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WO2014066686A1 - Cibles cérébrales pour facteurs neurotrophiques en vue de traiter une maladie neurodégénérative - Google Patents

Cibles cérébrales pour facteurs neurotrophiques en vue de traiter une maladie neurodégénérative Download PDF

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Publication number
WO2014066686A1
WO2014066686A1 PCT/US2013/066688 US2013066688W WO2014066686A1 WO 2014066686 A1 WO2014066686 A1 WO 2014066686A1 US 2013066688 W US2013066688 W US 2013066688W WO 2014066686 A1 WO2014066686 A1 WO 2014066686A1
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Prior art keywords
manf
ohda
subject
gdnf
striatum
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Inventor
John W. Commissiong
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Amarantus Bioscience Holdings Inc
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Amarantus Bioscience Holdings Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • a cause of Parkinson's disease is believed to be the death of dopaminergic (DAergic) neurons in the substantia nigra zona compacta (SNc) in the ventral midbrain.
  • DAergic dopaminergic
  • SNc substantia nigra zona compacta
  • the cell bodies of the DAergic neurons are located in the SNc, and their axons project to the striatum (caudate -putamen in man). This pathway is called the nigrostriatal projection.
  • MANF meencephalic astrocyte - derived neurotrophic factor
  • the invention provides a method of treating a subject with a condition, comprising contacting the substantia nigra of the subject with an amount of MANF effective to treat the condition.
  • the condition is a neurological disorder.
  • the neurological disorder is Parkinson's disease.
  • the subject is a mammal. In some embodiments, the
  • the method further comprises contacting the striatum of a subject with an amount of MANF effective to treat said condition.
  • the method further comprises administering one or more additional therapeutic agents.
  • the subject exhibits an improvement in their clinical scores after treatment.
  • the clinical score is a measurement of net ipsilateral rotations or total ipsilateral rotations.
  • the density of dopaminergic terminals in the putamen of the subject increases; the density of dopaminergic terminals in the putamen of the subject increases; the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the putamen of the subject both increase; the viability of cell bodies in the striatum of the subject increases; the percentage of dying dopaminergic neurons in the subject decreases; and/or the percentage of dead dopaminergic neurons in the subject decreases.
  • the amount of MANF administered is at least about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 ⁇ g. In certain embodiments, the amount of MANF administered is at least about 1.2, 1.4. 1.6. 1.8, or 2 mg.
  • the amount of MANF administered is less than about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 ⁇ g. In certain embodiments, the amount of MANF administered is less than about 1.2, 1.4. 1.6, 1.8, or 2 mg.
  • the amount of MANF administered is about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 ⁇ g. In certain embodiments, the amount of MANF administered is about 1.2, 1.4. 1.6. 1.8, or 2 mg.
  • the amount administered is selected to protect and/or
  • the method comprising administering a pharmaceutical composition comprising an effective amount of MANF to a brain region of the subject by convection enhanced delivery.
  • the neurological disorder is Parkinson's disease.
  • the brain region comprises the striatum, the substantia nigra, or a combination thereof.
  • the brain region comprises the striatum.
  • the brain region comprises the substantia nigra.
  • the brain region comprises the striatum and the substantia nigra.
  • the effective amount of MANF is from 1 ⁇ g to 1000 ⁇ g. In some embodiments, the effective amount of MANF is from 10 ⁇ g to 500 ⁇ g. In some embodiments, the effective amount of MANF is from 50 ⁇ g to 250 ⁇ g.
  • convection enhanced delivery is performed at a flow rate of from 0.1 ⁇ 7 ⁇ ⁇ 10 ⁇ 7 ⁇ .
  • convection enhanced delivery is performed acutely.
  • the convection enhanced delivery is repeated two or more times during a course of treatment.
  • convection enhanced delivery is performed chronically.
  • the pharmaceutical composition further comprises a tracer.
  • diffusion of the MANF in the brain region is monitored in by detection of the tracer.
  • monitoring comprises magnetic resonance imaging (MRI), computed tomography imaging, or X-ray computed tomography (CT).
  • the pharmaceutical composition further comprises a buffer.
  • the buffer is a citrate buffer.
  • the pharmaceutical composition further comprises a buffer.
  • composition has a pH of from 5.5 to 6.5.
  • the subject exhibits an improvement in a clinical score after
  • the percentage of viable dopaminergic neurons in the subject increases. In some embodiments, the density of dopaminergic terminals in the subject's putamen increases. In some embodiments, both the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the subject's putamen increase. In some embodiments, the viability of cell bodies in the striatum increases. In some embodiments, the percentage of dying dopaminergic neurons in the subject decreases. In some embodiments, the percentage of dead dopaminergic neurons in the subject decreases.
  • kits comprising: (a) a catheter, that can be stereotactically placed at or near the brain region; (b) a pump, for generating a positive pressure gradient between the catheter and the brain region; (c) a pharmaceutical composition comprising an effective amount of MANF to treat the neurological disorder.
  • kits comprising: (a) a catheter, that can be stereotactically placed at or near the brain region; (b) a pump, for generating a positive pressure gradient between the catheter and the brain region; (c) a pharmaceutical composition comprising an effective amount of MANF to treat the neurological disorder.
  • Some embodiments further comprise a delivery sheath to assist in placement of the catheter.
  • the catheter comprises multiple outlet ports.
  • the catheter comprises an outer tubing to provide structural rigidity to the catheter.
  • the pharmaceutical composition comprises from 1 ⁇ g to 1000 ⁇ g of the MANF. In some embodiments, the pharmaceutical composition comprises from 10 ⁇ g to 500 ⁇ g of the MANF. In some embodiments, the pharmaceutical composition comprises from 50 ⁇ g to 250 ⁇ g of the MANF.
  • the pharmaceutical composition further comprises a tracer.
  • the pharmaceutical composition further comprises a buffer.
  • the buffer is a citrate buffer.
  • the pharmaceutical composition has a pH of from 5.5 to 6.5.
  • the method comprising administering a pharmaceutical composition comprising an effective amount of MANF in a 10 mM citrate buffer, pH 6.1 to a brain region of the subject.
  • Figure 1 shows neurorestorative action of MANF and GDNF: MANF 3 and 10 ⁇ g, at 4-weeks, and GDNF, 10 ⁇ g at 8-weeks after 6-OHDA, caused a significant reduction in neurological deficits in the rodent model of PD, after a partial lesion of the nigrostriatal projection by the injection of 6-OHDA into the striatum unilaterally.
  • Order of Bars (1) Vehicle; (2) MANF 3 ⁇ g; (3) MANF 10 ⁇ g; (4) MANF 36 ⁇ 3 ⁇ 4 (5) GDNF 10 ⁇ g.
  • Figure 2 shows neurorestorative action of MANF but not GDNF: MANF 10 ⁇ g, at 2-weeks, and at 36 ⁇ g after 4-weeks, caused a significant reduction in neurological deficits in the rodent model of PD, after a partial lesion of the nigrostriatal projection by the injection of 6-OHDA into the SNc unilaterally.
  • GDNF had no effect at 10 ⁇ g, when tested at 2-weeks and 4-weeks after 6-OHDA.
  • Figure 3 shows the distribution of MANF in the striatum at different flow rates after 0 hours.
  • (C) 5 ⁇ / ⁇ . All infusions were 10 ⁇ g in 2 ⁇ _, PBS. Scale bar 250 ⁇ .
  • Figure 4 shows the distribution of MANF in the striatum at different flow rates after 7 days.
  • GDNF (1 & 3) and no infusion control (2 & 4) were completed at 2.5 ⁇ into the striatum, as were MANF (5) and no infusion control (6).
  • MANF (7, 9 & 1 1) or PBS (8)/ no infusion (10 & 12) controls were completed at 1.25 ⁇ into the striatum.
  • Scale bar 250 ⁇ .
  • Figure 5 shows calculated volume of distribution of MANF in the striatum at different flow rates after 7 days.
  • Figure 6 shows (A) an exemplary step-design cannula that can be used in
  • a delivery sheath that can function as a guide component for the step-design cannula
  • C an assembly of a step-design cannula wherein the upper portion of delivery sheath is removed.
  • Figure 7 illustrates an exemplary neurosurgical device that can be used in
  • convection enhanced delivery of therapeutic agents showing (A) a catheter and (B) the catheter inserted into a guide device.
  • Figure 8 illustrates 6-OHDA-induced dopaminergic denervation in the striatum.
  • A Densitometry data were acquired from rostral to caudal at four levels spaced by 320 ⁇ . A 3x3 grid was applied to define areas for spatial resolution (e.g. , temporal, medial, basal; dorsal, ventral) of densitometry data. Shown is a brain section generated from a vehicle/vehicle animal.
  • 6-OHDA treatment leads to partial denervation in the dorsomedial region.
  • C 6-OHDA-induced full denervation of the left striatum.
  • Figure 9 illustrates the study design for Example 3. Striatal 6-OHDA
  • administration at time 0. Unit of time is weeks. Single administration of growth factors at the indicated time-points. In the neuroprotection protocols the growth factors were administered 6h pre-6-OHDA. Behavioral testing in amphetamine- induced rotations at the indicated time-points. Tissue collections for biochemical and cell biological analyses immediately after the last behavioral test.
  • Figure 10 illustrates biochemical and cell biological analyses in Example 3.
  • the 6-OHDA and growth factor administration schedules were as described in Figure 9.
  • MANF and GDNF activities were tested after administration to the striatum or the substantia nigra.
  • Striatal administration analyses included counts of TH + neurons in the substantia nigra and measurement of dopamine levels in the striatum.
  • Nigral administration analysis included densitometry of TH + staining in the striatum, levels of dopamine and metabolites in the striatum and determination of the number of dopaminergic neurons in the substantia nigra by stereology.
  • Figure 12 shows dopaminergic neurons in the substantia nigra following striatal administration of MANF or GDNF in a neuroprotection protocol.
  • Number of TH + neurons per field (mean ⁇ SEM).
  • A Ipsilateral TH neurons
  • B Contralateral TH + neurons
  • C Ratio ipsilateral / contralateral.
  • Figure 13 shows striatal dopamine (DA) levels following striatal administration of MANF or GDNF in a neuroprotection protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ⁇ SD). Treatment groups as indicated. Statistical analysis by 1-way ANOVA. None of the treatment groups were different from 6- OHD A/vehicle. (A) Ipsilateral, (B) Contralateral.
  • DA striatal dopamine
  • DA striatal dopamine
  • Figure 17 shows amphetamine -induced rotational behavior following nigral
  • Figure 18 shows dopaminergic neurons in the substantia nigra determined by stereo logy following nigral administration of MANF or GDNF in a
  • Figure 19 shows dopaminergic terminals in the global, dorsal and ventral striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroprotection protocol. Density of terminals (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • 1-way ANOVA followed by Fisher's LSD. (**) P ⁇ 0.01, (*) P ⁇ 0.05 compared to 6-OHD A/vehicle; (o) P ⁇ 0.05 compared to 6- OHDA/GDNF 10 ⁇ g.
  • Global striatum (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral / contralateral; Dorsal striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral / contralateral; Ventral striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.
  • Figure 20 shows dopaminergic terminals in the temporal, medial and basal
  • Temporal striatum (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral / contralateral; Medial striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral / contralateral; Basal striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.
  • Figure 21 shows striatal dopamine (DA) levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • 1-way ANOVA followed by Fisher's LSD. (*) P ⁇ 0.05, (x) P ⁇ 0.1 (trend) compared to 6-OHDA / vehicle; (o) P ⁇ 0.05 compared to 6-OHDA/GDNF 10 ⁇ g.
  • Figure 22 shows striatal DOPAC levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of DOPAC shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • Figure 23 shows striatal HVA levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of HVA shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • Figure 24 shows amphetamine -induced rotational behavior following nigral
  • Figure 25 shows dopaminergic neurons in the substantia nigra determined by stereo logy following nigral administration of MANF or GDNF in a
  • Figure 26 shows dopaminergic terminals in the global, dorsal and ventral striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroregeneration protocol. Density of terminals (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • 1-way ANOVA followed by Fisher's LSD. (***) PO.001 , (*) P ⁇ 0.05 compared to 6- OHD A/vehicle; (o) P ⁇ 0.05, (x) P ⁇ 0.1 (trend) compared to 6-OHDA/GDNF 10 ⁇ g.
  • Figure 27 shows Dopaminergic terminals in the temporal, medial and basal
  • Basal striatum (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.
  • Figure 28 shows striatal dopamine (DA) levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • DA striatal dopamine
  • 1-way ANOVA followed by Fisher's LSD. **) P ⁇ 0.01, (x) P ⁇ 0.1 (trend) compared to 6- OHD A/vehicle.
  • A Ipsilateral
  • B Contralateral
  • C Ratio ipsilateral / contralateral.
  • Figure 29 shows striatal DOPAC levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of DOPAC shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • 1-way ANOVA followed by Fisher's LSD. (**) P ⁇ 0.01 compared to 6-OHD A/vehicle; (x) P ⁇ 0.1 (trend) compared to 6-OHD A/GDNF l( ⁇ g.
  • B Contralateral
  • C Ratio ipsilateral / contralateral.
  • Figure 30 shows striatal HVA levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of HVA shown as nmol/g wet tissue weight (mean ⁇ SEM) at week 4 post MANF / GDNF administration.
  • 1-way ANOVA followed by Fisher's LSD. (**) PO.01 and (***) PO.001 compared to 6-OHD A/vehicle.
  • A Ipsilateral
  • B Contralateral
  • C Ratio ipsilateral / contralateral.
  • A GDNF 2.5 ⁇ /min;
  • B No infusion control;
  • C MANF 2.5 ⁇ /min;
  • D No infusion control;
  • E MANF 1.25 ⁇ /min;
  • F No infusion control.
  • Figure 33 shows calculated volumes of distribution in the striatum for MANF and GDNF infusions at different flow rates after 7 days (mean ⁇ SEM).
  • Statistical analysis by 1-way ANOVA followed by Fisher's LSD. No statistically significant differences in distribution volumes between the treatment groups were found.
  • PD Parkinson's disease
  • DAergic dopaminergic
  • SNc substantia nigra zona compacta
  • the cell bodies of the DAergic neurons are located in the SNc, and their axons project to the striatum (caudate-putamen in man). This pathway is called the nigrostriatal projection.
  • the dopaminergic axonal terminals divide repeatedly and contact other neuronal cell bodies and nerve terminals in the striatum.
  • the SNc is one of a system of about eight related brain areas that control normal, slow movements like tying show laces, buttoning a shirt, raising food from plate to mouth and the hand shake greeting. These are some of the movements that are compromised in Parkinsonian patients.
  • Results from animal models of PD have shown that if the DAergic cell bodies in the SNc are viable, but the dopaminergic axonal terminals in the striatum are reduced significantly, then function is still compromised.
  • a strategy in treating PD is to find drugs that will both protect the remaining DAergic cell bodies, and stimulate sprouting of the dopaminergic axonal terminals in the putamen.
  • the drug GDNF (glial cell line-derived neurotrophic factor) is a standard in the field. However, when GDNF is placed in the SNc, it protects the DAergic cell bodies from death, but does not stimulate sprouting of dopaminergic axonal terminals in the striatum, and therefore does not correct neurological deficits. However, when GDNF is placed in the striatum, it does correct neurological deficits. Sprouting of DAergic terminals in the striatum can be important for the correction of neurological deficits.
  • the SNc is located deep in the ventral midbrain. Thus, based on results obtained to date (e.g. , with GDNF) the targeting of a drug to the SNc has been perceived as potentially risky, with little potential for benefit. Because of this, during clinical trials, drugs have been administered to the putamen, which is located
  • the ideal drug would be one that is effective when targeted to the putamen and/or the SNc. Such a drug could then be targeted to the putamen and/or the SNc in a treatment, thereby increasing the therapeutic benefit.
  • the additional risk of administering a drug to the SNc (alone or in combination with administration to the putamen) will be justified by the potential reward of a more robust clinical outcome versus then the drug is administered to the putamen alone.
  • MANF can have a dual action in treatment: (1) to increase in the number of viable DAergic neurons in the substantia nigra over time and (2) to increase in the density of dopaminergic terminals in the putamen over time, e.g., by stimulating neurite outgrowth.
  • MANF can be effective when delivered to either the striatum or the substantia nigra.
  • compositions, methods, and kits for treating a neurological disorder e.g., by increasing survival of neurons
  • MANF mesencephalic astrocyte-derived neurotrophic factor
  • MANF is a neurotrophic factor that selectively protects DA neurons in vitro and corrects the neurological deficits caused by the degeneration of dopaminergic neurons (DA) neurons in the brain in vivo.
  • DA dopaminergic neurons
  • MANF is expressed at a high level in the ventral mesencephalon, the same region of the brain where the DA neurons that die in PD are located.
  • the actions of MANF indicate that it can be used to treat neurological disorders such as Parkinson's Disease (PD).
  • PD Parkinson's Disease
  • the invention features a method of treating a patient having a
  • the method including administering to the patient an effective amount of MANF.
  • the effective amount can be an amount required to promote dopaminergic neuronal survival or neurite outgrowth.
  • the effective amount can be an amount required to reduce one or more symptoms of the disease or disorder.
  • the effective amount can be an amount required to delay the progression of the disease or disorder.
  • MANF neurotrophic factor
  • Dopaminergic neurons of the mesencephalon die in patients having Parkinson's disease.
  • the invention thus provides a treatment of Parkinson's disease.
  • the use of MANF in the treatment of disorders or diseases of the nervous system in which the loss of dopaminergic neurons is present or anticipated is included.
  • the MANF can be substantially purified MANF.
  • substantially purified is meant that a polypeptide (e.g., a MANF polypeptide) has been separated from the components that naturally accompany it.
  • the polypeptide is substantially purified when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the polypeptide can be at least 75%, at least 90%, or at least 99%), by weight, pure.
  • a substantially purified polypeptide can be obtained, for example, by extraction from a natural source (e.g., a neural cell), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state.
  • a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • substantially purified polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.
  • polypeptide or “protein” is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or
  • pharmaceutically acceptable excipient an excipient, carrier, or diluent that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the polypeptide with which it is administered.
  • exemplary pharmaceutically acceptable carrier is physiological saline solution.
  • physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippencott Williams & Wilkins.
  • the term "dopaminergic neuronal survival-promoting activity" can indicate that the presence of the compound increases survival of dopaminergic neurons by at least two-fold in a dopaminergic neuronal survival assay relative to survival of dopaminergic neurons in the absence of the compound.
  • the increase in the survival of dopaminergic neurons can be, e.g. , by at least three-fold, at least fourfold, or at least five-fold.
  • the assay can be an in vitro assay or an in vivo assay.
  • VMCL-1 mesencephalic origin
  • the results of the SDS-PAGE analysis revealed a distinctive protein band in the 20 kDa range in the lane from the active fraction.
  • the "active" band was excised and subjected to tryptic digest, and the molecular mass and sequence of each peptide above background were determined by mass spectrometry analysis.
  • the following two peptide sequences were identified: DVTFSPATIE and QIDLSTVDL.
  • a search of the database identified a match for human arginine-rich protein and its mouse orthologue.
  • the predicted protein encoded by the mouse EST sequence is about 95% identical to the predicted human protein.
  • rat EST database A search of the rat EST database revealed two sequences, one (dbEST Id: 4408547; EST name: EST348489) having significant homology at the amino acid level to the human and mouse proteins.
  • the full-length rat sequence was not in the GenBank database.
  • MANF unless the context indicates otherwise, is meant to encompass both the secreted and pro forms of the protein, and any biologically functional fragments thereof.
  • MANF can be from any organism, e.g., mammals (e.g., humans, non-human primates, rodents such as mice or rats, dogs, cats, horses, etc.) or insects (e.g., Drosophila species).
  • mammals e.g., humans, non-human primates, rodents such as mice or rats, dogs, cats, horses, etc.
  • insects e.g., Drosophila species.
  • MANF can be administered to a subject to treat a neurological disorder in the subject.
  • a subject is treated to enhance maintenance and/or promote growth (e.g., neurite outgrowth) of dopaminergic neurons.
  • the neurological disorder can be Parkinson's disease, or any other neurological disorder associated with the loss of dopaminergic neural function.
  • Various dosage formulations are contemplated for use in the methods disclosed herein.
  • a therapeutic approach disclosed herein can involve administration of MANF, either directly to the site of a potential or actual cell loss (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique).
  • MANF can be effective when delivered to either the striatum or the substantia nigra.
  • MANF has been identified in numerous species, including human, rat, mouse, and cow.
  • the source of MANF used e.g., the species from which it was derived
  • the source of MANF used can depend upon the species being treated. It is contemplated that MANF derived from the same species to be treated will be used in the treatment. It is also contemplated that MANF derived from one species can be used in the treatment of another species.
  • Any protein having dopaminergic neuronal survival-promoting activity and encoded by a nucleic acid that hybridizes to the cDNA encoding human ARP is considered part of the invention.
  • MANF or any other protein therapeutic agent, can be modified prior to administration to a subject.
  • Modified proteins can possess deletions and/or substitutions of amino acids
  • a protein with a deletion a protein with a substitution
  • a protein with a deletion and a substitution are modified proteins.
  • these modified proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example.
  • Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, for example, to reduce its immunogenicity/antigenicity, reduce any side effects in a subject, or increase its efficacy.
  • Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or
  • An antigenic region of a polypeptide may be substituted for a less antigenic region; the less antigenic region may contain residues that are identical to the corresponding residues in the native protein, yet also contain some conservative substitutions and/or
  • a modified protein may possess an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of a native polypeptide are included, provided the biological activity of the protein is maintained.
  • a modified protein may be biologically functionally equivalent to its native counterpart.
  • a biologically functional equivalent of MANF is administered in a therapeutically effective amount.
  • encoding a therapeutic agent can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • additional residues such as additional N- or C-terminal amino acids or 5' or 3' sequences, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5 ' or 3' portions of the coding region or may include various internal sequences, e.g., introns, which are known to occur within genes.
  • amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, binding sites to substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below.
  • a proteinaceous molecule has "homology” or is considered “homologous” to a second proteinaceous molecule if one of the following "homology criteria" is met: 1) at least 30% of the proteinaceous molecule has sequence identity at the same positions with the second proteinaceous molecule; 2) there is some sequence identity at the same positions with the second proteinaceous molecule and at the nonidentical residues, at least 30% of them are conservative differences, as described herein, with respect to the second proteinaceous molecule; or 3) at least 30%> of the
  • proteinaceous molecule has sequence identity with the second proteinaceous molecule, but with possible gaps of nonidentical residues between identical residues.
  • homologous may equally apply to a region of a proteinaceous molecule, instead of the entire molecule.
  • homology or “homologous” is qualified by a number, for example, “50% homology” or “50% homologous,” then the homology criteria, with respect to 1), 2), and 3), is adjusted from “at least 30%” to "at least 50%.” Thus it is contemplated that there may homology of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%), 85%o, 90%), 95%), or more between two proteinaceous molecules or portions of proteinaceous molecules.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • hydrophilicity of a protein correlates with a biological property of the protein.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Mimetics are peptide - containing molecules that mimic elements of protein secondary structure.
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • a specialized kind of insertional variant is the fusion protein comprising
  • MANF MANF, or another therapeutic agent.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of an
  • immunologically active domain such as an antibody epitope or other tag
  • immunologically active domain such as an antibody epitope or other tag
  • 6X His and GST glutthione S transferase
  • MANF or another therapeutic agent
  • conjugated polypeptides such as translated proteins, polypeptides and peptides that are linked to at least one agent to form an conjugate.
  • antibody conjugates in which the antibody portion targets the agent to a particular site. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g. , cell growth activity or cytotoxic activity.
  • Non-limiting examples of effector molecules which have been attached to antibodies include growth agents, toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides.
  • a reporter molecule is defined as any moiety that may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffmity molecules, colored particles or ligands, such as biotin.
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.
  • Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, and may be termed "immunotoxins.”
  • MANF, or another therapeutic agent can be joined in a fusion as described above or through a linker or coupling agent as follows.
  • linker types that can be used to conjugate MANF, or another therapeutic agent, include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH or susceptible to cleavage by proteases, such as cathepsins (e.g., cathepsins B, C, D).
  • multiple peptides or polypeptides may be joined via a biologically-releasable bond, such as a selectively-cleavable linker or amino acid sequence.
  • peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated.
  • Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
  • peptides or polypeptides may be joined to an adjuvant Amino acids such as selectively-cleavable linkers, synthetic linkers, or other amino acid sequences may be used to separate proteinaceous moieties.
  • MANF including endogenous polypeptides and peptides and recombinant polypeptides and peptides are disclosed herein.
  • these techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using
  • chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • the conditions under which such techniques are executed may be affect characteristics, such as functional activity, of the purified molecules.
  • isolated or purified protein or peptide as used herein, is
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%>, about 90%>, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, about 99.4%, about 99.6%, about 99.8%, about 99.9% or more of the proteins in the
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in
  • compositions of the invention is also contemplated.
  • a tag takes advantage of an interaction between two polypeptides.
  • a portion of one of the polypeptides that is involved in the interaction may be used as a tag.
  • GST glutathione S transferase
  • An epitope tag which an amino acid region recognized by an antibody or T cell receptor, may be used.
  • the tag may be encoded by a nucleic acid segment that is operatively linked to a nucleic acid segment encoding a modified protein such that a fusion protein is encoded by the nucleic acid molecule.
  • Other suitable fusion proteins are those with .beta.-galactosidase, ubiquitin, hexahistidine (6X His), or the like.
  • MANF or biologically functional fragments thereof are tagged with a fluorescence tag (e.g., GFP, eGFP), which are conventionally known and utilized in detection and monitoring of proteins.
  • a fluorescence tag e.g., GFP, eGFP
  • a protein-based therapy disclosed herein can include a combination of
  • MANF and one or more additional therapeutic agent.
  • additional therapeutics include, but are not limited to MANF2 (see, U.S. Patent Application Publication No. 20060084619), CDNF, interferon gamma, nerve growth factor, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), neurogenin, brain derived neurotrophic factor (BDNF), thyroid hormone, bone morphogenic proteins (BMPs), leukemia inhibitory factor (LIF), sonic hedgehog, glial cell line-derived neurotrophic factor (GDNFs), vascular endothelial growth factor (VEGF), interleukins, interferons, stem cell factor (SCF), activins, inhibins, chemokines, retinoic acid and ciliary neurotrophic factor (CNTF) or a combination thereof.
  • MANF2 see, U.S. Patent Application Publication No. 20060084619
  • CDNF interferon gamma
  • EGF epidermal growth factor
  • compositions of the protein therapeutic agents e.g., a pharmaceutical composition of the protein therapeutic agents (e.g., a pharmaceutical composition of the protein therapeutic agents).
  • compositions can comprise MANF, another therapeutic agent, or MANF and one or more additional therapeutic agents, which are administered to a subject to effect prophylactic or therapeutic effect.
  • compositions comprise a therapeutically or
  • prophylactically effective amount of MANF or functional fragments thereof examples include but are not limited to proteins that are or nucleic acids encoding proteins that are,
  • Neurotrophic factors are growth factors that promote differentiation, maintain a mature phenotype and provide trophic support, promoting growth and survival of neurons. Neurotrophic factors reside in the nervous system or in innervated tissues. The following have been described as neurotrophic factors: basic fibroblast growth factor, (bFGF), acidic fibroblast growth factors (aFGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), NT4/5, insulin-like growth factor (IGF-1), IGF-II, NT-4, IL- ⁇ ⁇ , TNFa, transforming growth factor ⁇ (TGF- ⁇ , TGF- ⁇ 1), MANF (NTN), persephin (PSP), artemin, and AL-1.
  • bFGF basic fibroblast growth factor
  • aFGF acidic fibroblast growth factors
  • CNTF ciliary neurotrophic factor
  • neurotrophic factors are well known to those of skill in the art and can be found, for example, in U.S. Patent Publication No. 2001001 1 126, U.S. Pat. Nos. 6,284,540, 6,280,732, 6,274,624, 6,221 ,676, which are incorporated by reference herein.
  • the use of Par4 is well known to those of skill in the art, as disclosed in U.S. Pat. No. 6, 1 1 1 ,075, which is hereby incorporated by reference.
  • methods of treating a neurological disorder are contemplated within the scope of the present invention, where MANF or functional fragments thereof are co-administered to a subject with one or more neurogrophic factor.
  • a pharmaceutical composition comprising MANF or functional fragments thereof, may further contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents [such as ethylenediamine tetraacetic acid (EDTA)]; complexing agents (such as caffeine,
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • antimicrobials such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite
  • buffers such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids
  • bulking agents such as mannitol or glycine
  • polyvinylpyrrolidone beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin
  • fillers monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or
  • immunoglobulins coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity
  • compositions may include one or more inert
  • excipients which include water, buffered aqueous solutions, surfactants, volatile liquids, starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, acids, bases, salts, emulsions, such as oil/water emulsions, oils such as mineral oil and vegetable oil, wetting agents, chelating agents, antioxidants, sterile solutions, complexing agents, disintegrating agents and the like.
  • Excipients can include: water, phosphate buffered saline solutions,
  • perfluorocyclobutane available under the tradename octafluorocyclobutane (from PCR Gainsville, Fla.); polyethylene glycol available under the tradename EG 400 (from BASF Parsippany, N.J.); menthol (from Pluess-Stauffer International Stanford, Conn.); propylene glycol monolaurate available under the tradename lauroglycol (from Gattefosse Elmsford, N.Y.), diethylene glycol monoethylether available under the tradename Transcutol (from Gattefosse); polyglycolized glyceride of medium chain fatty adds available under the tradename Labrafac Hydro WL 1219 (from Gattefosse); alcohols, such as ethanol, methanol and isopropanol; eucalyptus oil available (from Pluses-Stauffer International), and mixtures thereof.
  • EG 400 from BASF Parsippany, N.J.
  • menthol
  • Compounds of the present invention include amino acid derivatives.
  • a preferred surfactant may be the sodium salt form of the compound, which may include the monosodium salt form.
  • Suitable sodium salt surfactants may be selected based on desirable properties, including high speed of polymerization, small resultant particle sizes suitable for delivery, good polymerization yields, stability including freeze-thaw and shelf-life stability, improved surface tension properties, and lubrication properties.
  • Surfactants which can be used in pharmaceutical compositions and dosage forms include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.
  • aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride.
  • Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p- hydroxybenzoate.
  • Chelating agents which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, ethylene diaminetetraacetic acid (EDTA), EDTA disodium, calcium disodium edetate, EDTA trisodium, albumin, transferrin, desferoxamine, desferal, desferoxamine mesylate, EDTA tetrasodium and EDTA dipotassium, sodium metasilicate or combinations of any of these.
  • EDTA ethylene diaminetetraacetic acid
  • EDTA disodium calcium disodium edetate
  • EDTA trisodium albumin
  • transferrin desferoxamine, desferal, desferoxamine mesylate
  • EDTA tetrasodium and EDTA dipotassium sodium metasilicate or combinations of any of these.
  • Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g. , peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof.
  • Thickening agents which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, isopropyl myristate, isopropyl palmitate, isodecyl neopentanoate, squalene, mineral oil, C 12 - Ci5 benzoate and hydrogenated polyisobutene. Particularly preferred are those agents which would not disrupt other compounds of the final product, such as non- ionic thickening agents. The selection of additional thickening agents is well within the skill of one in the art.
  • antimicrobial agents to prevent spoilage upon storage, e.g., to inhibit growth of microbes such as yeasts and molds.
  • Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, purite, peroxides, perborates and combinations thereof.
  • Preservatives which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, purite, peroxides, perborates, imidazolidinyl urea, diazolidinyl urea, phenoxyethanol, alkonium chlorides including benzalkonium chlorides, methylparaben, ethylparaben and propylparaben.
  • Formulations include, but are not limited to, purite, peroxides, perborates, imidazolidinyl urea, diazolidinyl urea, phenoxyethanol, alkonium chlorides including benzalkonium chlorides, methylparaben, ethylparaben and propylparaben.
  • formulations of the one or more therapeutic agents e.g.,
  • parenteral formulations e.g. , for infusion, convection enhanced delivery, acoustic targeted drug delivery
  • parenteral formulations can be in the form of liquid solutions or suspensions
  • formulations may be in the form of tablets or capsules
  • intranasal formulations in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors.
  • Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • the present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate dopaminergic neuronal survival in neurological diseases, or peptidase or protease inhibitors.
  • phrases "pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of an pharmaceutical composition that contain MANF or additional active ingredient can be determined by the skilled artisan, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards, or other regulatory body.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is
  • the therapeutic agents may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • therapeutic compositions of the invention can be administered intraocularly, intravenously, intradermally, intraarterially, intraperitoneally, intracranially, topically, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, orally, topically, locally, by inhalation (e.g.
  • aerosol inhalation injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), by convection enhanced delivery, by acoustic targeted drug delivery, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • the methods of preparation can include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium can be buffered if necessary and the liquid diluent can be rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • the composition can be stable under the conditions of manufacture and storage, or preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination can be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • An approach for stabilizing solid protein formulations of the invention is to increase the physical stability of purified, e.g. , lyophilized, protein. This will inhibit aggregation via hydrophobic interactions as well as via covalent pathways that may increase as proteins unfold.
  • Stabilizing formulations in this context often include polymer-based formulations, for example a biodegradable hydrogel formulation/delivery system.
  • polymer-based formulations for example a biodegradable hydrogel formulation/delivery system.
  • proteins are relatively stable in the solid state with bulk water removed.
  • solid therapeutic protein formulations may become hydrated upon storage at elevated humidity or during delivery from a sustained release composition or device. The stability of proteins generally drops with increasing hydration.
  • Water can also play a significant role in solid protein aggregation, for example, by increasing protein flexibility resulting in enhanced accessibility of reactive groups, by providing a mobile phase for reactants, and by serving as a reactant in several deleterious processes such as beta-elimination and hydrolysis.
  • Protein preparations containing between about 6% to 28% water can be unstable. Below this level, the mobility of bound water and protein internal motions can be low. Above this level, water mobility and protein motions approach those of full hydration. Up to a point, increased susceptibility toward solid-phase aggregation with increasing hydration has been observed in several systems. However, at higher water content, less aggregation is observed because of the dilution effect.
  • composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • agents delaying absorption such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • formulations comprising therapeutic agents of the invention comprise a stabilizing or delivery vehicle.
  • vehicle in this context refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein.
  • exemplary vehicles include an Fc domain as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched- chain polymer (See, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tarn, issued Jul.
  • lipid a lipid
  • cholesterol group such as a steroid
  • carbohydrate or oligosaccharide a natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.
  • this invention provides for at least one peptide to be attached to at least one vehicle (F ls F 2 ) through the N-terminus, C-terminus or a side chain of one of the amino acid residues of the peptide(s).
  • vehicle F ls F 2
  • Multiple vehicles may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a side chain.
  • An Fc domain can be used as a vehicle.
  • the Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. See, for example WO 97/34631 and WO 96/32478.
  • the inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids.
  • An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a receptor or target molecule on a target cell.
  • a vehicle e.g., a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al.
  • Peptides could also be selected by phage display for binding to the FcRn salvage receptor.
  • salvage receptor-binding compounds are also included within the meaning of "vehicle” and are within the scope of this invention.
  • Such vehicles should be selected for increased half-life (e.g. , by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g. , by favoring non-immunogenic sequences, as discovered in antibody
  • a vehicle is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG group may be of any convenient molecular weight and may be linear or branched.
  • the average molecular weight of the PEG will preferably range from about 2 kiloDalton ("kDa") to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about 10 kDa.
  • kDa kiloDalton
  • the average molecular weight of the PEG will be from about 10 kDa to about 20 kDa, or 20 kDa to 30 kDa or 30 kDa to 40 kDa or 40 kDa to 50 kDa.
  • the PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).
  • a useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other.
  • the peptides can be easily prepared with conventional solid phase synthesis as known in the art.
  • the peptides are "preactivated” with an appropriate functional group at a specific site.
  • the precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC.
  • Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification.
  • Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by a 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa.
  • Dextran is a suitable water-soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc).
  • dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference.
  • Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a vehicle in accordance with the present invention.
  • the buffer can be any buffer known in the art. In one embodiment, the buffer is citrate buffer.
  • the concentration of the buffer can be from 0.01 mM to 100 mM.
  • the concentration of the buffer can be 0.01-100 mM, 0.01-50 mM, 0.01-10 mM, 0.01-5 mM, 0.01-1 mM, 0.01-0.1 mM, 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 0.1-5 mM, 0.1-1 mM, 1-100 mM, 1-50 mM, 1-10 mM, 1-5 mM, 5-100 mM, 5-50 mM, 5-10 mM, 10-100 mM, 10-50 mM, or 50-100 mM.
  • the concentration of the buffer can be about 10 mM.
  • the pH of the solution can be from 4 to 9.
  • the pH can be 4-9, 4-8, 4-7, 4-6.5, 4-6, 4-5.5, 4-5, 5-9, 5-8, 5-7, 5-
  • the pH can be about: 4, 4.1, 4.2, 4.3, 4.4, 4.5,
  • a buffered solution of MANF can be stable at 4°C for a period of time.
  • the period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.
  • the buffer is citrate buffer.
  • the citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM).
  • the citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).
  • a buffered solution of MANF can be stable at room temperature (e.g., from 20°C to 25 °C) for a period of time.
  • the period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.
  • the buffer is citrate buffer.
  • the citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM).
  • the citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).
  • a buffered solution of MANF can be stable at -20°C for a period of time.
  • the period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.
  • the buffer is citrate buffer.
  • the citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM).
  • the citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).
  • the actual dosage amount of a composition e.g., an effective amount
  • the practitioner responsible for administration can determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose of a pharmaceutical composition or formulation can comprise from about 1 ng/kg/body weight, about 5 ng/kg/body weight, about 10 ng/kg/body weight, about 50 ng/kg/body weight, about 100 ng/kg/body weight, about 200 ng/kg/body weight, about 350 ng/kg/body weight, about 500 ng/kg/body weight, 1 ⁇ g/kg/body weight, about 5 ⁇ g/kg/body weight, about 10 ⁇ g/kg/body weight, about 50 ⁇ g/kg/body weight, about 100 ⁇ g/kg/body weight, about 200 ⁇ g/kg/body weight, about 350 ⁇ g/kg/body weight, about 500 ⁇ g/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 200 milligram/kg/
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 ⁇ g/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • composition may comprise various ingredients
  • antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g. , methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g. , methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • methods of treatment include administration of therapeutic agents of the invention (e.g., MANF, or
  • MANF neurotrophic factor
  • a certain time period such as a therapeutically effective time period.
  • the administration formulations of the invention or a nucleic acid encoding MANF or biologically functional fragments is specifically contemplated. It is
  • the treatment may be administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 hours or 1 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 1 1 , 12 months or more.
  • Chronic administration is contemplated for 1 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 1 1 , 12 months or more.
  • Multiple administrations are also contemplated. In some embodiments the administration is given at least twice (repeated at least once).
  • a subject may be administered different amounts of therapeutic agents.
  • the amount of a formulation of the invention e.g. , protein or nucleic acid
  • the amount is between 1 and 1000 ng per kilogram body weight per hour. In other embodiments, the amount is between 1 and 100 ng per kilogram body weight per hour or between 1 and 10 ng per kilogram body weight per hour.
  • dosages maybe 1 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,
  • the formulation can be from 1 ⁇ g to 1000 ⁇ g.
  • the effective amount can be 1-1000 ⁇ & 1 -500 ⁇ & 1-250 ⁇ & 1-150 ⁇ & 1-100 ⁇ & 1-75 ⁇ & 1-50 ⁇ & 1 -25 ⁇ g, 1-10 ⁇ g, 1-5 ⁇ g, 5-1000 ⁇ g, 5-500 ⁇ g, 5-250 ⁇ g, 5-150 ⁇ g, 5-100 ⁇ g, 5-75 ⁇ g, 5-50 ⁇ g, 5-25 ⁇ g, 5-10 ⁇ g, 10-1000 ⁇ g, 10-500 ⁇ g, 10-250 ⁇ g, 10-150 ⁇ g, 10-100 ⁇ g, 10-75 ⁇ g, 10-50 ⁇ g, 10-25 ⁇ g, 25-1000 ⁇ g, 25-500 ⁇ g, 25-250 ⁇ g, 25-150 ⁇ g, 25-100 ⁇ g, 25-75 ⁇ g, 25-50 ⁇ g, 50-1000 ⁇ g, 50-500 ⁇ g, 50-250 ⁇ g, 50-150 ⁇
  • the effective amount is from 10 ⁇ to 250 ⁇ g. In some embodiments the effective amount of MANF is from 50 ⁇ g to 150 ⁇ g. In some embodiments the effective amount of MANF is from 10 ⁇ to 500 ⁇ g.
  • the effective amount of MANF can vary depending upon the species of a subject being treated.
  • the effective amount of MANF for treatment of a rodent such as a mouse or rat
  • the effective amount for treatment of a human or non-human primate can be lower than the effective amount for treatment of a human or non-human primate.
  • the subject is a rodent and the effective amount is from about 1 ⁇ g to about 50 ⁇ g.
  • the subject is a human or non-human primate and the effective amount is from about 10 ⁇ g to about 500 ⁇ g.
  • the volume of a pharmaceutical composition or formulation comprising the effective amount of MANF (or other therapeutic agent) that is administered to a subject can depend upon the species or size of the subject.
  • the volume administered to a rodent can be smaller than the volume administered to a larger mammal such as a human or non-human primate.
  • the effective amount can be in a volume that is from about 1 ⁇ ⁇ to about 2000 ⁇ .
  • the effective amount can be in a volume that is 1 -2000 ⁇ , 1-1500 ⁇ , 1-1000 ⁇ , 1- 750 ⁇ ,, 1-500 ⁇ ,, 1-250 ⁇ ,, 1-100 ⁇ ,, 1-50 ⁇ ,, 1-10 ⁇ ,, 10-2000 ⁇ ,, 10-1500 ⁇ ,, 10-1000 ⁇ ,, 10-750 ⁇ ,, 10-500 ⁇ ,, 10-250 ⁇ ,, 10-100 ⁇ , 10-50 ⁇ ,, 50- 2000 ⁇ ,, 50-1500 ⁇ ,, 50-1000 ⁇ ,, 50-750 ⁇ ,, 50-500 ⁇ ,, 50-250 ⁇ ,, 50-100 ⁇ ,, 100-2000 ⁇ ,, 100-1500 ⁇ ,, 100-1000 ⁇ ,, 100-750 ⁇ ,, 100-500 ⁇ ,, 100-250 ⁇ , 250-2000 ⁇ ,, 250-1500 ⁇ ,, 250-1000 ⁇ ,, 250-750 ⁇ ,, 250-500 ⁇ ,, 500- 2000 ⁇ ,, 500-1500 ⁇ , ⁇ ,
  • Penetration of active ingredients into the central nervous system after systemic administration can be limited because of the blood-brain barrier and metabolism of the active ingredient (e.g., by the liver). Improvements in treatment efficacy can be realized by direct administration of active ingredients into the cerebrospinal fluid (CSF) or by infusion directly into brain regions that are target sites for the active ingredient.
  • CSF cerebrospinal fluid
  • intrathecal or intraventricular administration can be used to bypass the blood-brain barrier.
  • diffusion- dependent methods which include intrathecal or intraventricular administration, can be limited by nontargeted distribution, nonuniform dispersion, and ineffective volumes of distribution.
  • Convection Enhanced Delivery can be a highly technical process that involves stereotactic placement of one or more catheters through cranial burr holes directly into brain tissue.
  • a therapeutic agent can be continuously administered through the catheters by a microinfusion delivery system to create a positive pressure gradient at the catheter tip. As the pressure is maintained, it creates fluid convection or flow to supplement diffusion through the extracellular spaces and enhance the distribution of the therapeutic agent to the targeted area.
  • the goals of CED can be to provide homogenous distribution of a therapeutic agent to a larger volume of brain tissue; provide higher drug concentrations directly to the tissue; and to utilize therapeutic agent in treatment that may not cross the blood brain barrier (BBB).
  • BBB blood brain barrier
  • Convection enhanced delivery can be chronic delivery, acute delivery, or a combination thereof.
  • the methods of treatment can include chronic delivery of MANF to a brain region, wherein the MANF is delivered at a continuous infusion rate over a time period of days, weeks, months or years.
  • the methods of treatment can include acute delivery of MANF wherein the MANF is delivered in discrete boluses over the course of minutes or hours.
  • the methods of treatment can include a combination of chronic and acute delivery wherein MANF is delivered at a continuous first infusion rate over a time period of days, weeks, months or years interspersed at regular or irregular intervals of limited duration infusions at a second, faster rate.
  • CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof can comprise an infusion rate of from about 0.1 ⁇ / ⁇ to about 20 ⁇ / ⁇ .
  • CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof can comprise an infusion rate of greater than about: 0.1 ⁇ / ⁇ , 0.5 ⁇ / ⁇ , 0.7 ⁇ / ⁇ , 1 ⁇ / ⁇ , 1.2 ⁇ / ⁇ , 1.5 ⁇ / ⁇ , 1.7 ⁇ / ⁇ , 2 ⁇ / ⁇ , 2.2 ⁇ / ⁇ , 2.5 ⁇ / ⁇ , 2.7 ⁇ / ⁇ , 3 ⁇ / ⁇ , 3.5 ⁇ / ⁇ , 4 ⁇ / ⁇ , 5 ⁇ / ⁇ , 7.5 ⁇ / ⁇ , 10 ⁇ / ⁇ , or 15 ⁇ / ⁇ .
  • CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof can comprise, or further comprise, an infusion rate of less than about: 25 ⁇ / ⁇ , 20 ⁇ / ⁇ , 15 ⁇ / ⁇ , 12 ⁇ / ⁇ , 10 ⁇ / ⁇ , 7.5 ⁇ / ⁇ , or 5 ⁇ / ⁇ .
  • CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof can comprise, or further comprise incremental increases in flow rate, referred to as "stepping", during delivery.
  • Stepping can comprise infusion rates of between about 0.1 ⁇ / ⁇ and about 20 ⁇ / ⁇ .
  • stepping CED can comprise one or more increases in infusion rate in steps of about: 0.1 ⁇ / ⁇ , 0.2 ⁇ / ⁇ , 0.3 ⁇ / ⁇ , 0.4 ⁇ / ⁇ , 0.5 ⁇ / ⁇ , 0.6 ⁇ / ⁇ , 0.7 ⁇ / ⁇ , 0.8 ⁇ / ⁇ , 0.9 ⁇ / ⁇ , 1 ⁇ / ⁇ , 1.25 ⁇ / ⁇ , 1.5 ⁇ / ⁇ , 2 ⁇ / ⁇ , or more.
  • the formulation administered by CED can be from 1 ⁇ g to 1000 ⁇ g.
  • the effective amount can be 1-1000 ⁇ g, 1-500 ⁇ g, 1-250 ⁇ g, 1-150 ⁇ g, 1-100 ⁇ g, 1-75 ⁇ ⁇ , 1-50 ⁇ ⁇ , 1-25 ⁇ ⁇ , 1-10 ⁇ ⁇ , 1-5 ⁇ ⁇ , 5-1000 ⁇ ⁇ , 5-500 ⁇ ⁇ , 5-250 ⁇ ⁇ , 5-150 ⁇ ⁇ , 5-100 ⁇ g, 5-75 ⁇ g, 5-50 ⁇ g, 5-25 ⁇ g, 5-10 ⁇ g, 10-1000 ⁇ g, 10-500 ⁇ g, 10-250 ⁇ g, 10-150 ⁇ g, 10-100 ⁇ g, 10-75 ⁇ g, 10-50 ⁇ g, 10-25 ⁇ g, 25-1000 ⁇ g, 25-500 ⁇ g, 25- 250 ⁇ 3 ⁇ 4 25-150 ⁇ & 25-100 ⁇ 3 ⁇ 4 25-75 ⁇ 3 ⁇ 4 25-50 ⁇
  • the effective amount of MANF is from 50 ⁇ g to 150 ⁇ g. In some embodiments the effective amount of MANF is from 10 ⁇ g to 500 ⁇ g.
  • the effective amount of MANF administered by CED can be in a volume o: 1-2000 ⁇ , 1-1500 ⁇ , 1-1000 ⁇ , 1-750 ⁇ , 1-500 ⁇ , 1-250 ⁇ , 1-100 ⁇ , 1-50 ⁇ , 1-10 ⁇ , 10-2000 ⁇ , 10-1500 ⁇ , 10-1000 ⁇ , 10-750 ⁇ , 10-500 ⁇ , 10-250 ⁇ , 10-100 ⁇ , 10-50 ⁇ , 50-2000 ⁇ , 50-1500 ⁇ , 50-1000 ⁇ , 50-750 ⁇ , 50-500 ⁇ , 50-250 ⁇ , 50-100 ⁇ , 100-2000 ⁇ , 100-1500 ⁇ , 100-1000 ⁇ , 100-750 ⁇ , 100-500 ⁇ , 100-250 ⁇ , 250-2000 ⁇ , 250-1500 ⁇ , 250-1000 ⁇ , 250-750 ⁇ ,, 250-500 ⁇ ,, 500-2000 ⁇ ,, 500-1500 ⁇ , 500-1000 ⁇ ,, 500-750 ⁇ , 750-2000 ⁇ ,
  • An advantage of convection enhanced delivery can be the ability to use imaging technology to allow drug distribution to be seen during infusion.
  • Gadolinium- and iodine-based imaging compounds can be used as tracers to safely and accurately track active ingredient distribution in real-time using, for example, magnetic resonance imaging or computed tomography imaging. These tracers can show the distribution of both small- and large-molecular- weight compounds with similar convective properties during infusion.
  • the pharmaceutical composition or formulation can comprise a tracing agent.
  • the tracing agent can enable monitoring the distribution of the tracing agent as it moves through the CNS, and ceasing delivery of the
  • the tracing agent can have a mobility in CNS tissue that is substantially similar to the therapeutic agent (e.g., MANF), and delivery can be ceased when the tracing agent is observed to reach a desired region or achieve a desired volume of distribution, or to reach or nearly reach or exceed the borders of the target tissue.
  • MRI magnetic resonance imaging
  • CT X-ray computed tomography
  • the desired volume may correspond to a particular region of the brain that is targeted for therapy (e.g., the striatum or substantia nigra).
  • the desired volume of distribution can be "substantially similar" to the volume of distribution observed for a tracing agent that is being monitored to follow the infusion.
  • substantially similar refers to a difference in volume of less than 20%. More preferably, the difference in volume is less than 15%, more preferably less than 10%, more preferably less than 5%.
  • the desired volume of distribution can be determined, for example, by using imaging software that is standard in the art, e.g., iFLOWTM. See also, for example, Krautze et al., Brain Res. Protocols, 16:20-26, 2005; and Saito et al., Exp. Neurol, 196:3891-389, 2005, each of which is incorporated herein by reference in its entirety.
  • imaging software that is standard in the art, e.g., iFLOWTM. See also, for example, Krautze et al., Brain Res. Protocols, 16:20-26, 2005; and Saito et al., Exp. Neurol, 196:3891-389, 2005, each of which is incorporated herein by reference in its entirety.
  • the tracer can comprise a paramagnetic ion for use with MRI.
  • Suitable metal ions include those having atomic numbers of 22-29 (inclusive), 42, 44 and 58-70 (inclusive) and have oxidation states of +2 or +3. Examples of such metal ions are chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).
  • the tracer may comprise a radiopaque material.
  • Suitable radiopaque materials include, but are not limited to, iodine compounds, barium compounds, gallium compounds, thallium compounds, and the like. Specific examples of radiopaque materials include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosumetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotriroic acid, ipodate, meglumine
  • Treatment methods disclosed herein can comprise or involve preoperative diagnosis.
  • Preoperative diagnosis can include neuroimaging, for example, by PET,
  • SPECT SPECT
  • MRI MRI
  • X-ray computed tomography or a combination thereof.
  • the neuroimaging can be used for target localization and guided cannula placement.
  • a stereotactic holder can be used in conjunction with neuroimaging to provide for guided cannula placement at or proximal to a target neuronal population.
  • any suitable device can be used in the methods disclosed herein wherein a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, is administered to a brain region by CED.
  • a delivery device can comprise a pump that is capable of delivering a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, by CED.
  • the pump can be an osmotic pump or an infusion pump.
  • the device can comprise, or can be used in
  • a catheter or cannula that facilitates localized delivery to a brain region of a subject (e.g., striatum or substantia nigra).
  • the catheter or cannula can comprise multiple outlet ports.
  • the catheter or cannula can comprise an outer tubing to provide structural rigidity to the catheter or cannula.
  • the catheter or cannula can be a reflux-free step-design cannula.
  • One or more catheters or cannuli can inserted into or near one or more brain regions of a subject (e.g., striatum or substantia nigra).
  • Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, Mich. Positioning can be conducted by using anatomical maps obtained by CT and/or MRI imaging of the subject's brain to help guide the injection device to the chosen target.
  • CED can be performed with the use of a CED-compatible reflux-free step- design cannula, such as that disclosed in Krauze et al, J Neurosurg. 2005
  • the step-design cannula is compatible with chronic administration. In another embodiment, the step-design cannula is compatible with acute administration. In another embodiment, the step-design cannula is compatible with a combination of chronic administration interspersed with periods of acute administration at a higher infusion rate or with a higher level of the therapeutic agent.
  • Figure 6A is a diagram of an exemplary step-design cannula 10 that can be used in the methods, systems, and kits disclosed herein.
  • the cannula 10 can comprise a tube 12 having a substantially uniform inner diameter (ID) that defines a central lumen.
  • ID inner diameter
  • OD outer diameter
  • the cannula can comprise a tube made from titanium or other biocompatible material having an ID of, for example, 0.286 mm (29 gauge) and a length of, for example, 234 mm. As illustrated, the OD of the tube decreases from 5 mm at its proximal end 14 to 0.33 mm at its distal end 16 in four steps, thus providing a cannula that has four segments.
  • the length of the first segment 18 is 40 mm with an OD of 5 mm
  • the length of the second segment 20 is 124 mm with an OD of 2.1 mm
  • the length of the third segment 22 is 10 mm with an OD of 0.64 mm
  • the length of the fourth segment 24 (needle tip) is 10 mm with an OD of 0.33 mm.
  • the measurements given are exemplary and can be modified by the skilled practitioner according to treatment needs.
  • FIG. 6B illustrates a delivery sheath 100 that can function as a guide component for the step-design cannula 10, for example, to assist in placement of the infusion cannula.
  • the system can be used in combination with an infusion pump that is either externally or subcutaneously placed.
  • the delivery sheath 100 comprises a tubular member 102 which has, at one end, a coupling 104 that is adapted to be connected to a stereotaxic frame for support and placement.
  • the tubular member 102 can be fabricated from a non-ferromagnetic flexible material such as a biocompatible plastic or a metal such as titanium.
  • the coupling 104 is a conventional coupling that is adapted for connection to a stereotaxic frame at its proximal end 106.
  • the connection between tubular member 102 and coupling 104 can be made using a conventional biocompatible bonding technique such as gluing the components together.
  • a central lumen 108 runs through at least a portion of tubular member 102 for receiving an infusion cannula. Accordingly, the inner diameter of central lumen 108 would typically be larger than the largest outer diameter of cannula 14 that will be inserted through the lumen.
  • the distal end 110 of tubular member 102 is open so that the infusion cannula can extend therethrough.
  • a longitudinal passageway or slot 112 communicates between the outer surface 114 of the tubular member and the central lumen for insertion of the infusion cannula into the delivery sheath.
  • a plurality of openings 116 are positioned adjacent the perimeter of passageway 112 though which sutures can be placed.
  • Figure 6C illustrates an assembly of a step-design cannula wherein the upper portion of delivery sheath 100 is removed, for example, by cutting the sheath just above where the infusion cannula 200 bends over.
  • a holding bracket 216 and bone screw 218 can be used to affix the assembly in place.
  • a tubing 208 is coupled to the proximal end of the tubular body 202, which can be connected to an infusion pump (not shown).
  • a set screw 220 can be used to lock delivery sheath 100 and cannula 200 in position to prevent relative movement.
  • CED can be performed with the use of a neurosurgical apparatus
  • Figure 7 illustrates a neurosurgical apparatus suitable for CED in the
  • the neurosurgical apparatus can comprise a guide device and a catheter.
  • the guide device can comprise an elongate tube having a head at its proximal end.
  • the elongate tube can be inserted into the brain towards a target brain region ⁇ e.g., striatum or substantia nigra) via a hole formed in the skull.
  • the head can be used to securely attach the guide device to the skull. This insertion may be performed using a stereoguide or surgical robot based technique.
  • An internal channel is provided through the head and bore of the tube. The catheter can then be passed down this channel and into the brain in the vicinity of the selected target.
  • the catheter 40 comprises a hub 42, a fine tube
  • the hub 42 comprises a body portion 48, a sealing element in the form of a tube having a tapered surface 50 and a pair of protruding wings 52.
  • the wings 52 have apertures 54 formed therein for receiving bone screws.
  • a catheter 40 of the type described is shown when inserted into a guide device 60.
  • the catheter 40 comprises a bore or lumen 62 that runs through the connector tube 46, a hub 42 and a fine tube 44.
  • the internal diameter of the lumen 62 is substantially constant through the catheter 40, although it could be varied if required.
  • the guide device 60 comprises a head 64 and an elongate tube 66.
  • the head 64 comprises external ridges 68 that allow it to be attached to a hole formed in the skull by a press fit action.
  • the guide device 60 comprises a passageway 70 through the head 64 in which the catheter 40 can be located.
  • the passageway 70 of the head 64 comprises an internally tapered region
  • Figure 7B shows the arrangement after the catheter has been bent through ninety degrees in the slot formed in the head 64.
  • the wings 52 of the catheter are, when the tapered region 72 of the guide device 60 engages the tapered surface 50 of the catheter 40, arranged to be located very close (e.g. within 0.5mm) to the head 64 of the guide device 60. This provides a visual indication that the tapered surfaces have engaged to form the fluid seal.
  • the wings 52 and head 64 will also engage if further insertion of the catheter is attempted, thereby acting as an insertion limiter or safety stop to prevent buckling or other damage to the catheter.
  • kits or devices comprising a pump that is capable of effecting delivery of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, by CED.
  • the kits or devices further comprise a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, such as any of those disclosed herein.
  • the kits or devices further comprises a CED-compatible cannula or catheter.
  • the cannula or catheter can be compatible with chronic or acute administration.
  • kits for the treatment of a CNS are provided.
  • kits can comprise one or more pharmaceutical compositions or formulations comprising MANF, another therapeutic agent, or a combination thereof.
  • the kit can further comprise a delivery device useful for CED.
  • the delivery device can comprise a pump.
  • the device can comprise one or more catheters or cannuli, such as those illustrated in Figure 6 and Figure 7.
  • the cannuli can comprise a step-design reflux-free cannula, such as illustrated in Figure 6.
  • Kits can further comprise guide devices for insertion of the cannuli or cather, such as those illustrated in Figure 6 and Figure 7.
  • Kits may additionally comprise connecting parts, tubing, packaging material, instruction pamphlets, and other materials useful for practicing CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, to one or more brain regions of a subject having a CNS disorder (e.g., Parkinson's Disease).
  • a CNS disorder e.g., Parkinson's Disease
  • the invention provides a method of treating a
  • a condition comprising contacting the substantia nigra of the subject with an amount of MANF effective to treat the condition.
  • the condition is a neurological disorder.
  • the neurological disorder is Parkinson's disease.
  • the subject is a mammal.
  • the mammal is a human, rat, mouse, monkey, and rabbit.
  • the method further comprises contacting the
  • striatum of a subject with an amount of MANF effective to treat said condition.
  • the method further comprises administering one or more additional therapeutic agents.
  • the subject exhibits an improvement in their clinical scores after treatment.
  • the clinical score is a measurement of net ipsilateral rotations or total ipsilateral rotations.
  • the percentage of viable dopaminergic neurons in a subject increases; the density of dopaminergic terminals in the putamen of the subject increases; the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the putamen of the subject both increase; the viability of cell bodies in the striatum of the subject increases; the percentage of dying dopaminergic neurons in the subject decreases; and/or the percentage of dead dopaminergic neurons in the subject decreases.
  • the amount of MANF administered is at least about
  • the amount of MANF is 1, 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 ⁇ g. In certain embodiments, the amount of MANF
  • administered is at least about 1.2, 1.4. 1.6. 1.8, or 2 mg.
  • the amount of MANF administered is less than about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 ⁇ g. In certain embodiments, the amount of MANF administered is less than about 1.2, 1.4. 1.6, 1.8, or 2 mg.
  • the amount of MANF administered is about 3, 5,
  • the amount of MANF administered is about 1.2, 1.4. 1.6. 1.8, or 2 mg.
  • the amount administered is selected to protect
  • the neurological disorder is Parkinson's disease.
  • the brain region comprises the striatum, the substantia nigra, or a combination thereof. In some embodiments, the brain region comprises the striatum. In some embodiments, the brain region comprises the substantia nigra. In some
  • the brain region comprises the striatum and the substantia nigra.
  • the effective amount of MANF is from 1 ⁇ g to 1000 ⁇ g. In some embodiments, the effective amount of MANF is from 10 ⁇ g to 500 ⁇ g. In some embodiments, the effective amount of MANF is from 50 ⁇ g to 250 ⁇ g.
  • convection enhanced delivery is performed at a flow rate of from 0.1 ⁇ 7 ⁇ ⁇ 10 ⁇ 7 ⁇ .
  • convection enhanced delivery is performed acutely.
  • the convection enhanced delivery is repeated two or more times during a course of treatment.
  • convection enhanced delivery is performed chronically.
  • the pharmaceutical composition further comprises a tracer.
  • diffusion of the MANF in the brain region is monitored in by detection of the tracer.
  • monitoring comprises magnetic resonance imaging (MRI), computed tomography imaging, or X-ray computed tomography (CT).
  • the pharmaceutical composition further comprises a buffer.
  • the buffer is a citrate buffer.
  • the pharmaceutical composition further comprises a buffer.
  • composition has a pH of from 5.5 to 6.5.
  • the subject exhibits an improvement in a clinical score after
  • the percentage of viable dopaminergic neurons in the subject increases. In some embodiments, the density of dopaminergic terminals in the subject's putamen increases. In some embodiments, both the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the subject's putamen increase. In some embodiments, the viability of cell bodies in the striatum increases.In some embodiments, the percentage of dying dopaminergic neurons in the subject decreases. In some embodiments, the percentage of dead dopaminergic neurons in the subject decreases.
  • kits comprising: (a) a catheter, that can be stereotactically placed at or near the brain region; (b) a pump, for generating a positive pressure gradient between the catheter and the brain region; (c) a pharmaceutical composition comprising an effective amount of MANF to treat the neurological disorder.
  • Some embodiments further comprise a delivery sheath to assist in placement of the catheter.
  • the catheter comprises multiple outlet ports.
  • the catheter comprises an outer tubing to provide structural rigidity to the catheter.
  • the pharmaceutical composition comprises from 1 ⁇ g to 1000 ⁇ g of the MANF. In some embodiments, the pharmaceutical composition comprises from 10 ⁇ g to 500 ⁇ g of the MANF. In some embodiments, the pharmaceutical composition comprises from 50 ⁇ g to 250 ⁇ g of the MANF.
  • the pharmaceutical composition further comprises a tracer.
  • the pharmaceutical composition further comprises a buffer.
  • the buffer is a citrate buffer.
  • the pharmaceutical composition has a pH of from 5.5 to 6.5.
  • the method comprising administering a pharmaceutical composition comprising an effective amount of MANF in a 10 mM citrate buffer, pH 6.1 to a brain region of the subject.
  • MANF and GDNF were administered at various time points after treatment with 6-OHDA, and the effect on neurorestoration was evaluated.
  • the objective of the study was to evaluate the degree to which damaged DAergic neurons can be rescued from death by each drug.
  • the animals were re-anesthetized with Isofluran and mounted in the Kopf stereotaxic apparatus.
  • Results showed that MANF significantly reduced neurological deficits in the rodent model of PD when targeted to the striatum or the SNc.
  • MANF or GDNF were targeted to the striatum, each caused a significant reduction in the neurological deficits in the restorative 6-OHDA model of PD ( Figure 1).
  • SNc as target suggests that MANF was effective at 10 ⁇ g and 36 ⁇ g.
  • GDNF 10 ⁇ g had no effect at 2-weeks or 4-weeks.
  • MANF at a dose of 36 ⁇ g had no effect with the striatum as target.
  • MANF had a significant effect at 4- weeks.
  • the positive action of 36 ⁇ g MANF at 4-weeks, but not at 2-weeks does suggest that dopaminergic neuronal repair can occur over time.
  • MANF may therefore have a dual action in treatment:
  • CED Convection-enhanced delivery
  • CED neurosurgical technique which has the potential to achieve more effective coverage of the putamen and other brain regions.
  • CED describes a direct method of drug delivery to the brain through very fine microcatheters. By establishing a pressure gradient at the tip of the infusion catheter, CED confers several advantages over conventional drug injection techniques, in particular,
  • Bilateral burr holes were drilled using a 2 mm drill. All CED procedures were performed using a custom-made catheter with an outer diameter of 0.22 mm and inner diameter of 0.15 mm, composed of fused silica with a laser cut tip. The cannula was attached to a 1 mL syringe (Hamilton, Bonaduz,
  • CED procedures were performed at an infusion rate of 0.1 ⁇ / ⁇ , 1.25 ⁇ / ⁇ , 2.5 ⁇ / ⁇ , or 5.0 ⁇ / ⁇ .
  • the cannula was left in situ for 10 minutes to minimize reflux, then withdrawn at a rate of 1 mm/ minute.
  • the wound was closed with 4/0 Vicryl, and a dose of intramuscular buprenorphine (Centaur Services, Castle Cary, UK) was administered (30 ⁇ g/kg).
  • the anaesthetic was reversed with 0.1 mg/kg i.p.
  • atipamezole hydrochloride in recovery procedures. Rats were euthanised by anaesthetic overdose with an intraperitoneal injection of 1 mL pentobarbital (Euthatal; Merial Animal Health, Harlow, UK) at pre-defined time -points following CED (0, 3, 24 hours or 7 days).
  • IHC immunohistochemical analysis
  • Rat brains were cut into 35 ⁇ thick coronal sections using a Leica
  • CM1850 cryostat Leica Microsystems, Wetzlar, Germany
  • CM1850 cryostat Leica Microsystems, Wetzlar, Germany
  • fluorescent immunohistochemistry fixed sections were mounted on gelatine- subbed slides. Once dry, the sections were washed with PBS for 5 minutes x 3. Sections were blocked in PBS plus 0.1% triton-X-100 containing 10% normal donkey serum (Sigma Aldrich, UK) for 1 hour at room temperature (RT). They were then washed with 0.1% triton-X-100 in PBS for 5 minutes.
  • MANF was delivered via CED at flow rates of 0.1 ⁇ min, 1.25 ⁇ 7 min, and 5 ⁇ min into the striatum. MANF was successfully detected at 0 hours at all flow rates by IHC ( Figure 3).
  • Parkinson's Disease to provide evidence and confirmation of MANF' s activities and experimental support for use of MANF for treatment of PD.
  • an additional object was to compare MANF and GDNF activities under the same experimental conditions.
  • MANF expression was induced by addition of Isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and cells were harvested 4 h post induction using a continuous flow centrifuge. Cell paste was stored at -80° C.
  • IPTG Isopropyl ⁇ -D-l-thiogalactopyranoside
  • the chromatography system and the columns for MANF purification were sanitized by soaking in 0.5 M NaOH, rinsed with low endotoxin water, and equilibrated in buffers prepared with low endotoxin water.
  • One hundred grams of cells from the fermentor run were resuspended in 1 liter of 20 mM NaH2P04, 0.25 M NaCl, pH 8 with a hand held homogenizer and passed through a micro fluidizer three times at approximately 15,000 psi.
  • the lysate was clarified by centrifugation and filtration.
  • the clarified lysate was applied to a 35 ml IMAC fast flow (FF) column (2.6 cm by 6.3 cm) equilibrated in Buffer NA (20 mM
  • the pooled fractions containing MANF were dialyzed against 2 liters of 20 mM NaH2P04, 50 mM NaCl, 0.1 % Tween 20, pH 7.5 at room temperature.
  • the dialysate was diluted to OD280 ⁇ 2 with 20 mM NaH2P04, 50 mM NaCl, 0.1 % Tween 20, pH 7.5, CaC12 was added to 2 mM, and 160 units enterokinase
  • EKMax Invitrogen
  • Digestion proceeded at room temperature for 8 hours and terminated by addition of EDTA to 5 mM final concentration.
  • the solution was stored at 4°C overnight.
  • the pH of the EK-digested MANF was adjusted to 6 with HC1 and filtered through a 0.22 ⁇ cellulose acetate filter (Corning).
  • This pool was combined with the pool from a previous purification run using a similar protocol and was dialyzed against 10 mM Na citrate, 150 mM NaCl, pH 6.0.
  • the dialysate was passed through a Mustang E filter and concentrated using a sanitized Amicon Ultra-15 (10 kDa molecular weight cut off) to an OD280 of 10.
  • the endotoxin level of this protein preparation was less than 10 EU per mg of protein using a Pyrosate® LAL clot assay kit (Cape Cod Associates).
  • the biological activity of MANF was verified in a dopaminergic cell culture assay.
  • Recombinant human GDNF was obtained from Peprotech (Catalog # 450-
  • the biological activity of GDNF was tested in a rat C6 cell proliferation assay with an ED50 of ⁇ 0.1 ng/ml (Peprotech technical information).
  • MANF and GDNF were diluted in filter sterilized 10 mM Na citrate buffer, pH 6.8. The solutions were prepared in siliconized, 1.5 ml Eppendorf tubes just prior to use.
  • This study consisted of a neuroprotection and neuroregeneration protocol and each protocol included 5 experimental groups (6-OHDA / Vehicle; 6-OHDA / MANF 3 ⁇ & 6-OHDA / MANF 10 ⁇ g; 6-OHDA / MANF 36 ⁇ & 6-OHDA / GDNF 10 ⁇ g) with 12 animals assigned to each group.
  • 6-OHDA 6-hydroxyacetylcholine
  • animals were re-anesthetized with isoflurane and injected with desipramine (15 mg/kg, i.p.) to protect noradrenergic neurons.
  • 6-OHDA 8 ⁇ g free base dissolved in 4 ⁇ of filter sterilized PBS / 0.02% ascorbic acid
  • the neuroregeneration protocol followed the same surgical procedures but differed in two important aspects.
  • the amount of striatally administered 6- OHDA was 20 ⁇ g.
  • (2) MANF and GDNF were administered four weeks after the 6-OHDA injection.
  • This study consisted of a neuroprotection and neuroregeneration protocol and each protocol included 6 experimental groups (Vehicle / Vehicle; 6-OHDA / Vehicle; 6-OHDA / MANF 3 ⁇ g; 6-OHDA / MANF 10 ⁇ 3 ⁇ 4 6-OHDA / MANF 36 ⁇ g; 6-OHDA / GDNF 10 ⁇ g) with 12 animals assigned to each group.
  • the administration of 6-OHDA, MANF and GDNF in this Phase 2 of the study followed the same general surgical procedures as presented in the previous section.
  • the amount of striatally administered 6-OHDA was 8 ⁇ g for the neuroprotection protocol and 20 ⁇ g for the neuroregeneration protocol.
  • MANF 3, 10 or 36 ⁇ g, single administration
  • GDNF 10 ⁇ g, single administration
  • the growth factors were administered to the substantia nigra using the following coordinates relative to bregma and the skull surface: Caudal 4.9 mm, lateral ⁇ 2.0 mm, ventral 8.3 mm.
  • D-amphetamine sulphate 2.5 mg/kg free base, i.p. was administered to animals of the treatment groups in Phases 1 and 2 of this study and circling behavior was monitored over a 2-h period using a video tracking system.
  • TH+ striatal dopaminergic
  • a total of 18 sections (40 ⁇ thickness) were cut through the substantia nigra and mounted on pre-cleaned, Superfrost Plus glass slides (6 sections per slide). The slides were air dried on the lab bench for at least 4 hours prior to staining. The sections were hydrated (lx PBS, 3x 10 min), endogenous peroxidase quenched (0.3% H 2 0 2 in 50% MeOH, 20 min), washed (PBS, 3x lOmin) and blocked (5% normal horse serum in PBS/ 0.3% Tween for 2 hrs, at room temperature).
  • the primary anti-tyrosine hydroxylase (TH) monoclonal antibody (Sigma, T-2928, 1 :2000, in 5% normal horse serum/lx PBS), was applied over night, at 4 °C (in the refrigerator), followed by washing (3x PBS, 3x 10 min).
  • the peroxidase complex (ABC: Vector Lab), made up 45 mins previously according to the instructions of the manufacturer, was applied for 1 hr, at room temperature, followed by washing (4x 0.1% tween in lx PBS). Finally, the freshly prepared diaminobenzadiene (DAB) substrate was applied, and the dark deposit in the substantia nigra developed within 5 to 8 minutes. The sections were rinsed in tap water, and dehydrated through 65%, 80 %, 95% and 100% changes of ethanol.
  • the sections were then cleared in HistoClear, mounted in VectMount (Vector Labs), cover slipped, and dried in the hood for 3 hours, before microscopic analysis.
  • the number of TH + cells per field in the dorsolateral region of the substantia nigra was determined by counting. This region was selected because the density of neurons is lower in this region and accurate cell counts can be made.
  • Rat brains were treated overnight with 20% glycerol and 2%
  • each block was rapidly frozen by immersion in isopentane, chilled to -70°C with crushed dry ice and mounted on an AO 860 sliding microtome.
  • Each MultiBrain® block was cut coronally to generate sections of 40 ⁇ thickness. All sections were collected sequentially in containers filled with antigen preserve solution (50% PBS pH 7.0, 50% ethylene glycol, 1% polyvinyl pyrrolidone).
  • TH + neurons of the substantia nigra were quantified by stereology of
  • Sections stained for TH and AgNOR were incubated in HC1 to enhance permeabilization, bleached to avoid non-specific silver staining, and incubated with a one-quarter strength concentration of the TH antibody to provide optimal contrast with the AgNOR stain while retaining robust pigmentation of TH -positive structures.
  • TH+ neurons in the substantia nigra was performed. Every 8th section containing the substantia nigra was selected and the substantia nigra was carefully outlined by using an atlas. TH- AgNORstained cells within these boundaries were quantified using the optical fractionator method and the Stereologer software package (Stereology Resource Center, Chester MD & Tampa- St.Petersburg, FL). A Nikon Eclipse 80i microscope was used which was coupled to a Sony 3CCD color digital video camera and operated an Advanced Scientific Instrumentation MS-2000 motorized stage with input into a Dell Precision 650 server and a high-resolution plasma monitor. The areas of interest were first identified using 4x / 1.3 aperture dry lenses and the stereology was performed at high magnification with 100 x / 1.4 aperture oil immersion lenses which allowed for clear visualization of the nucleoli and precise definition of the cell boundaries.
  • TH-stained brain sections For each animal, four 40 ⁇ thick sections of the ipsilateral and contralateral sides of the substantia nigra / striatum were analyzed from rostral to caudal, spaced by 320 ⁇ . The sections were then stained free-floating. The dopaminergic terminals were stained using a TH- specific antibody (Pel-Freez Biologicals; 1 : 1600 in cold TBS) and the Vectastain Elite ABC kit (Vector Laboratories). The immunohistochemical staining for TH was followed by a thionine Nissl counterstain and sections were then mounted on gelatinized glass slides.
  • TH-specific antibody Pel-Freez Biologicals; 1 : 1600 in cold TBS
  • Vectastain Elite ABC kit Vector Laboratories
  • a 3 x 3 grid was placed on each section relative to a landmark ( Figure 8) to collect densitometry data from the temporal (positions 1, 2, 3 (left) and 7, 8, 9 (right)), medial (positions 4, 5, 6 (left and right)) and basal (positions 7, 8, 9 (left) and 1, 2, 3 (right)) striatum ( Figure 1).
  • data for the dorsal (positions 1, 4, 7 (left and right)) and ventral (positions 3, 6, 9 (left and right)) striata were collected.
  • the optical densities were measured at the 4 rostrocaudal levels for the temporal, medial and basal striatum with a digital camera and a constant illumination table. To estimate the specific TH staining density, the optical density readings were corrected for the non-specific density as measured on the completely denervated parts of the striatum.
  • Striatal levels of DA and dopamine metabolites DOPAC and HVA were determined by negative chemical ionization / mass spectrometry.
  • the frozen brains were allowed to thaw on ice, the striata were removed and placed on a pre- cooled piece of aluminum foil and quickly placed back on dry ice.
  • the striatal samples were weighed using a microbalance with a sensitivity of 10 ⁇ g, transferred to 1.6 ml Eppendorf tubes, then sonicated on ice in 500 ⁇ 0.1 N HC1, and centrifuged at 15,000x g for 10 min. The supernatants were transferred to micro centrifuge tubes and frozen at -80°C.
  • Hslnternal Standards for each of the three analytes were added to each sample.
  • the samples were dried under a stream of N2 in a 96-well format dryer operated at 60°C and further dried in a vacuum (25 mm Hg) at 70°C for 30 min.
  • PFPA pentafluoropropionic anhydride
  • HFIP hexafluoroisopropanol
  • OHDA model of PD The study consisted of two phases with administration of the growth factors to the striatum (Phase 1) or the substantia nigra (Phase 2), respectively. Both phases included a neuroprotection and a neuroregeneration protocol in which the growth factors were administered shortly before (6h) or weeks after the 6-OHDA administration, respectively (Figure 9).
  • treatment groups ⁇ e.g., 6-OHDA/vehicle, 6-OHDA/MANF 3 ⁇ g, 6-OHDA/MANF l( ⁇ g, 6-OHDA/MANF 36 ⁇ g, 6-OHDA/GDNF l( ⁇ g) with a planned inclusion of 12 animals per group with a total number of animals of 60.
  • MANF has been tested in an almost identically designed protocol and a significant improvement compared to vehicle was demonstrated at weeks 2 and 4 after the 6-OHDA lesion. The strongest response to treatment was seen with the MANF 10 ⁇ g dose level. Moreover, GDNF has been tested in similar protocols and a reduction in net ipsilateral rotations was observed at weeks 2 and 4 and week 6 post-6-OHDA.
  • the TH + cells were counted in the substantia nigra at week 8 after administration of 6-OHDA and compared between the different treatment groups ( Figure 12).
  • the TH + cell counts were analyzed for the ipsilateral ( Figure 12 A) and contralateral (Figure 12B) sides of the lesion, and ratios between ipsilateral and contralateral sides ( Figure 12C) were calculated.
  • the treatment with 6-OHDA led to a reduction of TH cell counts to about 55% on the ipsalateral side compared to the contralateral side, which is in very good agreement with similarly designed studies.
  • MANF or GDNF were administered by a single striatal injection 4 weeks after the unilateral 6-OHDA lesion.
  • This design allowed for an amphetamine -induced rotations test prior to MANF or GDNF administration at week 3 post 6-OHDA to identify and exclude animals that did not display the expected rotational behavior.
  • This procedure led to the exclusion of 6 animals in the 6-OHD A/vehicle, 3 animals in the 6-OHDA/MANF 3 ⁇ g, 2 animals in the 6-OHDA/MANF 36 ⁇ g and 2 animals in the 6-OHD A/GDNF 10 ⁇ g groups.
  • GDNF decreased amphetamine-induced ipsilateral rotations compared to vehicle over a 12 week observation period.
  • the lack of a significant treatment effect by MANF or GDNF in the neuroregeneration protocol of this present study may be due to the comparatively rapid and almost complete spontaneous recovery of the vehicle treated animals.
  • TH + neurons in the substantia nigra using computer assisted stereology, quantification of striatal dopaminergic terminals by densitometry and measuring levels of dopamine and its metabolites DOPAC and HVA in the striatum.
  • the stereology provides for a measure of surviving dopaminergic cell bodies in the substantia nigra while the densitometry and dopamine level quantification provides for a measure of functional and structural improvement, respectively, of dopaminergic terminals in the striatum.
  • MANF 10 ⁇ g displayed an increase in rotational behavior compared to 6-OHDA/vehicle. While the net ipsilateral rotations in the 6- OHD A/vehicle group decreased from week 2 to week 4, the values for MANF 10 ⁇ g and GDNF 10 ⁇ g remained elevated. The GDNF 10 ⁇ g values were significantly higher compared to 6-OHDA/vehicle while the values for MANF 10 ⁇ g displayed a trend towards a difference to 6-OHDA/vehicle.
  • GDNF has been administered to the substantia nigra in several 6-OHDA studies in a neuroprotection design. One did not observe an effect on net ipsilateral rotations 5 months after GDNF treatment. In contrast, another observed an increase in net ipsialateral rotations at week 6 after GDNF was administered to the substantia nigra, similar to the effect observed in this study. Hence, this study and the literature provide evidence that GDNF and MANF increase net ipsilateral rotations in the 6-OHDA model when administered to the substantia nigra in a neuroprotection protocol.
  • substantia nigra in a 6-OHDA model and in which survival of nigral TH + cells was quantified.
  • GDNF protected TH + cells to a significant extent, ranging from 60% protection to complete protection.
  • TH + cells seem to be protected but remained in an atrophic state thereby preventing a substantial functional recovery.
  • GDNF administration to the substantia nigra and its effects on dopamine levels in the substantia nigra and the striatum were investigated in a 6-OHDA model.
  • Administration of 6-OHDA to the striatum led to a significant decrease of dopamine levels on the ipsilateral side in both the striatum and the substantia nigra which was almost completely prevented by prior administration of GDNF to the substantia nigra.
  • DOPAC levels were significantly different between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral but not on the contralateral side, and for the ipsilateral/contralateral ratio. Neither MANF 3 ⁇ g nor MANF 36 ⁇ g were different from 6-OHDA/vehicle but MANF 10 ⁇ g and GDNF 10 ⁇ g had their DOPAC levels significantly reduced. Similar differences were also observed for the ipsilateral/contralateral ratios. The DOPAC levels largely paralleled the effects seen across the treatment groups for the dopamine levels.
  • the vehicle/vehicle and 6-OHDA/vehicle groups are significantly different, the MANF 10 ⁇ g and GDNF 10 ⁇ g differ from the 6-OHDA/vehicle group and the MANF 36 ⁇ g differs from the GDNF 10 ⁇ g group.
  • HVA levels were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral ( Figure 23 A) but not on the contralateral side ( Figure 23B), and for the ipsilateral/contralateral ratio ( Figure 23C).
  • MANF 3 ⁇ g, MANF 10 ⁇ g and GDNF 10 ⁇ g had their HVA levels significantly reduced compared to the 6-OHDA/vehicle group. These differences were also observed for the ipsilateral/contralateral ratios but in addition, the MANF 36 ⁇ g level was also lower than 6-OHDA/vehicle. Again, the HVA levels largely parallel the effects seen across the treatment groups for the DOPAC and dopamine levels.
  • the vehicle/vehicle and 6-OHDA/vehicle groups are significantly different, the MANF 10 ⁇ g and GDNF 10 ⁇ g differ from the 6-OHDA/vehicle group and the MANF 36 ⁇ g differs from the GDNF 10 ⁇ g group.
  • MANF or GDNF were administered by a single injection into the substantia nigra 2 weeks after the unilateral 6-OHDA lesion to the striatum.
  • This design allowed for an amphetamine -induced rotations test prior to MANF or GDNF administration at week 1 post 6-OHDA to identify and exclude animals that did not display the expected rotational behavior.
  • This procedure led to the exclusion of 1 animal in the vehicle/vehicle, 2 animals in the 6-OHDA/vehicle, 2 animals in the 6-OHDA/MANF 10 ⁇ g, 2 animals in the 6-OHDA/MANF 36 ⁇ g and 1 animal in the 6-OHDA/GDNF 10 ⁇ g groups.
  • 6-OHDA 6-OHDA model and it is therefore not possible to compare the results of this current study with independently generated data.
  • GDNF was administered to the substantia nigra in a model in which 6-OHDA was injected into the basal forebrain bundle.
  • GDNF was given as single injections of 100 ⁇ g or 1000 ⁇ g, respectively, at 9 weeks post 6-OHDA. At week 10, the turning behavior was fully restored, an effect that was sustained until the end of the study at week 20.
  • the number of TH + neurons in the substantia nigra was quantified by stereo logy.
  • the numbers of TH + neurons were determined for the ispilateral and contralateral sides of animals from all treatment groups ( Figure 25A and B).
  • the ipsilateral to contralateral ratios were calculated at the individual animal level ( Figure 25C).
  • TH+ terminals of TH+ neurons were assessed by TH immunoreactivity densitometry of the ispilateral and contralateral striata of animals in all treatment groups at the end of the observation period.
  • the numbers of dopaminergic (e.g., TH+ ) terminals in the striatum were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral side for the entire striatum ( Figure 26A) and the dorsal (Figure 26D) and ventral (Figure 26G) striata.
  • 6-OHDA treatment significantly reduced the number of dopaminergic terminals in the striatum.
  • MANF 36 ⁇ g showed a trend towards normalization of dopamine levels in the striatum.
  • the striatal dopamine levels of GDNF were not different from 6-OHD A/vehicle. This lack of striatal dopamine level restoration was also observed in an independent study of GDNF assessing its neuroregenerative potential in a 6-OHDA medial forebrain bundle lesion.
  • DOPAC levels were significantly different between the vehicle/vehicle and 6-OHD A/vehicle groups on the ipsilateral ( Figure 29A) and for the ipsilateral/contralateral ratio (Figure 29C), but not on the contralateral side ( Figure 29B).
  • the MANF 36 ⁇ g group showed a trend towards a difference from 6-OHD A/vehicle on the ipsilateral side but none of the other treatment groups was different from 6-OHD A/vehicle.
  • the effect by MANF 36 ⁇ g on DOPAC mirrored the results obtained for dopamine.
  • HVA levels were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral ( Figure 3 OA) and for the
  • CED Convection-enhanced delivery
  • CED describes a direct method of drug delivery to the brain through very fine micro catheters. By establishing a pressure gradient at the tip of the infusion catheter, CED confers several advantages over conventional drug injection techniques, in particular, homogeneous drug distribution through large and clinically-relevant brain volumes.
  • the primary objective of this study was to determine whether a single infusion of MANF could be distributed via CED and whether MANF could be detected after 7 days.
  • MANF (10 ⁇ ) was delivered via CED at flow rates of 0.1 ⁇ / min, 1.25 ⁇ / min, and 5 ⁇ / min into the striatum. MANF was detected at 0 hours after the infusion at all flow rates by immunohistochemistry (Figure 31).
  • MANF's potent neuroprotective activity was confirmed in this present study both at the behavioral and structural level.
  • a single striatal administration of MANF prevented 6-OHDA-induced behavioral deficits and dopaminergic cell death in the substantia nigra.
  • a comparison of striatal and nigral administration of MANF revealed an intriguing mechanism of distal action of this growth factor.
  • MANF was administered to the substantia nigra, striatal dopaminergic terminal densities increased and when the striatum was the target of MANF administration an increase of TH + cells in the substantia nigra was detected.
  • MANF (and GDNF) administration to the substantia nigra resulted in an increase of ipsilateral rotations and we hypothesize that this effect might be due to activation of contralateral circuits. Therefore, MANF is a potent neuroprotective agent, MANF activity is site specific and MANF may cause effects in nigral neurons of the ipsilateral and contralateral projections to the striatum.
  • MANF normalized the 6-OHDA-induced ipsilateral rotations as early as 2 weeks after a single administration and this effect was sustained at 4 weeks post growth factor treatment.
  • the active doses ranged from 3 ⁇ g to 10 ⁇ g and are in good agreement with the literature. It appears likely that lower doses would be active in this paradigm; the minimally effective dose remains to be determined.
  • MANF may counteract some of the known intracellular effects of 6- OHDA.
  • 6-OHDA is relatively selective for monoaminergic neurons, resulting from preferential uptake by dopaminergic and noradrenergic transporters.
  • 6-OHDA Once inside the neurons 6-OHDA accumulates in the cytosol, generates reactive oxygen species and quinones with the latter leading to the inactivation of biological macromolecules by attack of nucleophilic groups.
  • the mechanism of cell death in mesencephalic neurons involves caspase-dependent pathways.
  • 6-OHDA but not MPP + , induced caspase-3 and caspase-9 enzymatic activity in the MN9D dopaminergic neuronal cell line.
  • Application of a caspase inhibitor (zVAD-fmk) prevented 6-OHDA-induced TH + cell death.
  • the specific neuronal toxicity of 6- OHDA thus involves several mechanisms including the activation of apoptotic pathways and the generation of reactive oxygen species.
  • MANF may counteract the toxicity of 6-OHDA on both levels. MANF decreased caspase-3 activation in serum starved cardiomyocytes. It is thus conceivable that MANF could prevent 6- OHDA-induced caspase activation in dopaminergic neurons and thereby promote the survival of these cells.
  • MANF protected cardiomyocytes from reperfusion injury in vivo. It is well known that a key mechanism of reperfusion injury is the generation of reactive oxygen species. MANF could thus counteract the effects of 6-OHDA-generated reactive oxygen species. Finally, MANF might have a more general role in counteracting cellular stress and in particular endoplasmatic stress, as exemplified by its protective activity in thapsigargin- and tunicamycin-induced cell death. These anti-stress effects of MANF may contribute to the observed in vivo effects of MANF in the 6-OHDA model of PD.
  • MANF displayed a strong neuroprotective activity that manifested itself in a reduced rotational behavior and a rescue of TH + neurons in the substantia nigra. Remarkably, this protective effect was not paralleled by a functional
  • MANF dopaminergic terminals in the striatum were assessed by densitometry in the current study.
  • MANF could facilitate outgrowth of fibers from sick or surviving neurons. This could be because of a neuroprotective MANF effect on neurons and their projections, and/ or by a sprouting of new fibers into previously occupied striatal terminal fields from surviving neurons or those unaffected by the 6-OH-dopamine lesion.
  • GDNF GDNF
  • administration to the substantia nigra in the neuroprotection protocol was remarkable but not unprecedented.
  • a reduction in TH + fiber density in the striatum after administration of GDNF to the substantia nigra has been observed.
  • aberrant local sprouting in the ventral thalamus was observed and the combination of these effects may have resulted in more severe motor impairments.
  • an increased TH density in the striatum but no data was collected on aberrant sprouting close to the growth factor injection site.
  • An alternative explanation for the increased behavioral abnormalities observed could involve activation of contralateral circuits by striatal
  • MANF vascular endothelial growth factor
  • Neuronal circuits that may be involved in this mechanism are the interhemispheric nigrostriatal and corticostriatal pathways.
  • 6-OHDA injection of 6-OHDA into the striatum spares these interhemispheric nigrostriatal neurons. It is thus conceivable that nigral administration of MANF could affect the activity of interhemispheric neurons by increasing the
  • contralateral striatal dopaminergic terminals Since those terminals are located in an environment conducive to neuronal activity, increased TH protein levels may represent increased active enzymatic activity resulting in increased striatal dopamine levels which could then lead to an increase of ipsilateral rotations.
  • increased TH protein levels may represent increased active enzymatic activity resulting in increased striatal dopamine levels which could then lead to an increase of ipsilateral rotations.
  • both the terminal densities and the dopamine levels on the contralateral side of MANF treated animals were slightly increased.

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Abstract

La présente invention concerne une méthode de traitement au moyen de MANF. Selon certains modes de réalisation, l'invention concerne une méthode de traitement de la maladie de Parkinson par administration de MANF au locus niger.
PCT/US2013/066688 2012-10-24 2013-10-24 Cibles cérébrales pour facteurs neurotrophiques en vue de traiter une maladie neurodégénérative Ceased WO2014066686A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017085362A1 (fr) 2015-11-18 2017-05-26 Herantis Pharma Plc Compositions comprenant du cdnf ou du manf pour une utilisation dans le traitement par voie intranasale de maladies du système nerveux central
WO2019218983A1 (fr) * 2018-05-16 2019-11-21 上海市同济医院 Vecteur de virus adéno-associé recombinant et son utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009133247A1 (fr) * 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009133247A1 (fr) * 2008-04-30 2009-11-05 Licentia Oy Facteur neurotrophique manf et ses utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LINDHOLM, P. ET AL.: "Novel CDNF/MANF Family of Neurotrophic Factors", DEVELOPMENTAL NEUROBIOLOGY, vol. 70, no. 5, 2010, pages 360 - 371 *
PETROVA, P.S. ET AL.: "MANF: A New Mesencephalic, Astrocyte-Derived Neurotrophic Factor with Selectivity for Dopaminergic Neurons", JOURNAL OF MOLECULAR NEUROSCIENCE, vol. 20, 2003, pages 173 - 187 *
VOUTILAINEN, M.H. ET AL.: "Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease", EXPERIMENTAL NEUROLOGY, vol. 228, 2011, pages 99 - 108 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017085362A1 (fr) 2015-11-18 2017-05-26 Herantis Pharma Plc Compositions comprenant du cdnf ou du manf pour une utilisation dans le traitement par voie intranasale de maladies du système nerveux central
WO2019218983A1 (fr) * 2018-05-16 2019-11-21 上海市同济医院 Vecteur de virus adéno-associé recombinant et son utilisation

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