Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/48—Nerve growth factor [NGF]

Definitions

• the present invention relates to the use of NGF for the preparation of a medicament for the cure and/or prevention of reactive gliosis and therapeutic methods for the cure and/or prevention of reactive gliosis by NGF administration.
• Astrocytes constitute one of the main components of the glia, and are the most abundant cells in the Central Nervous System (CNS) ; their numeric ratio with respect to nervous cells increases with the increasing complexity of the CNS.
• CNS Central Nervous System
• glial cells have received scarce attention in the neuroscience field.
• Studies conducted in the last decade have highlighted a key role of these elements within the scope of both physiological and pathological processes.
• Glial cells moreover, both within the developing nervous system and in the mature one, play a central role in synapse formation, maintenance and function.
• astrocytes participate in the propagation of the nervous impulse by releasing transmitters (gliotransmission) such as glutamate, ADP, D-serine and eicosanoids, able to modulate synaptic activity. Moreover, by releasing such substances astrocytes exert different effects on neighbouring neurons, on the same glia and on blood vessels. In fact, glial cells express a pool of receptors, transporters and ionic channels, by which they can receive and integrate neuronal messages and modulate the efficacy of synapses. Therefore, it can be stated that glia actively participates to brain function. For a long time, astrocytes have erroneously been considered as support cells.
• astrocytes are organized in separate territories, yet forming a syncytium, and that they are communication elements within the brain. Therefore, they can generate various regulatory signals and have an interface and connection function between neurons and between neurons and vessels. These findings have led to the concept of the "tripartite" synapse. This type of contact occupies a privileged position in the central nervous system at the interface between capillaries and neurons. Involved in cerebral metabolism, astrocytes may, therefore, account for the modifications of the cerebral blood flow, an indirect index of the cortical activity which is recorded by functional imaging techniques (fMRI) .
• fMRI functional imaging techniques
• Astrocytes play a specific and essential role not only in normal cerebral functions, but also in the development and progression of neurodegenerative processes.
• Astrocytes are arranged in an, orderly way and each cell covers a well-delimited territory, located so as to allow local interactions. Astrocytes interface with the microvasculature and might contact several neurons, many nerve fibers and hundreds to thousands of synapses.
• glial response known as reactive gliosis, characterized by cellular hypertrophy, increase in the expression of cytoskeleton proteins such as vimentin and GFAP (glial fibrillary acidic protein) , upregulation of intermediate filament (IF) proteins, reexpression of nestin and, in the first week, also cellular division (Okada, S. et al, 2006), and upregulation of factors participating in the formation of the IF network.
• cytoskeleton proteins such as vimentin and GFAP (glial fibrillary acidic protein)
• IF intermediate filament
• astrocyte activation is to date rather enigmatic but is now regarded as a reaction of the glia, with specific functional and structural characteristics.
• Activated astrocytes are present in various pathological conditions of the central nervous system, such as stroke, trauma, growth of a tumor, or neurodegenerative disease ' such as Alzheimer' s disease, Parkinson's disease, triplet diseases, amyotrophic lateral sclerosis, multiple sclerosis.
• Gliosis therefore, according to the region in which it onsets, would seem to be at the basis of the development of many pathologies: from neurodegenerative disorders (cerebellar gliosis in Spinocerebellar Ataxia type 1 - SCAl -, mesencephalic-striatal gliosis in Parkinson' s disease, cortical gliosis in Alzheimer' s disease) , to neuropathic pain.
• Hypertrophic and activated glial cells can thus determine a sort of compression of the surrounding parenchyma by inducing a suffering thereof and, concomitantly to the disregulation of the above-indicated proteins, by actively contributing to create a substrate for the development of pathologies.
• Gliosis beside fostering the onset of neurodegenerative pathologies and inhibiting CNS regeneration, is also involved in neuropathic pain associated to traumas to the CNS.
• a large number of scientific publications report evident proofs of the fact that gliosis at spinal cord level is to be accounted for the onset and persistence of pathological pain related to inflammations, neuropathies and spinal immune activation. Indeed, it has been demonstrated in experimental models that reactive gliosis is necessary to create pathological pain states in laboratory animals.
• NGF neurotrophic factor
• NGF or substances mimicking its function when administered in the site of the lesion, are able not only to prevent the appearance of reactive gliosis, but even to revert such a process, by restoring a no more hypertrophic glia, preventing or even making gliosis-correlated pathologies disappear in the involved compartment.
• the first object of the invention is the
• NGF factor or its functionally active NGF-equivalent or
• NGF agonist fragments or derivatives for use in the curative and/or preventive therapeutic treatment of reactive gliosis.
• the second object of the invention is the use of NGF, or its functionally active fragments or derivatives, i.e. NGF complete or partial agonists, or substances mimicking its function (agonists) , for the preparation of medicaments for the prevention and/or the treatment of reactive gliosis.
• Object of the invention is also a therapeutic method comprising the administration, at pharmaceutically effective doses, of NGF or its functionally active derivatives or fragments, i.e. NGF agonists or partial agonists, or e# substances mimicking its function (agonists) , in or nearby the lesion.
• FIGURE Ia represents three histopathologic specimens of spinal cord removed from animals made neuropathic by sciatic nerve constriction and treated, in the left-to- right order, as disclosed in example 2, with NGF, ACSF, and, lastly, healthy control animals, on which analysis of the expression of the reactive gliosis marker, GFAP, was carried out.
• the histogram reported below represents the respective quantitation of marker GFAP determination; values for the NGF-treated animal are similar to values measured on the healthy control animals, indicating that NGF inhibits the appearance of reactive gliosis in the animals, carrying the sciatic nerve constriction only.
• FIGURE Ib represents three histopathologic specimens of spinal cord removed from animals made neuropathic by sciatic nerve constriction, treated, in the left-to-right order, as described in example 2, with NGF, ACSF, and, lastly, healthy control animals on which it was carried out an analysis of the expression of the marker representative of the number of astrocytes, SlOO ⁇ .
• NGF or its fragments or derivatives pharmacologically active as NGF agonists can be used for the regression and/or prevention of reactive gliosis and for the preparation of medicaments suitable therefor. Since reactive gliosis is a phenomenon preceding the onset of disease, NGF can be used for the prevention of the onset of pathologies that follow gliosis. Such pathologies comprise: stroke, injuries from trauma, growth of a tumor or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, triplet diseases, amyotrophic lateral sclerosis, multiple sclerosis and neuropathic pain.
• pathologies comprise: stroke, injuries from trauma, growth of a tumor or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, triplet diseases, amyotrophic lateral sclerosis, multiple sclerosis and neuropathic pain.
• NGF can be used also for the cure of said manifestations. It can be used for human or animal use.
• functionally equivalent derivatives it is meant molecules capable of promoting at least one of the biological responses commonly associated with NGF.
• NGF agonists capable of supporting at least one of the biochemical or morphological NGF responses, such as interaction with its own receptors, and/or activation of TrkA receptor (auto- phosphorylation) , neuronal survival, or neurite outgrowth
• NGF agonists capable of supporting at least one of the biochemical or morphological NGF responses, such as interaction with its own receptors, and/or activation of TrkA receptor (auto- phosphorylation) , neuronal survival, or neurite outgrowth
• functionally equivalent it is meant NGF fragments or derivatives maintaining the physiological function of NGF and that therefore might replace the native molecule.
• the beta rhNGF disclosed in Pat. Appln. PCT/IB2005/053159 having the following characteristics.
• the beta rhNGF disclosed in said patent application, and suitable for the carrying out of the invention exhibits biological activities comparable to those exhibited by equal doses of 2.5S mNGF at least in the following tests: a. evaluation of PC12 pheochromocytoma cells differentiation into sympathetic-like neurons induced by incubation with the beta rhNGF of the invention, as compared to incubation with equal amounts of 2.5S mNGF; b.
• DRG dorsal root ganglia
• Said biological activities shall be higher than 76% of those given by equal doses of 2.5S mNGF in all the aforementioned tests.
• said activity will be advantageously comprised between 80 and 100% of the 2.5S mNGH used as reference.
• said activity will be comprised between 90 and 100% in all the tests described above.
• said tests can be carried out according to all the modes known to a person skilled in the art, provided that they are always performed by using equal amounts and/or concentrations (for example expressed as Molarity) of the beta rhNGF of the invention and 2.5S mNGF.
• the beta rhNGF having the aforementioned properties and suitable for the carrying out of the present invention can be prepared according to a process comprising the following steps: i) the construction of an expression vector suitable for expression in mammalian cells and comprising a cDNA sequence encoding the exon 3 of the human NGF gene, said cDNA sequence including a sequence encoding the beta chain of mature human NGF (120 aa) , a sequence encoding the prosequence of the beta chain of human NGF (103 aa) and a sequence encoding the signal sequence of the beta chain of human NGF (18 aa) ; ii) the transformation of mammalian cells with said vector; iii) the selection of cellular clones able to secrete beta rhNGF having biological activities higher than 76% of those given by the 2.5S subunit of the native murine NGF in the following tests: a.
• rhNGF recombinant human Nerve Growth Factor
• mNGF murine Nerve Growth Factor (murine 2.5S NGF or beta-NGF)
• rhNGF recombinant human Nerve Growth Factor
• mNGF murine Nerve Growth Factor (murine 2.5S NGF or beta-NGF)
• rhNGF recombinant human Nerve Growth Factor
• mNGF murine Nerve Growth Factor (murine 2.5S NGF or beta-NGF) ;
• TrkA high affinity NGF receptor
• DRG Dorsal Root Ganglia
• SCG Superior Cervical Ganglion
• SP Substance P.
• Sympathetic-like neurons cells exhibiting some characteristics of sympathetic neurons, such as neurite processes similar to those of the sympathetic neurons and some neurotransmitters like dopamine and norepinephrine.
• the biological activity in vitro can be analyzed by using PC12 pheochromocytoma cells (Greene LA & Tischler
• Said cells represent the in vitro neuronal system generally used to analyze the NGF signal transduction and the biochemical and morphological responses induced by this neurotrophic factor.
• DRG dorsal root ganglia
• paravertebral sympathetic ganglia prepared from 10, 11 or 12 days old chick embryos.
• the test at point a. may be carried out by- evaluating the differentiation of the aforementioned PC12 cells into sympathetic-like neurons with formation of long neurite processes. Differentiation can be expressed, for example, as percentage of the number of cells with neurite processes within a certain interval of time, or in terms of effectiveness on differentiation always within a certain time, as compared to a control system.
• the activity of the 2.5S mNGF in the same experimental conditions was used as positive control, i.e. maximum NGF activity equal to 100%.
• the differentiation response can be induced after incubation of cells with concentrations of beta rhNGF, or 2.5S mNGF, comprised between 1 and 100 ng/ml, in particular, between 5 and 20 ng/ml, and can be observed after an incubation time between 8 and 48 hours, in particular for a time comprised between 16 and 24 hours.
• the test at point b.
• DRG dorsal root ganglia
• the test at point c. could be carried out by evaluating the phosphorylation of the high affinity TrkA receptor by immunoprecipitation experiments.
• the peak of receptor activation can be observed after short times between 1 and 10 min, but in particular after 5 min, and with concentrations of rhNGF, or 2.5S mNGF, comprised between 1 and 100 ng/ml, for example concentrations of 5- 10 ng/ml could be used.
• the biological activity of the rhNGF at issue may be analyzed both in the conditioned culture medium of the rhNGF-producing cells properly diluted in the culture medium of PC12 cells or in ganglia to give the desired final concentrations. It can also be analyzed in the medium of the middle/large scale production systems in order to verify that the biological activity of the rhNGF produced in said systems is comparable, according to the indications given above, to those of the 2.5S mNGF.
• Production systems suitable for middle or large scale can be, for example, commercially available production systems like MiniPERM® (Greiner Bio-One, Germany) , Roller Bottles or any other bioreactor system known to the technician of the field for middle or large scale production of recombinant proteins in mammalian cells growing in adhesion.
• mice For the assay at point d. regarding the in vivo biological activity, newborn mice can be used. After injection of equal doses of beta rhNGF or 2.5S mNGF, usually about 5 ⁇ g/gr of body weight for 5 consecutive days in parallel experiments, the biological activity of the recombinant neurotrophin produced on: superior cervical ganglia (hypertrophy) and cutaneous tissue at the injection site (mast cell activation and regulation of Substance P and TrkA levels) can then be evaluated.
• a histological analysis can be carried out, comparing the effect of the beta rhNGF used to that of the murine neurotrophin by standard techniques for preparation fixing and staining, well known to the technician of the field, such as for example toluidine blue staining.
• the cDNA of interest may be cloned by means of standard PCR techniques using, for example, primers that can be obtained with standard programs, able to amplify the region of interest (exon 3) , using the published human NGF sequences to design the primers. Then, the cDNA of interest (exon 3 of the human NGF gene, published in the literature, e.g., Ullrich et al .
• the vector may be any vector known in the literature and/or commercially available able to express the inserted protein in mammalian cells.
• the pTRE vector TetOff System, Clontech
• any other vector comprising a strong inducible promoter such as for example the vectors of the pT-REx series (Invitrogen) .
• the choice of a vector containing a strong promoter, such as for example the CMV promoter offers the advantage of guaranteeing high production of the protein of interest in eukaryotic cells.
• a tetracycline- regulated vector guarantees maximum expression levels, much higher than those that can be obtained with a vector containing a constitutive CMV promoter.
• the pTRE vector (TetOff System, Clontech) contains, upstream of the minimal CMV promoter, seven repeats of a tetO sequence for binding of the regulatory protein tTA encoded by the regulatory pTet-Off plasmid (Gossen M & Bujard H, Proc. Natl. Acad. Sci . USA 89: 5547-5551, 1992) .
• This regulatory system ensures expression levels of the recombinant protein even higher than o'ther inducible expression systems containing, beside the promoter, enhancer regions that are responsive to heavy metals or steroid hormones .
• vectors suitable for expression in mammalian cells include the RheoSwitch system (NewEngland BioLabs), macrolide-inducible vectors, such as pTRIDENT, pDuoRex, pMFl89, pMF229 (Weber W et al., Biotechnol. Bioeng. 80: 691, 2002), ecdysone-inducible vectors such as the pEGSH (Stratagene) .
• RheoSwitch system NewEngland BioLabs
• macrolide-inducible vectors such as pTRIDENT, pDuoRex, pMFl89, pMF229 (Weber W et al., Biotechnol. Bioeng. 80: 691, 2002
• ecdysone-inducible vectors such as the pEGSH (Stratagene) .
• the amplified construct described above can be subcloned in a pTRE vector, (TetOff System, Clontech) , downstream of the pCMV promoter present in the commercial vector, thus generating the pTRE-hNGF construct .
• pTRE vector TetOff System, Clontech
• the mammalian cells for the cloning can belong to any mammalian cell line, known to the skilled person, suitable for production of human proteins.
• mammalian cell line known to the skilled person, suitable for production of human proteins.
• these merely by way of a non-limiting example, are the HeLa, MEF, CHO, COS, BHK, HEK293, VERO cells, W138 and MDCK cell lines, or L929 fibroblasts, 3T3 fibroblasts, or other stabilized mammalian cell lines.
• the cells shall be genetically modified to constitutively express, besides the plasmid vector comprising the human NGF cDNA, also the regulatory protein required by the inducible system of choice.
• Transformation of the mammalian cells with the appropriate expression vector as indicated above can be carried out by any one of the transformation methods known to the technician of the field, such as for example, electroporation, transfection by calcium- phosphate precipitation or liposomal complexes.
• the selection of suitable cells could be performed by verifying the abundant presence of beta rhNGF in the culture medium, and by analyzing said beta rhNGF with the tests indicated above.
• the clones obtained coul be selected depending on the properties of the beta rhNGF produced and their ability to secrete said beta rhNGF.
• the beta rhNGF described in the aforementioned patent could be recovered, according to the production process of the invention, directly from the cell culture medium without the need of extraction from cells and thus greatly limiting the likelihood of contamination of the protein with cellular materials, such as, for example, unprocessed forms of NGF.
• the protein so obtained can be, when required, purified by means of standard techniques known to the expert of the field.
• NGF derivative or fragment there can be used, e.g., the peptides disclosed in European Patent Appln. No. 05012643.2.
• Said peptides having a structure characterized by the presence of two loops constrained in cyclic structure by the presence of covalent bonds between amino acid side chains, wherein the amino acid sequences of the first and second loop of said molecules are, respectively, substantially homologue to those of loop 1 (loop 1, residues 29-38) and of loop 4 (loop 4, residues 92 and 97) of NGF.
• Said peptides can be represented by formula
• AAl and AA2 are selected from the group comprising Cys, Asp, GIu, Lys, Orn, Pen or Dap and are linked through an amide or S-S bond between chemical functions on their side chains;
• AA3 and AA4 are selected from the group comprising Cys, Asp, GIu, Lys, Orn, Pen or Dap and are linked through an amide or S-S bond between chemical functions on their side chains;
• L is a linker sequence formed by 2-4 amino acid residues, an organic linker or a mixed sequence formed by amino acid residues and organic linker;
• (Yaa)m is an amino acid sequence formed by 4 - 8 residues
• (Zaa)m is an amino acid sequence formed by 4 - 8 residues
• (Xaa)n is an amino acid sequence in which n is an integer between 0 and 22.
• L may be the TGA sequence.
• An example of a suitable organic linker is the di-oxoethylene group.
• (Yaa)m may be a sequence homologue to that of loop 1 (residues 29-38) of NGF, like, e.g., the sequence TDIKGK.
• (Zaa)m may be a sequence homologue to that of loop 4 (residues 92-97) of NGF, like, e.g., the sequence DGKQ.
• (Xaa)n may represent a sequence homologue to that of N-terminus of NGF, e.g. the sequence HPIFHRGEFSVADSVSVWVGD .
• the aforeindicated peptides may be free or acetylated in the N-terminal position, they may be in carboxylic free form or in amide form in the C-terminal position; one, two or more amino acid residues may be added on the C-terminal end.
• the position of the first and the second loop along the polypeptide chain can also be reversed.
• Said peptides may also be a dimeric form of those described in the above formula. In this case, a covalent bond is present between the monomeric peptides .
• NGF or a fragment or derivative thereof having NGF agonist function is formulated in a composition suitable for administration in the compartment or in the neighbourhood of the glial compartment of interest.
• NGF may be administered topically, subcutaneously, intravenously, intraventricularly, intrathecally, intranasally, by aerosol or ophthalmic eyewash for CNS- involving glioses, as it has been demonstrated • that NGF administered by aerosol is able to pass through the hematoencephalic barrier, by liposomes, or even by gene therapy by the insertion of suitable molecules transformed so as to express said molecule. NGF may also be administered conjugated to antibodies such as an antibody directed against the transferrin receptor (OX- 26) abundantly expressed on the hemato-encephalic barrier and capable of mediating its transit.
• OX- 26 transferrin receptor
• the NGF or its derivatives indicated above could be prepared by standard formulations ' known to the technician of the field at pharmacologically effective dosages with suitable excipients.
• fibroblasts such as fibroblasts, stem cells and other suitable cells genetically modified in order to express NGF.
• NGF fibroblasts
• TetOff system tetracycline-regulated promoter
• NGF-containing liposomes As those described by Xie et al . 2005, (J. Control release 105.1-2, pplO6-119) . Administration should be performed so as to provide a pharmacologically effective dose of the molecule.
• a weight-proportional dose can be hypothesized, owing to the diversity of drug volume of distribution between animal and human. Moreover, dosage also varies depending on the administration means used, since pharmacokinetics (drug metabolization) varies.
• Example 1 Evidence of presence of reactive gliosis resulting in peripheral neuropathy in rats.
• Presence of reactive gliosis was measured by evaluating in situ GFAP expression.
• the deeply anesthesized animals were perfused cardially with saline solution (0.1M TRIS HCl, 10 mM EDTA) followed by 4% paraformaldehyde added to 0.1% glutaraldehyde in 0.01 M phosphate-buffer (PB), pH 7.4 at 4°C.
• PB phosphate-buffer
• Removed spinal cords were post-fixed two hours in the same fixative, balanced in 30% sucrose PBS and cryoconserved in chilled isopentane on dry ice. 25- ⁇ m thick serial sections obtained at the cryostat were first treated with 10% ' normal serum in PB added with 0.25% Triton® for Ih. Then, the sections were reacted with an anti-GFAP antibody
• Densitometric analysis of GFAP expression in the dorsal horn of the spinal cord was accomplished by using a computer-assisted image analysis system (MCID 7.0; Imaging Res. Inc, Canada). The value expressed the total target area as relative percent to the scanned area. The averages were obtained from five randomly selected spinal cord sections for each animal, and compared with the values of control groups. The data were exported and converted to a frequency distribution histogram using the Sigma-Plot 8.0 program (SPSS Erkrath Germany) . The NGF group was compared to the control group by a paired t-test .
• Example 2 Reactive gliosis presence, or lack thereof, in peripherally neuropathic rats treated with NGF or ACSF only.
• osmotic system Alzet 2001® (Alza Corp., Cupertino, CA, USA) .
• the system held a solution of artificial cerebral spinal fluid (ACSF) , as vehicle containing 1 mg/ml rat serum albumin and rat recombinant beta-NGF at a concentration of 125 ng/ ⁇ l.
• the osmotic pump released the content at a rate of 1 ⁇ l/h for 7 days.
• an intrathecal infusion equal to 125 ⁇ g/h was produced.
• the system was attached to a PElO catheter, the catheter was inserted into the subarachnoid space, to the lumbar enlargement.
• ACSF group rats There were treated also rats in parallel with the same system, but with administration of ACSF only, called ACSF group rats.
• gliosis reached its maximum expression in control animals only (ACSF group) .
• rats belonging to the ACSF group showed, in the dorsal horn of the spinal cord, a marked gliosis as expressed by an intense reaction for GFAP (2.345 ⁇ 0.16) when compared to the modest reaction found in NGF group rats (1.365 + 0.16) therefore not showing any difference with a CTR group of control rats (1.44 ⁇ 0,15) .
• the marked gliosis found in the dorsal horn of the spinal cord of the ACSF group is due to a transformation of the astrocytes from the protoplasmic to the fibrillary type, a reaction due more to the hypertrophy of single cells rather than to a significant increase of the total number of glial cells, as showed by the analysis of SlOO ⁇ expression (yielded data are reported in FIGURE Ib) .
• the SlOO ⁇ expression is only slightly higher compared to that of the group treated with NGF and the CTR group (1.945 ⁇ 0.15 and 1.82 ⁇ 0.23 and 1.56 ⁇ 0.2, respectively).

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