Patent Application
20230257480
The present invention relates to methods and compositions for increasing blood-brain barrier permeability in a subject to enhance blood-brain barrier drug transport and methods and composition for identifying agents that regulate blood-brain barrier permeability.
The blood-brain barrier (BBB) maintains the necessary environment for proper brain function, which is thought to occur via the BBB's impermeability to circulating macromolecules. Vascular endothelial cells of the BBB are among the most specialized in the body. With support from abluminal mural cells, the BBB endothelium exhibits unique properties believed to be critical for optimal central nervous system (CNS) function, such as the display of tight junctions and minimal vesicular trafficking (Obermeier et al., Nature Medicine (2013) doi:10.1038/nm.3407; Ballabh et al., Neurobiol. Dis. (2004) doi:10.1016/j.nbd.2003.12.016; Chow, B. W. & Gu, C., Trends Neurosci. 38, 598-608 (2015); and Blood, T., Barrier, B., Daneman, R. & Prat, A. The Blood-Brain Barrier, 1-23 (2015)). This impermeability throughout healthy life has been reported with various exogenous tracers (Reese, T. S. & Karnovsky, M. J Cell Biol. (1967) doi:10.1083/jcb.34.1.207; Andreone et al., Neuron, 94(3): 581-594 (2017); Park et al., Neuron, 100(1):167-182.e9 (2018); Yao et al., Nat. Commun., 5: 3413 (2014); Janzer, R. C. & Raff, M. C., Nature, 325(6101): 253-257 (1987); and Saunders et al., Frontiers in Neuroscience, 9: 385 (2015). Several studies have found that the circulatory environment can influence brain function, and specifically regulate neurogenesis, synaptic plasticity, and cognitive performance (Conboy et al., Nature, 433: 760-764 (2005); Villeda et al., Nat. Med. (2014). doi:10.1038/nm.3569; Tingle et al., Nature (2017). doi:doi:10.1038/nature22067; Wyss-Coray, T., Nature, 539: 180-186 (2016); Katsimpardi, L. Science (80-.). (2014). doi:10.1126/science.1251141; Karnavas et al., J. Exp. Med. (2017). doi:10.1084/jem.20171320; and Gan, K. J. & Südhof, T. C., Proc. Natl. Acad. Sci., 16(25): 12524-12533 (2019). The canonical impermeability of the BBB to exogenous tracers has stimulated exploration regarding the mechanisms of such influence by the periphery on the CNS. At the same time, with each heartbeat, the BBB is endogenously met by a new wave of diverse blood plasma proteins, likely forming a dynamic interface teeming with binding, signaling, and trafficking events.
There remains a need for methods to identify regulators of blood-brain barrier permeability, particularly as regards the aging brain and delivery of therapeutics to treat central nervous system diseases.
The disclosure provides methods and compositions for increasing blood-brain barrier permeability in a subject. The disclosure further provides methods and compositions for identifying agents that regulate blood-brain barrier permeability.
In some embodiments, the disclosure provides methods and compositions for increasing blood-brain barrier permeability in a subject by administering to the subject an agent that inhibits the activity of alkaline phosphatase protein (ALPL).
The disclosure also provides method for delivering a therapeutic agent to the brain of a subject, which comprises administering to the subject: (a) an agent which inhibits the activity of alkaline phosphatase protein (ALPL); and (b) the therapeutic agent.
The disclosure further provides a method for identifying a protein or other biomolecule that regulates blood-brain barrier permeability in a mammal, which method comprises one or more or each of the steps of: (a) detectably labeling the proteome of blood plasma isolated from a first mammal; (b) introducing the isolated blood plasma comprising the labeled proteome into a second mammal; (c) isolating brain endothelial cells from the second mammal that form the blood-brain barrier; (d) measuring plasma uptake in each of the isolated brain endothelial cells using flow cytometry to produce a population of sorted brain endothelial cells; (e) detecting expression of genes that correlate with plasma uptake in each of the sorted brain endothelial cells; and (f) selecting a protein or other biomolecule encoded by a gene whose expression correlates with increased or decreased plasma uptake in a brain endothelial cell, whereby the protein or other biomolecule regulates blood-brain barrier permeability in the subject.
The present disclosure is predicated, at least in part, on the development of a screening method to identify proteins and other biomolecules that regulate permeability of the blood-brain barrier. Using the whole blood plasma proteome as a novel discovery tool, as described herein, endogenous proteins and other biomolecules can be directly visualized readily permeating the BBB-protected, healthy adult brain parenchyma. This process is highly regulated by transcriptional programs unique to the brain endothelium, and uptake varies significantly by vessel segment. With age, much of the active regulators of plasma uptake are downregulated, resulting in a shift from receptor-mediated to nonspecific caveolar transcytosis in the BBB, as shown schematically in
To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.
As used herein, the terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably and refer to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The terms encompass any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogenous or homogenous in composition, may be isolated from naturally occurring sources, or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. The terms “nucleic acid” and “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”).
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids comprising at least two or more contiguous amino acids, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely alleviates or cures an injury, disease, and/or an adverse symptom attributable to the injury or disease. Similarly, a “therapeutic agent,” is any substance, molecule, or compound that is capable of alleviating or curing an injury, disease, and/or adverse symptom when administered to a subject in need thereof. To this end, the methods described herein desirably comprise administering a “therapeutically effective amount” of a therapeutic agent. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the injury severity, age, sex, and weight of the individual, and the ability of therapeutic agent to elicit a desired response in the individual.
The terms “biomolecule” and “biological molecule,” as used herein, refer to any molecule or compound produced by a living organism or cell. Biomolecules typically are organic, and include, for example, large macromolecules (or polyanions) such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products.
The term “macromolecule,” as used herein, refers to a large polymeric molecule generated by the polymerization of two or more monomers. Macromolecules typically are comprised of thousands of atoms or more. Examples of macromolecules include biopolymers (e.g., nucleic acids, proteins, and carbohydrates) and large non-polymeric molecules (e.g., lipids and macrocycles). Synthetic macromolecules include, for example, common plastics, synthetic fibers, and carbon nanotubes.
The term “immunoglobulin” or “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (κ) or lambda (λ), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.
The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.
The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding fragment” of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Any antigen-binding fragment of the antibody described herein is within the scope of the invention. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab′ fragment, which results from breaking the disulfide bridge of an F(ab′)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
The term “recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may act to modulate production of a desired product by various mechanisms. Alternatively, DNA sequences encoding RNA that is not translated may also be considered recombinant. Thus, the term “recombinant” nucleic acid also refers to a nucleic acid which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, the artificial combination may be performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term “recombinant” polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a “recombinant” polypeptide is the result of human intervention, but may comprise a naturally occurring amino acid sequence.
The term “small molecule,” as used herein, refers to a low molecular weight (<900 daltons) organic compound that may regulate a biological process, with a size typically on the order of 1 nm. Small molecules exhibit a variety of biological functions and may serve a variety applications, such as in cell signaling, as pharmaceuticals, and as pesticides. Examples of small molecules include, but are not limited to, amino acids, fatty acids, phenolic compounds, alkaloids, steroids, bilins, retinoids, etc.
The term “proteome,” as used herein, refers to the complete set of proteins expressed by an organism or a particular set of proteins produced at a specific time in a particular cell or tissue type. The proteome of a particular organism, cell, or tissue actively changes in response to various factors, including the developmental stage of the organism, cell, or tissue, and both internal and external conditions.
The terms “subject” and “patient” are used interchangeably herein and refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human.
The disclosure provides a method for increasing blood-brain barrier permeability in a subject, so as to enhance delivery of macromolecules (e.g., therapeutic agents) to the brain of a subject suffering from a central nervous system (CNS) disease or disorder.
The term “blood-brain barrier (BBB)” is used to describe the unique properties of the microvasculature of the central nervous system. The BBB is a tight-knit layer of endothelial cells (ECs) that coats 400 miles of capillaries and blood vessels in the brain and forms the lumen of the brain microvasculature (Ransohoff et al., Nature Rev. Immun., 3: 569-581 (2003); Abbott et al., Neurobiol. Dis., 37: 13-25 (2010)). The BBB achieves a barrier function through tight junctions between endothelial cells that regulate the extravasation of molecules and cells into and out of the central nervous system (CNS) (Abbott et al., supra). The presence of specific transport systems within the capillary endothelial cells assures that the brain receives, in a controlled manner, all of the compounds required for normal growth and function. In many instances, these transport systems consist of membrane-associated proteins, which selectively bind and transport certain molecules across the barrier membranes. These transporter proteins are known as solute carrier transporters. This heavily restricting barrier capacity allows BBB ECs to tightly regulate CNS homeostasis, which is critical to allow for proper neuronal function, as well as protect the CNS from toxins, pathogens, inflammation, injury, and disease.
The restrictive nature of the BBB provides an obstacle for drug delivery to the CNS, and major efforts have been made to modulate or bypass the BBB for delivery of therapeutics (Larsen et al., Curr Top Med Chem., 14(9): 1148-60 (2014); and Daneman R. and Prat A., Cold Spring Harb Perspect Biol, 7(1): a020412 (2015)). Indeed, it has been estimated that more than 98% of small-molecule drugs less than 500 Da in size do not cross the BBB (Pardridge, “Brain Drug Targeting: the Future of Brain Drug Development,” Cambridge University Press, Cambridge, UK (2001); Pardridge, NeuroRx, 2: 3-14 (2005); and U.S. Patent Application Publication 2013/0224110). Current strategies for CNS drug-delivery fall into three broad categories: chemical BBB disruption, physical BBB disruption, and drug modification.
Loss of some or most of the BBB barrier properties during neurological diseases including stroke, multiple sclerosis (MS), brain traumas, and neurodegenerative disorders, is a major component of the pathology and progression of these diseases (Zlokovic, B. V., Neuron, 57(2): 178-201 (2008); and Daneman R, Ann Neurol. November; 72(5):648-72 (2012)). BBB dysfunction also can lead to ion dysregulation, altered signaling homeostasis, as well as the entry of immune cells and molecules into the CNS, all of which can lead to neuronal dysfunction and degeneration.
In some embodiments, the disclosure provides a method for increasing blood-brain barrier permeability in a subject, which comprises administering to the subject an agent which inhibits the activity of alkaline phosphatase protein (ALPL) in the brain. In addition to the endothelial cells of the blood capillaries that make up the BBB, the epithelial cells of the choroid plexus (“CP”), which separate the blood from the cerebrospinal fluid (“CSF”) of the central nervous system (“CNS”), together function as the CNS barrier. Thus, in some embodiments, the method for increasing BBB permeability in a subject may increase the permeability of the BBB, the CP, and/or the CNS barrier.
Alkaline phosphatases are membrane-bound glycoproteins that hydrolyze various monophosphate esters at a high pH (Weiss et al., Proc. Nat. Acad. Sci., 83: 7182-7186 (1986)). Liver/bone/kidney alkaline phosphatase, also known as tissue-nonspecific alkaline phosphatase (TNAP), acts physiologically as a lipid-anchored phosphoethanolamine (PEA) and pyridoxal-5-prime-phosphate (PLP) ectophosphatase. Tissue-nonspecific alkaline phosphatase is encoded by the ALPL gene (Weiss et al., supra). The deduced 524-amino acid ALPL protein has a presumed signal peptide of 17 amino acids and a predicted molecular mass of 57.2 kD. The ALPL protein shares 52% sequence identity with placental alkaline phosphatase. The ALPL protein is a key effector of bone calcification and is essential for normal skeletal development, as hypomorphic mutations in ALPL lead defective mineralization in hypophosphatasia patients (Sheen et al., J Bone Miner Res., 30(5): 824-836 (2015); Murshed et al., Genes Dev., 19(9): 1093-1104 (2005); Lomashvili et al., Kidney Int., 85(6): 1351-1356 (2014); Savinov et al., J Am Heart Assoc., 4(12): e002499 (2015); Romanelli et al.; PLoS One, 12(10): e0186426 (2017); and Whyte et al., N Engl J Med., 366(10): 904-913 (2012)). As demonstrated herein (see Examples), ALPL is upregulated in the BBB of aged brains and inhibition of ALPL enhances brain uptake of plasma as well as transferrin and a transferrin receptor antibody. The nucleic acid sequence of the ALPL gene is publicly available from, for example, the National Center for Biotechnology Information's (NCBI) GenBank database under Accession No. NG_008940.1. ALPL amino acid sequences are publicly available from, e.g., the GenBank database under Accession Nos. NP_001356734.1, NP_001356733.1, and NP_001356732.1.
Inhibition of ALPL activity may increase BBB permeability by any suitable amount or degree. For example, ALPL inhibition may increase BBB permeability by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25-fold, or a range defined by any two of the foregoing values. The increase in BBB permeability may occur for any suitable duration. For example, the increase in BBB permeability may last for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 24 hours, or longer.
The phrase “inhibiting ALPL,” as used herein, refers to the ability of a substance or method to interfere with the expression and/or biological activity or function of ALPL. The degree of inhibition may be partially complete (e.g., 10% or more, 25% or more, 50% or more, or 75% or more), substantially complete (e.g., 85% or more, 90% or more, or 95% or more), or fully complete (e.g., 98% or more, or 99% or more). Inhibition of ALPL as disclosed herein may involve interfering with or inhibiting the biological activity of ALPL. ALPL biological activity may be inhibited using any suitable agent. In one embodiment, for example, inhibiting ALPL comprises contacting the subject with an agent that inhibits activity of the ALPL protein. Any suitable agent that inhibits alkaline phosphatases may be used in the disclosed method. Such agents include, but are not limited to, phosphate derivatives, phosphonates, vanadate, arsenate, and homoarginine (Fernley, H. N. and Walker, P. G., Biochem. J., 104(3): 1011-1018 (1967); Shirazi et al., Biochem J., 194(3): 803-809 (1981)) or other small molecules that inhibit enzyme activity. Small molecules that inhibit tissue-nonspecific alkaline phosphatase (i.e., the ALPL protein) include, but are not limited to, aryl sulfonamides, such as those described in, e.g., Dahl et al., J. Med. Chem., 52(21): 6919-6925 (2009) and WO 2013/126608, each of which is herein incorporated by reference in its entirety. Other inhibitors include, for example, small molecules containing pyrazole, triazole, or imidazole scaffolds (see, e.g., Chung et al., Molecules, 15.5: 3010-3037 (2010)), small molecules that interfere with ALPL dimerization, and small molecules linked to degrons to induce protein degradation. ALPL inhibitors also include antibodies directed against ALPL (e.g., monoclonal or polyclonal antibodies, or antigen-binding fragments thereof, raised in any suitable animal species).
In some embodiments, a combination of two or more agents that inhibit ALPL may be administered to a subject. For example, any of the small molecule inhibitors described herein may be administered simultaneously or sequentially with other protein ligands that have receptors or transporters on the blood brain barrier (e.g., leptin or recombinant antibodies). Anti-ALPL antibodies, which are commercially available from a variety of sources, may also be administered simultaneously or sequentially with other protein ligands that have receptors or transporters on the blood brain barrier.
In other embodiments, inhibiting ALPL in the subject comprises inhibiting expression of ALPL. Inhibition of ALPL expression can be at the mRNA or protein level and can result from decreased synthesis, increased degradation, or both. In certain embodiments, the method comprises inhibiting expression of the gene encoding ALPL. ALPL gene expression may be inhibited using any suitable agent and/or method known in the art. For example, ALPL gene expression may be inhibited using an inhibitory nucleic acid molecule, including, for example, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), an antisense oligonucleotide, an aptamer, or a ribozyme. Alternatively, ALPL gene expression may be inhibited by introducing one or more mutations (e.g., insertion or deletion of nucleic acids or point mutation) into the ALPL gene which impairs or abolishes transcription. In some embodiments, all or part of the ALPL gene is deleted. Any number of nucleic acids may be deleted from the ALPL gene, so long as the deletion is sufficient to obliterate or impair gene transcription or gene function. Desirably, the entire ALPL gene is removed or deleted in suitable cells (e.g., brain endothelial cells). Any suitable “knock-out” technology for inactivating genes may be used to inhibit ALPL gene expression, a variety of which are known in the art. Such methods include, but are not limited to, homologous recombination, site-specific nucleases (e.g., CRISPR/Cas9 systems, zinc-finger nucleases), and conditional knock-out systems (e.g., Cre/lox technology). Gene editing technology is further described in, e.g., Appasani, K. (ed.), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery, 1st ed., Cambridge University Press (2018)).
The disclosure also provides a method for delivering a therapeutic agent to the brain of a subject in need thereof, which comprises administering to the subject: (a) an agent which inhibits the activity of alkaline phosphatase protein (ALPL) in the brain; and (b) the therapeutic agent. The therapeutic agent may be administered to the subject simultaneously with the agent that inhibits ALPL activity in the brain. Alternatively, the therapeutic agent may be administered to the subject before or after administration of the agent that inhibits ALPL activity in the brain. When the therapeutic agent and the agent that inhibits ALPL activity are administered separately, administration of each agent may be separated by up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
Any suitable therapeutic agent may be administered to the subject. Ideally, the therapeutic agent is indicated for the treatment of a disease or disorder of the central nervous system (CNS). In some embodiments, the therapeutic agent is a macromolecule. The macromolecule may be a biopolymer, such as a nucleic acid sequence, a protein or polypeptide, or a carbohydrate. A macromolecular therapeutic agent may be of any suitable size. For example, a macromolecular therapeutic agent may be about 150 kDa to about 70,000 kDa in size (e.g., about 200 kDa, about 500 kDa, about 1,000 kDa, about 2,000 kDa, about 5,000 kDa, about 10,000 kDa, about 15,000 kDa, about 20,000 kDA, about 30,000 kDa, about 40,000 kDa, about 50,000 kDa, or about 60,000 kDa in size, or a range defined by any two of the foregoing values).
In certain embodiments, the macromolecular therapeutic agent may be a bioactive protein or peptide. Examples of such proteins include antibodies, enzymes, steroids, growth hormone and growth hormone-releasing hormone, gonadotropin-releasing hormone and its agonist and antagonist analogues, somatostatin and its analogues, gonadotropins, peptide T, thyrocalcitonin, parathyroid hormone, glucagon, vasopressin, oxytocin, angiotensin I and II, bradykinin, kallidin, adrenocorticotropic hormone, thyroid stimulating hormone, insulin, glucagon, and analogs or derivatives of any of the foregoing molecules. The protein or peptide can be a synthetic or a naturally occurring peptide, including a variant or derivative of a naturally occurring peptide. A peptide therapeutic agent can be a linear peptide, cyclic peptide, constrained peptide, or a peptidomimetic.
In some embodiments, a protein or peptide therapeutic agent may specifically bind to a target protein or structure associated with a neurological condition. Thus, in accordance with the present disclosure, a protein or peptide therapeutic agent may be useful for the selective targeting of a target protein or structure associated with a neurological condition (e.g., for diagnosis or therapy) (see, e.g., U.S. Patent Application Publication 2009/0238754). Protein or peptide therapeutic agents that specifically bind to a target protein or structure associated with neurological conditions, include, but are not limited to, Aβ-peptide in amyloid plaques of Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and cerebral vascular disease (CVD); α-synuclein deposits in Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in frontal temporal dementia and Pick's disease; superoxide dismutase in amylotrophic lateral sclerosis; and Huntingtin in Huntington's disease and benign and cancerous brain tumors such as glioblastoma's, pituitary tumors, or meningiomas.
In other embodiments, the macromolecular therapeutic agent may be an antibody, such as a monoclonal or polyclonal antibody (as described above). The antibody may specifically bind to a target protein or structure associated with a neurological condition, such as a target protein or structure (such as a specific conformation or state of self-aggregation) associated with an amyloidogenic disease. Such antibodies include, for example, the anti-amyloid antibodies 6E10 and NG8 (see, e.g., Hunter S., Brayne C., J Negat Results Biomed., 16(1): 1 (2017)). Other anti-amyloid antibodies are known in the art, as are antibodies that specifically bind to proteins or structures associated with other neurological conditions, any of which may be used in the methods disclosed herein.
In certain embodiments, the macromolecular therapeutic agent is a monoclonal antibody. Suitable monoclonal antibodies include, but are not limited to, 6E10, PF-04360365, 131I-chTNT-1/B MAb, 131I-L19SIP, 177Lu-J591, ABT-874, AIN457, alemtuzumab, anti-PDGFR alpha monoclonal antibody IMC-3G3, astatine At 211 monoclonal antibody 8106, bapineuzumab, bevacizumab, cetuximab, cixutumumab, daclizumab, Hu MiK-beta-1, HuMax-EGFr, iodine I 131 monoclonal antibody 3F8, iodine I 131 monoclonal antibody 8106, iodine I 131 monoclonal antibody 8H9, iodine I 131 monoclonal antibody TNT-1/B, LMB-7 immunotoxin, MAb-425, MGAWN1, Me1-14 F(ab′)2, M-T412, natalizumab, neuradiab, nimotuzumab, ofatumumab, panitumumab, ramucirumab, ranibizumab, SDZ MSL-109, solanezumab, trastuzumab, ustekinumab, zalutumumab, tanezumab, aflibercept, MEDI-578, REGN475, muromonab-CD3, abiximab, rituximab, basiliximab, palivizumab, infliximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, tositumomab-I131, efalizumab, abciximab, certolizumab pegol, eculizumab, AMG-162, zanolimumab, MDX-010, antiOMRSA mAb, pexelizumab, mepolizumab, epratuzumab, anti-RSV mAb, afelimomab, catumaxomab, WX-G250, or combinations thereof. Therapeutic antibodies for the treatment of brain disorders are further described in, e.g., Freskgård P. O., Urich E., Neuropharmacology, 120: 38-55 (2017); and Yu Y. J., and Watts R. J., Neurotherapeutics, 10(3): 459-72 (2013).
In other embodiments, the macromolecular therapeutic agent may be a neurotrophic protein. Suitable neurotrophic proteins include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), insulin-like growth factors (IGF-I and IGF-II), glial cell line derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), glia-derived nexin (GDN), transforming growth factor (TGF-α and TGF-β), interleukin, platelet-derived growth factor (PDGF), and S100β protein, and analogs and derivatives thereof.
In other embodiments, the macromolecular therapeutic agent may be a protein associated with membranes of synaptic vesicles, such as calcium-binding proteins and other synaptic vesicle proteins. Calcium-binding proteins include, for example, cytoskeleton-associated proteins such as caldesmon, annexins, calelectrin (mammalian), calelectrin (torpedo), calpactin I, calpactin complex, calpactin II, endonexin I, endonexin II, protein II, synexin I, and enzyme modulators, such as p65. Other synaptic vesicle proteins include inhibitors of mobilization (such as synapsin Ia,b and synapsin IIa,b), synaptophysin, and proteins of unknown function such as p29, VAMP-1,2 (synaptobrevin), VAT1, rab 3A, and rab 3B.
Macromolecular therapeutic agents also include α-, β- and γ-interferon, epoetin, fligrastim, sargramostin, CSF-GM, human-IL, TNF, and other biotechnology drugs. The macromolecular therapeutic agent may also be a peptide, protein, or antibody obtained using recombinant biotechnology methods.
In other embodiments, the therapeutic agent may be a small molecule drug. Any suitable small molecule drug may be administered to the subject. Ideally, the small molecule drug is an agent that is capable of treating a disease or disorder of the central nervous system. Suitable small molecule drugs for treating a disease, disorder, or condition of the CNS include, but are not limited to, acetaminophen, acetylsalicylic acid, acyltransferase, alprazolam, amantadine, amisulpride, amitriptyline, amphetamine-dextroamphetamine, amsacrine, antipsychotics, antivirals, apomorphine, arimoclomol, aripiprazole, asenapine, aspartoacyclase enzyme, atomoxetine, atypical antipsychotics, azathioprine, baclofen, beclamide, benserazide, benserazide-levodopa, benzodiazepines, benztropine, bleomycin, brivaracetam, bromocriptine, buprenorphine, bupropion, cabergoline, carbamazepine, carbatrol, carbidopa, carbidopa-levodopa, carboplatin, chlorambucil, chlorpromazine, chlorprothixene, cisplatin, citalopram, clobazam, clomipramine, clonazepam, clozapine, codeine, COX-2 inhibitors, cyclophosphamide, dactinomycin, dexmethylphenidate, dextroamphetaine, diamorphine, diastat, diazepam, diclofenac, donepezil, doxorubicin, droperidol, entacapone, epirubicin, escitalopram, ethosuximide, etoposide, felbamate, fluoxetine, flupenthixol, fluphenazine, fosphenytoin, gabapentin, galantamine, gamma hydroxybutyrate, gefitinib, haloperidol, hydantoins, hydrocordone, hydroxyzine, ibuprofen, ifosfamide, IGF-1, iloperidone, imatinib, imipramine, interferons, irinotecan, KNS-760704, lacosamide, lamotrigine, levetiracetam, levodopa, levomepromazine, lisdexamfetamine, lisuride, lithium carbonate, lypolytic enzyme, mechlorethamine, mGluR2 agonists, memantine, meperidine, mercaptopurine, mesoridazine, mesuximide, methamphetamine, methylphenidate, minocycline, modafinil, morphine, N-acetylcysteine, naproxen, nelfinavir, neurotrin, nitrazepam, NSAIDs, olanzapine, opiates, oseltamivir, oxaplatin, paliperidone, pantothenate kinase 2, Parkin, paroxetine, pergolide, periciazine, perphenazine, phenacemide, phenelzine, phenobarbitol, phenturide, phenyloin, pimozide, Pinkl, piribedil, podophyllotoxin, pramipexole, pregabalin, primidone, prochlorperazine, promazine, promethazine, protriptyline, pyrimidinediones, quetiapine, rasagiline, remacemide, riluzole, risperidone, ritonavir, rivastigmine, ropinirole, rotigotine, rufinamide, selective serotonin reuptake inhibitors (SSRIs), selegine, selegiline, sertindole, sertraline, sodium valproate, stiripentol, taxanes, temazepam, temozolomide, tenofovir, tetrabenazine, thiamine, thioridazine, thiothixene, tiagabine, tolcapone, topiramate, topotecan, tramadol, tranylcypromine, tricyclic antidepressants, trifluoperazine, triflupromazine, trihexyphenidyl, trileptal, valaciclovir, valnoctamide, valproamide, valproic acid, venlafaxine, vesicular stomatitis virus, vigabatrin, vinca alkaloids, zanamivir, ziprasidone, zonisamide, zotepine, zuclopenthixol, and analogs or derivatives of any of the foregoing.
Other therapeutic agents or compounds that may be administered according to the present invention may be of any class of drug or pharmaceutical agent for which crossing the BBB is desired. Such therapeutics include, but are not limited to, antibiotics, anti-parasitic agents, antifungal agents, anti-viral agents, and anti-tumor agents.
A CNS disease, disorder, or condition is any disease, disorder, or condition that affects the brain and/or spinal cord (collectively known as the central nervous system (CNS)). CNS disease, disorder, or conditions that may be treated in accordance with the inventive methods include, but are not limited to, addiction, arachnoid cysts, autism, catalepsy, encephalitis, locked-in syndrome, meningitis, multiple sclerosis (MS), myelopathy, metabolic disease, a behavioral disorder, a personality disorder, dementia, a cancer, a neurodegenerative disorder (e.g., Alzheimer's disease, Huntington's disease, and Parkinson's disease), stroke, pain, a viral infection, a sleep disorder, epilepsy/seizure disorders, acid lipase disease, Fabry disease, Wernicke-Korsakoff syndrome, attention deficit/hyperactivity disorder (ADHD), anxiety disorder, borderline personality disorder, bipolar disorder, depression, eating disorder, obsessive-compulsive disorder, schizophrenia, Barth syndrome, Tourette's syndrome, Canavan disease, Hallervorden-Spatz disease, Lewy Body disease, Lou Gehrig's disease, Machado-Joseph disease, restless Leg syndrome, traumatic brain injury (TBI), and autoimmune disease.
The therapeutic agent and the agent that inhibits the activity of ALPL may be formulated together as a single composition. Alternatively, the therapeutic agent and the agent that inhibits the activity of ALPL may be formulated as separate compositions which may be administered simultaneously or sequentially as described herein. In either case, the composition desirably is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the therapeutic agent and/or the ALPL inhibitor. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The carrier typically will be liquid, but also can be solid, or a combination of liquid and solid components. The choice of carrier will be determined, at least in part, by the location of the target tissue and/or cells, and the particular method used to administer the composition.
The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and/or its end use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention. The following formulations and methods are merely exemplary and are in no way limiting.
Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit dose or multi dose sealed containers, such as ampules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. For example, the composition may contain preservatives, such as, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives optionally may be used. In addition, buffering agents may be included in the composition. Suitable buffering agents include, for example, glutamic acid (glutamate), citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents optionally may be used.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The composition may be formulated for oral administration. Suitable oral formulations include, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The composition may be formulated for parenteral administration, e.g., intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, etc. The compounds or agents of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the compounds of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The composition also may be administered in a non-pressurized form such as in a nebulizer or atomizer. Methods for preparing compositions for pharmaceutical use are known to those skilled in the art and are described in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The disclosure also provides a method for identifying a protein or other biomolecule that regulates blood-brain barrier permeability in a mammal (e.g., a mouse, rat, primate, or human). In some embodiments, the method comprises: (a) detectably labeling the proteome or other expressed biomolecules (RNAs) of blood plasma isolated from a first mammal; (b) introducing the isolated blood plasma comprising the labeled proteome or other expressed biomolecules into a second mammal; (c) isolating brain endothelial cells from the second mammal that form the blood-brain barrier; (d) measuring plasma uptake in each of the isolated brain endothelial cells using flow cytometry to produce a population of sorted brain endothelial cells; (e) detecting expression of genes that correlate with plasma uptake in each of the sorted brain endothelial cells; and (f) selecting a protein or other biomolecule encoded by a gene whose expression correlates with increased or decreased plasma uptake in a brain endothelial cell, whereby the protein or other biomolecule regulates blood-brain barrier permeability in the subject. In some embodiments, such identified molecules are utilized for research or clinical purposes to regulate blood-brain barrier permeability. For example, the expression or activity of such molecules is manipulated, as described above for ALPL, to effectuate the desired result.
Blood collection and plasma separation/isolation from whole blood may be accomplished using any desired method. It will be appreciated that serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a pipette. Plasma is produced when whole blood is collected in tubes that are treated with an anticoagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma, is carefully removed from the cell pellet using a pipette. Systems and methods for blood collection and plasma separation are commercially available from a variety of sources, any of which may be used in the methods described herein.
Methods for detectably labeling the proteome or other biomolecules of blood plasma isolated from a mammal are known in the art and can be used in connection with the methods described herein. For example, chemical labeling with isobaric tandem mass tags, such as isobaric tags for relative and absolute quantification reagents (iTRAQ) and tandem mass tag (TMT) reagents, has been employed in a wide range of different clinically orientated serum and plasma proteomics studies (see, e.g., Moulder et al., Mass Spectrometry Reviews, 37(5): 583-606 (2018)). Fluorescent labeling methods also may be used, such as those described in, e.g., Liu et al., Proteomics, 12(14): 2258-70 (2012); Leclerc et al., Bioconjugate Chem., 29(8): 2541-2549 (2018); Volke, D. and R. Hoffmann, Electrophoresis, 29(22): 4516-4526 (2008); and Obermaier et al., Methods Mol Biol., 1295: 153-65 (2015), herein incorporated by reference in their entireties. In certain embodiments, the plasma proteome is detectably labeled using fluorescent labeling methods.
Once the proteome of the plasma isolated from the first mammal has been detectably labeled, the isolated blood plasma comprising the labeled proteome is then introduced into a second mammal to allow for visualization and measurement of BBB permeability. The second mammal and first mammal desirably are the same type of mammal (e.g., both mice, rats, or non-human primates). The isolated blood plasma comprising the labeled proteome is introduced into the second mammal under conditions that allow for uptake of the plasma comprising the labeled proteome into endothelial cells of the BBB of the second mammal. Following a period of time sufficient for uptake of the plasma comprising the labeled proteome by the BBB of the second mammal, brain endothelial cells that form the blood-brain barrier may be isolated from the second mammal using any suitable method known in the art. Brain endothelial cell isolation protocols are described in, e.g., Assmann et al., Bio Protoc., 7(10): e2294 (2017); Welser-Alves et al., Methods Mol Biol., 1135: 345-56 (2014); Luo et al., Methods Mol Biol., 1135: 357-64 (2014); and Navone et al., Nature Protocols, 8: 1680-1693 (2013), herein incorporated by reference in their entireties.
Plasma uptake by brain endothelial cells of the BBB may be measured using in vitro or in vivo methods that are used in the art for measuring drug transport across the BBB. In vivo methods include, for example, intravenous injection/brain sampling, brain perfusion, quantitative autoradiography, microdialysis, and CSF sampling. In vitro methods include, for example, analysis of fresh isolated brain microvessels and endothelial cell culture. Methods for measuring drug and protein transport across the BBB are described in further detail in, e.g., Bickel, U., NeuroRx, 2(1): 15-26 (2005); and Feng, M. R., Curr Drug Metab., 3(6): 647-57 (2002). In some embodiments, plasma uptake in each of the isolated brain endothelial cells may be measured using flow cytometry to produce a population of sorted brain endothelial cells. Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters (e.g., from 1 up to 30 or more parameters). Visible light scatter is measured in two different directions: (1) the forward direction (Forward Scatter or FSC) which can indicate the relative size of the cell and (2) at 90° (Side Scatter or SSC) which indicates the internal complexity or granularity of the cell. Light scatter is independent of fluorescence. Samples are prepared for fluorescence measurement through transfection and expression of fluorescent proteins (e.g., green fluorescent protein, GFP), staining with fluorescent dyes (e.g., propidium iodide, DNA) or staining with fluorescently conjugated antibodies (e.g., CD3 FITC) (see, e.g., McKinnon, K. M., Curr Protoc Immunol., 120: 5.1.1-5.1.11 (2018)). Flow cytometry methods for analysis of the blood-brain barrier in particular are described in, e.g., Williams et al., Cytometry A, 87(10): 897-907 (2015), herein incorporated by reference in its entirety.
Performing flow cytometry on the isolated brain endothelial cells results in a population of flow cytometry-sorted (also referred to as “sorted”) brain endothelial cells, which are then analyzed to detect expression of genes that correlate with plasma uptake in each of the sorted brain endothelial cells. In this regard, brain endothelial cells may be sorted, and fluorescence recorded for each sorted cell. mRNA expression in each sorted cell may then be detected and quantified using any suitable method known in the art for measuring gene expression. Such methods include, but are not limited to, quantitative or real-time RT-PCR (qRT-PCR), microarray analysis, RNA sequencing, in situ hybridization, or Northern blot. In certain embodiments, RNA sequencing (also referred to as “RNA-Seq”) is used to detect expression of genes that correlate with plasma uptake. RNA-Seq uses next-generation sequencing (NGS) to detect and quantify RNA in a biological sample at a particular moment, allowing for the analysis of the continuously changing cellular transcriptome (Chu, Y. and Corey D. R., Nucleic Acid Therapeutics, 22 (4): 271-274 (2012); and Wang et al., Nature Reviews Genetics, 10(1): 57-63 (2009)). RNA-Seq facilitates the ability to examine alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/single nucleotide polymorphisms (SNPs) and changes in gene expression over time (Maher et al., Nature, 458(7234): 97-101 (2009)). In addition to mRNA transcripts, RNA-Seq can be used to analyze different populations of RNA such as small RNA, miRNA, tRNA, and ribosomes. RNA-Seq methods and techniques are described in, e.g., Maekawa et al., Methods Mol Biol., 1164: 51-65 (2014), and techniques for single-cell RNA-Seq are described in, e.g., Chen et al., Front. Genet., 10: 317 (2019).
The expression of a specific gene may be correlated with increased or decreased plasma uptake of proteins or other biomolecules in a particular brain endothelial cell. In this regard, upregulation or downregulation of expression of a specific gene may directly or indirectly lead to increased plasma uptake of proteins (i.e., increased BBB permeability) or to decreased plasma uptake of proteins (i.e., decreased BBB permeability). Expression of a gene is downregulated if the expression is reduced by at least about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) as compared to a reference or control level. Expression of a gene is upregulated if the expression is increased by at least about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) as compared to a reference or control level. A protein or other biomolecule encoded by a gene whose upregulation or downregulation correlates with increased or decreased plasma uptake may be selected as a regulator of blood-brain permeability.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
The following materials and methods were used in the experiments described in the Examples.
Aged C57BL/6 mice (20-24 months old) were obtained from the National Institute on Aging rodent colony. Young male C57BL/6 mice (3 months old) were obtained from Jackson Laboratories or Charles River Laboratories. All experiments used male mice. All mice were kept on a 12-h light/dark cycle and provided ad libitum access to food and water. All animal care and procedures complied with the Animal Welfare Act and were in accordance with institutional guidelines and approved by the V.A. Palo Alto Committee on Animal Research and the institutional administrative panel of laboratory animal care at Stanford University.
Blood was collected with 250 mM EDTA (Millipore Sigma) as anticoagulant by terminal intracardial bleeding. EDTA-plasma was isolated by centrifugation at 1,000 g for 15 min at 4° C. before pooling, aliquoting, flash freezing in liquid nitrogen, and storage at −80° C. Hemolyzed plasma was discarded. Before labeling, frozen plasma was thawed on ice, gently mixed, and inspected for precipitates. Plasma protein molarity was approximated to be 750 μM. Labeling relied on amine-reactive NHS ester moieties, and labeling ratios and times were determined empirically for each specific label: for radiotracing, NHS-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Macrocyclics) was added at a 10× molar ratio; for fluorescence, NHS-Atto 647N (Millipore Sigma, ATTO-TEC) added at 1.4× molar ratio; and for characterizing the labeled plasma proteome, NHS-biotin (Thermo Fisher) and NHS-trans-Cyclooctene (Click Chemistry Tools) added at 17.5× molar ratio. NHS-Atto 647N was incubated with plasma for 1.5 h at room temperature before PBS dialysis overnight. The next day, 50 mM Tris pH 8.0 was added for 10 minutes at room temperature, and the labeled plasma washed extensively three times with Amicon Ultra-15 Centrifugal Filter Unit, 3 KDa cutoff (Millipore Sigma, 10 KDa for NHS-Atto 647N), and subsequently washed four times with Zeba Spin Desalting Columns, 7K MWCO cutoff (Thermo Fisher) in chilled PBS. Other conjugations proceeded overnight at 4° C. before washing. These steps expanded on washing that consistently yielded >99% radiochemical purity of 64Cu-labelled DOTA (below) to minimize contamination from residual free label. Throughout labeling and washing steps, plasma samples were monitored for signs of aggregation and flocculation. Plasma concentrations were measured using the Nanodrop spectrophotometer (Thermo Fisher), and approximately 10-15 mg (0.5 mg/g body weight, in line with prior studies using horseradish peroxidase5,6,126) was administered intravenously (retro-orbital) per mouse in a volume less than or equal to 150 μL.
For characterization by mass spectrometry, labeled plasma was depleted of albumin and IgG using the ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit (Millipore Sigma), enriched with tetrazine agarose overnight at 4° C. (Click Chemistry Tools), and extensively washed, as previously described127. In parallel, unlabeled plasma was processed using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3), as previously described128. Plasma was fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher) and concatenated fractions cleaned using C18-based STAGE Tips and lyophilized. Peptides were analyzed on an LTQ Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Peptides were separated by capillary reverse-phase chromatography for 180 min on a 24 cm reversed-phase column (100 μm inner diameter, packed in-house with ReproSil-Pur C18-AQ 3.0 m resin (Dr. Maisch)). A four-step linear gradient was achieved using a Dionex Ultimate 3000 LC-system (Thermo Fisher Scientific) as follows: 97% A+3% B for 15 min, 75% A+25% B for 135 min, 55% A+45% B for 15 min, and 5% A+95% B for 15 min, where buffer A is 0.1% formic acid in water and buffer B is 0.1% formic acid in acetonitrile. Full MS scans were acquired in the Orbitrap mass analyzer at a resolution of 120,000 (FWHM) and m/z scan ranges 340-1540 in a data-dependent mode. The AGC targets were 4*105 and the maximum injection time for FTMS1 were 50 ms. The most intense ions were then selected for sequencing and fragmented in the Orbitrap mass analyzer using higher-energy collisional dissociation (HCD) with a normalized collision energy of 30% and resolution of 15,000 (FWHM). Monoisotopic precursor selection was enabled and singly charged ion species and ions with no unassigned charge states were excluded from MS2 analysis. Dynamic exclusion was enabled with a repeat count of 2 and ions within ±10 ppm m/z window around ions selected for MS2 were excluded from further selection for fragmentation for 30 sec. AGC target were 5*104 and maximum injection time of 200 ms. The raw data files were processed and analyzed using MaxQuant and Perseus (Max Planck)129,130. In brief, spectra were matched to a Mus musculus database downloaded from uniprot.org, and a contaminant and decoy database. Precursor mass tolerance was set to 4.5 p.p.m., fragment ion tolerance to 10 p.p.m., with fixed modification of Cys residues (carboxyamidomethylation +57.021 Da) and variable modifications of Met residues (Ox +15.995 Da), Lys residues (acetylation +42.011 Da), Asn and Gln residues (deamidation +0.984 Da) and of N termini (carbamylation +43.006 Da). Peptide identifications were calculated with FDR<0.01, and for protein quantification, minimum ratio count was set to one, with both unique and razor peptides used for quantification.
For characterization by protein microarrays, ELISA-grade antibodies against several hundred secreted and potentially cleaved transmembrane mouse or human signaling proteins were obtained from commercial sources and printed in five replicates onto SuperEpoxy2 glass slides (Arrayit) with a robotic microarrayer (NanoPrint LM210, Arrayit), as previously describedl3. Arrays were then blocked with 3% (w/v) casein solution before incubation with biotinylated plasma samples overnight at 4° C. Following washes, arrays were incubated with Alexa Fluor 647-conjugated streptavidin secondary antibodies (Thermo Fisher) and fluorescent signal detected using a GenePix4400A scanner and GenePix Pro? software (Molecular Devices). Data processing and analysis followed previously described methods13.
Mice were euthanized with 2.5% (v/v) Avertin and transcardially perfused with at least 50 ml of chilled PBS. Perfusion was performed using a peristaltic pump, with the flow rate not exceeding 10 ml/min to approximate the physiological pressure of the mouse's circulatory system131-133. Mice with perfusate leaking out of the nostrils were not processed further for analysis. For immunohistochemistry (below), mice were subsequently perfused with 4% paraformaldehyde except when hemibrains were taken for flow cytometry (below).
Brain tissue processing, immunohistochemistry, and immunofluorescence experiments were performed as described previously12,13. Hemibrains were isolated and post-fixed in 4% (w/v) paraformaldehyde overnight at 4° C. before preservation in 30% (w/v) sucrose in PBS. Hemibrains were sectioned coronally or sagittally at a thickness of 50 μm on a freezing-sliding microtome, and sections were stored in cryoprotective medium at −20° C. Free-floating sections were blocked with appropriate serum before incubation at 4° C. with primary antibodies at the following concentrations for confocal microscopy: goat monoclonal anti-CD31 (1:100, AF3628, R&D), Fluorescein-labeled Lectin (1:200, Vector Laboratories), rabbit monoclonal anti-Aquaporin 4 (1:500, AB2218, Millipore Sigma), rat anti-CD13 (1:100, MCA2183EL, Bio-Rad), goat anti-Alpl/ALPL (1:100, AF2909, R&D), mouse anti-NeuN (1:400, MAB377, Millipore), goat anti-albumin (1:100, NB600, Novus), rabbit anti-Collagen I (1:100, ab21286, Abcam), and goat anti-Iba1 (1:500, ab5076, Abcam). Sections were washed, stained with Alexa Fluor-conjugated secondary antibodies (1:250), mounted and coverslipped with ProLong Gold (Life Technologies) before imaging on a confocal laser-scanning microscope (Zeiss LSM880). Age-related autofluorescence was quenched with 1 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5), as previously described73. National Institutes of Health ImageJ software was used to quantify the percentage of vasculature (CD31 or AQP4) covered by CD13, AQP4, or ALPL, as described previously63. All analyses were performed by a blinded observer. Alizarin red staining was performed as described previously83, with minor adaptations: sections were incubated for 1 h in 40 mM alizarin red in PBS (pH 7.4) at room temperature, and extensively washed overnight with PBS prior to mounting. Images of brain sections were acquired by conventional light microscopy to detect calcified nodules. Sections with biotinylated plasma were blocked overnight in 6% BSA at room temperature, detected with streptavidin-Alexa Fluor 647 (1:1500, Thermo Fisher) for 2 hours, and washed overnight before mounting. Sections containing L-azidohomoalanine-labeled plasma were blocked overnight in 6% BSA at room temperature, incubated in 45 mM iodoacetamide (Millipore Sigma) in 100% methanol for 1 hour, washed, detected with 1.2 μM sDIBO (Thermo Fisher Scientific) in 100% methanol, and washed overnight before mounting. Wole brain coronal and sagittal sections were processed at 100 μm, incubated in Focusclear (CellExplorer Labs), and imaged in tiles. Vascular ALPL activity was measured using the Red Alkaline Phosphatase Substrate Kit (SK-5100, Vector Laboratories) with 20-minute incubation.
Conjugation of rat IgG2a (400501, BioLegend) and albumin/IgG plasma with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was performed using metal-free buffers, as previously described13,134,135. Conjugations proceeded overnight at 4° C. with a 10× molar ratio of DOTA-NHS ester in HEPES buffer (0.1 mol 1-1, pH 8) and quenched with 50 mM Tris-HCl (Millipore Sigma). Excess DOTA-NHS was removed by Zeba Spin Desalting Columns (0.5 ml, 7K molecular weight cut-off, Thermo Fisher), and the resulting solution was buffer-exchanged into ammonium acetate buffer (0.1 M, pH 5.5) for Cu-64 labelling. DOTA-conjugate solutions were concentrated by ultrafiltration (Amicon 0.5 ml, Millipore Sigma) and kept on ice. Radiolabeling with 64Cu (half-life=12.7 h) was performed as previously describedl3. In brief, DOTA-IgG2a or DOTA-plasma (in 25 μg aliquots in 50 μl) was mixed with pH-balanced [64Cu]C12 solution (pH 4.5-5.5, University of Wisconsin, Madison) at 37° C. with gentle shaking at 400 r.p.m. After incubation (30 min for IgG2a-DOTA and 60 min for plasma-DOTA), 0.1 M EDTA (0.5 M, pH 8.0) was added to a final concentration of 0.01 M and incubated at ambient temperature for 15 min to scavenge unchelated [64Cu]Cl2. Purification was achieved by G25 Sephadex size-exclusion purification (GE Life Sciences). Radiochemical purity was determined by instant thin-layer chromatography with TEC-Control Chromatography strips (Biodex Medical Systems), developed in saline, and size exclusion liquid chromatography with a SEC 3000 column (Phenomenex).
Mice were injected intravenously with 7.7±1.5 MBq of 64Cu-labelled DOTA-IgG2a or 64Cu-labelled DOTA-plasma (radiochemical purity >99%). After 20 h, mice were placed in a dual microPET/CT scanner (Inveon, Siemens) to capture static images (10 min) for subsequent analysis with VivoQuant software (version 4.0, inviCRO), as previously described136,137. After anaesthetization, blood samples (100-200 μl) were collected by cardiac puncture immediately prior to transcardial perfusion. Tissue-related radioactivity (dose and decay-corrected to time of injection) in blood and brain was assessed following the literature13 procedure with an automated gamma counter (Hidex Oy, calibrated using aliquots of the initial administered dose as standards). Activity in cardiac blood samples at time of sacrifice was used to correct for differential in vivo stability and clearance of plasma vs. IgG. Brain tissue was embedded in optimal-cutting temperature compound (Tissue-Tek), and coronal sections (40 μm) were obtained for ex vivo autoradiography. Autoradiography was conducted using previously described methods13,138: 40 μm sections were mounted, air-dried for 10 min, and then exposed to a digital storage phosphor screen (Amersham Biosciences) for 72 hours at −20° C. The image plate was analyzed using a Typhoon 9410 Variable Mode Imager (Perkin Elmer). Slides were then Nissl stained and scanned using a Nanozoomer 2.0-RS (Hamamatsu) to enable anatomical co-localization. Images were visualized, processed, and quantified blinded with ImageJ. For quantification, at least 10 consistently-sized hippocampal and cortical areas were drawn for each mouse. Mean pixel intensity was dose and decay corrected for each mouse.
CNS cell isolation adopted previously described methods42,139,140. Briefly, cortices and hippocampi were microdissected, minced, and digested using the Neural Dissociation Kit (Miltenyi). Suspensions were filtered through a 100 μm strainer and myelin removed by centrifugation in 0.9 M sucrose. The remaining myelin-depleted cell suspension was blocked for ten minutes with Fc preblock (CD16/CD32, BD 553141) on ice and stained for 30 minutes with antibodies to distinguish brain endothelial cells (CD31+/CD45−), astrocytes (ACSA-2+), and neurons (NeuN+ or CD90.2/Thy1.2+). For analysis of plasma uptake, at least 1,000 cells of each population of interest were analyzed per sample. Antibody dilutions: rat anti-CD31-PE/CF594 (1:100, clone MEC 13.3, BD, cat. No. 563616), rat anti-CD45-PE/Cy7 (1:200, clone 30-F11, Biolegend, cat. no. 103114), rat anti ACSA-2-PE (1:200, clone IH3-18A3, Miltenyi, cat. no. 130-102-365), mouse anti-NeuN-PE (1:100, clone A60, Millipore Sigma, cat. no. FCMAB317PE), and rat anti-CD90.2-FITC (1:100, clone 30-H12, Biolegend, cat. no. 105305).
Cell lysis, first-strand synthesis and cDNA synthesis was performed using the Smart-seq-2 protocol as described previouslyl39-141 in 384-well format, with some modifications. Briefly, brain endothelial cells (CD31+/CD45−) were sorted using SH800S (Sony) sorters on the highest purity setting (‘Single cell’). Plasma-647 fluorescence was recorded for each cell and corresponding sorted well. cDNA synthesis was performed using the Smart-seq2 protocol140-142. After cDNA amplification (23 cycles), concentrations were determined via the PicoGreen quantitation assay. Cells passing quality control were selected through custom scripts and cDNA concentrations normalized to ˜0.2 ng/μL using the TPPLabtech Mosquito HTS and Mantis (Formulatrix) robotic platforms. Libraries were prepared and pooled using the Nextera XT kits (Illumina), following the manufacturer's instructions. Libraries were then sequenced on the Nextseq or Novaseq (Illumina) using 2×75 bp paired-end reads and 2×8 bp index reads with a 200-cycle kit (Illumina, 20012861). Samples were sequenced at an average of 1.5M reads per cell. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, and gene counts made using HTSEQ version 0.6.1p1. Downstream analysis was performed as previously described58, and plasma fluorescence of each cell correlated (Spearman) with gene expression and zonation59.
Mice were treated with six doses of ALPL inhibitor (613810, Millipore Sigma; 2,5-Dimethoxy-N-(quinolin-3-yl)benzenesulfonamide, Tissue-Nonspecific Alkaline Phosphatase Inhibitor, MLS-0038949; C17H16N2O4S; CAS #496014-13-2) or ‘Vehicle’ control over three days, injected intraperitoneally twice per day (8.75 mg/kg). ‘Vehicle’ treatments consisted of phosphate-buffered saline (PBS) with a matching concentration of DMSO (less than 4% v/v). After the sixth treatment, mice were injected intravenously with 150 μL of Atto 647N-labeled plasma (as above), human holo-transferrin (T4132, Millipore Sigma, 40 mg/kg), transferrin receptor antibody (BE0175, BioXcell, 20 mg/kg), or 3-kDa dextran-FITC (10 mg/kg, Thermo Fisher). Plasma, human holo-transferrin, and transferrin receptor antibody were infused 20 h before sacrifice, while 3-kDa dextran-FITC was given 2 h before sacrifice, as previously described143. After extensive perfusion, CNS cells were isolated as described above. Cell pellets were additionally incubated in Red Blood Cell Lysis Buffer (Millipore Sigma, R7757) for 2 minutes to ensure high CNS cell purity. Brain endothelial cells were distinguished by CD3 I+/CD45− staining and parenchymal cells by CD31−/CD45− staining.
For brain microvessel western blotting of TFRC and CAV1, cortical and hippocampal microvessels were isolated as previously described51,55,144. Protein lysates were prepared by incubating cell pellets on ice in 1% SDS in 50 mM HEPES with complete protease inhibitor cocktail (Thermo Fisher Scientific) and spun at 13,000×g for 10 minutes. The supernatant was collected, and protein concentration measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). An aliquot containing 10-20 ug of protein from each sample was mixed with 4× loading buffer (Thermo Fisher Scientific) and boiled for 5 minutes at 95° C. before being subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was first blocked with 5% milk and stained overnight at 4° C. with primary antibodies at the designated concentrations: rabbit anti-Caveolin-1 (1:1000, D46G3, CST), rabbit anti-Transferrin Receptor (1:1000, ab214039, Abcam), and rabbit anti Histone H3 (1:4000, ab1791, Abcam). The membrane was washed, stained with IRDye conjugated secondary antibodies (1:15,000, LI-COR) and imaged on the Odyssey CLx (LI-COR). Images were analyzed for band intensities with the ImageStudio software (LI-COR).
Brain microvessels were isolated from young (3 m.o.) and old (20 m.o.) mice as previously described51,55,144-148, after overnight fasting (˜12 h). Sixteen mice of each age were used, with microvessels from 4 mice pooled in each group (n=4 groups). Lipids were extracted in a randomized order via biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water, as previously described149. Briefly, 260 μl of methanol and 40 μl of water were added to the brain microvessels and vortexed for 20 s. A lipid internal standard mixture was spiked in each sample (Equi SPLASH LIPIDOMIX, Avanti Polar Lipids (cat #: 330731), and d17-Oleic acid, Cayman chemicals (cat #: 9000432)) to control for extraction efficiency, evaluate LC-MS performance and normalize LC-MS data. Samples were diluted with 1,000 μl of MTBE, vortexed for 10 s, sonicated for 30 s three times in a water bath, and incubated under agitation for 30 min at 4° C. After addition of 250 μl of water, the samples were vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20° C. The upper phase containing the lipids was collected and dried down under nitrogen. The dry extracts were reconstituted with 150 μl of 9:1 methanol:toluene.
Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific) as previously described150. Each sample was run twice in positive and negative ionization modes. Lipids were separated using an Accucore C18 column 2.1×150 mm, 2.6 μm (Thermo Fisher Scientific) and mobile phase solvents consisted in 10 mM ammonium acetate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 10 mM ammonium acetate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The gradient profile used was 30% B for 3 min, 30-43% B in 2 min, 43-55% B in 0.1 min, 55-65% B in 10 min, 65-85% B in 6 min, 85-100% B in 2 min and 100% for 5 min. Lipids were eluted from the column at 0.4 ml/min, the oven temperature was set at 45° C., and the injection volume was 5 μl. Autosampler temperature was set at 20° C. to prevent lipid aggregation. The Q Exactive was equipped with a HESI-II probe and operated in data dependent acquisition mode for all the samples. To maximize the number of identified lipids, the 100 most abundant peaks found in blanks were excluded from MS/MS events. External calibration was performed using an infusion of Pierce LTQ Velos ESI Positive Ion Calibration Solution or Pierce ESI Negative Ion Calibration Solution.
LC-MS peak extraction, alignment, quantification and annotation was performed using LipidSearch software version 4.2.21 (Thermo Fisher Scientific). Lipids were identified by matching the precursor ion mass to a database and the experimental MS/MS spectra to a spectral library containing theoretical fragmentation spectra. The most abundant ion for each lipid species was used for quantification i.e. [M+H]+ for LPC, LPE, PC, SM and Cer, [M−H]− for PI, PS, PA and PG, and [M+NH4]+ for CE, DAG and TAG. To reduce the risk of misidentification, MS/MS spectra from lipids of interest were validated as follows: 1) both positive and negative mode MS/MS spectra match the expected fragments, 2) the main lipid adduct forms detected in positive and negative modes are in agreement with the lipid class identified, 3) the retention time is compatible with the lipid class identified and 4) the peak shape is acceptable. The fragmentation pattern of each lipid class was experimentally validated using lipid internal standards. Data were normalized using (i) class-specific internal standards to control for extraction efficiency and (ii) median of all annotated lipids to correct for differential quantity of starting material.
SP3-prepared and STAGE tip cleaned brain microvessel peptides were resuspended in 0.1% formic acid and analyzed by online capillary nanoLC-MS/MS. Samples were separated on an inhouse made 20 cm reversed phase column (100 μm inner diameter, packed with ReproSil-Pur C18-AQ 3.0 μm resin (Dr. Maisch GmbH)) equipped with a laser-pulled nanoelectrospray emitter tip. Peptides were eluted at a flow rate of 400 nL/min using a four-step linear gradient including 2-4% buffer B in 1 min, 4-25% buffer B in 120 min and 25-40% B in 30 min, 40-98% buffer B in 2 min (buffer A: 0.2% formic acid and 5% DMSO in water; buffer B: 0.2% formic acid and 5% DMSO in acetonitrile) in a Dionex Ultimate 3000 LC-system (Thermo Fisher Scientific). Peptides were then analyzed using an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Data acquisition was executed in data dependent mode with full MS scans acquired in the Orbitrap mass analyzer with a resolution of 60000 and m/z scan range of 340-1600. The top 20 most abundant ions with intensity threshold above 500 counts and charge states 2 and above were selected for fragmentation using collision-induced dissociation (CID) with isolation window of 2 m/z, normalized collision energy of 35%, activation Q of 0.25 and activation time of 5 ms. The CID fragments were analyzed in the ion trap with rapid scan rate. Dynamic exclusion was enabled with repeat count of 1 and exclusion duration of 30 s. The AGC target was set to 1000000 and 5000 for full FTMS scans and ITMSn scans, respectively. The maximum injection time was set to 250 ms and 100 ms for full FTMS scans and ITMSn scans, respectively. The raw files were analyzed as above, but with a precursor mass tolerance set to 20 p.p.m., fragment ion tolerance set to 0.6 Da, and for protein quantification, minimum ratio count was set to two, with unique peptides used for quantification.
Following intravenous injections of 150 μL of saline or plasma (prepared as above), tracers were injected 20 h later as previously described90,139,143. Briefly, mice were injected with fixable 3-kDa dextran-FITC (10 μg/g, Thermo Fisher), 70-kDa dextran-TMR (100 μg/g, ThermoFisher), or 2-mDa dextran-FITC (100 μg/g, Thermo Fisher) dissolved in saline. After 2 h (for 3-kDa dextran-TMR) or 4 h (for 70-kDa dextran-TMR), mice were anesthetized and perfused, and cortices and hippocampi microdissected. Frozen tissue was thawed and suspended in 300 μL of a custom Lysis Buffer (200 mM Tris, 4% CHAPS, 1M NaCl, 8M Urea, pH 8.0). Tissues were then homogenized using a Branson Digital Sonifier sonicator set to 20% amplitude for 3 seconds, allowed to rest for 30 secs on ice, and repeated 3 times. Samples were centrifuged at 14,000 g for 20 minutes at 4° C., and supernatant was extracted for analysis. Fluorescence for FITC and Texas Red were measured with a Varioskan Flash microplate reader (Thermo Fisher Scientific). Fluorescence signal was standardized to quantity of protein per sample using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Brains from separate mice were processed for immunohistochemistry as described above, and endogenous mouse IgG imaged and quantified. As a positive control, cortical mild traumatic brain injury (mTBI, also referred to as concussion) injury was induced in mice using a Benchmark Stereotaxic Impactor (MyNeurolab), as previously described151.
This example describes experiments which demonstrate that plasma proteins permeate the healthy adult mouse brain.
The BBB excludes nearly 100% of exogenous macromolecules (Pardridge, W. M.
NeuroRx (2005). doi:10.1602/neurorx.2.1.3; Abbott et al., Neurobiol. Dis. (2010). doi:10.1016/j.nbd.2009.07.030; Banks, W. A. BMC Neurology (2009). doi:10.1186/1471-2377-9-S1-S3; St-Amour et al., J. Cereb. Blood Flow Metab. (2013). doi:10.1038/jcbfm.2013.160), from tracers used in preclinical research to therapeutic immunoglobulins (IgG). However, the extent to which the BBB excludes the thousands of endogenous blood proteins it is constitutively exposed to remains largely unknown (Anderson, N. L. & Anderson, N. G., Mol. Cell. Proteomics (2002). doi:10.1074/mcp.R200007-MCP200; and Hood et al., J. Proteome Res. (2005). doi:10.1021/pr050107r). To address this, the mouse blood plasma proteome was chemoselectively labeled (Bragg, P. D. & Hou, C., Arch. Biochem. Biophys. (1975). doi:10.1016/0003-9861(75)90467-1; Sélo, I. et al., J. Immunol. Methods (1996). doi:10.1016/S0022-1759(96)00173-1; and Anderson et al., J. Am. Chem. Soc. (1964). doi:10.1021/ja01063a037) with a variety of small tags to enable a panel of studies upon transfer into a recipient young or aged mouse (
To study plasma uptake at a cellular resolution, plasma was labeled with the small but bright far-red Atto 647N fluorophore used in super-resolution imaging (Dempsey et al., Nat. Methods (2011). doi:10.1038/nmeth.1768; Han et al., Nat. Commun. (2017). doi:10.1038/s41467-017-01503-6). Fluorescence imaging bore a strong resemblance to autoradiography (
At greater magnification, two cardinal features were observed: a punctate vasculature and plasma-containing (plasma+) parenchymal cells (
Together, these data show that endogenous circulatory proteins constitutively enter and permeate the healthy adult brain.
This example demonstrates the regulation of plasma uptake by the brain vasculature.
Due to the unexpectedly high amount of plasma uptake by the brain, the complex system of genetic regulators (Jefferies et al., Nature (1984). doi:10.1038/312162a0; Broadwell, R. D.; Acta Neuropathologica (1989). doi:10.1007/BF00294368; Daneman, R., Ann. Neurol. (2012). doi:10.1002/ana.23648; and Zlokovic, B. V., Neuron (2008). doi:10.1016/j.neuron.2008.01.003) that plasma harnesses for enhanced BBB transport was investigated. A ‘functional transcriptomics’ platform was developed that records plasma uptake by endothelial cells via flow cytometry and index sorts them for deep, single-cell RNA sequencing (scRNA-seq, average 1.5 million reads per cell). Linking transcriptomic data and plasma uptake for every cell enabled an unbiased and high-throughput correlation between each gene's expression and degree of plasma uptake. 745 brain endothelial cells were processed from healthy adult mice four hours after administration of fluorescently-labeled plasma to ensure measurement of transcytosis, as previously described (Niewoehner, supra; Zuchero et al., Neuron (2016). doi:10.1016/j.neuron.2015.11.024; and Friden et al., Proc. Natl. Acad. Sci. (2006). doi:10.1073/pnas.88.11.4771). Across the 745 brain endothelial cells and 19,899 genes sequenced, a hierarchy of plasma uptake suggestive of regulatory control was observed (
Although the vast majority of the 19,899 sequenced genes showed no effect, specific genetic correlates of enhanced or inhibited uptake were identified, as shown in Table 1 and
The brain vasculature follows an arteriovenous zonation (Vanlandewijck et al., Nature, 554: 475-480 (2018); Chen et al., bioRxiv (2019). doi:10.1101/617258; Simionescu et al., J. Cell Biol. (1976). doi:10.1083/jcb.68.3.705) (
Together, these data suggest that in health, BBB-specific and therapeutically relevant transcriptional programs actively regulate plasma transport and are unique to each vessel segment.
This example describes an age-related shift in BBB transcytosis.
With age, the BBB is ‘leakier’ to exogenous tracers (Montagne et al., Neuron (2015). doi:10.1016/j.neuron.2014.12.032). IgG brain uptake was detected at levels consistent with prior work (Poduslo et al., Proc. Natl. Acad. Sci., 91: 5705-5709 (1994)), and concordantly, an increase in IgG accumulation with age was observed by both autoradiography and quantitative gamma counting (
To investigate the mechanisms responsible for the age-related impairment in plasma transport, the scRNA-seq data was combined with a previously published brain endothelial cell transcriptomic ageing dataset (Yousef et al., Nat. Med. (2019). doi:10.1038/s41591-019-0440-4) (
To functionally validate this age-related shift in endothelial transcytosis, three independent approaches were employed: lipidomics, proteomics, and flow cytometry. Recent work has shown that the unique lipid composition of brain endothelial cells underlies their low rates of caveolar transcytosis; and that Mfsd2a maintains these properties by embedding omega-3 fatty acid docosahexaenoic acid (DHA)-containing phospholipids into the luminal plasma membrane to preclude the formation of caveolae (Andreone et al., Neuron (2017). doi:10.1016/j.neuron.2017.03.043, Ben-Zvi et al., Nature (2014). doi:10.1038/nature13324; and Nguyen et al., Nature (2014). doi:10.1038/nature13241). Consistent with diminished Mfsd2a expression with age, unbiased lipidomic analysis revealed an age-related reduction in DHA-containing phospholipid species in the lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) classes (
Transferrin and albumin are canonical ligands for protein RMT and caveolar transcytosis, respectively (Fishman et al., J. Neurosci. Res. (1987). doi:10.1002/jnr.490180206; Roberts et al., J. Cell Sci. (1993); Lajoie, J. M. & Shusta, E. V., Annu. Rev. Pharmacol. Toxicol. (2014). doi:10.1146/annurevpharmtox-010814-124852; and Schubert et al., J. Biol. Chem. (2001). doi:10.1074/jbc.C100613200). By mass spectrometry-based proteomics, a decrease in endogenous transferrin and a concomitant increase in albumin abundance was observed within aged microvessels (
Finally, since an age-related change in functional transport was measured consistent with the predicted shift from receptor-mediated (exemplified by transferrin, leptin) to caveolar (IgG, albumin, HRP) transcytosis, the frequency of clathrin and caveolar vesicles was visualized and quantified with age. Through high-resolution microscopy of antibody-stained microvessels, a greater density of clathrin vesicles was observed in the young brain endothelium but more caveolar vesicles were observed in the aged brain (
In contrast to RMT, which was hierarchical across endothelial cells (
The data described above suggest that the aged BBB undergoes a shift from receptor-mediated to caveolar transcytosis, and that this results in both an altered composition of BBB-permeable proteins and an overall decrease in plasma uptake. A model of the shift in BBB transcytosis with age is schematically depicted in
This example describes age-related calcification in the CNS and that ALPL inhibition restores BBB transcytosis.
To discover overt anatomical changes associated with the age-related shift in brain endothelial transport, vascular density, astrocyte coverage, and pericyte coverage were assessed with age (
Several major drug and clinical development programs to enhance CNS drug delivery rely on BBB RMT, with the majority targeting the transferrin receptor, TFRC (Atwal et al., Sci. Transl. Med. (2011). doi:10.1126/scitranslmed.3002254; Yu et al., Sci. Transl. Med. (2011). doi:10.1126/scitranslmed.3002230; Zuchero et al., Neuron (2016). doi:10.1016/j.neuron.2015.11.024; Sonoda et al., Mol. Ther. (2018). doi:10.1016/j.ymthe.2018.02.032; Bien-Ly et al., J. Exp. Med. (2014). doi:10.1084/jem.20131660; and Oller-Salvia et al., Chemical Society Reviews (2016). doi:10.1039/c6cs00076b). Having observed a loss of BBB RMT with age, a search for molecular targets to enhance aged BBB transcytosis (
Yet, Alpl's canonical role is the key effector of bone calcification (Sheen et al., J. Bone Miner. Res. (2015); Murshed et al., Genes Dev. (2005). doi:10.1101/gad.1276205; Villa-Bellosta, R. & O'Neill, W. C., Kidney International (2018). doi:10.1016/j.kint.2017.11.035; Savinov et al., J. Am. Heart Assoc. (2015). doi:10.1161/JAHA.115.002499; Romanelli, F., PLoS One (2017). doi:10.1371/journal.pone.0186426)). Remarkably, alizarin red staining detected calcification (bone-dense nodules) in normally aged brains (
Consistent with the non-overlapping expression of Alpl and Tfrc in capillaries (
Administration of a selective ALPL inhibitor (Dahl et al., J. Med. Chem. (2009). doi:10.1021/jm900383s; herein incorporated by reference in its entirety) decreased ALPL activity and thus, mineralization activity in the aged brain vasculature (
To validate a pleiotropic role for Alpl in BBB protein uptake, an injection paradigm from prior work (Armulik et al., Nature, 468: 557-561 (2010) was adopted (
Because existing transferrin receptor antibodies in clinical development similarly cross the BBB via RMT, it was investigated whether ALPL inhibition could exemplify a new class of general “adjuvants” to boost brain uptake. Indeed, increased parenchymal uptake was observed after ALPL inhibition for both human holo-transferrin and a high affinity, commercially-available transferrin receptor antibody (
The results of this example show that brain calcification is an attribute of normal ageing, and that inhibition of age-upregulated, calcification-promoting ALPL in the vasculature can enhance uptake of plasma and therapeutically relevant biologics.
Gene correlates of plasma uptake were determined for brain capillaries, veins, and arterioles in younger and older subjects, and the results are shown in
This example demonstrates that ALPL inhibition upregulates blood-brain barrier iron transporters Tfrc and Slc40a1, and provides a therapeutic approach for treating restless legs syndrome (RLS).
Restless Legs Syndrome (RLS) is a serious movement-related sleep disorder affecting 7-10% of the general population, with 3% requiring therapeutic intervention (Allen et al., Arch. Intern. Med. (2005). doi:10.1001/archinte.165.11.1286; Innes, K. E., Selfe, T. K. & Agarwal, P. Sleep Medicine (2011). doi:10.1016/j.sleep.2010.12.018; Ohayon et al., Sleep Medicine Reviews (2012). doi:10.1016/j.smrv.2011.05.002). Both prevalence and severity are exacerbated with age (Milligan, S. A. & Chesson, A. L., Drugs and Aging (2002). doi:10.2165/00002512-200219100-00003). Although the exact pathogenesis of RLS is unclear, clinical studies consistently indicate that brain iron insufficiency is associated with RLS. Yet, peripheral iron supplementation is hampered by iron's limited permeability across the blood-brain barrier (BBB), necessitating facilitated delivery via dedicated transporters (Mills et al., Future Medicinal Chemistry (2010). doi:10.4155/fmc.09.140; and Chiou, B. et al., PLoS One (2018) doi:10.1371/journal.pone.0198775). Recent research has revealed impaired expression of the very genes mediating homeostatic iron transport across the BBB in RLS patients (Mizuno et al., J. Sleep Res. (2005). doi:10.1111/j.1365-2869.2004.00403.x; and Connor et al., Brain (2011). doi:10.1093/brain/awr0127,8). Thus, there remains a significant unmet need for therapeutics that can promote iron delivery to the brain to restore iron insufficiency.
It has been found that intravenous administration of a selective ALPL inhibitor employing the methodologies described herein is sufficient to enhance the iron transport pathway across the BBB (shown schematically in
These findings demonstrate that ALPL inhibition provides a pharmacologic approach to upregulate the determinants of iron transport into the brain, offering a therapeutic approach to RLS.
In some embodiments, ALPL inhibition is used in combination with other treatments or therapies for RLS. For example, in some embodiments, ALPL inhibition is combined with ropinirole (REQUIP), rotigotine (NEUPRO), pramipexole (MIRAPEX), gabapentin (NEURONTIN, GRALISE), gabapentin enacarbil (HORIZANT), pregabalin (LYRICA), tramadol (ULTRAM, CONZIP), codeine, oxycodone (OXYCONTIN, ROXICODONE), hydrocodone (HYSINGLA ER, ZOHYDRO ER), muscle relaxants, and sleep medications.
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 “at least one” 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 use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), 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 Application No. 62/943,610, filed on Dec. 4, 2019, and U.S. Provisional Application No. 63/044,048, filed on Jun. 25, 2020, the contents of both of which are incorporated by reference herein.
This invention was made with Government support under grant numbers DP1 AG053015 and R01 AG059694 awarded by the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/063112 | 12/3/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63044048 | Jun 2020 | US | |
| 62943610 | Dec 2019 | US |
