EP0463082A1 - Facteur neurotrope ciliaire purifie - Google Patents

Facteur neurotrope ciliaire purifie

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Publication number
EP0463082A1
EP0463082A1 EP19900905302 EP90905302A EP0463082A1 EP 0463082 A1 EP0463082 A1 EP 0463082A1 EP 19900905302 EP19900905302 EP 19900905302 EP 90905302 A EP90905302 A EP 90905302A EP 0463082 A1 EP0463082 A1 EP 0463082A1
Authority
EP
European Patent Office
Prior art keywords
cntf
neurons
highly purified
sds
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19900905302
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German (de)
English (en)
Inventor
Marston Manthorpe
Silvio Varon
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University of California
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University of California
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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to growth factors and more specifically to a newly purified growth factor which supports parasympathetic motor neurons of the ciliary ganglion.
  • Neuronotrophic factors are protein which dramatically affect the performance of nerve cells.
  • the first neuronotrophic factor, Nerve Growth Factor (NGF) was discovered and purified almost 40 years ago.
  • NGF Nerve Growth Factor
  • NGF is a 26 kD basic di eric protein present in minute amounts in most, if not all tissues, and is required for the survival of developing peripheral sensory and sympathetic neurons in vitro and in vivo. It also affects cholinergic neurons in the brain that are thought to be involved in cognitive processes.
  • NGF is provided to the neuron in vivo by certain cells to which the neuron connects (e.g., muscle or nerve cells) and/or by neighboring glial cells (e.g., Schwann cells or astroglia) to which the neuron is apposed. NGF is thought to be required not only for the survival of the neurons but also for the maintenance and stimulation of neurite growth (axonal and dendritic processes) and the synthesis of function-related neuronal enzymes.
  • certain cells to which the neuron connects e.g., muscle or nerve cells
  • neighboring glial cells e.g., Schwann cells or astroglia
  • NGF acts on developing peripheral (“PNS") and central (“CNS”) nervous system neurons and may thus provide useful treatments of related developmental deficits in the human fetus or child.
  • NGF has recently been shown to work on mechanically- or age-impaired cholinergic neurons in the adult rat CNS, pointing to its potential use for i) regeneration of damaged CNS nerve cells, ii) functional improvements of age-related memory deficits, and iii) prevention and/or reversal of nerve cell degeneration in patients with Alzheimer's, Parkinsons and related disorders.
  • neuronotrophic growth factors or growth factors having neurotrophic activities, have also been identified, including the Fibroblast Growth Factors, and Epithelial Growth Factor. For the past ten years these studies have prompted investigators to predict that other neuronotrophic factors exist which act on neurons other than those acted on by NGF.
  • CG neurons chick embryo cholinergic parasympathetic motor neurons of the ciliary ganglion
  • Chick Eye CNTF was shown to be a 204 kD protein with an isoelectric point of 5.0 which supported the survival not only of CG neurons but cultured avian and mammalian sensory neurons and avian sympathetic neurons as well.
  • CNTF isolated from rat sciatic nerves has been isolated and has been reported to have been "purified" (Manthorpe et al., Brain Res. 367:282-286 (1986)) . This 24 kD protein was found- to be distinct from chick eye CNTF. However, further work on rat nerve CNTF revealed that the preparation described in Manthorpe (1986) , supra , is in fact, heavily contaminated with other chemically related inactive proteins.
  • Growth factors particularly those exhibiting nuerontrophic activities, have great potential utility for promoting the growth or regeneration of specific populations of cells. In order to be therapeutically useful, however, such growth factors must be highly purified and must retain their activity in the purified state. There thus exists a need for methods of purifying neurotrophic growth factors and for characterizing these purified factors.
  • the present invention satisfies these needs and provides related advantages as well.
  • the present invention provides highly purified mammalian Ciliary Neurotrophic Factor (CNTF) having a specific activity greater than 2 x 10 7 TU/mg protein and a method for obtaining such highly purified CNTF.
  • highly purified CNTF is characterized by a molecular weight of about 23 to 28 kD as determined by SDS-PAGE under either reducing or non-reducing conditions; a PI of about 5.0 to about 5.4; not being inactivated by anti-NGF antibodies; not being inactivated by SDS or reducing agents; and supporting in vitro survival of E8 chick ciliary ganglion neurons, E12 sympathetic ganglion neurons, ElO chick dorsal ganglion neurons and neonatal mouse dorsal root ganglion neurons but not E8 chick dorsal root ganglion neurons; and being sensitive to trypsin and chymotrypsin digestion.
  • the highly purified CNTF may be attached to a solid support, which
  • Figure 1 shows the quantitative survival of purified chick embryo neurons selectively on the purified 28 kilodalton CNTF band on a Western blot after SDS-PAGE.
  • the present invention relates to a highly purified neuronotrophic factor termed Ciliary Neuronotrophic Factor, or CNTF.
  • the highly purified CNTF has a specific activity of at least 2 x 10 7 TU/mg protein, and retains activity after submission to SDS-PAGE and Western blotting.
  • Highly purified CNTF can be obtained from mammalian peripheral nerve tissue, such as rat sciatic nerve. Because it is present in such low concentration, a large amount of tissue must be used.
  • An aqueous extract of the tissue was prepared, as by homogenation, preferably in the presence of proteolytic inhibitors. The extract was then submitted to SDS-PAGE and the band in the 25 to 30 kD region cut out and the protein extracted, as by electroelution. Those eluates containing peak CNTF activity, as determined by bioassay, were pooled. Extraneous matter, such as residual pieces of gel, was removed from the pooled fractions, as by centrifugation and the pooled fractions submitted to reverse phase high performance liquid chromatography. Fractions exhibiting high activity were again pooled and submitted to SDS-PAGE, resulting in a band at approximately 28 kD which exclusively exhibited CNTF activity. The material obtained from the 28 kD band is highly purified CNTF.
  • Highly purified CNTF retains its biological activity after submission to SDS-PAGE and Western blotting.
  • Highly purified CNTF was heated to 90°C for 10 minutes in the presence of SDS and ⁇ -mercaptoethanol.
  • Highly purified CNTF was then subjected to SDS-PAGE and Western blotting, using methods well known in the art.
  • the highly purified CNTF exhibits one sharp protein band corresponding to a molecular mass of 28 kD on analytical SDS gels and Western blots. This band possesses high biological activity after being either eluted directly from the SDS gel and provided to test neurons in soluble form or after being electrophoretically transferred onto nitrocellulose paper and being provided to test neurons in an immobilized form.
  • Test neurons supported by this processed CNTF included embryonic chick day 8 CG, day 10 dorsal root ganglia (DRG) and 11 sympathetic ganglia (SG) and neonatal mouse DRG.
  • the specific activity of the highly purified CNTF for E8 CG was 2 x 10 7 TU/mg protein.
  • Bovine heart Ciliary Neurontrophic Factor bhCNTF: Watters and Hendry, J. Neurochem 49:705-713 (1987).
  • FGF Bovine basic Fibroblast Growth Factor
  • NGF Mouse Submaxillary Nerve Growth Factor
  • BDNF Bovine brain-derived Neuronotrophic Factor
  • Table I presents a comparison of the cells supported by these neuronotrophic factors. Values given are the percentage of neurons in the ganglion tissues whose neuronal survival is supported by the indicated factor.
  • the specific activity of the highly purified CNTF is greater than 2 x 10 7 TU/mg protein.
  • bhCNTF has been reported to have an activity of 4.2 x 10 4 TU/mg protein.
  • This highly purified CNTF is over 200 times as potent, and possibly also purer, than bhCNTF.
  • highly purified CNTF retained biological activity after being subjected to heat and SDS detergent.
  • bhCNTF has been reported to be completely inactivated by such treatment. Watters and Hendry, J. Neurochem. 49 ⁇ OS- VIS (1987).
  • CNTF can be attached to a solid support, such as for example, to nitrocellulose paper or a plastic prosthesis.
  • a solid support such as for example, to nitrocellulose paper or a plastic prosthesis.
  • the material is useful, for example, as a neural bridge.
  • CNTF can be attached to a solid support, such as by Western blotting to nitrocellulose paper after SDS- PAGE (Carnow et al., J. Neuroscience 5:1965-1971, 1985; Rudge et al., Dev. Brain Res. 32:103-110, 1987, which are incorporated herein by reference) , as is for example, the prototype neuronotrophic factor, Nerve Growth Factor (Pettmann, et al., J. Neuroscience 8:3624-3630, 1988, which is incorporated herein by reference) . In this form the material is useful as a neuronal bridge.
  • tissue materials such as fetal brain tissue, adult rat sciatic nerve or placental amnion membrane have been grafted into experimentally injured adult rat brain or nerve tissue and shown to support the limited regeneration of damaged nerve cells (Kramer et al., Brain Research 210:153-172, 1981; David and Aguayo, Science 214:931-934, 1981; Davis et al., Science 236:1105-1109, 1987; Danielsen et al., Dev. Brain Res. 39:39-50, 1988, which are incorporated herein by reference) .
  • uncoated nitrocellulose paper has been grafted into damaged adult rat brain and spinal cord tissue and shown to serve to a limited extent as a nerve bridge.
  • Nitrocellulose coated with CNTF can provide bridges for the stimulation of brain regeneration.
  • One procedure for preparing CNTF bridges is to submit the purified CNTF to SDS-PAGE and Western blotting by standard methods.
  • the 28 kD CNTF band region is cut out, and the remaining protein binding sites on the paper blocked with a biologically inactive protein such as purified albumin.
  • the CNTF coated and blocked paper is inserted between the stumps of a regenerating peripheral nerve (such as has been done with Fibroblast Growth Factor, Danielsen et al., J. Neuroscience Res. in press, 1989, which is incorporated herein by reference) or within a damaged central nervous system pathway.
  • the nitrocellulose itself has been shown to be relatively inert and does not elicit an inflammatory reaction.
  • the immobilized CNTF can stimulate the apposing damaged nerves to regrow axons across the paper and into the denervated regions.
  • This Crude Extract was then submitted to preparative SDS-PAGE using a l80 X 120 X 6 mm thick 7.5-20% polyacrylamide gradient slab gel.
  • the gel lane (including the 20-30 kD protein band region) then cut into about 10 X 2-3mm long slices. Each slice in the appropriate region, i.e., 25 to 30 kD, was electroeluted into 1 ml of 0.1% SDS using an electroelution chamber (CBS Scientific, Del Mar, CA) .
  • Bioassays using embryonic day 8 chick ciliary ganglion (CG) nerve cells were then performed to determine which eluates contain the peak of CNTF biological activity, described in detail in a recent review (Manthorpe, M. , et al. (1989) Ciliary Neuronotrophic Factors, In “Nerve Growth Factors,” R.A. Rush, ed. , John Wiley and Sons, Ltd., New York, pp. 31-56, which is incorporated herein by reference) .
  • the cultures are maintained at 37 ⁇ C in a 5% C0 2 -95% air humidified incubator for 16 hours when 5 ⁇ l of the vital dye, MTT (Sigma Cat. # M2128; 1.5 mg/ml in culture medium) was added and the culture incubated for an additional 8 hours.
  • MTT Sigma Cat. # M2128; 1.5 mg/ml in culture medium
  • the blue crystals within the remaining viable neurons were solubilized (0.08 N hydrochloric acid in isopropanol) .
  • the intensity of the blue solution which is proportional to the number of surviving neurons in each well, was then measured and plotted using a microplate reader interfaced with a computer.
  • One Trophic Unit (TU)/ml is defined as that amount of CNTF activity per ml of culture medium (i.e., the final dilution) supporting half-maximal neuronal survival (i.e., optical density) . Those fractions exhibiting the highest activity were pooled. Usually there were two to three peak active fractions representing 2 to 3 ml of eluate from each batch of 50 nerves.
  • Each 5 ml pool was thawed, centrifuged for 10 minutes at 1000 X g to sediment any small residual pieces of polyacrylamide gel carried over from the electroelution step and the supernate collected.
  • Each 5 ml of gel-free supernate was then submitted to reverse phase high performance liquid chromatography on a 250 cm X 4.6 mm C-4 300A microbore column (e.g., HiPoreTM Reverse Phase Column #RP304; Bio-Rad, Inc., Cat No. 125-0550 or VydacTM Cat No. 214TP54) using a 30 minute ramp to 45% acetonitrile followed by a 180 minute 45-60% acetonitrile gradient in 0.1% trifluoroacetic acid.
  • HiPoreTM Reverse Phase Column #RP304 e.g., HiPoreTM Reverse Phase Column #RP304; Bio-Rad, Inc., Cat No. 125-0550 or VydacTM Cat No. 214TP54
  • the HPLC eluate pool was submitted to analytical SDS-PAGE using a 15-25% polyacrylamide gradient in a low tris buffer system.
  • the prep at this stage contains two prominent bands, one at about 30 kD and the other at about 28 kD, as well as a few minor bands in the region.
  • CNTF biological activity as determined by two techniques (by elution from the gel and assay of the eluate and by a cell blot technique, as described below) was exclusively associated with the 28 kD band.
  • the CNTF was then collected free of the 30 kD and the few other minor bands by cutting 1 cm X l mm regions out of the appropriate region 23 to 28 kD and eluting them into 200 ⁇ l of water; the eluates were assayed for CNTF activity and the active bands pooled; the peaks were then pooled and the pool concentration by SpeedvacTM (Savant, New York, NY) to about 100 ⁇ l. This fraction was called "Highly Purified CNTF.”
  • CNTF and NGF activities were determined using purified microcultures set up as described in Example I, except that either purified E8 chick ciliary or dorsal root or Ell-12 chick sympathetic or neonatal mouse dorsal root ganglion neurons were used as test cells.
  • test neurons were presented with 50 TU/ml of the indicated trophic factor and serial dilutions of the test antibody (i.e., rabbit anti-mouse Nerve Growth Factor or rabbit anti-bovine basic or acidic Fibroblast Growth Factor) and neuronal survival quantified spectrophotometrically as in the trophic factor assays. Typical measured activities were greater than 10 8 TU/mg protein.
  • test antibody i.e., rabbit anti-mouse Nerve Growth Factor or rabbit anti-bovine basic or acidic Fibroblast Growth Factor
  • 0.88 ml acetone, 0.05 ml triethylamine, 0.05 ml acetic acid and 0.02 ml H 2 0 were combined and added to the residual in an eppendorf centrifuge tube. After 2 hours at -20°C, the sample was centrifuged at 15,000 rpm for 9 minutes. The supernatant was removed and 0.5 ml cold acetone was again centrifuged at 15,000 rpm for 9 minutes and the supernatant removed.
  • Trypsin digestion was carried out as follows. The protein pellet was dried and resuspended in 10 ⁇ l of 0.1% SDS and heated to 100°C for 3 minutes after which 100 ⁇ l trypsin buffer (2.5 mM CaCl 2 , 0.1 M Tris:HCl; pH 8.0) and 0.3 ⁇ g trypsin were added. The reaction was incubated at 37°C overnight after which 0.2 ⁇ g of trypsin was added and the sample incubated for an additional 4 hours at 37°C.
  • trypsin buffer 2.5 mM CaCl 2 , 0.1 M Tris:HCl; pH 8.0
  • Tryptic fragments were separated by reverse phase HPLC (Hewlett Packard #HP109Q) using a Vydac C-18 column (2 mm x 15 cm) . Fragments were eluted in a gradient of 5-65% acetonitrile in 0.1% trifluoroacetic acid. A peak eluting at 24.5 minutes (approximately 22% acetonitrile) was selected for sequence analysis by the Edman degradation technique using an automated sequenator (Applied Biosystems model 477A) . The fragment gave a single, unambiguous amino acid sequence indicating that the peak contained a single, homogenous peptide. The amino acid sequence of this peptide was as follows:
  • CNTF On SDS-PAGE, highly purified CNTF exhibits one band having a molecular mass between about 23,000 and 28,000 da, depending on the molecular mass standard proteins used to calibrate the SDS-PAGE system.
  • Phosphorylase b 97,400 da
  • bovine serum albumin 66,200 da
  • ovalbumin 42,699 da
  • soybean trypsin inhibitor 21,500
  • lysozyme 14,400
  • bhCNTF is reported to exhibit two bands, at 22 and 23 kDs, but neither of these bands exhibited activity towards cCG8 neurons. Watters and Hendry, supra.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Compounds Of Unknown Constitution (AREA)

Abstract

L'invention concerne un facteur neurotrope ciliaire (FNC) mammifère hautement purifié ayant une activité spécifique supérieure à 2x107 TU/mg de protéine, ainsi qu'un procédé permettant d'obtenir ledit FNC hautement purifié. Outre le fait qu'il a une activité supérieure à 2x107, le FNC hautement purifié est caractérisé par une masse moléculaire d'environ 28 kD déterminée selon SDS-PAGE dans des conditions soit de réduction soit de non-réduction; le FNC présente un PI compris entre environ 5,0 et environ 5,4; il n'est pas inactivé par des anticorps anti-NGF; il n'est pas inactivé par SDS ou des agents de réduction; il assure la survie in vitro de neurones de ganglions ciliaires de poussin E8, de neurones de ganglions sympathiques E12, de neurones de ganglions dorsaux de poussins E10 et de neurones de ganglions de racine dorsale de souris néonatales, mais pas de neurones de ganglions de racine dorsale de poussins E8; et il est sensible à la digestion de la trypsine et de la chymotrypsine. On peut fixer le FNC hautement purifié à un support solide pouvant à son tour être utilisé, par exemple, pour stimuler la survie de neurones.
EP19900905302 1989-03-15 1990-03-14 Facteur neurotrope ciliaire purifie Withdrawn EP0463082A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32376189A 1989-03-15 1989-03-15
US323761 1989-03-15

Publications (1)

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EP0463082A1 true EP0463082A1 (fr) 1992-01-02

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EP19900905302 Withdrawn EP0463082A1 (fr) 1989-03-15 1990-03-14 Facteur neurotrope ciliaire purifie

Country Status (5)

Country Link
EP (1) EP0463082A1 (fr)
JP (1) JPH04503811A (fr)
AU (1) AU5341790A (fr)
CA (1) CA2047718A1 (fr)
WO (1) WO1990010647A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT405835B (de) * 1989-01-05 1999-11-25 Synergen Inc Aufgereinigter ziliärer neurotropher faktor
US5141856A (en) * 1989-01-05 1992-08-25 Synergen, Inc. Expression of purified ciliary neurotrophic factor
IE903130A1 (en) * 1989-09-15 1991-03-27 Regeneron Pharma Ciliary neurotrophic factor
GR900100691A (el) * 1989-09-15 1992-01-20 Max Planck Gesellschaft Νευροτροφικός παράγοντας ακτινωτού.
EP0596034A4 (fr) * 1991-07-23 1995-05-24 Syntex Inc Purification du facteur neurotrophique ciliaire recombine et du facteur neurotrophique ciliaire tronque a terminaison c et procedes de traitement des lesions de nerfs peripheriques.
US5593857A (en) * 1991-08-23 1997-01-14 Scios Inc. Production of homogeneous truncated CNTF
EP1517988B1 (fr) * 2002-06-20 2013-05-08 Augustinus Bader Utilisation de l'érythropoïétine pour la régénération de tissus in vivo
US20050222101A1 (en) * 2004-03-30 2005-10-06 Jeffrey Hutterer Method and composition for treatment of skin conditions

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5128449A (en) * 1988-07-05 1992-07-07 The University Of Tennessee Research Corporation Polypeptide and a method for its production

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9010647A1 *

Also Published As

Publication number Publication date
WO1990010647A1 (fr) 1990-09-20
AU5341790A (en) 1990-10-09
JPH04503811A (ja) 1992-07-09
CA2047718A1 (fr) 1990-09-16

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