WO2006082609A1 - Human ngf-like protein isolated from eisenia foetida an uses thereof - Google Patents

Abstract

The invention refers to a novel protein identified in Eisenia foetida having homology from 98 to 100 % with β subunit of the huma mature nerve growth factor (NGF), isolation method and uses thereof.

Description

HUMAN NGF-LIKE PROTEIN ISOLATED FROM EISENIA FOETIDA AND USES THEREOF

*_****_***********_* The present invention relates to an eisenia foetida isolated human NGF-like protein and uses thereof.

Nerve growth factor (NGF) belongs to the family of neurotrophins, growth factors playing a key role in the neuronal development in mammalians, identified only in vertebrates but thought to be essential in the evolution of quite complex nervous systems of several invertebrates, particularly invertebrate species more long-lived than well known Caenorhabditis elegans and Drosophila melanogaster (Jaaro H. et al. 2001).

NGF consists of three subunits named α, β e γ; particularly NGF biological activity was attributed to β subunit, defined 2,5S NGF to indicate β subunits isolated from mouse salivary glands by using a method which can cause limited proteolytic processes within terminal portion of the molecule, or β-NGF or simply NGF. First the factor was isolated from mouse connective tumour tissue, subsequently from snake venom and finally from adult male mouse salivary glands which up now are the main extraction source of the factor. High, i.e. from 87 to 90 %, homology between NGF coding human and murine gene, allowed to begin experimental pre-clinical and clinical studies about NGF against various human pathologies wherein it is involved. Several studies carried out in the last fifty years provided evidence of NGF pleiotropicity which is not synthesized only by neurons (key role for the growth and differentiation of certain neuronal cell populations) but also by endocrine and immune system cells. Inevitably these facts lead to experiment various uses in order to treat central and peripheral nervous system (degenerative diseases, like Alzheimer or Parkinson, neuropathies), ocular (corneal neurotophic ulcer), autoimmune (multiple sclerosis, rheumatoid arthritis, Lupus erythematous) pathologies (Aloe L. et al., 1999).

From above it results that the NGF production, particularly of the growth factor β subunit, in high amounts is very interesting from the economic point of view. In fact in addition to the usual NGF extraction process from mouse salivary glands the NGF production via recombinant route in Escherichia coli bacterium is described in the patent applications EP 0121338 and US 5288622. This technique allow the production of a protein without other extract contaminating mammalian proteins which exert undesired biological activities. Other recombinant techniques used different expression systems like baculovirus transformed insect cells (patent application EP 037171) for the NGF expression. Protein production techniques via recombinant route allow, in fact, to provide NGF with high purity grade and without immunogenicity because the sequence is homologous to human one. However above mentioned techniques of course suffer in that they are very expensive and laborious (long times). Furthermore clinical experiments carried out by inoculating human recombinant NGF did not satisfy expectations resulting from NGF treatment of human pathologies in animal models (Yasuda H. et al. 2003). On the contrary surprising clinical results were obtained by topical heterologous NGF application (murine NGF) which proved to be effective and resolving therapy for the treatment of ophthalmic pathologies thought incurable for the humans like, for example, corneal neurotophic ulcers and for local treatment of cutaneous ulcers both in chronic human pathologies and pets (Aloe L., 2004).

In view of above it would desirable to provide a new NGF source able to furnish high protein yields by rapid, cheap and large scale usable extraction methods. Further the availability of a protein having high homology with human NGF could also be helpful for more effective strategies for the systemic or intracerebral administration of the molecule, possibly suitable carriers conjugated or microsphere encapsulated (Benoit J.-P et al. 2000; Xu X. et al., 2003).

Rainworms are long considered a source of biologically active possibly useful in medical field compounds, as in the case of EFEa fibrinolytic enzyme, extracted form Eisenia foetida, able to degrade fibrin and activate the plasminogen (Tang et al., 2002). The authors of the present invention now identified ed isolated from Eisenia foetida a NGF-like protein which within the sequenced portion shows a 100 % homology with human NGF β subunit.

Protein natural expression in very common, easily available organisms and which can be brought without operating difficulties, offers various advantages, first of which the NGF availability at low costs. Further the E. foetida naturally extracted protein is suitable for a large scale production because the reproducibility and maintenance easiness of extraction source.

Identification in Eisenia foetida of a protein having 100 % homology with a substantial portion of human NGF (77.4 % of the whole molecule: portion wherein murine NGF homology is 88 %), as alternative to a NGF production recombinant method, allows to provide NGF without contaminating proteins present in extract from murine salivary glands. Furthermore homology grade of the protein in comparison to human NGF is higher (100 % versus 88 % for mouse).

It is therefore an object of the present invention a NGF-like protein isolated from Eisenia foetida having within the amplified portion homology between 98 and 100 % with respect to β subunit of mature human nerve growth factor (NGF) in a portion corresponding to about % of molecule. Isolation of above said protein is carried out preferably from Eisenia foetida coelomocytes.

According to a preferred embodiment of the present invention said protein includes the following amino acid sequence:

VaI Trp VaI GIy Asp Lys Thr Thr Ala Thr Asp lie Lys GIy Lys GIu VaI Met VaI Leu GIy GIu VaI Asn lie Asn Asn Ser VaI Phe Lys GIn Tyr Phe Phe GIu Thr Lys Cys Arg Asp Pro Asn Pro VaI Asp Ser GIy Cys Arg GIy lie Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe VaI Lys Ala Leu Thr Met Asp GIy Lys GIn Ala Ala Trp Arg Phe lie Arg lie Asp Thr Ala Cys VaI Cys (SEQ ID No:2). Oligonucleotide sequences encoding for above described protein are a further object of the present invention. In a particular embodiment of the present invention oligonucleotide sequence comprises the following encoding sequence: gtg tgg gtt ggg gat aag ace ace gcc aca gac ate aag ggc aag gag gtg atg gtg ttg gga gag gtg aac att aac aac agt gta ttc aaa cag tac ttt ttt gag ace aag tgc egg gac cca aat ccc gtt gac age ggg tgc egg ggc att gac tea aag cac tgg aac tea tat tgt ace acg act cac ace ttt gtc aag gcg ctg ace atg gat ggc aag cag get gcc tgg egg ttt ate egg ata gat acg gcc tgt gtg tgt g (SEQ ID No:1). These oligonucleotide sequences can be desoxyribonucleotidic or ribonucleotidic or complementary sequences. The present invention further refers to a method for the extraction of the protein according to the invention comprising the following steps: a) isolation of coelomocytes from rainworms by treatment with extrusion solution; b) preparation of proteic extract from coelomocytes; c) protein purification by chromatography.

Further object of the present invention is the protein achievable according to above described extraction method. It is a further object of the present invention the protein as above defined for use in medical field, particularly for the treatment of pathologies in connection with NGF defective activity.

Therefore it is an object of the present invention the use of the protein as above defined for the preparation of a medicament for the treatment of neurodegenerative diseases of central, peripheral or autonomic nervous system like Alzheimer's disease, elderly immunodefieincy, Parkinson's disease, amiotrophic lateral sclerosis, epilepsy, Down's syndrome, nervous deafness, peripheral neuropathies, Huntington's disease, secondary neural injuries from hypoxia, ischaemia, infections (HIV, poliomyelitic virus) or trauma (including surgical ones).

The present invention refers also to the use of the protein for the preparation of a medicament for the treatment of ocular pathologies like, for example, glaucoma, retinal degeneration, etiologically different keratites (post-traumatic or post-infective conditions) and neurotrophic corneal ulcers.

The present invention refers additionally to the use of the protein for the preparation of a medicament for the topical treatment of cutaneous ulcers both associated with human pathologies and detectable in animals and also for the preparation of a medicament for the treatment of pathologies concerning hair loss like alopecia.

Furthermore the protein according to the invention can be used for the preparation of a medicament for the treatment of pathologies wherein endothelial growth or neoangiogenesis process is to be stimulated. These pathologies can be selected from the group consisting of ictus, aneurysms, gastrointestinal ulcers, myocardial infarction and peripheral vasculopathies. Further object of the present invention is the use of the protein as a factor for the promotion of growth and/or survival in vitro or ex-wVo of neural cells (as dopaminergic, cholinergic, sensorial neurons, striatal, cortical cells, as well as cells from corpus striatum, hippocampus, cerebellum, olfactive bulb, moto neurons, sympathetic neurons, neuronal staminal cells) and also for the maturation and differentiation of haematopoietic staminal cells, progenitors of immune system cells both of myeloid and lymphoid line and male reproduction system cells.

Finally an object of the present invention is a pharmaceutical composition comprising the protein object of the present invention together with pharmaceutically acceptable excipients, carriers and/or adjuvants.

Administration of said pharmaceutical composition can be carried out via any conventional administration route, for example systemically using intravenous or parenteral injections as injectable solutions or suspensions, intramuscularly, intraparenchimally, topically or orally

The present invention now will be described by way of illustration, but not limitation, according to some preferred embodiments thereof with particular reference to the figures of enclosed drawings wherein: figure 1 depicts RT-PCR amplification products of conserved actin gene in Eisenia foetida coelomocytes; figure 2 depicts primer design of a nested RT-PCR for the mature portion of NGF molecule; figure 3 shows agarose gel electrophoresis of the external RT- PCR products; figure 4 shows agarose gel electrophoresis of the internal RT- PCR products; figure 5 shows the homology level between the amplified portion on retrotranscribed DNA from E. foetida total RNA and mature human NGF (β subunit)

EXAMPLE 1 : Method for the production and extraction of NGF- iike proteins from Eisenia foetida

Animals reared in the dark at 17°C, closed within terracotta containers with air permeable covers in humidified, fertilized and periodically enriched with vegetables, coffee grounds and dehydrated foods, in pellets for pounded animals were used (rabbits, mice, rats). At least a week before the use an appropriate number of animals is transferred and maintained in terracotta pots (about 1 animal/10 cm²) a 25°C. Extrusion of coelomocytes (free cells contained within the liquid of coelomatic cavity and involved in defensive mechanisms of organisms) is carried out from sexually mature animals using the modified Eyambe method (Eyambe S. G. et al., 1991).

Particularly in the present method animals were previously "purged" to eliminate earth grains and excrements. To this they were put in 10 cm diameter Petri dishes in number of three animals/dish and placed between two filter paper disks which together with some further added paper chips were imbibed with tap water (3 ml). Then the animals were maintained in wet environment during three days replacing the dish at the second day.

At time of the use any animal is washed with saline (NaCI, 0.85 %) at ambient temperature and then transferred in Petri dishes (3 cm diameter) containing 1.5 ml of extrusion solution. This solution consists of 5 % ethanol and 95 % NaCI solution containing 2.5 mg/ml of EDTA and 10 mg/ml of glyceryl ether guaiacol mucolytic agent, adjusted to pH 7 with NaOH; ethanol is added at the time of the use. Animals were maintained in the extrusion solution for two minutes and then removed. Cells contained in the coelomatic extruded liquid were immediately transferred in ice cooled tubes containing PBS 0.01 M or NaCI 0.85 %, both sterile and at 4°C, in a volume of 12-13 ml or 38 ml for individual or multiple samples containing coelomatic liquid from four animals, respectively. In any case finally the cells are washed thrice at 4°C by centrifugation for 5 minutes at 150xg.

From the extrusion about 5-8x10⁵/animal are collected. The presence of NGF-like proteins in coelomocytes was confirmed by the authors of the present invention using both immunocytochemical studies with confocal microscopy and flux cytometry and Western blotting analysis (as outlined in Davoli et al. 2002 and described in Goos H.J.T. et al., 2001).

EXAMPLE 2: Study of the gene expression of NGF-like molecules in Eisenia foetida coelomocytes by RT-PCR In three independent experiment 10 animals were sacrificed for the separation and analysis of the coeloma cellular population, I.e. coelomocytes. From any animal were obtained about 10⁵ cells which were washed in cold PBS, centrifuged at 1800 rpm for 5 minutes and pellet thereof was re-suspended in 500 μl of guanidine thiocyanate (GTC) 4M solution. GTC is very potent denaturing agent able not only to lyse coeloma cells but also to denaturate intracellular RNases which are responsible of the rapid RNA messenger degradation.

Total RNA of coelomocytes was then extracted according to the Chomczynski and Sacchi (Chomczynski P., Sacchi N, 1987) method, its integrity was verified on agarose gel electrophoresis and the obtained amount was measured by spectrophotometric reading of the optical density.

For every sample two 1 g aliquots of total RNA were digested with DNAse, Amplification Grade (RNAse-free), enzyme, to eliminate potential genomic DNA traces in the RNA sample. Thus digested RNA was then retro-transcribed in DNA copy (cDNA), by addition of inverse transcriptase enzyme (MMU-LV-RT) to one aliquot of every sample while in the other instead of the enzyme only water as a control was added. This control, defined no-RT, serves to detect the permanency of trace amounts of genomic DNA in the RNA sample, also after DNAses digestion.

Thus obtained cDNAs were subjected to PCR using Eisenia foetida conserved gene specific primers, like actin, to verify the cDNA successful synthesis and that cDNA relative amounts were comparable for various samples (figure 1).

All conserved gene positive cDNAs were then subjected to a nested PCR (selected because high sensitivity of the method) for the amplification of the NGF molecule mature portion.

Primers for a nested PCR, consisting of a first gene region (external) amplification and subsequent re-amplification using primers designed on a region belonging to the first (internal) were selected within the mature portion of murine β-NGF sequence (Figure 2). The selection of murine NGF, which shows high homology with human one, was carried out because NGF immunologically correlated proteins were detected in the present animal by the use of anti-NGF murine polyclonal antibodies which are also able to react with homologous human neurotrophins (Davoli C, et al. 2002). Sequenced portions corresponds to about 75 % of the human mature NGF molecule as primers used for the amplification were selected depending on the cysteines present in the mature protein which are conserved in NGF of all up to now examined species. It is noteworthy that in this portion 5 out of 6 cysteines are present and remaining one was contained in the A1 and B1 primers used for nested PCR (Table 1).

Sequences of the used primers and their relative position in the mature human NGF sequence (reference sequence: gi|34192368|gb|BC032517.2|) are reported in Table 1

Table 1

Amplification conditions used in PCR protocol were the following: 1 μl cDNA from every sample was amplified in 20 μl final volume containing 2 mM MgCI_∑. 0.05 mM dNTPS and 0.5 μM primers, denaturated for 5 minutes at 95°C and 35 cycles consisting of 1 minute at

95⁰C, 1 minute at 62⁰C and 1 minute and 30 seconds at 72°C followed by a 10 minute extension at 72°C. RT-PCR products were analyzed by agarose gel electrophoresis (Figure 4) and external PCR positive samples were re-amplified using primers for the DNA region internal to the first one.

All internal RT-PCR positive samples were purified and sequenced. Readable portion of 274 base pairs has 100 % homology with human β-NGF sequence. Figure 5 depicts the alignment of two sequences and shows homology level as high as 100 % between the amplified portion on E. foetida total RNA and mature human NGF (β subunit)

EXAMPLE 3: Method for the extraction and purification of proteins from Eisenia foetida

For the extraction and purification of NGF-like proteins at least 40-50 g of rainworm extruded coelomocytes, washed with NaCI 0.85 % and stored at -20⁰C till at time of use obtaining 1 g of coelomocytes from about 30 animals.

For the isolation of coelomocytes animals are pre-treated according to the following method: stabling of sexually mature animals at 25°C, transferring in plate and fasting in wet environment at 25°C for 3 days with plate replacing; coelomocytes collecting; washing of any animal with saline solution at ambient temperature, transferring into plate and preservation in extrusion solution (Eyambe S. G. et al., 1991) for 2 minutes, collecting of extruded cells and their transferring in sterile buffer or saline solution at 4°C with subsequent washings by centrifugation at 150xg for 5 minutes.

As to the preparation of the proteic extract from coelomocytes cell lysate obtained by re-suspending coelomocytes in buffer containing protease inhibitors (Davoli C. et al. 2002) or alternatively without inhibitors is homogenized in Wheaton potter at 4°C and centrifuged at 12000xg for 2 minutes at 4⁰C with separation soluble (super) extract from precipitate.

For the purification it is appropriate to compare various processes, resulting from alternative combinations of chromatographic methods.

Following scheme summarizes various purification steps of two processes (respectively A and B) to be compared:

Frozen Coelomocytes at -20⁰C

Soluble extract Soluble extract

(supernatant from streptomycin sulfate precipitation)

Lyophilization 1 Ion exchange chromatography (CM-Sepharose/ DEAE- Sepharose FF) Sephadex G-100

Dialysis

Ion exchange Sephadex G-50 chromatography (CM 52 - Cellulose)

Process indicated by A letter corresponds to the conventional purification method used for the preparations of NGF from mouse salivary glands with pre-treatment of the extract using streptomycin sulfate (Bocchini V., Angeletti P. U., 1969); in such a case for a better purification a further chromatographic run in CM-cellulose can be necessary (Graiani G. et al., 2004).

Process indicated by B letter is based on chromatographic separations used for snake venom obtained NGF for which recently most appropriate purification method has been stated (Kostiza T. et al., 1995; Bian L.-J. et al., 2004). In this method chromatographic separation can be by ion, cation or anion exchange.

In both methods (A and B) elution buffer equilibrated gel filtration columns are used, while in ion exchange chromatography columns are equilibrated with initial buffer using then NaCI gradient for elution of column adsorbed proteins.

In any process step the fractions chromatografically isolated and SDS monitored by absorption at 280 nm are analyzed by SDS-PAGE to detect protein components while the presence of NGF-like proteins is pointed out by Western blotting, using cross-reactive polyclonal antibodies. Immunoreactive fractions are pooled and subjected to the following chromatography or, when sufficiently purified and dyalized, if desired, stored at -20⁰C or lyophilized and stored at 4°C.

Biological activity is evaluated both for the ability to provoke the emission of nervous fibers by dosage in PC-12 cells or in sympathetic nervous ganglions isolated from chicken embryos (Kostiza T. et al., 1995; Bian L.-J. et al., 2004) and for the effects on immunocompetent cells (La SaIa et al., 2000). BIBLIOGRAPHY

- Jaaro H., Beck G., Conticello S.G.,Fainzilber M. 2001. Trends Neurosci. 24: 79-85.

- Aloe L., Bracci Laudiero L. 1999. Le Scienze 369: 46-52. - EP 0121338.

- US 5288622. - EP 0370171.

- Yasuda H., Terada M., Maeda K., Kogawa S., Sanada M., Haneda M., Kashiwagi A., Kikkawa R. 2003. Prog. Neurobiol. 69: 229-285. - Aloe L. 2004. Prog. Brain Res. 146 : 515-522.

- Benoit J.-P., Faisant N., Venier-Julienne M.- C₁ Menei P. 2000. J. Control. Release 65: 285-296.

- Xu X., Yee W.-C, Hwang P. Y.K., Yu H., Wan A. C.A., Gao S., Boon K,- L., Mao H.-Q., Leong K. W., Wang S. 2003. Biomaterials 24: 2405-2412. - Tang Y., Liang D., Jiang T., Zhang J., Gui L. Chang W. 2002. J. MoI. Biol. 321 :57-68.

- Davoli C, Marconi A., Serafino A., lannoni C, Marcheggiano A., Ravagnan G. 2002. Cell MoI. Life Sci. 59: 527-539.

- Eyambe S. G., Goven AJ. , Fitzpatrick L.C., Venables BJ. , Cooper E. L. 1991. Lab. Anim. 25: 61-67.

- Goos HJ.T. et al. 2001. Perspectives in Comparative Endocrinology: Unity and Diversity, pp. 879-885, Monduzzi, Bologna.

- Chomczynski P., Sacchi N. 1987. Anal. Biochem. 162: 156-159.

- Bocchini V., Angeletti P.U. 1969. Proc. Nat. Acad. Sci. USA 64: 787-794. - Graiani G., Emanueli C, Desortes E., Van Linthout S., Pinna A.,

Figueroa CD., Manni L., Madeddu P. 2004. Diabetologia 47: 1047-1054.

- Kostiza T., Dahinden CA., Rihs S., Otten U., Meier J. 1995. Toxicon 33: 1249-1261.

- Bian L-J., Wu P., Yang X.-Y. 2004. J. Chromatogr. B 805: 119-125. - La SaIa A., Corinti S., Federici M., Saragovi H. U., Girolomoni G. 2000. J. Leukocyte Biol. 68:104-110.

Claims

1. NGF-like protein isolated from Eisenia foetida having homology from 98 to 100 % with respect to β subunit of mature human nerve growth factor (NGF) within a trait corresponding to about ³A of the molecule

2. Protein according to claim 1 wherein said protein is isolated from Eisenia foetida coelomocytes.

3. Protein according to anyone of preceding claims comprising the following amino acid sequence: VaI Trp VaI GIy Asp Lys Thr Thr Ala Thr Asp lie Lys GIy Lys GIu

VaI Met VaI Leu GIy GIu VaI Asn lie Asn Asn Ser VaI Phe Lys GIn Tyr Phe Phe GIu Thr Lys Cys Arg Asp Pro Asn Pro VaI Asp Ser GIy Cys Arg GIy He Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe VaI Lys Ala Leu Thr Met Asp GIy Lys GIn Ala Ala Trp Arg Phe lie Arg lie Asp Thr Ala Cys VaI Cys (SEQ ID No:2).

4. Oligonucleotide sequence encoding for the protein as defined in claims from 1 to 3.

5. Oligonucleotide sequence according to claim 4 comprising the following sequence: gtg tgg gtt ggg gat aag ace ace gee aca gac ate aag ggc aag gag gtg atg gtg ttg gga gag gtg aac att aac aac agt gta ttc aaa cag tac ttt ttt gag ace aag tgc egg gac cca aat ccc gtt gac age ggg tgc egg ggc att gac tea aag cac tgg aac tea tat tgt ace acg act cac ace ttt gtc aag gcg ctg ace atg gat ggc aag cag get gcc tgg egg ttt ate egg ata gat acg gcc tgt gtg tgt g (SEQ ID No:1).

6. Oligonucleotidic sequence according to anyone of claims 4 and 5 wherein said sequences are desoxyribonucleotidic or ribonucleotidic or complementary sequences thereto.

7. Method for the extraction of the protein according to anyone of claims 1 and 2 comprising the following steps: a) isolation of coelomocytes from rainworms by treatment with extrusion solution; b) preparation of proteic extract from coelomocytes; c) protein purification by ion exchange chromatography. 8. Protein achievable according to method as defined in claim

7. 9. Protein according to anyone of claims form 1 to 3 or claim 8 for use in medical field.

10. Use of the protein according to claim 9 for the preparation of a medicament for the treatment of neurodegenerative diseases of central, peripheral or autonomic nervous system.

11. Use according to claim 10 wherein said diseases are selected form the group consisting of Alzheimer's disease, Parkinson's disease, amiotrophic lateral sclerosis, peripheral neuropathies, Huntington's disease, secondary neural injuries from hypoxia, ischaemia or trauma.

12. Use of the protein according to claim 9 for the preparation of a medicament for the treatment of ocular pathologies.

13. Use according to claim 12 wherein said ocular pathologies are selected from the group consisting of glaucoma, keratites and neurothophic corneal ulcers.

14. Use of the protein according to claim 9 for the preparation of a medicament for the treatment of pathologies wherein endothelial growth or neoangiogenesis are to be stimulated.

15. Use of the protein according to claim 14 wherein said pathologies are selected from the group consisting of ictus, aneurysms, gastrointestinal ulcers, myocardial infarction and peripheral vasculopathies.

16. Use of the protein according to claim 9 for the preparation of a medicament for the treatment of cutaneous ulcers. 17. Use of the protein according to claim 9 for the preparation of a medicament for the treatment of alopecia.

18. Use of the protein according to claim 9 as reagent for the promotion of growth and/or survival in vitro or ex-vivo of neural cells.

19. Pharmaceutical composition comprising the protein according to anyone of claims from 1 to 3 o claim 8 together with pharmaceutically acceptable excipients, carriers and/or adjuvants..