Patent Application
20190194324
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 26, 2017, is named P13931-03_SL.txt and is 6,197 bytes in size.
Parkinson's disease (PD) is the second most common neurodegenerative disorder that is characterized clinically by motor dysfunction and pathologically by the aggregation and accumulation of α-synuclein (α-syn). Emerging evidence suggests that α-syn spreads from neuron to neuron via self-amplification, propagation, and transmission in the pathogenesis of PD. In the brains of PD patients, α-syn aggregates seem to spread in a stereotypical and topographical pattern. Postmortem examination of fetal grafts in patients with PD found α-syn positive Lewy bodies suggestive of spread of α-syn from host to graft. Other proteins such as β-amyloid and tau in Alzheimer's disease are also thought to propagate and spread and contribute to the onset and progression of this disorder. Pathological α-syn has been shown to spread among neighboring cells and/or anatomically connected brain regions. Recently recombinant α-syn pre-formed fibrils (PFF) provide a model system enabling the study of the transmission of misfolded α-syn from neuron to neuron both in vitro and in vivo. How pathological α-syn exits cells and enters neighboring neurons is not known, but entry into neurons is thought to occur through an active endocytic process. Understanding this process would enable the development of drugs for the treatment or prevention of Parkinson's disease.
One embodiment of the present invention is a method of inhibiting neurodegeneration in a subject comprising administering to the subject an agent that prevents α-syn PFF from binding to its receptor. The methods of the present invention may be used to treat or prevent Parkinson's disease, Diffuse Lewy Body Disease (DLB), dementia with Lewy Bodies, multiple atrophy, or other neurodegenerative disease. The agent may be a small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide. A suitable agent may be a vector that expresses antisense LAG3 mRNA in the subject. An agent may also be a capture molecule such as an aptamer, monoclonal antibody, antibody, or portion thereof that binds to a target molecule such as α-syn PFF. A suitable α-syn PFF receptor is lymphocyte-activation gene 3 (LAG3), Neurexin1β, Neurexin2β, Neurexin3β, or a combination thereof. Alternatively a receptor maybe an amyloid precursor-like protein 1 (APLP1). The receptor is found within a subject. A suitable subject of the present invention is a human. Alternatively, the agent may bind to α-syn PFF receptor such as LAG3. For example, a capture molecule may bind to LAG3 and prevent the binding of α-syn PFF with LAG3. An agent, such as a capture molecule, may bind to the LAG3 D1 domain specifically to amino acids 81-109, amino acids 52-80, or to both sites. An agent may also inhibit the phosphorylation of α-syn at serine 129 in a subject. Subjects suitable for the present invention comprises α-syn PFF and endocytosis of α-syn PFF is inhibited in the subject when the methods of the present invention are performed. Agents of the present invention may also inhibit the misfolding of α-syn protein in a subject. The methods of the present invention may treat and prevent Parkinson's disease, or neurological disease, in subjects.
Another embodiment of the present invention is a method of drug screening comprising the steps of: providing one or more agent(s); applying the one or more agents to LAG3; and identifying those agents that prevent α-syn PFF from binding to LAG3.
Another embodiment of the present invention is a method of drug screening comprising the steps of: providing one or more agent(s); applying the one or more agents to cells, and identifying those agents that prevent α-syn PFF from binding to LAG3 or that inhibit the phosphorylation of α-syn PFF at serine 129.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.”
By “APLP1” is meant amyloid beta precursor like protein 1. An APLP1 protein is expressed from an APLP1 gene such as a human APLP1 gene including NCB1 Gene ID: 333, as an example. An example of an APLP1 human protein sequence includes NCBI reference sequences NP-001019978.1 and NP_005157.1.
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By “LAG3 gene” is meant lymphocyte activation gene 3 gene and an example of such a gene is a Homo sapiens LAG 3 gene sequence having an NCBI Gene ID 3902 and a NCBI Reference Sequence number NC_00012.12.
By LAG3 protein” is meant a protein, a polypeptide, or a fragment thereof having at least about 90% amino acid identity to a LAG 3 gene. An example of a Homo sapiens LAG3 protein having NCBI Reference Sequence NP_002277.4 (SEQ ID NO: 1) is shown below:
By “α-synuclein gene” is meant a nucleic acid sequence able to express an α-synuclein protein, a polypeptide, or a fragment thereof including the human DNA sequence at the NCBI Gene ID 6622.
By “α-synuclein protein” is meant a protein, a polypeptide, or a fragment thereof having at least about 90% amino acid identity to a α-synuclein gene. An example of an Rattus norvegicus α-synuclein protein is the sequence at NCBI GenBank Number AAS55695.1 (SEQ ID NO: 2) is shown below:
By “LAG3 antibody” is meant an antibody that selectively binds a LAG3, preferably at the LAG3 α-syn PFF receptor binding site.
By “anti-α-syn PFF antibody” is meant an antibody that selectively binds a α-syn PFF.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include pancreatic cancer.
By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
“Diagnostic” means identifying the presence or nature of a pathologic condition, i.e., pancreatic cancer. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the “false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. The term “biomarker” is used interchangeably with the term “marker.”
The term “measuring” means methods which include detecting the presence or absence of marker(s) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein, including but not limited to immunoassay. Any suitable methods can be used to detect and measure one or more of the markers described herein. These methods include, without limitation, ELISA and bead-based immunoassays (e.g., monoplexed or multiplexed bead-based immunoassays, magnetic bead-based immunoassays).
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins. The terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or there between.
“Immunoassay” is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
The term “antibody,” as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless of whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term “intact,” as in “intact antibodies,” for the purposes of this disclosure, the term “antibody” also includes antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind, for example, a α-syn PFF receptor site such as LAG3 or to α-syn PFF. Typically, such fragments would comprise an antigen-binding domain.
The terms “antigen-binding domain,” “antigen-binding fragment,” and “binding fragment” refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as “epitope” or “antigenic determinant.” An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.
Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab′, F(ab′)2, Fv, and single-chain antibodies. An antibody other than a “bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical. Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as “Fab” fragments, and a “Fc” fragment, having no antigen-binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in the a F(ab′)2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab′)2 fragment has the ability to crosslink antigen. “Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. “Fab” when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
The term “mAb” refers to monoclonal antibody. Antibodies of the invention comprise without limitation whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
As used herein, the term “sensitivity” is the percentage of subjects with a particular disease.
As used herein, the term “specificity” is the percentage of subjects correctly identified as having a particular disease i.e., normal or healthy subjects. For example, the specificity is calculated as the number of subjects with a particular disease as compared to non-cancer subjects (e.g., normal healthy subjects).
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 mug/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
Such treatment (surgery and/or chemotherapy) will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for pancreatic cancer or disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like). In particular embodiments, determination of subjects susceptible to or having a pancreatic cancer is determined by measuring levels of at least one of the markers of the invention (e.g., CA19-9, MIA, MIC-1, CEACAM-1, OPN, SPON1, HSP27, POSTN, or LGALS3BP). In particular embodiments, a subject determined susceptible to or having a pancreatic cancer is selected for surgery.
The term “activity” refers to the ability of a gene to perform its function such as ZnT8 (a zinc transporter) being able to transport zinc.
The term “express” refers to the ability of a gene to express the gene product including for example its corresponding mRNA or protein sequence (s).
The term “reference” refers to a standard or control conditions such as a sample (human cells) or preoteolipisomes with a zinc transporter ZnT8 free, or substantially free, of agent.
As used herein, the term “subject” is intended to refer to any individual or patient to which the method described herein is performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
α-Syn was synthesized and conjugated to biotin (α-syn-biotin), and then aggregated over seven days followed by sonication to form PFF. Size exclusion chromatography was used to separate PFF from α-syn monomers (
The selectivity of LAG3, Neurexin1β and APLP1 and related transmembrane proteins for α-syn-biotin PFF versus α-syn-biotin monomers was determined via the ratio of Kd values (
Like other major histocompatibility complex (MHC) class II molecules, LAG3 contains an ectodomain composed of four Ig-like domains (D1-D4). To determine the α-syn-biotin PFF binding domain, we sequentially deleted each domain of LAG3 and performed the cell surface binding assay with overexpression of the LAG3 deletion mutants. These experiments reveal that α-syn-biotin PFF preferentially bind to the D1 domain whereas deletion of the D2, D3 or the intracellular domain (ICD) substantially weakens binding, but not the D4 domain (
To determine whether LAG3 is required for the endocytosis of α-syn PFF, pHrodo red was conjugated to α-syn PFF. pHrodo red is a pH dependent dye that increases in fluorescence as pH decreases from the neutral cytosolic pH to the acidic pH of the endosome. Conjugation of α-syn PFF with pHrodo red does not appreciably change the properties of the α-syn PFF as assessed by immunoblot and atomic force microscopy (
The Rab5 GTPase is an early endosomal marker and helps mediate endocytosis. As such, the inventors sought to confirm the endocytosis of α-syn-biotin PFF into endosomes by measuring the intensity of internalized α-syn-biotin PFF that is co-localized with Rab5. The inventors find that internalized α-syn-biotin PFF is co-localized with Rab5 in wild type cortical neurons (
Microsomes, which contain endosomes were isolated via differential centrifugation from wild type and LAG3 knockout cultures following treatment with α-syn-biotin PFF (
We then asked whether knocking out LAG3 prevents the pathology induced by α-syn PFF. Phosphorylation of α-syn at serine 129 (P-α-syn) and misfolded α-syn are associated with pathology in α-synucleinopathies. Their levels increase following administration of α-syn PFF to neuronal cultures. Accordingly, we administered α-syn PFF to wild type and LAG3 knockout cortical cultures at seven days in vitro (DIV). Ten days later the levels of P-α-syn are markedly increased in wild type cultures, while the levels of P-α-syn in LAG3 knockout cultures is barely detectable (
Immunoblots from lysates sequentially extracted in 1% TX-100 (TX-soluble) followed by 2% SDS (TX-insoluble) of α-syn and P-α-syn 12 days after α-syn PFF treatment of cortical neurons were examined. α-Syn PFF leads to an accumulation of α-syn and P-α-syn in the TX-insoluble fraction in wild type cultures, while there is significantly less accumulation in LAG3 knockout cultures (
To examine the transmission of α-syn PFF and to establish the role of LAG3 in the intemeuron transmission of α-syn, we used a microfluidic neuronal culture device with three chambers connected in tandem by a series of microgrooves separating the chambers. The medium volume in chamber 1 (C1) is 50-μL lower than the one in chamber 2 (C2), and 100-μL lower than the one in chamber 3 (C3) to prevent diffusion of α-syn PFF to adjacent chambers. Cortical neurons were cultured in each chamber. To ensure that α-syn PFF cannot diffuse between chambers, primary wild type cortical neurons in C1 were treated with α-syn-biotin PFF. 14 days post-treatment, the neurons were fixed in 4% paraformaldehyde (PFA) and stained with streptavidin-568 fluorescence dye. Only neurons in C1 exhibit immunofluorescence, indicating that α-syn-biotin PFF cannot transmit from chamber to chamber through diffusion (
α-Syn transmission from C1 to C3 requires neurons in C2, since α-syn PFF administered to C1 fails to induce P-α-syn accumulation in C3 when C2 was left empty (
Treatment of wild type cortical cultures with α-syn PFF cause neuronal cell death as previously described (
To determine whether LAG3 is necessary for α-syn PFF transmission and toxicity in vivo, α-syn PFF were stereotactically injected into the dorsal striatum of wild type and LAG3 knockout mice (
Recently, the Toll-like receptor 2 (TLR2) on microglia was shown to be involved in the activation of microglia due to exposure to oligomeric α-syn from conditioned neuronal media. On the other hand, LAG3 appears to mediate the transmission of misfolded α-syn from neuron to neuron. According to the Allen Brain Atlas, it is localized to neurons throughout the central nervous system including dopamine neurons. The function of LAG3 in the CNS is not known and whether misfolded α-syn activates downstream signaling following engagement of LAG3 requires further study.
Recent studies have revealed that lymphocytes can extract surface molecules from antigen presenting cells through a process called trogocytosis. Lag3 is enriched in lymphocytes and binds to major histocompatibility complex (MHC) class II from neighboring cells where it may participate in trogocytosis. Trogocytosis has been proposed a mechanism for intercellular communication either through endocytic vesicles or through a membrane bridge. We propose a novel mechanism cell-to-cell transmission of misfolded α-syn that involves the endocytosis of exogenous α-syn PFF by the engagement of LAG3 on neurons similar to Lag3 facilitated trogocytosis by binding to MHC class II molecules.
In summary, the interaction between LAG3 and α-syn PFF provides a new target for the development of therapeutics designed to slow the progress of PD and related α-synucleinopathies.
Embodiments of the disclosure concern methods and/or compositions for treating and/or preventing a neurological disorder in which modulation of the α-syn PFF transmission pathway is directly or indirectly related. In certain embodiments, individuals with a neurological disorder such as Parkinson's disease (PD) are treated with an agent that acts as a modulator of the pathway, and in specific embodiments an individual with PD is provided an agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF.
In certain embodiments, the level to which an agent inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be any level so long as it provides amelioration of at least one symptom of the neurological disorder, for example PD. The level of inhibition may increase by at least 2, 3, 4, 5, 10, 25, 50, 100, 1000, or more fold compared to the level in a standard (where the agent is not applied), in at least some cases.
An individual known to have PD, suspected of having PD, or at risk for having PD may be provided an effective amount of an agent of the present invention. Those at risk for PD may be those individuals having one or more genetic factors, may be of advancing age, and/or may have a family history, for example.
In particular embodiments of the disclosure, an individual is given a second or third agent for PD therapy in addition to the one or more agents that inhibit α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF. Such additional therapy may include L-DOPA or dopamine receptor agonists and/or deep brain stimulation, for example. When combination therapy is employed with one or more agents that inhibit α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF the additional therapy may be given prior to, at the same time as, and/or subsequent to the agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF.
Certain methods of the disclosure provide for methods of diagnosing PD prior to the therapeutic methods of the disclosure, and such diagnosis may occur by any methods or means, including at least genetic marker assay, single-photon emission computed tomography, olfactory system testing, autonomic system testing, or a combination thereof.
Pharmaceutical compositions of the present invention comprise an effective amount of one or more agents that inhibit α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF dissolved or dispersed in a pharmaceutically acceptable carrier. The 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 a pharmaceutical composition that comprises at least one agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. As used herein, “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 pharmaceutical compositions is contemplated.
The agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF 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. The present compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, 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), 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).
The agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like. Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various 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. In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include an agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.
One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, 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. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/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 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In one embodiment of the present disclosure, the agents that inhibit α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
For oral administration the compositions of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
In further embodiments, agents that inhibit α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be administered via a parenteral route. As used herein, the term “parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
Sterile injectable solutions are 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. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
In other preferred embodiments of the invention, the active compound or agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a “patch”. For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF may be comprised in a kit.
The kits may comprise a suitably aliquoted agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The agent that inhibits α-syn PFF from binding to its receptor and/or neural transmission of α-syn PFF (s) may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation.
α-Syn purification and PFF preparation. Recombinant α-syn was purified as published. Assembly reactions were agitated in a transparent glass vial with a magnetic stirrer (350 rpm at 37° C.). After 7 days incubation and then sonication, α-syn monomer/PFF was separated by HPLC and kept in −80° C. To characterize α-syn PFF receptors, recombinant α-syn monomer was purified and labeled with sulfo-NHS-LC-Biotin (Thermo Scientific, EZ-link Sulfo-NHS-LC-Biotin, 21435). The mole ratio of biotin to α-syn was 2-3. After conjugation, α-syn-biotin monomer/PFF is prepared as mentioned above.
Expression cloning and SH-SY5Y cell surface binding assays. We performed a directed experiment to identify α-syn PFF receptor(s): a library consisting of 352 individual preparations of cDNAs encoding transmembrane proteins (TMGW10001, GFC-transfection array panel, Origene) was transfected into SH-SY5Y cells. Two days after transfection, the cells were incubated with α-syn-biotin PFF (1 μM total α-syn-biotin monomer concentration) in DMEM media with 10% FBS at 22° C. for 2 h. Next, unbound α-syn-biotin PFF was removed by extensive washing with DMEM with 10% FBS. The cells were fixed with 4% paraformaldehyde in PBS, washed three times with PBS, blocked for 30 min with 10% goat serum and 0.1% Triton X-100 in PBS, and incubated for 16 h with alkaline-phosphatase-conjugated streptavidin in PBS supplemented with 5% goat serum and 0.05% Triton X-100. Finally, bound alkaline-phosphatase was visualized by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium reaction. Quantification of bound α-syn-biotin PFF to LAG3-transfected SH-SY5Y cells was performed with ImageJ. We also tested similarly several candidate receptors; the cDNA plasmids were obtained from Addgene.
Primary neuronal cultures, α-syn PFF transduction and neuron binding assays. Primary cortical neurons were prepared from E15.5 and cultured in Neurobasal media supplemented with B-27, 0.5 mM L-glutamine, penicillin and streptomycin (all from Invitrogen) on tissue culture plates coated with poly-L-lysine. The neurons were maintained by changing medium every 3-4 days. α-syn PFF transduction was performed at 7 DIV and α-syn PFF was kept for 10-21 days for biochemical experiment or toxicity assay. Each experiment was performed in duplicate and repeated 3-6 times. Transduced neurons were harvested for indirect immunofluorescence and sequential extraction. To determine bound α-syn-biotin PFF in wild type and LAG3 knockout culture, α-syn-biotin PFF with different indicated concentrations were used. Quantification of bound α-syn-biotin PFF to wild type and LAG3 knockout neurons were performed with ImageJ.
ELISA analysis. The binding affinity between α-syn-biotin PFF and LAG3 were analyzed using a sandwich ELISA kit (Sigma) according to manufacturer instructions. The lyophilized human LAG3 protein was added into a human LAG3 antibody-coated ELISA plate and left overnight at 4° C. with gentle shaking. After the extensive wash, different concentrations of α-syn-biotin PFF (0.1 nM to 100 nM) were added to each well and were incubated for 2 hours at 22° C. with gentle shaking. HRP-streptavidin solution was incubated for 45 min at 22° C. with gentle shaking and follows with the extensive wash. Finally, ELISA colorimetric TMB Reagent was incubated for 10 min at 22° C. in the dark with gentle shaking.
Plasmids. Human and mouse LAG3 cDNA clones were kindly obtained from Dr. Charles Drake at the Johns Hopkins University, School of Medicine. APLP1 cDNA clone was obtained from Dr. Yasushi Shimoda at Nagaoka University of Technology and Dr. Gopal Thinakaran at The University of Chicago. Neurexin cDNA clones were obtained from Dr. Thomas C. Sildhof at Stanford University and Dr. Peter Scheiffele at Basel University. The rest cDNA plasmids were obtained from Addgene. Deletion mutants. LAG3 deletion mutants were constructed by PCR using herculase polymerase (Agilent Technologies) and primers flanking the sequences to be deleted. The DNA was separated on a 1% agarose gel and the appropriate band was isolated using a gel extraction kit (Qiagen). 100 ng of DNA was phosphorylated at the 5′ end using T4 polynucleotide kinase (Invitrogen) for 30 mins at 37° C. and ligated overnight at room temperature using T4 DNA ligase (Invitrogen). Reactions were purified with a PCR purification kit (Qiagen) and transformed into competent Stbl3 cells (Invitrogen).
Live images and confocal microscopy. α-Syn PFF was labeled with pHrodo red (Invitrogen). pHrodo red is weakly fluorescent at neutral pH but increasingly fluorescent as the pH drops. α-syn-pHrodo PFF was directly added to LAG3 wild type (WT) and knockout (KO) neuron groups. For the WT+LAG3 and KO+LAG3 groups, neurons were transfected with LAG3 expression vector 2 days prior to the addition of α-syn-pHrodo PFF. Live images were observed every minute for 20 minutes using confocal microscopy. To confirm the endocytosis of α-syn PFF mediated by LAG3, neurons were fixed 2 hour after α-syn-biotin PFF treatment using 4% paraformaldehyde in PBS.
Microsome enrichment. α-syn-biotin PFF was administrated into the neuron (12 DIV) cultures and incubated for 1.5 h. To clear up the bound α-syn-biotin PFF, trypsin was added for 30 seconds and follows with 3 times medium wash. The neurons were harvested with PBS and prepared with the lysis buffer (250 mM sucrose, 50 mM Tris-C1 (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA) with inhibitor cocktail. The suspended cell lysates were pipetted for 6 times and syringed for 20 times. The microsomes were harvest in the third pellet following by three steps of centrifuges 1st (1000 g, 10 min), 2nd (16,000 g, 20 min) 3rd (100,000 g, 1 h) for immunoblot directly.
Biochemical analysis. Dissected brain regions of interest or culture samples were prepared with RIPA buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton-100, and 2% SDS) containing protease and phosphatase inhibitor (Roche). Samples were sonicated and centrifuged with 20,000 g for 20 min. Protein concentrations were determined using the BCA assay (Pierce) and samples (10 g total proteins) were separated on SDS-polyacrylamide gels (13.5%) and transferred onto nitrocellulose membranes. Blots were blocked in 5% non-fat milk or 7.5% BSA in TBST and probed using various primary antibodies. Target antigens were detected using ImageQuant LAS 4000mini scanner or film following incubation with the appropriate infrared secondary antibodies.
In vivo co-immunoprecipitation. Transgenic mice overexpressing human A53T α-synuclein, as previously characterized (9), and wild type littermate controls were sacrificed at 4 months and 8 months of age. The brainstem was removed and lysates prepared with brain lysis buffer containing 50 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, and protease inhibitors (Roche). Samples were frozen and thawed three times, followed by centrifugation at 14,000 rpm for 20 min. Protein concentration of the supernatants were determined using the BCA assay (Pierce). Aliquots of the samples containing 500 μg of protein were pre-cleared with 10 μL of Dynabeads® Protein G (Life Technologies) for one hour. Simultaneously, 50 μL of Dynabeads® were incubated for one hour with 4 μL of either rabbit α-synuclein antibody (Cell Signaling) or rabbit Igg (Santa Cruz). Pre-cleared samples were incubated with Dynabead®-antibody/Igg overnight at 4° C. The immunocomplexes were washed five times with IP buffer and then denatured by adding 2× Laemlli Buffer plus β-mercaptoethanol, followed by boiling for five minutes.
Microfluidic chambers. Triple compartments microfluidic devices were obtained from Xona Microfluidic (TCND 1000). Glass coverslips were prepared and coated as described before being affixed to microfluidic devices (6). Approximately 100,000 neurons were plated per chamber. At 4 DIV, WT+LAG3 and KO+LAG3 groups were transduced with lenti-virus LAG3. At 7 DIV, 0.5-μg α-syn PFF was added into chamber 1. To control for direction of flow, a 75-μL difference in media volume was maintained between Chamber 1 and Chamber 2, Chamber 2 and Chamber 3 according to the manufacturers' instructions. Neurons were fixed 14 days after α-syn PFF treatment using 4% paraformaldehyde in PBS. The devices were then ready to be used for immunofluorescence staining.
Mouse strains. C57BL6 and CD1 mice were obtained from the Jackson Laboratories (Bar Harbor, Me.). LAG3 knockout mice were kindly obtained from Dr. Drake in Johns Hopkins University and were maintained on a C57BL6 background. All housing, breeding, and procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the Johns Hopkins University Animal Care and Use Committee.
Injection material and stereotaxic injections. Purification of recombinant of α-syn proteins and in vitro fibril assembly was performed as published (8). Assembly reactions were agitated in a transparent glass vial with a magnetic stirrer (350 rpm at 37° C.) and PFF harvested after 7 days. Preparations were diluted in sterile PBS and sonicated briefly with a hand held probe before intracerebral injection. Mice between 2 and 3 months of age were anesthetized with pentobarbital (diluted 1:4 in sterile saline, 250 μL for 25 mg mice) and stereotactically injected in one hemisphere with recombinant α-syn PFF (5 μg). Control animals received sterile PBS. A single needle insertion (coordinates: +0.2 mm relative to Bregma, +2.0 mm from midline) into the right forebrain was used to target the inoculum to the dorsal neostriatum (+2.8 mm beneath the dura). Injections were performed using a 2 μL syringe (Hamilton, Nev.) at a rate of 0.1 μL per min (2.5 μL total per site) with the needle in place for ≥5 min at each target. Animals were monitored regularly following recovery from surgery, and sacrificed at various pre-determined time points (30 or 180 dpi) by overdose with pentobarbital. For histological studies the brains were removed after transcardial perfusion with PBS and 4% PFA, and underwent overnight fixation in 4% PFA, cryoprotected in 30% sucrose. For biochemical studies, tissues were immediately frozen after removal and stored at −80° C. until used.
Behavioral Analysis. To evaluate the effects of transmissible α-syn pathology on motor skills, mice were tested with three behavioral tests 1-week prior to sacrifice. The order of tests was randomized and an experimenter blinded to treatment group conducted all tests. All tests were conducted between 10:00-16:00 in the lights-on cycle. Mice were habituated to the testing room 1 day before tests, and the apparatus were cleaned with 70% ethanol in between animals to minimize odor cues.
Table S2. cDNA Library Data not Shown.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims the benefit of U.S. Provisional Patent Application Nos. 62/378,436, filed on Aug. 23, 2016, and 62/401,315 filed on Sep. 29, 2016 both of which are hereby incorporated by reference for all purposes as if fully set forth herein.
This invention was made with government support under grant no. NS038377 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/047878 | 8/22/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62378436 | Aug 2016 | US | |
| 62401315 | Sep 2016 | US |
