EP0566630A1

EP0566630A1 - Process for the synthesis and purification of human ciliary neuronotrophic factor (cntf) from escherichia coli

Info

Publication number: EP0566630A1
Application number: EP92902811A
Authority: EP
Inventors: Francesco Della Valle; Lanfranco Callegaro; Alessandro Negro
Original assignee: Fidia SpA
Current assignee: Fidia SpA
Priority date: 1991-01-21
Filing date: 1992-01-21
Publication date: 1993-10-27
Also published as: WO1992013072A2; ITPD910013A1; ITPD910013A0; IT1247150B; AU1165692A; WO1992013072A3

Abstract

Est décrit un procédé pour la synthèse et la purification du facteur neuronotrophique ciliaire (CNTF) humain par l'emploi de cellules transformées renfermant un vecteur d'expression constitué du gène pour le CNTF humain.A process for the synthesis and purification of human ciliary neuronotrophic factor (CNTF) is described by the use of transformed cells containing an expression vector consisting of the gene for human CNTF.

Description

PROCESS FOR THE SYNTHESIS AND PURIFICATION OF

HUMAN CILIARY NEURONOTROPHIC FACTOR (CNTF)

FROM ESCHERICHIA COLI

FIELD OF THE INVENTION The present invention concerns a process for the synthesis and purification of human ciliary neuronotrophic factor (CNTF) from Escherichia coli by the application of recombinant DNA technology. In this way it is possible to obtain sufficient quantities of homogeneous protein to permit experiments in clinical trials with the CNTF by itself, with derivatives thereof, as well as each in association with other therapeutic materials.

BACKGROUND OF THE INVENTION A. THE CILIARY NEURONOTROPHIC FACTOR

The ciliary ganglion (CG) contains two populations of neurons, ciliary and choroid, both of them cholinergic. CG neurons innervate the intrinsic muscle structures of the eye in the choroid, the ciliary body and the iris. During embryonic development in chicks, about half the CG neurons die between days E8 and E15 when their axons establish connection with the intraocular innervation territory. This neuronal death is strongly favored by removal of the eye, while it is significantly impeded by the implantation of a supplementary eye bud. On the basis of this observation it has been hypothesized that the innervation territories contain trophic factors for their own specific neurons. Extracts from various chick embryonic tissues at day E8 have proved to have trophic activity on neurons dissociated from the ciliary ganglia of chick embryos at the same stage of development (Adler, R. et al. 1979, Science 204, 1434-1436) .

This trophic activity was subsequently named ciliary neuronotrophic factor (CNTF) (Barbin G. et al 1984, J. Neurochem. 43, 1468-1478).

A third of all the CNTF trophic activity in the embryo is found in the eye and most of this was localized in the choroid and in the intrinsic muscle structure of the eye.

Neurons dissociated from chick embryo ciliary ganglia at day E8, seeded on a suitable substrate and with a suitable culture medium, do not survive unless a special supplement is added to the medium, making a model for biological tests for ciliary neuronotrophic factors in general, and in particular for those of a proteic nature. When various extracts of chick embryo tissue at day E8 are compared in this regard, a third of the total CNTF activity of the embryo is found in the eye, and three quarters of the activity of the eye itself is localized in a portion of the choroid, the iris and the ciliary body (intrinsic muscle structure of the eye) as well as the pigmented epithelium (CIEP) . Assessments of survival showed that a soluble protein, trophically active for CG neurons, (i) is found at high concentrations in the eye territory which they innervate, and (ii) reaches its highest specific activity between E8-E15, the very developmental stage at which the fate of these neurons is decided in vivo . The purification of E15 chick embryonic CNTF involves the selective subdissection of CIEP tissues and also sequential treatment of the relative extract by ion exchange chromatography, ultracentrifugation in sucrose gradient, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) .

Purification of this protein by ion exchange chromatography, ultracentrifugation in a sucrose gradient and SDS-PAGE under reducing conditions revealed a molecular weight of approximately 21Kd and a net negative charge. The trophic activity exercised by the purified protein on chick CG neurons at stage E8, on chick DRG neurons at stage E10 and on sympathetic neurons at stage Ell is in the order of 10"¹¹ 10"^I2M and therefore of the same order as another neuronotrophic factor, namely Nerve Growth Factor (NGF) , purified from submaxillary glands of male mice, with regard to its DRG and sympathetic neuronal targets.

Chick embryo eye extract, like the CIEP extract, has a trophic activity (i) on E10 (but not E8) chick embryonic DRG neurons and newborn mouse neurons, and (ii) on sympathetic neurons from Ell chick and newborn rat ganglia - not surprisingly, considering that the innervation territories of the sensitive and sympathetic neurons are partly the same as those of the CG neurons. DRG and sympathetic neurons on the other hand are sensitive to the trophic effect of purified chick eye CNTF, despite the fact that CNTF was chosen for its trophic activity on CG neurons.

Like NGF, CNTF supports the growth of adrenal chromaffin cells in culture although these two factors differ in the extent of their effects on tyrosine hydroxylase and phenylethanolamine N-methyltransferase enzymes. CNTF enhances CAT activity in chick retinal cell cultures, indicating a response of cholinergic neurons. CNTF has no trophic effects on cholinergic neurons of the rat embryonic basal forebrain or pontine region of the brain and cannot be considered a cholinergic neuronotrophic factor. Very recent

ERSATZBLATT - -

data indicate that the CNTF's action is not restricted to neuronal cells. CNTF has been found to induce the differentiation of glial progenitor cells of the rat optic nerve into type-2 astrocytes.

Among CNTF's other activities is one linked with the prevention of motoneuron degeneration following axotomy (Sendtner M. et al. 1990, Nature 345, 440-441).

To summarize, this new factor is represented by a protein which is active on at least three types of neuron, two of which are also sensitive to NGF. Bearing in mind its molecular weight, chick eye CNTF promotes half the maximum survival of CG neurons of E8 chick, DRG neurons of E10 chick and sympathetic neurons of Ell chick at a concentration of 10"^u—10"¹²M, with a specific activity of the same order as that of purified NGF from mouse submaxillary gland on its neuronal targets, DRG and sympathetic. None of the trophic activities of chick eye CNTF is blocked or inhibited by antibodies directed against beta subunit of NGF from mouse submaxillary gland.

Other Sources of CNTF

Trophic factors for CG neurons, as for DRG and sympathetic neurons, have been obtained from various other sources, ranging from tissue extracts and lesion fluids to media conditioned by various cell cultures. "Conditioned culture media" have an advantage over tissue extracts, namely that of facilitating the identification of "cell sources." On the other hand, due to the low concentrations of trophic activity which can be obtained from them, these conditioned media are not very suitable for the preparation of purified proteins with trophic activity. Indeed, conditioned media from heart muscle cultures were the first materials (closely followed by skeletal muscle cultures) in which the presence of CNTF was demonstrated. Bovine heart extracts have also

ERSATZBLAT been considered as a possible starting material, but the complete characterization of their CNTF has proven impossible. The presence of a CNTF in the heart and skeletal muscle leads to the assumption that it may be active on viscero- and somato-motor cholinergic neurons other than those (CG) which innervate the intrinsic muscles of the eye. However, a systematic comparison between CNTF of the eye, heart and skeletal muscles has not yet been carried out, either with regard to their molecular characteristics or range of neuronal targets.

Conditioned media from neuroglial cell cultures, both peripheral (Schwann) and central (astroglia) , also show trophic activities, different from those of NGF, on ciliary neurons and those of the dorsal and sympathetic root ganglia, decidedly favoring the hypothesis of a possible role of the neuroglia as a source of neuronotrophic factors in vivo .

It has also been demonstrated that extracts from adult rat sciatic nerve, unlike those of CNS tissues, have a very high CNTF content, with a specific activity equal to that of CIEP from E15 chick eye (about 20,000 trophic units/mg of protein) . CNTF's activity is also high in extracts from nerves or from pure motor roots or pure sensory roots, demonstrating that no differential criterion can justify correlating the activity of the nerve with the specific innervation territories, rather than with the Schwann cells contained within its structure, or together with them.

Manthorpe and co-workers (Manthorpe et al.. Ciliary Neuronotrophic Factors in Nerve Growth Factors, R.A. Rush Ed. , Ibro Handbook Series: Methods in the Neurosciences, Vol. 12, pp. 31-56) purified rat nerve CNTF at an analytical level, revealing a slightly inferior negative charge and a slightly higher molecular weight (24 Kd) than those of CNTF purified from chick eye.

CNTF has now also been isolated from rat sciatic nerve, bovine heart and neuroblastoma cells. Williams et al. (1984,

ERSATZBLATT Int. J. Dev. Neurosci. 2, 177-180) demonstrated a high CNTF content in the sciatic nerve of adult rats, with a specific activity equal to that of chick (20,000 TU/mg protein).

Two forms of CNTF have been identified, one with a molecular weight of about 25 Kd and another of about 28 Kd. By homogenous purification of quantities of CNTF from rabbit and rat sciatic nerve it has been possible to partially identify its amino acid sequence (Lin L-F, H. , J. Bio. Chem. , Vol. 265, No. 15, pp. 8942-8947, 1990). With this knowledge and by using the PCR (Polymerase Chain Reaction) technique the complete gene segment encoding the CNTF of these two animals was isolated (Stδckli et al., Nature, Vol. 342, pp. 920-923, 1989, Lin L-F.H. et al. , Science. Vol. 246, pp. 1023-1025, 1989). The molecular weight of the protein was 22.7 and 22.8 Kd, respectively, with an isoelectric point of 5.78 in the first case, as for the purified proteins.

Comparative analysis of the CNTF sequences of rat and rabbit has shown them to be highly homologous both in amino acid and nucleotide sequences. Some of the better preserved domains of this molecule have been successfully determined by computer analysis, allowing the identification and isolation of a significant portion of the human gene segment, corresponding to 80% of the complete sequence, and using the PCR technique some oligonucleotides were synthesized in these very domains. This portion of the gene sequence and the corresponding amino acid sequence are described in patent application Nos. 41557A/90 and 41655A/90.

Conventional techniques to isolate biologically active proteins include the isolation of the protein itself by purification from biological fluids or tissues, but the quantities of protein thus obtainable are not always sufficient to allow a study of their structures, functions and, above all, applications. The case of CNTF is one such example, indeed, only a small quantity of rat, rabbit, bovine and chick CNTF has been isolated to date and the human protein has been isolated from neuroblastoma cells.

B. RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology makes it possible to construct series of vectors able to express large quantities of proteins and allows molecular biologists to assemble DNA sequences to create hybrid molecules capable of producing the protein of interest. Various reactions are used, such as cutting with restriction enzymes, joining the fragments thus obtained with ligase, the chemical synthesis of oligonucleotides to be assembled and other methodologies devised at various laboratories working in this field (Maniatis, T. et al.. Molecular Cloninσ. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Laboratory NY, 1982) .

To obtain high levels of expression, the DNA elements to be assembled must present certain essential information. For example, a replication origin, a selection for antibiotics, an expression promoter, activators of the transcription of the gene of interest and other characteristics known to the expert in the art. Suitable combination of these elements results in a plasmid, if the gene of interest is inserted naturally with respect to the regulatory sequences of the transcription and translation and the resulting plasmid is called an expression plasmid. The expression plasmid or vector is thus able to express the protein in host cells, which may be of eukaryotic or prokaryotic origin. The protein can therefore be obtained by purification.

The elements (promoters) which naturally control the expression of many genes such as growth factors, are not strong in their expression and are activated only in suitable natural conditions which are often unknown. To this end, promoters with known activity are used, such as viruses of the Papovavirus series, or other known promoting gene

ERSATZBLATT sequences. The elements used for high levels of expression are therefore a combination of DNA of various origin (eukaryotic, bacterial, viral, etc.) constituted in the terminal part by different gene portions bound together to form a hybrid. The transcription activity of a gene depends on the distances between the regulatory and encoding sequences.

Conventional techniques to obtain genes situated in the close vicinity of regulatory sequences depend on there being suitable restriction sites which allow their cloning. If no compatible sites are nearby, but only different sites, it is possible to obtain the union of the segments by synthesis of an oligonucleotide (linker) containing such a restriction site. This system is very limiting to the molecular biologist. An alternative strategy is to use the Polymerase Chain Reaction (PCR) technique (Saiki R. K. et al 1988, Science 239, 487-489). By this technique it is possible to amplify a gene segment up to l x 106. The principle is based on the use of two oligonucleotides, each of which can be exactly paired onto one of the DNA strands to be amplified. The distance between the two oligonucleotides with respect to the gene sequence gives the dimensions of the molecule to be produced. These two oligonucleotides are so constructed that there is a restriction site within their sequence which allows subsequent cloning. This restriction site is either naturally present or is constructed ad hoc by degenerating the minimum number of nucleotides. This approach, which can be defined as site-directed mutagenesis, allows restriction sites to be constructed in positions previously decided on by the molecular biologist. The construction of sites compatible with other gene segments not only facilitates cloning but also makes it possible to specifically join different gene segments. This technique can be defined as cloning by direct mutagenesis. By this technique, other vectors have been prepared to obtain, by recombinant DNA technology, other neuronotrophic factors, described in patent applications No.s 48564A/89 and 41538A/90.

SUMMARY OF THE INVENTION The present invention, therefore, relates to the synthesis by Escherichia coli cells and the purification of human CNTF in quantities large enough and homogenous enough as to permit the production of commercially useful and biologically active amounts of the CNTF protein to allow for in vivo and in vitro experiments and therapeutic use.14

The invention also relates to pharmaceutical compositions comprised of human CNTF, either alone or in combination with gangliosides (including naturally existing gangliosides, ganglioside derivatives, and semisynthetic analogues of gangliosides) , polysaccharides (including natural and chemically modified polysaccharides) , and/or other growth factors.

The invention further relates to the therapeutic use of human CNTF, and the above noted pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts plasmid pT7.7 containing the human CNTF gene.

Figure 2 discloses the activity of the recombinant

CNTF's ability to maintain chick embryo dorsal root ganglia neuronal cells at day E10 in culture.

DETAILED DESCRIPTION OF THE INVENTION The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description of the invention should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by - 10 - those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

The contents of each of the references and patent applications cited in the present application are herein incorporated by reference in their entirety.

General Procedures

Some reactions and procedures are well known to those skilled in the art and are described, for example, in Molecular Cloning, Sambrook J. et al. (1989) and Mantiatis, T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Laboratory, N.Y. (1982) .

Cutting and attachment of the DNA segments with restriction enzymes is done according to the manufacturer's instructions. Generally, 1 μg of plasmid was cut with 1 U of enzyme in 20 μl of solution; incubation temperature and time dependent on which enzyme is used, but are generally 1 hour at 37°C. After incubation, the plasmids and gene segments are purified in all cases in an Agarose gel, LMP Agarose (BRL - United States of America) in 40 mM Tris HC1, 20 mM sodium acetate, 1 mM EDTA and then eluted from the agarose with a GENECLEAN™ kit (BIO 101 Inc. , La Jolla, CA - USA) .

Ligation is performed with T4 ligase at a concentration of 1 U per 0.5 μg of DNA in a reaction of 20 μl at 13°C for 12 hours. Analyses to confirm the correct plasmid sequence are affected by transfecting into HB101 cells and the transformed cells selected in agarose plates in LB (Luria Bertani) medium with 50 μg/ml of the antibiotic ampicillin. The plasmids contained in the HB101 cells are grown in LB, 100 μg/ml of ampicillin, and are purified, for both small and large preparations, with a kit supplied by Quiagen (DIAGEN GmbH, DUsseldorf - Germany) . The cloning vectors are

ERS_ATZBLATT prepared from bacterial cells according to the instructions from Quiagen.

The DNA for PCR reactions is prepared from human placenta at term as follows. A 0.4 cm³ piece of chorionic villi is finely chopped with scissors and suspended in 700 μl Of 50 mM Tris/HCl pH 7.8, 100 mM EDTA, 100 mM NaCl, 1% SDS. To this is then added 35 μl of proteinase K (100 μg/ml) and the whole is incubated overnight at 55°C. 20 μl of a 13 μg/ml solution of RNAase A is then added and incubated for another 2 hours. This is followed by two extractions with phenol and two with chloroform. The DNA is then precipitated onto a fine glass tube by the addition of 1 volume of isopropanol. At this point it undergoes several passages with 70% and 100% ethanol and is then dried. The DNA is dissolved in buffer (10 mM Tris/HCl pH 7.4, 1 mM EDTA) , under gentle stirring. A few hours later the dissolved DNA is ready for gene amplification. 0.1 μg of DNA normally proves sufficient for PCR.

The DNA for Southern blot is prepared as described hereafter from human placenta at term. A 0.4 cm³ piece of chorionic villi is finely chopped with scissors and suspended in 700 μl of 50 mM Tris/HCl pH 7.8, 100 mM EDTA, 100 mM NaCl, 1% SDS. To this are then added 35 μl of proteinase K (100 μg/ml) and the whole is incubated overnight at 55^βC. To this are then added 20 μl of a 13 μg/ml solution of RNAase A and it is incubated for another 2 hours. This is followed by two extractions with phenol and two with chloroform. The DNA is then precipitated onto a fine glass tube by the addition of 1 volume of isopropanol. At this point it undergoes several passages with 70% and 100% ethanol% and is then dried.

The DNA is dissolved in buffer (10 mM Tris/HCl pH 7.4, 1 mM EDTA) , under gentle stirring. It is then digested with restriction enzymes at 37°C overnight. The fragments are then separated in a 0.8% Agarose gel in TAE (Tris Acetate

ERSATZBLATT EDTA) . The gel is washed twice in 0.1 M HC1 and 1.5 M NaCl, twice in 1M NaOH 1.5M NaCl and twice in 2M Tris pH 7.4 0.6 M NaCl then transferred overnight to nitrocellulose (Hyboud N Amersham) . The nitrocellulose filter is exposed to ultraviolet rays for 3 minutes then prehybridized in 50% Formamide, 5 x Denhardt's, 5XSSC, 1 mg/ml of DNA from salmon sperm and incubated at 42°C for 2 hours. Hybridization is effected overnight in 50% Formamide 5 x Denhardt's, 5 x SSC, 0.250 μg/ml of DNA from salmon sperm and with 10% Dextran Sulfate Sodium. The filter is washed in 0.1% SDS 2 x SSC for 2 hours at 65°C and then auto-radiographed.

All oligonucleotides are synthesized in solid phase using a 380B DNA Synthesizer (Applied Biosystems - USA) according to the manufacturer's instructions. They are treated at 55°C for 12 hours in NH₃ and then treated in a vacuum-speed. They are resuspended in 2.5 M ammonium acetate and then precipitated with 3 volumes of cold ethanol (-20°C) . They are rewashed with cold 80% ethanol and resuspended in water. The concentration of oligonucleotides is assessed by spectrophotometer.

Amplification was affected on a Perkin Elmer Cetus DNA Thermal Cycler and the reagents used for amplification were those of the DNA™ Amplifier (Perkin Elmer-Cetus) .

In short, a mixture containing 200 uM of each oligonucleotide was used, 0.5 uM of each of the nucleotides dATP, dTTP, dCTP, dGTP and 0.1 ug of human DNA and reaction buffer in a total mixture of 100 ul with 0.5 U of TAQ polymerase, the whole being then covered with liquid paraffin to prevent evaporation.

Prokarvotic Expression Vector for Human CNTF

Following the basic procedures set forth in patent application No. 41655A90 under the section "Prokaryotic expression vectors for human CNTF", certain modifications were made to increase transcription and synthesis of the protein of interest. In particular:

The cDNA of the human CNTF cloned in plasmid pGEM7 CNTFh between sites Smal and EcoRI was modified by PCR. Two oligonucleotides were synthesized. In the first (A) some bases were changed so that the codons encoding the amino acids were those preferred by E. coli and other bases were changed to take advantage of the stability of the messenger in relation to the Shine-Dalgarno region of the expression vector (A)

ACCATATGGCTTTTACTGAACATTCA The second oligonucleotide (B) with the following sequence:

(B)

AAGTCGACAGAGGGACTAACTGCTACAT contains the Sal I restriction site to facilitate subsequent cloning. The amplified product obtained by PCR with the oligonucleotides A and B which contains the human CNTF gene was cloned in plasmid pT7.7 between sites Nde I and Sal I (Fig. 1.)

The human CNTF is thus controlled by the promoter of the T7 phage RNA polymerase dependent promoter. his expression plasmid was transfected in various E. coli lines, including BL 21 (D3) , to obtain expression of the protein.

Bacterial Growth and Propagation

A colony of E. coli of BL21(D3) containing plasmid pT77RCNTF is inoculated into 5 ml of LB medium containing 200 ug/ml of ampicillin. The cells were grown overnight at 30°C. An aliquot was then diluted at a ratio of 1/100 in M9 containing 200 ug/ml of ampicillin. The cells were grown in fermentation to an absorbancy of 0.8 OD at 590nm.At this point 4mM isopropyl thiogalactoside (IPTG) was added. The cells were left to grow for another 3 hrs after which they were centrifuged and resuspended in Tris/HCL pH8.0 lOmM EDTA and were then ready for the subsequent extractions.

Purification Procedure

The pellet obtained after centrifugation was washed at least 3 times with 50mM Tris pH 7.4 and 50 mM NaCl (C) , the pellet being recovered each time by centrifugation at 600 rpm for 10 min.

The final pellet was resuspended in buffer and passed at least twice through a Montain Gaulin at 800 psi.

The material was centrifuged again at 600 rpm for 10 min and resuspended in 8 M Urea 50 mM Tris pH 7.4, 1 mM PMSF, 2 mM EDTA at room temperature.

After centrifugation at 600 rpm for 10 min. the soluble material was dialysed against 1 M Urea.

After centrifugation for 15 min at 600 rpm it was loaded into an ion exchange column, such as a Mono Q column.

In these conditions the recombinant protein was eluted at 100 mM NaCl.

The material obtained was dialysed against 50 mM Ammonium Acetate and loaded into an inverse phase column.

The column used was a Vydac C18 7um (0.46 x 26 cm) , the eluents were (A) 0.05% TFA in water, (B) 0.05% TFA in acetonitryl.

The column was equilibrated with A and 10% B and in these conditions the protein was loaded.

A gradient was thus obtained where the percentage of buffer (B) reached 60% in 60 min. , after which the column was washed with 100% (B) for 10 min.

The recombinant protein eluted from the column as a single peak at a percentage of about 80% of acetonitryl. Determination of the Amino Acid Sequence

The amino terminal sequence of recombinant CNTF was determined by Edman degradation using an Applied Biosystem automatic protein sequencer model 477A.

The phenylthiohydantoins derived from the amino acids were directly determined using a protein sequencer.

The sequence of the first 14 amino acids starting from the N-terminal amino acid was as follows: Ala-Phe-Thr-Glu-His-Ser-Pro-Leu-Thr-Leu-His-Arg-Arg-Asp.

This sequence is correlated with that of the gene for CNTF (patent application No. 41655A/90) , where Met amino acid, usually present in the polypeptides obtained by recombinant DNA techniques in E. coli, is absent because CNTF, as second amino acid, contains an Ala, and in 98% of such cases, the initial Met is removed. (Hivel P-H. et al 1989, Proc. Natl. Acad. Sci. USA, 86, 8247-8251).

Determination of the Biological Activity

The activity of the recombinant protein was assessed by CNTF's ability to a maintain chick embryo dorsal root ganglia neuronal cells at day E10 in culture. These cells were removed from the chick embryo at day E10, dissociated with trypsin and enriched by subsequent preplating steps.

The neurons were then seeded in 96-well tissue culture dishes, treated with polyornithine (100 ug/ml in 15 mM borate, pH 8.4) and laminin (10 ug/ml).

The neurons were seeded at a concentration of 4000 neurons per well, the culture medium was Dulbecco minimal essential medium (DMEM) with 100 ug/ml of penicillin, 2 mM L-glutamine (v/v) and 10% inactivated fetal calf serum.

The surviving neurons were counted after 24 hours in culture (Skaper S. D. et al 1985, Dev. Brain Res. 24, 39-46) (Fig. 2).

The result of this experiment was that the specific activity calculated as ED50 was 5 x 10-11M. Pharmaceutical Compositions

As discussed above, the present invention also relates to the therapeutic use of CNTF and its administration via pharmaceutical compositions containing the factor, either alone or together with other active substances. The CNTF is useful for the maintenance of or to prevent the loss of nervous function, and to treat the loss of nervous function due to acute or chronic pathological conditions, including the treatment of acute conditions such as cerebrovascular, infective, inflammatory, compressive and metabolic deficiencies, and chronic or neurodegenerative conditions. The CNTF is also useful for the treatment of neuropathological conditions caused by aging of the nervous system or diseases affecting the immune system.

The pharmaceutical preparations may contain, as the active substances, one or more combinations of the growth factor CNTF and a natural ganglioside (or ganglioside derivatives or semisynthetic analogues) or a salt or associations between these and other growth factors or polysaccharides (either natural, chemically modified or transformed into finished products such as biomaterials. Such pharmaceutical preparations can be for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection and can therefore be prepared as solutions of the active compounds or as freeze-dried powders of the active compounds to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids. For local use, those preparations in the form of creams or ointments for topical use or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays, for example nose sprays, should be considered.

The preparations of the invention can be intended for administration to humans or animals. They contain preferably between 0.01% and 10% of active component in the case of solutions, sprays, ointments and creams and between 1% and 100% and preferably between 5% and 50% of active compound in the case of solid form preparations. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration, but daily dosages to humans by subcutaneous, intramuscular or intracerebral injection generally vary between 0.05 mg and 5 mg of active substance per kg of body weight.

The invention also embraces the therapeutic use of all the new complexes of gangliosides or hyaluronic acid derivatives or such derivatives with the growth factor CNTF for the aforesaid indications.

The pharmaceutical compositions containing the human CNTF molecule derived from recombinant DNA, without and possibly also with gangliosides, phospholipids, hyaluronic acid, can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the CNTF molecule is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles and their formulation containing other proteins are described, for example, in "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations."

On this basis, the pharmaceutical formulations include, albeit not exclusively, solutions of the CNTF growth factor or its freeze-dried powder in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso- osmotic with the physiological fluids. In the case of dried preparations, supporting excipients are, for example, mannitol or glycin, and suitable buffered solutions of the desired volume can be supplied to obtain suitable isotonic buffered solutions with the desired pH. Similar solutions can be used for the pharmaceutical compositions of the CNTF molecule obtained from recombinant DNA in isotonic solutions of the desired volume, and include, but not exclusively, buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain isotonic pharmaceutical preparations of the desired pH, for example, neutral pH.

The pharmaceutical formulations may also include suppositories for rectal administration with lipophilic excipients, for example, hydrosoluble, self-emulsifying excipients such as glycogelatin or others. In these preparations, the CNTF growth factor obtained from recombinant DNA can be present in quantities varying between 0.01% and 1% in weight of the total excipient. The suppositories can contain, but are not limited to these, suitable quantities of acetylsalicylate.

For purely descriptive and not limiting purposes, we report hereafter some examples of pharmaceutical compositions prepared according to the present invention.

A) EXAMPLES OF INJECTABLE SOLUTIONS

Preparation No. 1 - one 2-ml vial contains:

Active substance μg 1 (3,200 BU)

Sodium chloride mg 16

Citrate buffer pH = 7 ml 2 in water for injection Preparation No. 2 - one 2-ml vial contains:

Active substance μg 1 (32,000 BU)

Sodium chloride mg 16

Citrate buffer pH = 7 ml 2 in water for injection

Preparation No. 3 - one 2-ml vial contains: Active substance Sodium chloride Gangliosides as sodium salts

Citrate buffer pH = 7 in water for injection

Preparation No. 4 - one 2-ml vial contains:

Active substance μg 10 (32,000 BU)

Sodium chloride mg 16

Gangliosides as sodium salts mg 50

Citrate buffer pH = 7 ml 2 in water for injection

Preparation No. 5 - one 2-ml vial contains:

Active substance

Sodium chloride

Monosialotetrahexosylganglioside (GM_t) sodium salts

Citrate buffer pH = 7 in water for injection

Preparation No. 6 - one 2-ml vial contains:

Active substance

Sodium chloride

Monosialotetrahexosylganglioside (GMj) sodium salts

Citrate buffer pH = 7 in water for injection Preparation No. 7 a) one 2-ml ampoule contains:

Freeze-dried active substance μg 4 (12,800 BU) Glycine mg 30 b) one 2-ml ampoule of solvent contains:

Sodium chloride mg 16

Citrate buffer in water ml 2 for injection

Preparation No. 8 a) one 2-ml vial contains:

Freeze-dried active substance μg 4 (12,800 BU) Mannitol mg 40 b) one 2-ml ampoule of solvent contains:

Sodium chloride mg 16

Citrate buffer in water ml 2 for injection

Preparation No. 9 a) one 3-ml vial contains:

Freeze-dried active substance μg 10 (32,000 BU) Glycine mg 45 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection

Preparation No. 10 a) one 3-ml vial contains:

Freeze-dried active substance μg 10 (32,000 BU) Gangliosides as sodium salts mg 100 Glycine mg 45 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection Preparation No. 11 a) one 3-ml vial contains:

Freeze-dried active substance μg 10 (32,000 BU) Gangliosides as sodium salts mg 50 Glycine mg 45 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection

Preparation No. 12 a) one 3-ml vial contains:

Freeze-dried active substance μg 1 (3,200 BU)

Monosialotetrahexosylganglioside

(GMj) sodium salts mg 100

Glycine mg 45 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection

Preparation No. 13 a) one 3-ml vial contains:

Freeze-dried active substance μg 10 (32,000 BU)

Monosialotetrahexosylganglioside

(GMj) sodium salts mg 100

Glycine mg 45 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection Preparation No. 14 a) one 3-ml vial contains: Freeze-dried active substance 3-sn-phosphatidyl L-serine Lecithin Mannitol b) one 4-ml ampoule of solvent contains: Mannitol mg 60 Water for injection to vol. ml 4

Preparation No. 15 a) one 3-ml ampoule contains:

Freeze-dried active substance μg 10 (32,000 BU) Mannitol mg 60 b) one 3-ml ampoule of solvent contains:

Sodium chloride mg 24

Citrate buffer in water ml 3 for injection

B) EXAMPLES FOR SUBCUTANEOUS INJECTION

Preparation No. 16 a) one 2-ml vial contains:

Freeze-dried active substance μg 5 (16,000 BU)

Mannitol mg 30 b) one 2-ml ampoule of solvent contains:

Sodium chloride mg 16

Citrate buffer in water ml 2 for injection C) EXAMPLES OF SUPPOSITORIES FOR THE RECTAL ROUTE

Preparation No. 17

Active substance μg 10 (32,000 BU)

Cocoa butter mg 2.5

Preparation No. 18

Active substance μg 10 (32,000 BU)

Carbowax 1540 g 1.75

Carbowax 6000 g 0.75

Preparation No. 19

Active substance μg 10 (32,000 BU)

Tween 61 g 2.125

Lanolin g 0.25

Preparation No. 20

Active substance μg 10 (32,000 BU)

Glycerine g 1.5

Water g 0.25

Gelatine g 0.25

The invention being thus described, it is clear that these methods can be modified in various ways. Such modifications are not to be considered divergences from the spirit and purpose of the invention, and any modification which would be evident to an expert in the field comes within the scope of the following claims.

Claims

1. A process for the synthesis and purification of the human ciliary neuronotropic factor (CNTF) which comprises culturing a bacterial cell containing an expression vector containing the gene for CNTF, whereby CNTF is produced.

2. A process according to claim 1, wherein the bacterial cell is Escherichia coli , BL,21(D3) strain.

3. A process according to claim 1, wherein the CNTF that is produced is recovered.

4. A process according to claim 3, wherein the CNTF is recovered in the form of inclusion bodies.

5. A process according to claim 4, wherein the inclusion bodies containing CNTF are purified.

6. A process according to claim 5, wherein the inclusion bodies are treated by solubilizing the human CNTF therein, thereby releasing the CNTF.

7. A process according to claim 6, wherein the solubilized human CNTF is purified by treatment with an ion exchange resin.

8. A process according to claim 1 , wherein the CNTF obtained from the ion exchange resin treatment is further treated in a reverse-phase column.

9. A process according to claim 1, wherein the CNTF has the N-terminal amino acid sequence Ala-Phe-Thr-Glu-His-

Ser-Pro-Leu-Thr-Leu-His-Arg-Arg-Asp.

*

10. A process according to claim 8, wherein the CNTF has the N-terminal amino acid sequence

Ala-Phe-Thr-Glu-His-Ser-Pro-Leu-Thr-Leu- His-Arg-Arg-Asp.

11. A process according to claim 1, wherein the CNTF produced is not fused to other amino acids.

12. A process according to claim 1, wherein the CNTF is produced free from contaminating human proteins.

13. A pharmaceutical preparation containing as an active substance a complex between the CNTF growth factor and a natural ganglioside or one of its derivatives or semisynthetic analogues or one of its salts.

14. A pharmaceutical preparation containing as an active substance a complex between the CNTF growth factor and an acidic polysaccharide or its derivative or semisynthetic analogue.

15. A pharmaceutical preparation according to claim 14, wherein the polysaccaride is hyaluronic acid.

16. A pharmaceutical preparation according to claim 14, wherein the CNTF is compounded into a biomaterial.

17. A pharmaceutical preparation according to claim 16, wherein the biomaterial is in the form of microspheres or nanospheres.

18. A pharmaceutical preparation according to claim 16, wherein the biomaterial is in the form of membranes or films.

19. A pharmaceutical preparation according to claim 16, wherein the biomaterial is in the form of tubes or guides.

20. A pharmaceutical preparation according to any one of claims 13 to 19 for administering by the parenteral route.

21. A pharmaceutical preparation according to any one of claims 13 to 19 for administering by the intracerebral route.

22. A pharmaceutical preparation according to any one of claims 13 to 19 for administering by the oral or rectal route.

23. A pharmaceutical preparation according to any one of claims 13 to 19 for administering topically or by the intradermal route.

24. A pharmaceutical preparation according to any one of claims 13 to 19 for administering by subcutaneous route.

25. A pharmaceutical preparation according to any one of claims 13 to 19 for administering by inhalation.

26. Escherichia coli BL,21(D3) strain.