Patent Application
20190192629
The present invention relates to the field of neurotrophic factors and endoplasmic reticulum (ER) located proteins and more particularly to the field of treating central nervous system diseases. In particular, the present invention is directed to a composition suitable for intranasal administration for use in the intranasal treatment of a central nervous system disease, such as Alzheimer's disease, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease, traumatic brain injury and stroke.
Neurotrophic factors cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are currently the most efficient proteins for the treatment degenerating dopamine neurons in the rat 6-OHDA model of Parkinson's disease (Lindholm and Saarma, 2010). Both factors potently prevent the 6-OHDA-induced behavioral and histological symptoms of Parkinson's disease when applied before the toxin (Lindholm et al., 2007; Voutilainen et al., 2009). More importantly, post-treatment with either factor efficiently restored the normal motor behavior and dopaminergic innervations of the striatum when applied at the stage when the 6-OHDA-induced symptoms of the Parkinson's disease are already far-reaching (Lindholm et al., 2007; Voutilainen et al., 2011). CDNF protects and repairs dopamine neurons also in mouse MPTP model of Parkinson's disease (Airavaara et al., 2012), and in a severe 6-OHDA model it is more efficient than glial cell line-derived neurotrophic factor (GDNF)(Airavaara et al., 2012; Voutilainen et al 2011). The mechanisms behind the neuronal protection for these factors are not fully clear but it has been suggested they activate pathways, which aim at alleviating oxidative- and ER stress and depressing apoptotic cell death. Many pathophysiological conditions including diabetes mellitus and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are associated with ER stress. Accordingly, the effect of CDNF and MANF has been shown in various central nervous system diseases (WO2009133247; WO2007068803; and Airavaara et al, 2009). In addition, CDNF and MANF suppress neuroinflammation, which is involved in the pathophysiology of most if not all CNS diseases and injuries (Nadella et al, 2014; Zhao et al, 2013).
In WO2014066686, a convection-enhanced delivery of MANF to a brain region of a patient is disclosed. A neurosurgical device for this invasive method comprises a catheter that can be placed at or near the brain region and a pump for generating a positive pressure gradient between the catheter and the brain region.
Aly et al., 2015, reviews current progress in intranasal gene delivery methods for treating Parkinson's disease. In the abstract, it is stated that intranasal delivery provides a promising approach for development of a non-invasive gene therapy for Parkinson's disease. The authors also discuss that CDNF and MANF have demonstrated efficacy in animal models of Parkinson's disease but have not yet been tested by the intranasal route. However, it is notable that nose-to-brain penetration properties of viral particles used in gene delivery methods are not dependent on the gene sequence of the chosen neurotrophic factor. In contrast, the intranasal penetration ability of a high molecular weight protein is always a unique and unpredictable property.
U.S. Pat. No. 6,180,603 discloses a method and a pharmaceutical composition for transporting a neurologic agent to brain in which the pharmaceutical composition is administered intranasally. The document specifically discloses pharmaceutical compositions comprising the neurotrophic factor NGF.
Alcalá-Barraza et al., 2010, disclose the intranasal delivery of BDNF, CNTF, neurotrophin-4/5 (NT-4/5), and erythropoietin (EPO), resulting in nanomolar concentrations of the molecules in rat brain. In a corresponding way, Bender et al., 2015, disclose intranasal administration of GDNF, either in liposomal formulation or in PBS, to rat brain.
Pardeshi & Shailendra (2013) disclose that conventional routes of drug administration, such as oral administration, are not able to deliver a number of therapeutic agents to the brain efficiently. The blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCB) hinder the entrance of drugs from systemic circulation into the central nervous system (CNS). The authors review direct nose-to-brain-drug-delivery and conclude that a variety of neurotherapeutic agents can be delivered to the CNS by nasal route. However, one of major limitations of the intranasal delivery as listed by the authors is decreased permeability of high molecular weight drugs across nasal mucosa.
It is an aim of the present invention to provide compositions for intranasal administration for use in the intranasal treatment of central nervous system diseases.
It is another aim of the invention to provide novel uses of CDNF and MANF polypeptides.
In the present invention, it has been discovered that surprisingly chemical and biological characteristics of CDNF and MANF polypeptides facilitate the transport of the polypeptides to the central nervous system (CNS) when the polypeptides are intranasally administered to a patient. CDNF and MANF polypeptides are therefore able to bypass blood-brain-barrier through nasal route and reach the CNS.
Thus, in one aspect the invention provides a composition for intranasal administration comprising a CDNF polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof for use in the intranasal treatment of a central nervous system disease.
In another aspect, the invention provides a composition for intranasal administration comprising a MANF polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a functional fragment thereof for use in the intranasal treatment of a central nervous system disease.
As used herein, a “polypeptide” generally refers to a molecule having an amino acid sequence encoded by a polynucleotide. “Polypeptide” also refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic nonnaturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
As used herein, the term “fragment” as applied to a polypeptide, may ordinarily be at least about seven contiguous amino acids, typically, at least about fifteen contiguous amino acids, more typically, at least about thirty contiguous amino acids, typically at least about forty contiguous amino acids, preferably at least about fifty amino acids, even more preferably at least about sixty amino acids and most preferably the peptide fragment will be greater than about seventy contiguous amino acids in length. The term “functional fragment” refers to a fragment, which still retains biological activity of the intact polypeptide.
The present invention is related to a neurotrophic factor protein CDNF. CDNF polypeptides are the full-length human CDNF with a signal peptide having the total length of 187 amino acids and the mature human CDNF without the signal peptide having the total length of 161 amino acids (see Table 1) with the size of 18.5 kDa.
The present invention is also related to neurotrophic factor protein MANF. Particularly important MANF polypeptides are the full-length human MANF with a signal peptide having the total length of 179 amino acids and the mature human MANF without the signal peptide having the total length of 158 amino acids (see Table 1) with the size of 18 kDa. The MANF nucleic acid encoding mature MANF polypeptide without a signal peptide may be combined with a nucleic acid encoding another signal peptide to provide a full-length MANF polypeptide with a different signal peptide than the native MANF signal peptide.
Functional fragments of the MANF and CDNF polypeptides are also available for a skilled person as the production of fragments of whole polypeptides is known in the art. Accordingly, the MANF or CDNF protein may be a full length, naturally occurring form or may be a truncated or otherwise derivatised form. Examples of functional MANF or CDNF fragments are disclosed in WO2013034805, such as peptides comprising the sequence CKGC (SEQ ID NO:5) or CRAC (SEQ ID NO:6), preferably with the length of 4-40 amino acids. Further, Hellmann et al., 2011, disclose an active C-terminal fragment construct of MANF comprising residues 96-158 of SEQ ID NO:3.
In addition to naturally occurring allelic variants of MANF/CDNF, changes can be introduced by mutation into MANF/CDNF sequences that incur alterations in the amino acid sequences of the encoded MANF/CDNF polypeptide. Nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of a MANF/CDNF polypeptide. MANF/CDNF polypeptides or functional fragments thereof comprising one or more “non-essential” substitutions can be seen as equivalents to wild-type MANF/CDNF polypeptides disclosed herein. These equivalents are preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% homologous to said wild-type polypeptides.
A “non-essential” amino acid residue is a residue that can be modified in the wild-type sequences of MANF/CDNF without altering its biological activity, whereas an “essential” amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the MANF/CDNF molecules of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known in the art.
As discussed above, it has been found that CDNF and MANF polypeptides are able to bypass the blood-brain-barrier through the nasal route and to reach the central nervous system, CNS. Therefore, CDNF and MANF polypeptides can be intranasally administered to a patient and used for treatment of a number of central nervous system diseases.
Accordingly, in one embodiment a composition is provided for intranasal administration comprising a CDNF polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof for use in the intranasal treatment of a central nervous system disease. Particularly, in this embodiment, intranasal treatment of Alzheimer's disease, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease, traumatic brain injury and stroke is achieved.
A second embodiment provides for the use of a composition comprising a CDNF polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof for the manufacture of a medicament for use in the intranasal treatment of a central nervous system disease.
Another embodiment is a method for intranasal administration of a CDNF polypeptide to a subject suffering from a central nervous system disease, the method comprising the step of: intranasally administering to the subject in one or more doses a composition comprising a CDNF polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof.
An embodiment is directed to a composition for intranasal administration comprising a MANF polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a functional fragment thereof for use in the intranasal treatment of a central nervous system disease. Preferably, said central nervous system disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease, traumatic brain injury and stroke.
One further embodiment is the use of the composition comprising a MANF polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a functional fragment thereof for the manufacture of a medicament for use in the intranasal treatment of a central nervous system disease.
Another further embodiment is a method for intranasal administration of a MANF polypeptide to a subject suffering from a central nervous system disease, the method comprising the step of: intranasally administering to the subject in one or more doses a composition comprising a MANF polypeptide comprising the amino acid sequence of SEQ ID NO:3 or a functional fragment thereof.
Compositions suitable for intranasal administration comprising CDNF/MANF polypeptides as active agents can be in the form of a liquid suspension, a liquid dispersion, a powder, a liposome, an aqueous solution or combinations thereof. The composition may thus be dispensed intranasally as a powdered or liquid nasal spray, nose drops, a gel or ointment, through a tube or catheter, by syringe, by packtail, by pledget, or by submucosal infusion. The composition may be administered to the nasal cavity alone or in combination with other neurologically active agents. CDNF/MANF polypeptides may be combined with a carrier and/or other adjuvants to form a pharmaceutical composition. Preferably, the composition comprise absorption promoters and absorption modulator systems such as cyclopenta decalactones, alkylsaccharides, chitosan, low methylated pectin or polyglycol hydroxystearate esters. Lipophilic substances in the form of micelles may be added to the composition to enhance absorption of the neurologic agent across the olfactory epithelium. Among those substances that are preferred micellar additives are GM-1 gangliosides and phosphatidylserine (PS), which may be combined with the neurologic agent either alone or in combination. Said absorption promoters (i.e. absorption enhancers) and absorption modulator systems may increase the transport of CDNF/MANF polypeptides across the nasal membrane and thus promote sufficiently high therapeutic levels in the CNS. It is believed that absorption promoters enhance drug delivery via the transcellular route or alterations in junctional proteins resulting in promotion of drug translocation through the paracellular route. Other absorption enhancers suitable for the present compositions are known in the art, see e.g. Stolnik et al., 2009 and Shubber et al., 2014.
Recombinant MANF and CDNF polypeptides for the compositions can be produced as disclosed in WO2009133247 and WO2007068803. For intranasal delivery purified recombinant protein can simply be added to a pharmaceutically suitable buffer. For instance, in Hanson et al, 2012, a growth factor was administered intranasally in acidic buffer comprising 20 mM sodium acetate at pH 4.25. In a preferred embodiment, the proteins are freeze-dried or spray-dried to produce a powder for use in a nasal spray. The methods for freeze-drying are well-known in the art (see e.g. Tang and Pikal 2004).
The optimal concentration of the active CDNF/MANF polypeptide in the composition will necessarily depend upon the characteristics of the patient, and the nature of the disease or condition for which the treatment is to be used. Preferably, the composition is administered intranasally 1-5 times a day, more preferably 1-2 times a day, and the amount of CDNF/MANF polypeptide is between 10-1000 mg, more preferably 100-500 mg per dosage. The most acceptable dosing regimen would be one dose into one or both nostrils.
One example of suitable devices for intranasal administration is disclosed in WO2012119153. A suitable device is designed to deliver a drug in the form of a liquid suspension, a liquid dispersion, a powder, a liposome, an aqueous solution or combinations thereof. The device is preferably designed so that the administered nasal composition is delivered to the olfactory area in the upper third of the nasal cavity and particularly to the olfactory epithelium in order to promote transport of the active agents of the composition into the peripheral olfactory neurons. The device usually comprises a bottle, pump and actuator. Preferably, the device has a canister capable of containing a propellant, a diffuser in communication with the canister, a compound chamber for the drug in communication with the diffuser, and a nozzle in communication with the compound chamber. Devices are available for single dose or multiple dose systems. The powder formulations for the use in said device are preferably in aerosolised form.
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the scope of the invention as defined by the claims. For instance, the choice of protocols is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.
Having generally described the invention above, the same will be more readily understood by reference to the following Experimental Section, which is provided by way of illustration and is not intended as limiting.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
The present study was designed to evaluate brain and plasma distribution of intranasally administered radiolabeled recombinant human CDNF protein (rhCDNF). 125I-labelling of rhCDNF using the lactoperoxidase method and analysis were carried out as described in Voutilainen et al, 2011.
Animal use. 125I-labelling of rhCDNF using the lactoperoxidase method and analysis were carried out as described in Voutilainen et al, 2011. Six unlesioned Sprague Dawley rats were anesthetized and approximately 10 ng of 125I-labeled rhCDNF in 1% bovine serum albumin was administered intranasally under anesthesia. 20 min-4 hours later blood samples were collected together with brain regions olfactory bulb, cerebellum, cortex and non-cortical areas.
Gamma counter analysis. Using gamma counting, 2.9±1.3% (range 1.3-4.5%) of 125I-labeled rhCDNF was found in blood (
The present study was designed to evaluate brain and plasma distribution of intranasally administered radiolabeled recombinant human MANF protein (rhMANF).
Animal use. Two male Sprague Dawley rats were anesthetized and went through the procedure of middle cerebral artery occlusion (MCAO) together with common carotid artery occlusion for 60 min. Two days later rats were again anesthetized followed by intranasal administration of approximately 8 ng 125I-rhMANF (16 μl), labeled using the lactoperoxidase method (Voutilainen et al, Exp. Neurol. 2011) and 20 μg of unlaballed rhMANF (in 4 μl). One hour later blood samples were collected and rats were perfused transcardially with saline and brains were collected.
Gamma-counter analysis. Approximately 0.14-0.16% of 125I-labeled rhMANF total dose was found in the brain and in the blood (
The present study was designed to evaluate therapeutic effects of intranasally administered recombinant human MANF protein (rhMANF).
Animal use, clinical and histopathological assessment. Rats were briefly anesthetized with isoflurane on the previous night. 10 μl of PBS or MANF solution was administered into each nostril in approximately 70° angle. On the next day, rats were anesthetized with chloral hydrate (400 mg/kg) and intranasal administration was repeated just before the MCAO and right after reperfusion. MCA together with common carotid arteries were occluded for 60 minutes. Animals were necropsized 48 h post MCAO.
Triphenyltetrazolium chloride (TTC) staining of coronal brain sections showed that MANF reduced infarction volume as compared with the PBS treated animals (P=0.034, Student's t-test, n=17-24;
In another set of animals, MANF dose of total of 20 μg (3.3 μg per nostril; 0.33 μg/μl) was administered in similar manner as above and behavior of rats was monitored over 2 weeks to measure the functional recovery. Biased swing activity, Bederson's score, distance travelled and vertical activity were analyzed on post stroke days 2, 7 and 14. The two-way repeated measures ANOVA showed significant Treatment X Time interaction (F2.54)=9.34, P<0.001 for the body swing and F(2.54)=7.97, P<0.001 for the Bederson's score (
The present study was designed to evaluate brain penetration and kinetics of intranasally administered radiolabeled recombinant human CDNF protein (rhCDNF) in non-human primates. rhCDNF, labeled with 124I using the lactoperoxidase method, was administered (400 μg, 500 μCi; formulated in 1% BSA, 0.1% sodium ascorbate and 0.1% gentisic acid, pH 6.4) to the nasal cavity of cynomolgus macaques. Positron emission tomographic (PET) imaging was used to determine nose-to-brain penetration of the 124I-rhCDNF tracer.
Animal use. Four female cynomolgus (macaca fascicularis) monkeys (MPI Research) were used in this study. The monkeys were 2-4 years old and weighed 3.08±0.22 kg on the day of imaging. Animal rooms were set to maintain a temperature of 18-26° C., a relative humidity of 50±20%, both to the extent possible, and intermittent light and dark cycles of approximately 12 hours. Appropriate primate diet food and water ad libitum was provided. For various procedures where the animals were handled, e.g. physical examinations, the animals may have been sedated to effect with ketamine. Each animal was fasted for a minimum of 4 hours prior to induction of anesthesia with a ketamine dose (5-10 mg/kg IM), followed by isoflurane (1-2%) with oxygen (2%) for the duration of each scan. Animal welfare for this study was in compliance with the U.S. Department of Agriculture's (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3). Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011, was followed. The animal facility maintains an Animal Welfare Assurance statement with the National Institutes of Health Office of Laboratory Animal Welfare.
Device. A pressurized olfactory delivery device similar to that disclosed in U.S. Patent Application Publication No. US2012/027754 was used for intranasal delivery of rhCDNF.
Positron emission tomographic imaging and image analysis. Whole-body continuous bed motion and focused positron emission tomography (PET) and computed tomography (CT) data were acquired on a Focus220 microPET (Siemens Medical Systems, Knoxville, Tenn.) and CereTom CT (NeuroLogica Corp, Danvers, Mass.), respectively. Each monkey was anaesthetized, intubated and placed on the PET bed in the head first and prone position for scanning. Two doses per nostril were administered on the bed. Immediately after the final dose had been administered a dynamic focused brain PET scan was acquired for 225 minutes. An additional scan was performed 24 h post-dosing to evaluate kinetics of brain clearance of the tracer.
Emission data were acquired in 3D list mode and rebinned into 2D sinograms. PET images were reconstructed by a 2D Ordered Subset Expectation Maximization (OSEM2D) algorithm. Corrections were made for detector normalization, decay, dead time, randoms, and attenuation. The dynamic scan was binned into 45×5 minute frames and 1×225 minute frame. Each reconstructed CT image was resampled and interpolated to the same voxel resolution as the accompanying PET image (0.9 mm). Each PET image was co-registered to the corresponding CT image (
Rotating Maximum Intensity Projection (MIP) movies and 3-slice view images centered on the brain were generated from the preprocessed PET/CT images. A linear color scale was chosen for the 0.2 to 20% ID/mL PET intensity range. Regions of interest (ROIs) for the upper nasal cavity, throat and thyroid were defined by applying a combination of manual and automated segmentation thresholds to the PET or were hand drawn on the co-registered images. ROIs for various brain regions including the Entire Brain were acquired by fitting a proprietary 45-region Cynomolgus brain atlas to the brains of each monkey. A master spreadsheet was generated which included the volume, activity, and concentration (ActivityNolume) at each time point for each ROI generated.
The PET imaging data shows that recombinant human CDNF protein, as a drug substance, is capable of nose-to-brain penetration when aerosolized and delivered to the upper nasal cavity in healthy non-human primates (
The PET imaging data also shows that approximately 92±1% of brain-delivered rhCDNF is cleared from the brain within 24 hours (
The present study was designed to evaluate brain penetration of intranasally administered radiolabeled recombinant human CDNF protein (rhCDNF). rhCDNF, labeled with 124I using the lactoperoxidase method, was administered (400 μg, 500 μCi; formulated in 1% BSA, 0.1% sodium ascorbate and 0.1% gentisic acid, pH 6.4) to the nasal cavity of cynomolgus macaques. Autoradiography was used to determine nose-to-brain penetration of the 124I-rhCDNF tracer.
Animal use. The same animal procedures were used as in Example 4.
Device. The same intranasal dosing device and dosing procedure was used as in Example 4.
Autoradiography. At the scheduled termination time (after a 4-hour whole body imaging time point), one animal was euthanized and the head removed. The head was subsequently frozen in a hexane/dry ice bath and embedded in a 5% carboxymethylcellulose matrix. The embedded head was mounted onto a cryomacrotome stage maintained at approximately −10 to −30° C. and was sectioned to approximately 40 μm thick sections taken every 0.5 mm in the sagittal plane and captured on adhesive tape. Quantitative 124I autoradiography was performed with 52 slices and 40 μm slice thickness in a section of the brain encompassing the area between both eyes.
In particular the following regions were analyzed: nasal cavity, olfactory bulb, olfactory tract, olfactory cortex, thalamus, striatum, substantia nigra, hypothalamus and lateral ventricle. A white light image of the block face was taken at each section. A set of sections were mounted, exposed to phosphor imaging screens and scanned using the Storm 860 image acquisition system (
As shown in
To confirm the nose-to-brain delivery of rhCDNF in non-human primates, autoradiography was used to visualize radiolabeled rhCDNF in the sections of a non-human primate necropsized 4 hours after nasal administration of the tracer. The autoradiography data confirms that some labeled rhCDNF had penetrated to the brain from the upper nasal cavity. Signal was detected in various brain regions, including cerebellum, brainstem, thalamus, striatum, substantia nigra, hypothalamus and olfactory bulb. The presumed route of entry to lateral ventricles is via the cerebrospinal fluid (CSF) passages through the cribriform plate. These data suggest that intranasally administered rhCDNF gets access to the CSF allowing wide distribution in the CNS.
Aly, AE-E and Waszczac, B. 2015. Intranasal gene delivery for treating Parkinson's disease: overcoming the blood-brain barrier, Expert Opin. Drug Deliv. 12(12):1923-1941.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20155857 | Nov 2015 | FI | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FI2016/050813 | 11/18/2016 | WO | 00 |
