WO1999015192A1 - Agent de sauvetage neuronal - Google Patents

Agent de sauvetage neuronal Download PDF

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Publication number
WO1999015192A1
WO1999015192A1 PCT/NZ1998/000139 NZ9800139W WO9915192A1 WO 1999015192 A1 WO1999015192 A1 WO 1999015192A1 NZ 9800139 W NZ9800139 W NZ 9800139W WO 9915192 A1 WO9915192 A1 WO 9915192A1
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activin
neurons
insult
patient
neuronal
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PCT/NZ1998/000139
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English (en)
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Peter David Gluckman
Christopher Edward Williams
Dahao Wu
Paul Edmund Hughes
Maggie Lai
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Auckland Uniservices Limited
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Priority to AU93684/98A priority Critical patent/AU738192B2/en
Publication of WO1999015192A1 publication Critical patent/WO1999015192A1/fr
Priority to US11/493,883 priority patent/US20070015707A1/en

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    • 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
    • A61K38/22—Hormones

Definitions

  • This invention is directed to a new therapeutic use of activin and its .analogs. More p-articularly, it is directed to the use of activin and its analogs as neuronal rescue agents and/ or neuronal phenotype restoratives.
  • TGF- ⁇ tr.ansforming growth factor- ⁇ family display multiple roles as hormonal, paracrine and autocrine regulators of cellular function, growth and differentiation. Amongst them, activin is emerging as an important factor in an increasingly diverse range of biological processes including hormone production and secretion, modulation of testicular and ovarian cell function, induction of erythropoiesis, initiation of early embryonic development and more recently as a promoter of wound repair.
  • activin has been shown to be neuro- prophylactic and to promote survival of neurons subjected to a subsequent toxic event (see, for example, Krieglstein et al; "TGF- ⁇ superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP + toxicity" EMBO Journal Vol. 14 No. 4 pp 736-742 (1995)).
  • TGF- ⁇ superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP + toxicity
  • neuronal “rescue” is distinct from “prophylaxis”.
  • a neuronal “rescue” agent is one which, when administered after an insult prevents neurons from dying which would otherwise be destined to die.
  • a neuro- prophylactic agent is one which protects neurons against insult but only where the agent is present at the time of or before the insult.
  • activin is a neuronal rescue agent as well as being neuro-prophylactic which primarily underlies the present invention.
  • a number of known compounds .are neuro-prophylactic but are not neuronal rescue agents, eg. flunarazine (Gunn .and Gluckman, (1991)), GM1 ganglioside (Simon et al, (1993)), NGF- ⁇ (Rabizadeh et al, (1994)), bFGF (Mattson et al, (1993)), TNF (Barger et al, (1995)) .and TNF ⁇ (Barger et al, (1995)).
  • Other compounds are neuronal rescue agents without being neuro-prophylactic, eg.
  • IGF- 1 (Guan et al, (1993), Gwag et al, (1995)) and BDNF (Gwag et al, (1995)).
  • BDNF (Gwag et al, (1995)).
  • GPE GPE
  • the present invention provides a method of treating a patient to rescue neurons otherwise destined to die as the result of a prior neuronal insult which comprises administering to said patient activin or an analog thereof after said insult in an amount sufficient to prevent the neurons from dying.
  • the present invention provides a method of treating a patient to rescue neurons otherwise destined to die as the result of a prior neuronal insult which comprises increasing the active concentration of activin within said patient after said insult such that the neurons are prevented from dying.
  • Neuronal insult is used herein in its broadest possible sense and includes neuronal insults due to trauma, toxins, asphyxia, hypoxia-ischemia (HI) and disease.
  • Activin as used herein means activin A, activin B or activin AB of mammalian origin and preferably of human, porcine, bovine or murine origin.
  • Analog is used herein to mean a variant of activin through insertion, deletion or substitution of one or more amino acids but which retains at least substantially equivalent biological activity to activin.
  • the neuronal rescue role of activin is mediated through the activin type II receptor.
  • Specifically contemplated analogs are therefore those which bind to and activate the activin type II receptor.
  • the invention provides a method of treating a patient to rescue neurons otherwise destined to die as the result of prior neuronal insult which comprises activating the activin type II receptors of neuronal cells of a patient who has suffered a prior neuronal insult.
  • Activation can be through administration of a ligand which binds to, and activates, the receptor.
  • activin type II receptor activation is effected through administration of activin or an analog thereof.
  • the invention provides the use of activin or an analog thereof, or a ligand which binds to .and activates activin type II receptors, in the preparation of a medicament for rescuing neurons otherwise destined to die as a result of a prior neuronal insult.
  • the invention provides a method of treating a patient to restore the phenotype of neurons degenerating as a result of a prior neuronal insult which comprises administering to said patient activin or an analog thereof after said insult in an amount effective to restore the phenotype of said neurons.
  • the invention provides a method of treating a patient to restore the phenotype of neurons degenerating as a result of a prior neuronal insult which comprises increasing the active concentration of activin within said patient after said insult such that the phenotype of said neurons is restored.
  • the invention provides a method of treating a patient to restore the phenotype of neurons degenerating as a result of a prior neuronal insult which comprises activating the activin type II receptors of neuronal cells of a patient who has suffered a prior neuronal insult.
  • the invention provides the use of activin or an analog thereof, or a ligand which binds to and activates activin type II receptors, in the preparation of a medicament for restoring the phenotype of neurons degenerating as a result of a prior neuronal insult.
  • Figure 1 is a graph showing the rescue of striatal neurons by activin following HI
  • Figure 2 is a graph showing the rescue of striatal cholinergic phenotype neurons by activin after HI;
  • Figure 3 shows the results of immunoreactive staining of the activin-type II receptor in neurons of the striatum ( Figure 3A), the substantia nigra ( Figure 3B), thalamus ( Figure 3C) .and paraventricular nucleus ( Figure 3D);
  • Figure 4 is a graph showing the rescue of calbindin, cholinergic, NADPH- diaphorase and parvalbumin phenotypic neurons by activin following an intrastriatal quinolinic acid lesion ( Figures 4A to 4F);
  • Figure 5 shows the restorative effects of rhActivinA treatment on striatal interneurons immunostained for ChAT ( Figures 5A to 5D);
  • Figure 6 shows the areas of the rat brain analysed for the effect of activin and inhibin on neuronal rescue (Figure 6A: cortex and hippocampus; Figure 6B: striatum);
  • Figure 7 shows the comparative effects of activin A and inhibin A on the survival of neurons following HI ( Figure 7 A: Activin, Figure 7B: Inhibin); and Figure 8 shows the results of immunohistochemical staining of an Alzheimer's brain ( Figures 8A to 8G).
  • the present invention relates primarily to neuronal rescue. This is the maintenance of neuronal cells which would otherwise be destined to die as a result of a prior neuronal insult. The cells are therefore "rescued” from death and not merely protected prophylactically.
  • the invention also relates to phenotype restoration.
  • the applicants have found that degenerating neuronal cells which have lost their phenotype as the result of a prior neuronal insult can be phenotypically restored.
  • the applicants have found that neuronal rescue ⁇ phenotype restoration is able to be effected using two approaches.
  • the first approach is through a focus upon activin.
  • the applicants have found that increasing the effective concentration of activin within a patient following neuronal insult rescues neurons and/ or restores their phenotype.
  • activin itself is critical to this approach. There are three isoforms of activin, designated activin A, activin B and activin AB. Structural analysis shows that activins are disulphide linked dimers of two subunits, which are two distinct 14 kD ⁇ subunits ( ⁇ A and ⁇ B) (Ying, 1989, Vale et al, 1990). Activins (28 kD) are homodimers of the two ⁇ subunits. The mature ⁇ A or ⁇ B subunit has 116 or 115 amino acids respectively including 9 cysteines with no glycosylation sites. The two ⁇ subunits share about 85% homology within each species, and are also highly homologous across species. The mature ⁇ A subunits are completely identical across porcine, bovine, human and murine species and the mature ⁇ B subunit only has differences at four amino acid positions (Esch et al, (1987)).
  • activin ⁇ C, ⁇ D, ⁇ E subunits Recently, the molecular cloning of activin ⁇ C, ⁇ D, ⁇ E subunits has been reported (Fang et al, 1996). Activins made up from or including one or more of these subunits are in no way intended to be excluded. All of the above forms of activin are contemplated for use in this invention.
  • activin A The preferred form of activin for use in this invention is activin A. This is available from National Institute of Health, USA.
  • the effective concentration of activin will be increased through direct administration using either activin itself or an activin prodrug (a form which is cleaved within the body to release activin). It is however not the applicant's intention to exclude increasing activin concentration through administration of either activin agonists (substances which effect a direct increase in production or activity of activin within the body, eg.
  • FSH FSH
  • cAMP activator of protein kinase A activator
  • 12-O-tetradecanoylphorbol 13-acetate TGF- ⁇ , IL- l ⁇ and TNF- ⁇
  • inhibitors of activin antagonists include substances such as estradiol.
  • Follistatin is one such substance. It is a single chain glycosylated protein, which was first isolated from porcine and bovine follicular fluid. The amino acid sequence of follistatin is distinct from those of the activin subunits and any other proteins in the TGF- ⁇ family. However, across species, follistatin amino acid sequence is highly conserved with over 98% homology.
  • follistatin As a binding protein for activin, follistatin has been observed to have different actions on the biological activities of activin. Follistatin can directly bind to activin to neutralize its function in many systems (Mathews, 1994). However, it has also been suggested to have an ability to enhance activin action through either bringing activin to its receptors or maintaining a high local concentration of activin. Thus follistatin may exert a dual effect in mediating activin activities (Macconell et al, 1996) as both agonist and .antagonist.
  • Inhibin can also be regarded as an activin antagonist.
  • the mechanism by which this is achieved is not completely understood but is likely to include competitive binding to the activin receptor. Therefore, effecting a decrease in the production or action of inhibin is likely to increase the effective concentration of activin.
  • Another possibility is administration of a replicable vehicle encoding activin to the patient.
  • a vehicle which may be a modified cell line or virus which expresses activin within the patient
  • analogs can be employed in this invention.
  • analog means a protein which is a variant of activin through insertion, deletion or substitution of one or more amino acids but which retains at least substantial functional equivalency.
  • a protein is a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has at least substantially the same function as, the original protein.
  • the equivalent can be, for example, a fragment of the protein, a fusion of the protein with another protein or carrier, or a fusion of a fragment with additional amino acids.
  • activin analogs can also be readily screened for by reference to the ability of the analog to both bind to and activate the appropriate receptor.
  • the receptor is an activin type II receptor.
  • ActRII activin type I receptor
  • ActRIII activin type II receptor
  • ActRII was the first receptor identified for activin and for other members in the TGF- ⁇ superfamily (Mathews and Vale, 1991).
  • the mature ActRII is comprised of 494 amino acids which includes a small 116 amino acid extracellular ligand binding domain, a single transmembrane domain and an intracellular serine/threonine kinase domain, which is common in the TGF- ⁇ superfamily.
  • Over 90% sequence homology of ActRII has been observed across species, which is consistent to the high similarity of mature activin ⁇ A sequences in various species (Mathews, 1994).
  • ActRIIB A distinct but closely related activin receptor ActRIIB and its four isoforms have subsequently been characterized (Mathews, 1994; Mathews et al, 1992; Attisano et al, 1992). ActRII and ActRIIB are approximately 50-60% identical in the ligand-binding domain and 60-70% identical in the kinase domain. ActRII, ActRIIB and its isoforms all bind to activin with high affinity.
  • ActRII ActRIIB and their isoforms are referred to herein as "activin type II receptors”.
  • Activin type II receptors are distinct from Activin type I receptors (ActRI). ActRI and its isoforms have been cloned using PCR with oligonucleotides based on the ActRII sequence. These receptors also have highly conserved serine kinases. However, cells expressing ActRI but not ActRII cannot bind to activin alone (Mathews, 1994) and hence the capacity to bind to activin type II receptors is considered critical to this aspect of the invention.
  • ActRII initiates signal transduction across the membrane through both ActRI and ActRII which can form a stable complex with the ligand (Willis et al, 1996; Mathews, 1994). ActRII binds activin and then associates with a type I receptor. This is followed by auto- and trans-phosphorylation between the two receptors and the initiation of intracellular signalling (Smith J, 1995; Willis et al, 1996).
  • activin and its analogs are ligands which achieve this. Indeed, the use of activin and activin analogs represents a preferred aspect of the invention. However, it should be appreciated that this approach is not restricted to the use of activin and its analogs but also extends to any ligand which fulfils the functional requirement of both binding to and activating (stimulating) the activin type II receptor. Implicit in this will be the ability of the ligand to effect the association with ActRI needed to initiate intracellular signalling.
  • Such stimulatory ligands can be identified by a screening protocol employing at least the ligand binding domain of an activin type II receptor.
  • This screening method can, for example, utilize the expression of the Act II receptor in Xenopus oocytes using standard recombinant DNA methods and measurement of the Act II receptor-mediated signal transduction evolved by novel stimulatory ligands. Further classical "grind and bind" ligand-binding experiments can also be utilized. Here, whole brain or specific brain regions would be homogenized and the specific- binding of novel compounds to the Act II receptor characterized. This technique allows further characterization of specificity and affinity (potency) of the compound for the Act II receptor complex.
  • the active compound (activin, analog or ligand) will be formulated as a medicament.
  • the details of the formulation will ultimately depend upon the insult to be remedied and the route of administration, but will usually include combination of the active compound with a suitable carrier, vehicle or diluent.
  • Insults which can be treated in accordance with the invention include any prior neuronal insult. These include trauma, toxins, asphyxia, ischemia and disease, particularly neurodegenerative diseases such as Alzheimer's disease (including both early and late onset forms) Parkinson's disease, Huntington's disease and Lewy Body disease. Also included is peripheral neuropathy.
  • a variety of administration routes can be used. Examples include peripherally in conditions where the blood brain barrier is disrupted (ie. ischemia), intracerebroventricularly (ICV), intraventricular administration involving neurosurgical insertion of a ventricular cannula with an abdominal pump and reservoir and intraparenchymal (ie. at the site of action). Dosage rates will also be formulation- and insult-dependent. However, by way of example, the recommended dosage rate of activin A formulated for injection would be in the range of lng/ lOOg to lOO ⁇ g/ lOOg administered centrally.
  • HI injury was induced in weaned 21 day old Wistar rats using a modified version of the Levine rat preparation as described previously (Sirimanne et al, (1994)). Rats were maintained on a 12 hour cycle of light and dark and given free access to food and water throughout the study. Rats of both sexes weighing between 40-49g were selected, anaesthetized with halothane and underwent double ligation of the right carotid artery following exposure through a midventral neck incision.
  • Rats were euthanised 72 hours after hypoxia by sodium pentobarbital overdose and brains were collected for histological processing after in situ fixation by transcardial perfusion with 30ml saline (0.9%) then neutral buffered formalin (10%). Fixed brains were dehydrated through graded alcohols, defatted in chloroform and embedded in paraffin.
  • Double-labelling Immunohistochemistry Double immunohistochemical labelling to localise ActRII to specific neuronal subtypes was performed on rat brain sections at the level of striatum (cholinergic, parvalbumin, calbindin) or substantia nigra (dopaminergic). Brains were processed as described above. Antiserum generated towards a synthetic fragment of mActRII (482-494) was a generous gift of Professor Wylie Vale (The Salk Institute, San Diego, CA, USA). Sections were dewaxed, rehydrated and incubated for 30 minutes in 0.6% hydrogen peroxide to quench endogenous peroxidase activity.
  • ActRII antibody (1: 1000) diluted in 1.5% normal goat serum and phosphate buffered saline (PBS) was carried out overnight in a humidified environment. PBS-washed sections were then incubated with biotinylated secondary antiserum for 1 hour, washed, then incubated with an avidin-biotinylated horseradish peroxidase complex (Vector Labs, Burlingame, CA, USA) for a further 1 hour. Signal for ActRII was visualised using 0.05%, 3,3'diaminobenzidine tetrahydrochloride (Sigma Chemicals, St Louis, MO, USA).
  • Sections were then reincubated with either parvalbumin (1: 1000), calbindin D28K (1: 1000) or ChAT (1:50, identifies cholinergic neurons) or tyrosine hydroxylase (1: 1000 identifies dopaminergic neurons) as described above except that immunostaining for the second .antibody was visualised using benzidine dihydrochloride (BDHC, Sigma). Non-specific immunostaining was determined using normal rabbit serum, normal mouse serum and incubating sections in the absence of primary antibodies.
  • BDHC benzidine dihydrochloride
  • the total number of surviving neurons were counted in the CA1/2 region of the hippocampus, 3 selected areas of the cortex and 4 selected areas of the striatum or the total number of ChAT immunopositive neurons in the dorsolateral striatum were counted in the injured half brain of both treatment groups (vehicle and activin A) with a light microscope (Leica, Germany) and were compared. Results
  • Activin type II receptor immunoreactivity was found in cholinergic, parvalbumin and calbindin neurons of the striatum and in dopaminergic neurons of the substantia nigra. Staining was predominantly in the cell body, axons and processes of neurons but clearly did not show labelling in the nucleus. ActRII immunoreactivity was observed in a number of thalamic nuclei particularly; the lateral and ventral posterior thalamic nucleic, and reticular thalamic nucleus.
  • Activin A is applicable for the treatment of brain injury after HI. Since activin was significantly protective in the striatum and 95% of striatal neurons are GABAergic, activin is likely able to rescue GABAergic neurons in vivo, which has relevance for treating the loss of GABAergic neurons in the striatum seen in Huntington's disease.
  • Activin specifically rescues cholinergic neuronal phenotype after HI and activin type II receptor is coloca ⁇ zed on cholinergic neurons. This indicated an application for activin in treating the hypofunction of central cholinergic neurotransmission seen in the human neurodegenerative condition known as Alzheimer's disease. This further indicated another application for activin in delaying or preventing the loss of cholinergic neurons in the nucleus basalis of Meynert.
  • Activin type II receptor colocalizes with tyrosine hydroxylase (dopaminergic) neurons of the substantia nigra. This indicates an application for activin A in delaying or preventing the loss of dopamine neurons seen in the human neurodegenerative condition known as Parkinson's disease.
  • a 22-gauge guide cannula (Plastics One, USA) was permanently fixed into place 0.5 mm dorsal to the ventral coordinate (i.e., at +0.5, +3.0, -4.5).
  • Groups of 4-5 animals received daily 1 ⁇ l injections of either rhactivinA (0.73 ⁇ g/ ⁇ l/day, National Hormone and Pituitary Program, CA, USA) or phosphate-buffered saline (PBS, pH 7.4) vehicle beginning at the time of cannulation for 7 days following QA lesioning.
  • Sections were rinsed 3 times in PBS and incubated in primary GAD67 at 1:200 with 2% normal goat serum in PBS at 4°C for 3 days. Sections were then rinsed .and incubated in the secondary antibody, biotinylated anti-rabbit IgG (Amersham RPN480, USA), at 1: 1000 with 1% normal goat serum in PBS at 4°C overnight. Sections were rinsed and incubated in streptavidin biotinylated peroxidase complex (Amersham RPN1050) at 1: 100 in PBS at room temperature for 4 hours. Sections were rinsed and developed with diaminobenzidine (Sigma) in deionized water with 0.03% hydrogen peroxide for 5 minutes.
  • Acetylcholine neurons were stained using an antibody against human choline acetyl-transferase (ChAT) made in goat from Chemicon (#AB144P). Sections were stained as for GAD 67 , except that incubation in the secondary antibody was for 3 hours at room temperature and the solutions were as follows: the blocking solution was 2% rabbit serum in PBS, the primary antibody was diluted 1: 100 with 2% rabbit serum in PBS, the secondary antibody was anti-goat at 1:200 in PBS with 2% rabbit serum, the peroxidase complex was AB cocktail (Vector Laboratories, USA) at 9 ⁇ l of A plus 9 ⁇ l of B per ml of PBS, and the development solution was unchanged.
  • the blocking solution was 2% rabbit serum in PBS
  • the primary antibody was diluted 1: 100 with 2% rabbit serum in PBS
  • the secondary antibody was anti-goat at 1:200 in PBS with 2% rabbit serum
  • the peroxidase complex was AB cocktail (Vector Laborator
  • NADPH-diaphorase (NADPHd) cells were visualized by incubating sections in 1 mg/ml NADPH (ICN Biochemicals) and 0.1 mg/ml nitrotetrazolium blue (ICN) dissolved in PB with 0.3% triton X- 100 for 2 hours at 37°C.
  • Stained cells (ChAT, NADPHd, parvalbumin and calretinin) were counted at 100X magnification in a visual field 950 x 730 ⁇ m.
  • the location of the field was 465 ⁇ m below the cannula tip at the lesion center (0.5 mm anterior to Bregma), and extended rostrocaudally along a parallel to sections at +360 ⁇ m rostral to the lesion center and -360 ⁇ m caudal to the lesion center. This was done to assess the effects of radial diffusion of solutions from the cannula on the rostrocaudal plane while keeping a fixed distance along the dorsoventral and mediolateral planes.
  • Cell counts are presented as means of cells/mm 2 ⁇ SEM at each of the three levels (rostral, center, caudal) in the striatum for both the contralateral (control) and ipsilateral (lesion and treatment) sides from 4-5 animals per group.
  • ipsilateral values were divided by contralateral values for each animal and means were calculated for each of the two groups. Comparisons between phenotypic cell survival at each coronal level were performed on percentage values to standardize for the differing sizes of the populations examined.
  • ActivinA also conferred a significant protective effect on parvalbumin (to 87.5+7.7%, see Fig 4E) and NADPH- diaphorase (to 77.5+7.5%, see Fig 4D) interneuron populations but failed to prevent the phenotypic degeneration of calretinin neurons (to 56.6 ⁇ 5.5%, see Fig 4F).
  • Glutamate decarboxylase67 and calbindin-staining nerve cells represent largely overlapping populations and both identify striatal GABAergic projection neurons.
  • ActivinA significantly attenuated the loss in the numbers of neurons staining for calbindin (to 79.7+6.6%, see Fig 4B) at 7 days following quinolinic acid lesioning.
  • Exogenous administration of ActivinA rescues both striatal interneurons (labelled with choline acetyltransferase, parvalbumin, NADPH-diaphorase) and striatal projection neurons (labelled by calbindin) from excitotoxic lesioning with QA. It also restores the phenotype of degenerating neurons.
  • Activin A to rescue striatal GABAergic projection neurons from degeneration following QA lesioning indicates an application for activin A in delaying or preventing the loss of GABAergic projection neurons which are preferentially lost in the human neurodegenerative disease, Huntington's disease.
  • activin A can restore the ChAT neuronal phenotype after a QA lesion in the striatum. This indicates an application for activin A in restoring ChAT neurons in the human neurodegenerative condition Huntington's disease and also Alzheimer's disease.
  • Moderate HI brain injury (15 minutes hypoxia) was first induced in 21 -day-old Wistar rats. Two hours after hypoxia, all rats were lightly anaesthetised with intraperitoneal injection of Saffan. An infusion needle (3 ⁇ G x 25.4 mm) was placed into the right lateral cerebroventricle of the rat brain with the aid of a metal skull template as described by Jirikowski (Jirikowski, 1992). Each rat was injected either with the drug (activin A or inhibin A) diluted with vehicle solution in a total volume of 20 ⁇ l, or 20 ml vehicle solution. The injections into vehicle and treatment groups were performed simultaneously with a micro-infusion pump at a rate of 3 ⁇ l/minute. After infusion, the rats were left to recover in an incubator maintained at 34°C and relative humidity of 85-95%. Once awake, the rats were transferred into their holding cages and fed food and liquid ad libitum.
  • the rats were transcardially perfused with 0.9% saline followed by 4% PFA, and the brains removed and embedded in paraffin.
  • Symmetric serial coronal sections (4 mm) were cut and stained with thionin/ acid-fusion for live/dead neurons (Sirimanne et al, 1994).
  • the histological outcome of neuronal survival was examined with light microscopy (Leica) in the cortex, hippocampus and striatum in the injured half of the brain according to a reference of rat brain anatomy (Paxinos and Watson, 1982), as these areas suffer most of the neuronal loss in the moderate HI brain injury model (Sirimanne et al, 1994). Only cells with a morphology like live neurons were counted, while dead neurons or cells with morphology like glial were not included. For each of the above three brain areas, one coronal section was used for each brain.
  • One coronal section between 2.8mm to 3mm from Bregma was used for the analysis of cerebral cortex. Live neurons within three rectangular areas each measuring 1000 ⁇ m x 5000 ⁇ m were counted with a microscope grid ( Figure 6A). The areas selected covered layer II to layer V of the parietal cortex, where selective neuronal loss occurred.
  • the size of the areas were: Area 1-3: 1000 ⁇ m x 2000 ⁇ m, Area 4: 2000 ⁇ m x 2000 ⁇ m. Results
  • dead cells identified by the uptake of acid fusion were found in the ligated (right) hemisphere 3 days after hypoxia, particularly in regions such as the CA1/2 region of hippocampus, the upper two thirds of the striatum and layer III-IV of the cerebral cortex.
  • the severity of cell loss ranged from non-selective to massive in these areas. No cell loss was observed in the non-injured hemisphere.
  • Hippocampus All the live neurons in the hippocampal CA1/2 region in the injured (right) side were counted.
  • the mean number of surviving neurons increased to 107+39 as compared with 67+36 in the vehicle control group (mean+SEM, p ⁇ 0.05, Mann- Whitney rank sum test).
  • CA1/2 region, striatum and cerebral cortex as described in the activin A treatment study.
  • inhibin A decreased the number of surviving neurons in the dorsolateral striatum from 71+ 10 to 70+9 and in the cortex from 311+ 18 to 289+_19 while increased the number of surviving neurons in the hippocampal CA1/2 region from 100+26 to 123+31 (mean+SEM).
  • inhibin is not effective as a neuronal rescue agent. This is consistent with inhibin binding to but not activating the activin type II receptor and therefore being a functional antagonist of activin. This in turn supports the applicant's findings regarding the critical role activation or stimulation of the activin type II receptor plays in neuronal rescue.
  • follistatin peptide in the brain of an Alzheimer's sufferer was examined immunohistochemically with a Vectastain kit (Vector labs, USA). Serial formalin fixed and paraffin embedded post mortem human brain tissue (from the medial frontal gyrus) were cut at 4 mm, which were then dewaxed and rehydrated. Non-specific staining which arises from the endogenous peroxidase activity and non-specific binding sites on the brain sections were blocked by 0.3% H 2 0 2 and 1% normal goat serum respectively.
  • the sections were incubated in three steps with a polyclonal anti- human follistatin (1:500, a gift from Hiromu Sugino, the Institute for Enzyme Research, The University of Tokushima, Japan) at room temperature overnight, biotinylated goat-anti-rabbit-IgG (1: 100) at 37°C for 1 hour and horseradish-peroxidase (1: 100) at 37°C for 1 hour.
  • the sections were washed with 0.01M PBS three times after each of the above incubation.
  • the signals were washed with 0.01M PBS three times after each of the above incubations.
  • the signals on the sections were detected with a solution made with DAB tablets (Sigma, USA), which showed a brown colour.
  • the sections were counterstained with thionin, dehydrated and mounted with DPX.
  • a double immunohistochemistry method was used to identify brain cells which express follistatin.
  • the brown immunostaining for follistatin was first obtained on brain sections as described above.
  • the above three step immunostaining procedure with monoclonal anti-GFAP (1:500, Sigma) as the primary antibody was used to identify astrocytes.
  • the signals were detected with BDHC which showed a blue colour.
  • Cortical senile plaques which contain ⁇ -amyloid are characteristic of Alzheimer's disease.
  • a triple immunohistochemistry method was used. After dewaxing and rehydration, brain sections were treated with concentrated formic acid for 5 minutes, which can enhance the intensity of the immunostaining of senile plaques. The sections were thorough washed with water and blocked for possible non-specific staining. A monoclonal anti-human ⁇ -amyloid (1: 1000, Dako, Denmark) was used as the primary antibody in the three step immunostaining procedure and Ni-DAB tablets (Sigma) were used as a blue chromagen. The brain sections were then immunostained for follistatin and GFAP as described above.
  • Activin ⁇ A peptide expression and its spatial relationship to senile plaques was examined with a similar double immunohistochemistry method as described above. Briefly, the brain sections were first immunostained for ⁇ -amyloid positive plaques with Ni-DAB as the chromagen. The sections were then incubated with polyclonal anti-activin ⁇ A subunit (1:250, a gift from Professor Wylie Vale, The Salk Institute for Biological Studies, USA) as the primary antibody. DAB was used as the brown chromagen for this staining.
  • follistatin protein is increased in an Alzheimer's disease patient brain ( Figure 8B).
  • Double immunohistochemistry labelling showed that follistatin expression was mainly colocalised with glial fibrillary acidic protein (GFAP) positive astrocytes (blue staining) and also possibly microglia (Figures 8C and 8D).
  • Figure 8D also shows staining for ⁇ -amyloid (red brown staining).
  • Figure 8G shows activin type II receptor staining in neurons that ook' damaged in Alzheimer's disease brain tissue.
  • the black staining is ⁇ -amyloid.
  • follistatin upregulation an activin inhibitor
  • the upregulation of activin may indicate the induction of an endogenous neuronal rescue mechanism in the Alzheimer's disease brain.
  • the invention therefore provides new approaches to neuronal rescue and neuronal phenotype restoration. These involve firstly increasing the active concentration of activin in a patient following neuronal insult and secondly the activation of the activin type II receptors localized on neuronal cells, again following neuronal insult.
  • the approaches of the invention have application in the treatment of patients who have suffered neuronal insult particularly as the result of a neurodegenerative disease.
  • Two such diseases of considerable interest are Alzheimer's disease and Parkinson's disease. Patients suffering from these diseases will benefit greatly by a treatment protocol able to rescue damaged and dying neuronal cell populations.
  • the invention has application in the rescue of neurons destined to die following insult in the form of trauma, toxin exposure, asphyxia or hypoxia- ischemia.
  • the present methods also show the capability of restoring phenotypes in injured, degenerating and diseased neurons, particularly those of the following phenotypes:
  • ChAT ChAT, calbindin, NADPHd, parvalbumin, GABAergic and glutamatergic neurons.
  • Novel activin receptors distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors.
  • BDNF or IGF-1 potentiates free radical-mediated injury in cortical cell cultures. NeuroReport, 7(l):93-96.
  • Beta-amyloid precursor protein metabolites and loss of neuronal Ca 2+ homeostasis in Alzheimer's disease. Trends in Neurosciences, 16:409-414.

Abstract

L'invention concerne une nouvelle utilisation thérapeutique de l'activine et des analogues de celle-ci. Cette utilisation est notamment destinée à sauver des neurones, lesquels sans cette thérapie seraient destinés à mourir après la survenue d'un accident neuronal, et/ou à restaurer le phénotype d'une cellule neuronale dont la dégénérescence a été provoquée par un accident neuronal.
PCT/NZ1998/000139 1997-09-19 1998-09-18 Agent de sauvetage neuronal WO1999015192A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU93684/98A AU738192B2 (en) 1997-09-19 1998-09-18 Neuronal rescue agent
US11/493,883 US20070015707A1 (en) 1997-09-19 2006-07-26 Neuroprotective effect of activin in animals exposed to hypoxia/ischemia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ32879697 1997-09-19
NZ328796 1997-09-19

Related Child Applications (2)

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US10/157,542 Continuation US20030060398A1 (en) 1997-09-19 2002-05-28 Neuronal rescue agent

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000281A1 (fr) * 2001-06-22 2003-01-03 Neuronz Limited Neuroprotection et/ou neurorestoration via le recepteur de l'activine neurale de type iib
US7041314B2 (en) 2001-05-24 2006-05-09 Neuren Pharmaceuticals Ltd. GPE analogs and peptidominetics
WO2006108651A2 (fr) * 2005-04-12 2006-10-19 Develogen Aktiengesellschaft Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique
EP2011484A2 (fr) 1999-11-30 2009-01-07 Novartis Ag Utilisation d'une composition de formotérol sous forme de poudre séche pour le traitement de maladie chronique obstructive pulmonaire
US7605177B2 (en) 2001-05-24 2009-10-20 Neuren Pharmaceuticals Limited Effects of glycyl-2 methyl prolyl glutamate on neurodegeneration
US7714020B2 (en) 2001-05-24 2010-05-11 Neuren Pharmaceuticals Limited Treatment of non-convulsive seizures in brain injury using G-2-methyl-prolyl glutamate
US9271904B2 (en) 2003-11-21 2016-03-01 Intercontinental Great Brands Llc Controlled release oral delivery systems
EP3059245A1 (fr) * 2004-07-23 2016-08-24 Acceleron Pharma Inc. Polypeptides du récepteur actrii, procédés et compositions

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WO1991019508A1 (fr) * 1990-06-15 1991-12-26 Peter Wehling Regenerateur des nerfs

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WO1991019508A1 (fr) * 1990-06-15 1991-12-26 Peter Wehling Regenerateur des nerfs

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DERWENT ABSTRACT, Accession No. 91-322780/44, Class S03; & SU 1608584 A (ROST MEDICINE INST) 23 November 1990. *
DERWENT ABSTRACT, Accession No. 92-005183/01, Class B05; & SU 1623660 A (SEMIPALANTINSK MED) 30 January 1991. *
NEUROREPORT, Vol. 7, No. 11, 29 July 1996, TRETTER Y.P. et al., "Strong Induction of Activin Expression after Hippocampal Lesion", pages 1819-1823. *
NEUROREPORT, Vol. 8, No. 12, 18 August 1997, LAI M. et al., "Focal Brain Injury Increases Activin beta A mRNA Expression in Hippocampal Neurons", pages 2691-2694. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2011484A2 (fr) 1999-11-30 2009-01-07 Novartis Ag Utilisation d'une composition de formotérol sous forme de poudre séche pour le traitement de maladie chronique obstructive pulmonaire
US7041314B2 (en) 2001-05-24 2006-05-09 Neuren Pharmaceuticals Ltd. GPE analogs and peptidominetics
US7605177B2 (en) 2001-05-24 2009-10-20 Neuren Pharmaceuticals Limited Effects of glycyl-2 methyl prolyl glutamate on neurodegeneration
US7714020B2 (en) 2001-05-24 2010-05-11 Neuren Pharmaceuticals Limited Treatment of non-convulsive seizures in brain injury using G-2-methyl-prolyl glutamate
WO2003000281A1 (fr) * 2001-06-22 2003-01-03 Neuronz Limited Neuroprotection et/ou neurorestoration via le recepteur de l'activine neurale de type iib
US9271904B2 (en) 2003-11-21 2016-03-01 Intercontinental Great Brands Llc Controlled release oral delivery systems
EP3059245A1 (fr) * 2004-07-23 2016-08-24 Acceleron Pharma Inc. Polypeptides du récepteur actrii, procédés et compositions
WO2006108651A2 (fr) * 2005-04-12 2006-10-19 Develogen Aktiengesellschaft Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique
WO2006108651A3 (fr) * 2005-04-12 2007-06-28 Develogen Ag Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique

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