WO1998046254A1 - Facteur de croissance du tissu nerveux pour la prevention de la demyelinisation dans le systeme nerveux - Google Patents

Facteur de croissance du tissu nerveux pour la prevention de la demyelinisation dans le systeme nerveux Download PDF

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Publication number
WO1998046254A1
WO1998046254A1 PCT/EP1998/002029 EP9802029W WO9846254A1 WO 1998046254 A1 WO1998046254 A1 WO 1998046254A1 EP 9802029 W EP9802029 W EP 9802029W WO 9846254 A1 WO9846254 A1 WO 9846254A1
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WO
WIPO (PCT)
Prior art keywords
ngf
demyelination
nerve
human
nerve fibers
Prior art date
Application number
PCT/EP1998/002029
Other languages
English (en)
Inventor
Ilse Bartke
Jürgen Unger
Claude Genain
Stephen Hauser
Original Assignee
Roche Diagnostics Gmbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics Gmbh filed Critical Roche Diagnostics Gmbh
Priority to AU72149/98A priority Critical patent/AU7214998A/en
Priority to CA002286137A priority patent/CA2286137A1/fr
Publication of WO1998046254A1 publication Critical patent/WO1998046254A1/fr
Priority to US09/854,142 priority patent/US7282482B2/en

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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
    • 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
    • A61K38/55—Protease inhibitors

Definitions

  • NGF for the prevention of demyelination in the nervous system
  • the present invention concerns a process for prevention of demyelination in the nervous system by administering NGF.
  • the invention concerns a pharmaceutical composition for treating diseases in which a demyelination of nerve fibers occurs as well as a process for its production.
  • the covering of nerve fibers in the nervous system (NS) with myelin is essential for the function of neuronal signal transmission.
  • the myelin sheath is formed by oligodendrocytes (OL) in central nervous system (CNS) cells or Schwann cells which wrap these myelin sheaths around the axon of a nerve cell.
  • Oligodendrocytes are part of a separate non-neuronal cell population distributed throughout the CNS as part of the neuroglia.
  • NGF acts on different subtypes of neuronal cells.
  • NGF is a survival factor for sensory and sympathetic neurons
  • NGF NGF-induced central nervous system
  • NGF is a survival factor for cholinergic neurons and prevents the degeneration of cholinergic neurons in the basal forebrain (Gage et al. (1988) J. Comp. Neurol. 269: 147-155; Hefti et al. (1986) J. Neurosci. 6: 2155-2162; Hefti et al. (1986) J. Brain Res. 293: 305-311.).
  • NGF neurosci. Lett. 135: 219-223, and International Application No. WO 93/03140.
  • NGF induced proliferation and differentiation of oligodendrocytes, a non-neuronal cell -population.
  • oligodendrocytes are specialized and the only cells in the central nervous system which are capable of producing myelin and wrapping the myelin sheaths around the axons.
  • the oligodendrocytes and myelin sheaths are susceptible to attack by auto-immune processes, e.g., multiple sclerosis. Therefore, Althaus indicates that the induction of remyelination could be an important step in a therapeutic approach for multiple sclerosis.
  • NGF nerve fibers of the nervous system of a mammal, preferably of a human being, by influencing the immune system or the blood brain barrier (endothelial cells, T cells, macrophages, monocytes and microglia cells) At the moment the mechanism by which NGF is effective is not yet clear. But prevention of demyelination is a new and unexpected activity of NGF.
  • the effective amount of NGF or active NGF fragments is between 10 and 300 pg NGF/ml CSF (cerebrospinal fluid).
  • the object according to the present invention is achieved by a process for preventing the demyelination of nerve fibers in the nervous system of a human being, wherein said human being is treated with an amount of nerve growth factor (NGF) or active fragments of NGF elective to prevent demyelination.
  • NGF nerve growth factor
  • EAE allergic encephalomyelitis
  • C. jacchus common marmoset Callithrix jacchus
  • MS human multiple sclerosis
  • NGF or "active fragment of NGF” within the sense of the present invention refers to natural NGF, in particular mammalian NGF preferably natural human or murine NGF and all fragments or derivatives of NGF which have its desired biological activity, i.e., prevent the fiber demyelination of oligodendrocytes.
  • NGF molecules which are suitable for the process according to the present invention are for instance NGF- ⁇ , NGF 2.5S or NGF 7S from the submaxillary gland of the mouse. These NGF molecules can, for example, be obtained commercially from Sigma (St. Louis, USA) or Boehringer Mannheim GmbH (Mannheim, DE).
  • the process according to the present invention is preferably carried out with a human NGF, particularly preferably with human recombinant NGF- ⁇ .
  • a human NGF particularly preferably with human recombinant NGF- ⁇ .
  • the production of an active NGF fragment by tryptic digestion of NGF is described by Mercanti et al. in Biochim. Biophys. Acta 496 (1977) 412-419.
  • This fragment is composed of two linear oligopeptides which are linked by a disulfide bridge and contains the amino acid residues 10 to 25 and 75 to 88 of the amino acid sequence of NGF [according to the nomenclature of Angeletti and Bradshaw, Proc. Natl. Acad. Sci. USA 68 (1970) 2417-2421].
  • the present invention also concerns a pharmaceutical composition for the treatment of diseases in which a demyelination of nerve fibers occurs and which contains NGF or an active fragment thereof as the active substance together with the usual pharmaceutical vehicles, auxiliary substances, fillers and diluents.
  • the pharmaceutical composition preferably contains human NGF, especially human recombinant NGF- ⁇ .
  • the composition can contain one or several pharmaceutically tolerated protease inhibitors, for example, aprotinin, preferably in a kit wherein NGF and said inhibitor are located in separate containers.
  • composition according to the present invention can be processed with therapeutically acceptable vehicles.
  • Suitable vehicles for the production of such solutions are water, polyols, sucrose, invert sugar and glucose.
  • Suitable vehicles for injection solutions are water, alcohols, polyols, glycerol and vegetable oil.
  • the pharmaceutical preparations can contain preservatives, solvents, stabilizing agents, wetting agents, emulsifiers, salts for changing the osmotic pressure, buffers and, if desired, other therapeutic drugs.
  • Inflammatory toxic-metabolic or hypertoxic disorders may cause damage to the myelin sheaths.
  • disorders, or diseases are:
  • ADEM acute disseminated encephalomyelitis and perivenous encephalomyelitis
  • SSPE subacute sclerosing panencepohalitis
  • the Guillain-Barre syndrome (B. Vinken, Handbook of Clinical Neurology 7, Diseases of Nerves, Part I, Chapter 19 (1970) pp. 495 et seq.) is the most frequently observed type of peripheral polyneuritis.
  • treatment with NGF should be initiated immediately so as to prevent demyelination, as is described in the present invention.
  • an inflammation of the optic nerve is usually observed.
  • the present invention is also directed to the treatment of inflammatory diseases of the optic nerve.
  • the action of NGF on the optic nerve is shown in Fig. 3.
  • Diseases in which a demyelination of nerve fibers occurs and which can be treated with the aid of the pharmaceutical composition according to the present invention can, for example, be caused by inflammation, autoimmune processes, enzymes or toxins. Examples of such diseases are, for instance, Multiple Sclerosis, slow virus encephalitis, various forms of myelitis or heavy metal poisoning. According to the invention it is preferred to administer NGF immediately after an inflammatory disease (causing further a demyelination) of an optic nerve is recognized.
  • composition according to the present invention is preferably administered systemically.
  • administration can be carried out by methods familiar to a person skilled in the art, for example, intrathecally, intravenously or subcutaneously.
  • NGF can be dissolved, for example, in physiological saline.
  • protease inhibitors e.g., aprotinin
  • the preferred lower limit for the daily administered NGF dose is at a concentration between 0.05 ⁇ g and 5 ⁇ g/kg body weight .
  • the administration of NGF is preferably carried out over a longer time period, i.e., longer than a day, preferably at least 48 hours.
  • Figure la shows the experimental procedures for preventing acute EAE according to the present invention.
  • the NGF treated animals showed a significant amelioration of the clinical score versus placebo treated animals.
  • Figure lc shows the EAE (experimental allergic encephalomyelitis) reaction in four different marmosets. Marmosets 26.33.93 and 26.17.94 are treated on day 7 after immunization with placebo (cytochrome) whereas marmosets 26.29.94 and 26.30.92 are treated on day 7 after immunization with NGF.
  • Figure 2 is a photomicrograph of frontal sections through the brains of marmosets (Luxol Fast Blue Staining), depicting two representative areas from the prosencephalon and mesencephalon.
  • the -number of lesions (arrows point to examples in the photomicrographs) is lower in the NGF treated animal than in the marmoset receiving placebo- infusion of cytochrome C (magnification: 4x).
  • Figure 3 is a higher magnification of cross sections through the optic nerves of marmosets (Luxol Fast Blue-staining). Note the severe inflammation and demyelination in a cytochrome C-treated EAE-animal, in contrast to the absence of lesions in the marmoset receiving intraventricular NGF-infusion. Magnification: ca. 100X.
  • a technique for implanting indwelling cannulas in the brain ventricles of marmosets has been developed. Seven days prior to immunization for EAE, surgery is carried out after the animal is anaesthetized with ketamine/midazolam (20 mg/kg). The skin above the skull is shaved and surgically disinfected with surgical scrub and ethyl alcohol. The animal is positioned in a stereotaxic table on a K-pad and a sterile field is created over the skull. A skin incision is made and the skull exposed.
  • the dura meninges are exposed through a small hole drilled in the bone of the skull, in regard to the appropriate coordinates for the right lateral ventricle (according to a published atlas of marmoset brain anatomy).
  • a 25 gauge 5 mm- long stainless steel guide containing a 35 gauge polycarbonate cannula is then inserted in the cerebral ventricle, and secured to the bone with 2 lateral screws and a thin layer of dental cement.
  • a second skin incision is made in the right flank of the animal.
  • a 25 gauge polyvinyl catheter is connected with the ventricular cannula and then tunneled under the skin to the flank incision.
  • the animal For pump replacement, the animal is first sedated (ketamine/midazolam), the hair on one flank trimmed and the skin disinfected using surgical, sterile technique. The animal is placed on a K-pad to prevent hypothermia. A small skin incision (0.5 cm) is made on the flank and the pump connected with the intracerebroventrical catheter and inserted in the subcutaneous space, then the incision is closed with surgical staples. Although no infectious complication has been observed using this technique, Clamoxyl at 10 mg/kg intramuscularly x 1 is administered prophylactically at the time of pump insertion. The size of the pump suitable for marmosets is the 200 ⁇ l capacity model 2002, i.e. the smallest available.
  • EAE is induced with 100 ⁇ g of rat recombinant myelin/oligodendrocyte glycoprotein (MOG) in complete Freund's adjuvant followed by intravenous administration of 10 10 killed Bordetella Pertussis organisms on the day of immunization and again 48 hours later.
  • MOG myelin/oligodendrocyte glycoprotein
  • antigen and adjuvant are emulsified under sterile conditions.
  • the animal is then anesthetized with keamine/midazolam and 100 ⁇ l of the mixture is injected intradermally into four sites in the shoulder and hip areas. Prior to injection the sites are shaved, cleaned three times with surgical scrub and then twice with ethyl alcohol.
  • Bordetella Pertussis is injected slowly (over 5 minutes) after placement of a 21 gauge catheter in the popliteal vein using the same skin preparation technique. A second Bordetella Pertussis injection is given 48 hours later using the same technique.
  • the animals receive either placebo (cytochrome) or NGF delivered via a cannula implanted in the lateral ventricle and connected by a mini catheter to an osmotic Alza mini pump implanted under the skin of the flank; the pump containing NGF or placebo is implanted on day 0 (to account for the dead volume of the mini catheter) and delivers 6 ⁇ 1 ⁇ l/day until day 28.
  • the dose of NGF is 6 ⁇ g/day, determined on the basis of preliminary experiments in marmosets. Treatment is continued for a total of 21 days and the animals are euthanized ( Figure la and table 2).
  • Neuropatho logic examination of the brain and spinal cord is performed according to standard published techniques. Animals are euthanized at the end of the 28 day period and the nervous system perfused. Under deep pentobarbital anesthesia a thoracotomy is performed and a 14 gauge catheter is introduced and secured in the left ventricle. The right atrium is then opened and 200 ml of cold phosphate buffered saline are perfused through the heart. The descending aorta is then clamped in order to preserve spleen, inguinal lymph nodes, and the lower portion of the spinal cord as a supply of fresh or cryopreserved tissues for immunologic studies.
  • Immunohistochemistry is performed on fixed tissues or on cryopreserved specimens from the caudal spinal cord, including staining with anti-MOG, anti-PLP and anti-MAG antibodies. In addition to ultrastructural analysis, these studies provide a valid assessment of the remyelinating process, if any, in and around the inflammatory lesions.
  • TNF- ⁇ tumor necrosis factor
  • TNF- ⁇ lymphotoxin
  • IL-2 lymphotoxin
  • IL-6 transforming growth factor-
  • TGF- ⁇ transforming growth factor-
  • NGF NGF
  • 6 ⁇ g/day intracerebroventricularly b) Placebo (Cytochrome c), 6 g/day intracerebroventricularly
  • Treatments begin 7 days after immunization and are continued until day 28 after immunization.

Abstract

La présente invention a pour objet un procédé pour la prévention de la démyélinisation des fibres nerveuses, en particulier, des fibres nerveuses de l'homme. Selon ce procédé, les patients sont traités par le facteur de croissance du tissu nerveux, ou par des fragments de ce facteur. En outre, l'invention traite d'une composition pour traiter les maladies impliquant une démyélinisation des fibres nerveuses. Cette composition contient un facteur de croissance du tissu nerveux ou un fragment actif de ce dernier comme substance active, avec les excipients pharmaceutiques classiques, les substances auxiliaires, les charges et les diluants.
PCT/EP1998/002029 1997-04-11 1998-04-08 Facteur de croissance du tissu nerveux pour la prevention de la demyelinisation dans le systeme nerveux WO1998046254A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU72149/98A AU7214998A (en) 1997-04-11 1998-04-08 Ngf for the prevention of demyelination in the nervous system
CA002286137A CA2286137A1 (fr) 1997-04-11 1998-04-08 Facteur de croissance du tissu nerveux pour la prevention de la demyelinisation dans le systeme nerveux
US09/854,142 US7282482B2 (en) 1998-04-08 2001-05-10 NGF for the prevention of demyelination in the nervous system

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83395997A 1997-04-11 1997-04-11
US08/833,959 1997-04-11

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/854,142 Continuation-In-Part US7282482B2 (en) 1998-04-08 2001-05-10 NGF for the prevention of demyelination in the nervous system
US09529369 A-371-Of-International 2001-06-08

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WO1998046254A1 true WO1998046254A1 (fr) 1998-10-22

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AU (1) AU7214998A (fr)
CA (1) CA2286137A1 (fr)
WO (1) WO1998046254A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049313A1 (fr) * 1999-12-30 2001-07-12 Medscand Medical Ab Utilisation du facteur de croissance neuronale pour la production d'un medicament destine au traitement d'affections allergiques
CN1112215C (zh) * 1999-12-28 2003-06-25 潘晓东 神经生长因子在治疗有机溶剂中毒性周围神经病中的用途
WO2004091651A1 (fr) * 2003-04-15 2004-10-28 Scil Proteins Gmbh Prongf utilise en tant qu'agent pharmaceutiquement efficace pour traiter des maladies demyelinisantes
US7282482B2 (en) * 1998-04-08 2007-10-16 The Regents Of The University Of California NGF for the prevention of demyelination in the nervous system
EP1891966A1 (fr) * 2002-12-20 2008-02-27 Neuronicon ApS Modulation de l'activité des neurotrophines
US8748384B2 (en) 2006-12-21 2014-06-10 H. Lundbeck A/S Modulation of activity of proneurotrophins

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4125933A1 (de) * 1991-08-05 1993-02-11 Max Planck Gesellschaft Verbesserung der regeneration von oligodendrocyten
EP0731108A1 (fr) * 1995-03-10 1996-09-11 Boehringer Mannheim Gmbh Procédé de fabrication d'un agent thérapeutique pour la régénération d'oligodendrocytes
WO1997017087A1 (fr) * 1995-11-07 1997-05-15 Genentech, Inc. Formulation stabilisante pour le facteur humain de croissance nerveuse

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4125933A1 (de) * 1991-08-05 1993-02-11 Max Planck Gesellschaft Verbesserung der regeneration von oligodendrocyten
WO1993003140A1 (fr) * 1991-08-05 1993-02-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Amelioration de la regeneration d'oligodendrocytes
EP0731108A1 (fr) * 1995-03-10 1996-09-11 Boehringer Mannheim Gmbh Procédé de fabrication d'un agent thérapeutique pour la régénération d'oligodendrocytes
WO1997017087A1 (fr) * 1995-11-07 1997-05-15 Genentech, Inc. Formulation stabilisante pour le facteur humain de croissance nerveuse

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7282482B2 (en) * 1998-04-08 2007-10-16 The Regents Of The University Of California NGF for the prevention of demyelination in the nervous system
CN1112215C (zh) * 1999-12-28 2003-06-25 潘晓东 神经生长因子在治疗有机溶剂中毒性周围神经病中的用途
WO2001049313A1 (fr) * 1999-12-30 2001-07-12 Medscand Medical Ab Utilisation du facteur de croissance neuronale pour la production d'un medicament destine au traitement d'affections allergiques
EP1891966A1 (fr) * 2002-12-20 2008-02-27 Neuronicon ApS Modulation de l'activité des neurotrophines
US8066997B2 (en) 2002-12-20 2011-11-29 Anders Nykjaer Modulation of activity of neurotrophins
US8815808B2 (en) 2002-12-20 2014-08-26 H. Lundbeck A/S Modulation of activity of neurotrophins
US8986690B2 (en) 2002-12-20 2015-03-24 H. Lundbeck A/S Modulation of activity of neurotrophins
US9605073B2 (en) 2002-12-20 2017-03-28 H. Lundbeck A/S Modulation of activity of neurotrophins
WO2004091651A1 (fr) * 2003-04-15 2004-10-28 Scil Proteins Gmbh Prongf utilise en tant qu'agent pharmaceutiquement efficace pour traiter des maladies demyelinisantes
US8748384B2 (en) 2006-12-21 2014-06-10 H. Lundbeck A/S Modulation of activity of proneurotrophins
US9234036B2 (en) 2006-12-21 2016-01-12 H. Lundbeck A/S Modulation of activity of proneurotrophins

Also Published As

Publication number Publication date
AU7214998A (en) 1998-11-11
CA2286137A1 (fr) 1998-10-22

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