNF by acylation with activated MPEG derivatives The procedure of Example 5 was repeated, except when using an MPEG succinimidyl propionate having an average molecular weight of 20 kDa, and a six-fold molar excess of MPEG to Dimer of BDNF.

EXAMPLE 8 Preparation of conjugate of monoMPEG (20 kDa) -NT-3 with the binding site at the N-terminal a-amino residue To a stirred, cooled solution (4 ° C) of r-metHuNT-3 (4.77 mg / ml) in 20 mM sodium acetate, pH 4.0, containing 150 mM NaCl and 20 mM NaCNBH3, was added a three-fold molar excess of activated MPEG, which has an average molecular weight of 20 kDa. The degree of protein modification during the course of the reaction was monitored by size exclusion chromatography using a 6HR 10/30 Super-To column.

(Pharmacia) eluted at a flow rate of 0.4 ml / min with 10 mM sodium phosphate, pH 7.1, containing 150 mM NaCl. After ten hours, size exclusion chromatography indicated that all the protein (in solution as a dimer) had been converted to two possible forms of the N-terminally pegylated derivative: MPEG conjugated to one, or both, N-terminators of the dimer of NT-3. The reaction mixture was then diluted a total of five times with 20 mM sodium phosphate, pH 7.1, and applied to an ion exchange column of HiLoad 16/10 S Sepharose HP (Pharmacia) equilibrated with sodium phosphate buffer. mM, pH 7.1. The reaction mixture was loaded onto the column at a flow rate of 1 ml / min, and the unreacted MPEG aldehyde was eluted with three column volumes of the same buffer. A linear gradient from 0% to 100% of 20 mM sodium phosphate, pH 7.1, containing 0.4 M NaCl, was used to elute the two forms of the N-terminally pegylated NT-3 dimer. The fractions containing the MPEG-BDNF derivatives were pooled, concentrated, and sterile filtered. Additional MPEG-BDNF or MPEG-NT-3 conjugates can be obtained by modifying BDNF or NT-3 with MPEG aldehydes of different average molecular weights, for example, ranging from 5 kDa to 50 kDa, can be prepared in a similar manner. The homogeneity of the resultant PEGylated BDNF or NT-3 conjugates is determined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), using 10-20% or 4-20% of the prevailed gradient gels ( Integrated Separation Systems). To characterize the effective size (hydrodynamic radius) of each MPEG-BDNF or MPEG-NT-3 species, a 6 HR 10/30 Super6se gel filtration column (Pharmacia) is used. The proteins are detected by UV absorbance at 280 nm. The BIO-RAD gel filtration models serve as molecular weight indicators of the protein, globular. The molecular weights of the conjugates are determined by the analytical ultracentrifugation of the sedimentation equilibrium and by mass spectrometric analysis by desorption of the laser beam aided by the matrix. The structure of each N-terminal MPEG-BDNF or MPEG-NT-3 conjugate is confirmed using normal methods of N-terminal protein sequence and peptide tracing. The in vitro biological activity of the MPEG-BDNF conjugates prepared in Examples 1-7 are determined by their effect on 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- ( 4-sulfophenyl) 2H-tetrazolium, internal salt uptake by PC12 / pcDneo-trkB # 18 cells. These results as well as main reaction parameters used in the preparation of the conjugates are summarized in Table 1.

Table 1 Summary of the characteristics of the MPEG-BDNF conjugate and main parameters of the preparation reactions.

MPEG Conjugate Reagent Characteristic Conditions of the conjugate Reaction Type MW, MPEG pH to MW, b MPEGa Activ. kDa BDNE kDa BDNF in vitro or r-metHuBDNF N / AN / AN / AN / A 27 N / A 100% (1) mono-MPEG-BDNF Aldehyde 6 4 4 111.8 2 101 (2) mono-MPEG-BDNF Aldehyde 20 5 4 483.8 2 73 (3) poly-MPEG-BDNF Aldehyde 6 8 8 110.1 2.4 82 (4) poly-MPEG-BDNF Aldehyde 6 8 12 257.2 4.6 25 (5) poly-MPEG-BDNF ester of 6 8 8 298.0 4.6 2 NHS (6) poly-MPEG-BDNF ester of 6 8 2 113.2 2.3 28 NHS (7) poly-MPEG-BDNF this of 20 8 6 934.3 2.8 8 NHS mol / mol dimer of apparent BDNF MW determined by filtration in gel n / a - not applicable Example 9 Evaluation of the In Vivo Biological Activity of the MonoMPEG (20 kDa) -BDNF Conjugate on Motoneurones in Adult Rats BDNF has been previously shown to rescue the development of motoneurons from the death of cells that occur naturally and induced by axotomy; Yan et al. Nature 360: 753-755 (1992); Oppenheim et al., Nature 360: 755-757 (1992); Y Sendtner et al., Nature 360: 757-759 (1992).

It has also been shown that axotomized adult motor neurons respond to exogenous BDNF, and more specifically, that BDNF applied by various modes of administration, attenuates the axotomy-induced decrease in the immunoreactivity of colin acetyltransferase (ChAT) in the facial motor neurons of adult rats; Yan et al., J. Neurosci. 14 (9): 5281-5291 (1994). The decrease in ChAT activity is indicative of a loss in motoneuronal function, since ChAT is a vital neurotransmitter produced by motoneurons. Thus, these studies indicate that BPNF is useful as a potential therapeutic agent for adult motor neuron diseases. In the present study, the biological activity of the N-terminally pegylated and randomly pegylated BPNF species (see Examples 1 and 3) on adult motoneurons injured in vivo is evaluated.

Methods A. Surgery and treatment of the animal. The rats Female Sprague-Pawley, adults (total of 52 rats, n = 4 for each group) were anesthetized with a cocktail (43 mg / ml ketamine hydrochloride, 8.6 mg / ml xylazine and 1.43 mg / ml acepromazine) in a dose of 0.7 ml / kg of body weight. The right facial nerve was transected near the mastoid-like foramen. The animals were treated subcutaneously once a day for 7 days, starting on the day of surgery (day 0). The doses used were 0.1, 0.3, 1.0, and 5.0 mg per kg bodyweight of non-pegylated BDNF, conjugate of monoMPEG (29 kDa) -BDNF at the N-terminus, or conjugate of random polyMPEG-BDNF, each at PBS. A group of rats was treated with PBS only as a control. The body weights of the animals were measured daily.

B. Immunohistochemistry of ChAT. The rats were sacrificed for an overdose of anesthesia and a transcardial liquid was introduced with PBS, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2. The brain stems were removed, cryoprotected with 30% sucrose in PBS, frozen in the tip or holding piece of a sliding microtome, and serial coronal sections of 80 μl were cut through the partial core region. The sections were then processed for immunohistochemistry with mouse monoclonal antibody against ChAT (ascites, 1: 500, Chemicon, Temecula, CA), followed by 2 μg / ml of biotinylated, anti-mouse mouse antibody, secondary using the ABC method ( Vector Laboratories, Burlingame, CA) described by Yan and Hohnson in J. Neurosci. 8 (9): 3481-3498 (1988).

C. Quantification of immunohistochemical sections. A Quantimet 520 image analyzer (Leica, Inc., Deerfield, IL) coupled to a Nikon Optiphot-FXA microscope was used to quantify the relative intensity of the ChAT staining. A bandpass filter was used, narrow, 510 nm (Oriel Corp., Stratford, CT) with a Nikon-Plan 2X Apochromatic objective lens to produce high contrast images of the facial core region in the histology sections. The relative intensity of the ChAT immunoreactivity was determined by obtaining the average gray scale intensity for each outlined or outlined core minus the negative gray matter of ChAT adjacent to the background dyeing. For each animal, three or four sections containing the facial nucleus were used for quantification. Since treatment with BDNF did not affect the immunostaining with ChAT of the unwounded facial nucleus, the data were expressed as the ratio of the relative optical density of the injured facial nuclei to the non-injured of the same sections. The data were analyzed statistically by ANOVA followed by the Dunnett t test.

D. Results. Axotomy in adult rats causes a rapid and reproducible decrease in ChAT immunoreactivity in motoneurons; Lams et al., Brain Res. 475: 401-406 (1988) and Armstrong et al., J. Comp. Neurol. 304: 596-607 (1991). To evaluate the effects of non-pegylated BDNF and pegylated BDNF on adult motor neurons, the model or paradigm of facial nerve transection described above was used to study the influence of these polypeptides on the expression of ChAT in motoneurons. Seven days after the transection of the right facial nerve, ChAT immunoreactivity largely disappeared from the injured facial nuclei receiving PBS treatment (FIGURE 4, panel A, the facial nucleus on the right side). Subcutaneous treatment with natural BDNF (5 mg / kg, FIGURE 4, panel B), N-terminal monoMPEG-BNNF conjugate (0.3 mg / kg, FIGURE 4, PANEL C), and random polyMPEG-BDNF conjugate (0.3 mg / kg, FIGURE 4, panel D) significantly attenuated the injury-induced decrease in ChAT immunoreactivity. To quantify this phenomenon, the average optical densities of both the injured and non-injured facial nuclei of the sections immunostained with ChAT were measured. The conjugate of N-terminal mono-MPEG-BDNF and the natural random polyMPEG-BDNF conjugate reduced the injury-induced decrease in ChAT immunoreactivity in a dose-dependent manner (FIGURE 5). Natural BDNF in the 5 mg / kg dose showed a significant attenuation of the decrease in ChAT immunoreactivity induced by the lesion on vehicle control (p <0.01). Treatment with either conjugate of N-terminal monoMPEG-BDNF or random polyMPEG-BDNF conjugate resulted in a significant improvement over the control vehicle at each dose tested, and over natural BDNF in the lower conclusions tested (p <0.01). ). By titration, treatment with pegylated BDNF changed the dose response curve to the left approximately twenty times over natural BDNF (FIGURE 5), indicating an increase in the efficacy of pegylated BDNF over injured motor neurons.

EXAMPLE 10 Evaluation of the In Vitro Biological Activity of the N-terminal MonoMPEG-NT-3 Conjugate in an Embryo Chick DRG Bioassay The comparative biological activities of the non-pegylated NT-3 and the cojugado of monoMPEG (20 kDa) -NT- 3 N-termini were measured using the posterior or dorsal root ganglion (DRG) assay of the embryonic chick described by Lindsay et al in Dev. Biol. 112: 319-328 (1985). Briefly, five nodes of the posterior or dorsal root of the embryonic chick (E8) per well were cultured as explantations in a collagen matrix with 2 ml of F14 medium containing 5% normal horse serum. The effects of neurotrophic factor (both without pegylation and pegylation) were visually assessed under a phase microscope and were recorded on a scale of 0-5 (0 = result without neurite, 5 = maximum result of neurite). The results reported in Table 2, below, indicate that pegylated NT-3 did not suffer loss of activity compared to non-pegylated NT-3, a surprising result in view of the experience with other pegylated proteins tested in vitro that suffer decreases substantial in bioactivity.

Table 2 In Vitro Bioassay of Chick E8 DRG Concentration of the Sample Factor (ng / l) Result of Neurite NT-3 0.5 1, 2, 2, 3, 3 5 4, 4, 5, -, - 10 0.5, 1, 1, 1, 2 50 0.5, 0.5, 0.5 1, 1 NT-3 pegylated 5 4, 4 , 4, 2, 2 50 3, 3, 2, 2, 1 500 0, 0.5, 0.5, 1, 2 1000 3, 3, 2, 1, 0.5 Example 11 Additional Evaluation in the In Vivo Biological Action of the MonoPEG Conjugate (20 kDa) -N-Terminal BDNF in Adult Rats - Permeation through the Brain Tissue 1. Single Striatal Injection. One microliter of non-pegylated BPNF or a conjugate of N-terminal monoMPEG (20 kDa) -BDNF (1 mg / ml in phosphate buffered saline) was injected into the center of the right striatum of female rat brains, adult (n = 4) for 18-20 hours. Twenty hours later, the animals were sacrificed by means of an anesthetic overdose and a transcardial liquid was introduced with PBS, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2. The brains were removed, cryoprotected with 30% sucrose in PBS, frozen at the tip or point of attachment of a sliding microtome, and coronal sections were cut in series of 60 μm. Sections were then processed for immunohistochemistry with 1 μm / ml of rabbit anti-BDNF antibody, followed by 2 μm / ml biotinylated goat anti-BDNF antibody, secondary using the ABC method referred to above. A very intense staining was observed at the injection site of the striatum with both the pegylated and pegylated BDNF samples. Pegylated BDNF was then observed to diffuse into a larger area of tissue than non-pegylated BDNF, approximately 2.4 times as observed in FIGURE 6. There were few neurons in the substantia nigra that were positively labeled (not shown). 2. Infusion of seven days of the striatum. Female, adult rats (n = 4) received a daily infusion of 12 μg of either non-pegylated BPNF or N-terminal monoMPEG (20 kPa) -BPNF conjugate in the striatum for a period of seven days. The penetration of both forms of BPNF was even better, in this study, than for the individual injection protocol, above. The pegylated BPNF diffused in a much larger area than the natural BPNF, approximately 6.1 times, as seen in FIGURE 7. Many more neurons in the substantia nigra compact ("CNS") and in the ventral fragmental area ("VTH"). ") were positively marked followed by the infusion with pegylated BDNF (FIGURE 8, panels C and D) than with the non-pegylated BDNF (FIGURE 8, panels A and B). Under a higher energy increase, the BDNF immunoreactivity was dotted and within the pericarions, which indicated that the BDNF had been transported retrogradely from the nerve ending to the body of the cells. The positive staining observed in the middle ventral part of the reticulated nigra substance ("SNR") was in the neuropil and was not associated with any body of the cells. This staining was due to non-specific diffusion in place of the retrograde transport mediated by the receptor; Ferguson et al., J. Comp. Neurol. 313: 680-692 (1991). These results are very significant. Typically, after parenchymal administration to the brain in animals, BDNF was found to diffuse through the brain tissue very poorly. The present data shows a dramatic improvement in the ability to achieve such diffusion when pegylated BDNF is used, which is suggestive of potentially greater efficacy as a therapeutic agent at least for such forms of administration.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: Amgen Inc. (ii) TITLE OF THE INVENTION: DERIVATIVES OF BDNF AND NT-3 (iii) NUMBER OF SEQUENCES: 4 (iv) ADDRESS OF CORRESPONDENCE: (A) ADDRESS : Amgen Inc. (B) STREET: 1840 Dehavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) PAI S: USA (F) ZIP: 91320-1789 (v) READABLE FORM OF THE COMPUTER: ( A) AVERAGE TYPE: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patent in Release # 1.0, Version # 1.25 (vi) DATA OF THE APPLICATION CURRENT: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Mazza, Richard J. (C) REFERENCE NUMBER / REGISTRATION: A -298 (2) INFORMATION FOR IDEN. FROM THE SEC. NO: l: (i) CHARACTERISTICS OF THE SEQUENCES: (A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) DESCRIPTION OF THE SEQUENCE: IDEN. FROM THE SEC. NO: 1: His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser He 1 5 10 15 Ser Glu Trp Val Thr Wing Wing Asp Ly3 Lys Thr Wing Val Asp Met Ser 20 25 30 Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Glu Ser Lys Gly Gln 35 40 45 Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr 50 55 60 Lys Glu Gly Cys Arg Gly He Asx Lys Arg His Trp Asn Ser Gln Cys 65 70 75 80 Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys 85 90 95 Arg He Gly Trp Art Phe He Arg He Asp Thr Ser Cys Val Cys Thr 100 105 110 Leu Thr He Lys Arg Gly Arg 115 (2) INFORMATION FOR IDEN. FROM THE SEC. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: protein (ix) ) DESCRIPTION OF THE SEQUENCE: IDEN. FROM THE SEC. NO: 2: Met His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser 1 5 10 15 He Be Glu Trp Cal Thr Wing Wing Asp Lys Lys Thr Wing Val Asp Met 20 25 30 Ser Gly Gly Thr Val Thr Val Leu Glu Val Val Val Lys Gly 35 40 45 Gln Leu Lys Gln Tyr Phe Tyr Gly Thr Lys Cys Asn Pro Met Gly Tyr 50 55 60 Thr Lys Glu Gly Cys Arg Gly He Asp Lys Arg His Trp Asn Ser Gln 65 70 75 80 Cys Arg Thr Thr Gln Ser Tyr Asx Arg Ala Leu Thr Met Asp Ser Lys 85 90 95 Lys Arg He Gly Trp Arg Phe He Arg He Asp Thr Ser Cys Asx Cys 100 105 100 Thr Leu Thr He Lys Arg Gly Arg 115 120 (2) INFORMATION FOR IDEN. FROM THE SEC. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) DESCRIPTION OF THE SEQUENCE: IDEN. FROM THE SEC. NO: 3: Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val Thr Asp Lys Ser Be Wing 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 Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys 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 Tyr 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 IDEN. FROM THE SEC. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) DESCRIPTION OF THE SEQUENCE: IDEN. FROM THE SEC. NO: 4: Met Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp 1 5 10 15 Ser Glu Be Leu Trp Cal 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 40 45 Val Lys Gln Tyr Phe Tyr 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 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 It is noted that in relation to this date, the best method known to the applicant to carry the practice said 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 (24)

1. A derivative of BDNF, characterized in that it comprises a BDNF polypeptide connected to at least one water-soluble polymer.

2. An NT-3 derivative, characterized in that it comprises an NT-3 polypeptide connected to at least one water-soluble polymer.

3. A derivative according to claim 1 or 2, characterized in that the polypeptide is produced recombinantly in a bacterial cell.

4. A derivative according to claim 1 or 2, characterized in that the water-soluble polymer is selected from the group consisting of dextran, poly (N-vinyl pyrrolidone), polyethylene glycol, polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyols polyoxyethylated and polyvinyl alcohols.

5. A derivative according to claim 4, characterized in that the water-soluble polymer is polyethylene glycol.

6. A derivative according to claim 5, characterized in that the polyethylene glycol is a monomethoxy polyethylene glycol.

7. A derivative according to claim 5, characterized in that the polyethylene glycol is bound to a polypeptide by an acyl or alkyl bond.

8. A derivative according to claim 5, characterized in that the polyethylene glycol has a molecular weight of about 2 to about 100 kDa.

9. A derivative according to claim 8, characterized in that the polyethylene glycol has a molecular weight of about 5 kDa to about 50 kDa.

10. A method for attaching a water-soluble polymer to a polypeptide selected from the group consisting of BDNF and NT-3, wherein the water-soluble polymer has a reactive, individual aldehyde group, the method is characterized in that it comprises: (a) making reacting the polypeptide with a water soluble polymer under reductive alkylation conditions, at a sufficiently acidic pH to allow the a-amino group to be reactive at the amino terminus of the polypeptide; and (b) isolating the polypeptide linked to at least one water-soluble polymer.

11. A method for attaching a water-soluble polymer to a polypeptide according to claim 10, characterized in that it additionally comprises the step of (c) separating the polypeptide bound to at least one water-soluble polymer from the unreacted molecules.

12. A method according to claim 10, characterized in that the polymer is pharmaceutically acceptable.

13. A method according to claim 10, characterized in that the water-soluble polymer is selected from the group consisting of dextran, poly (N-vinylpyrrolidone), polyethylene glycol, polypropylene glycol, homopolymers, polypropylene oxide / ethylene oxide copolymers, polyols polyoxyethylated and polyvinyl alcohols.

14. A method according to claim 13, characterized in that the water-soluble polymer is polyethylene glycol.

15. A method according to claim 10, characterized in that the pH is between about 3 and about 9.

16. A method according to claim 10, characterized in that the reductive alkylation conditions involve the use of sodium cyanoborohydride as a reducing agent.

17. A method for attaching a polyethylene glycol molecule to the polypeptide selected from the group consisting of BDNF and NT-3, wherein the polyethylene glycol molecule has a reactive, individual aldehyde group, the method is characterized in that it comprises: (a) reacting the polypeptide with the polyethylene glycol molecule under reductive alkylation conditions, at a sufficiently acidic pH to allow the a-amino group to be reactive at the amino terminus of the polypeptide; and (b) obtaining the polypeptide linked to the polyethylene glycol.

18. A method for attaching a polyethylene glycol molecule to a polypeptide according to claim 17, further characterized in that it comprises the step of (c) separating the reaction product from the unreacted molecules.

19. A method according to claim 17, characterized in that the polyethylene glycol molecule has a molecular weight of about 2 kDa to about 100 kDa.

20. A conjugate of a water-soluble polymer and a polypeptide produced by the method of claim 17.

21. A substantially homogeneous preparation of monopegylated BDNF in the a-amino group in the N-terminus.

22. A substantially homogenous preparation of monopegylated NT-3 in the a-amino group in the N-terminus.

23. A method for improving the in vivo efficacy of BDNF for treating injured motor neurons, characterized in that it comprises using a pegylated derivative of BDNF.

24. A method for improving the migration of BDNF or NT-3 through brain tissue, characterized in that it comprises using pegylated BDNF or NT-3.