Patent Application
20170112787
The present disclosure is related to new uses of cannabinoid receptor ligands such as the endocannabinoids.
In autoimmune disease, the immune system attacks the body's own tissues. In many autoimmune diseases, the immune system attacks part of the gastrointestinal tract resulting in chronic gut inflammation. Autoimmune diseases with gastrointestinal inflammation include celiac disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, inflammatory bowel disease, and primary biliary cirrhosis. Even systemic autoimmune disorders such as lupus can result in gastrointestinal inflammation. While the treatment of autoimmune disease typically depends on the disease, a major goal of treatment is the reduction of inflammation. Thus, treatments that reduce gut inflammation would be helpful in treating a wide variety of autoimmune disorders.
What is needed are therapies useful to reduce gut inflammation and promote gut homeostasis in subjects with autoimmune diseases.
In one aspect, a method of improving immune homeostasis in the gut of a subject suffering from an autoimmune disease characterized by inflammation of the gut comprises administering to the subject an effective amount of a cannabinoid receptor ligand to improve immune homeostasis in the gut of the subject, wherein the gut includes the gastrointestinal tract as well as organs served by the blood supply to the gut.
In another aspect, a method of improving the symptoms of gut inflammation associated with diabetes mellitus Type I in a subject suffering from diabetes mellitus Type I comprises administering to the subject an effective amount of a cannabinoid receptor ligand to reduce the symptoms of gut inflammation in the subject.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
Disclosed herein are methods of improving immune homeostasis in the gut of a subject suffering from an autoimmune disease characterized by inflammation of the gut by administering, e.g., orally administering, to the subject an effective amount of a cannabinoid receptor ligand to improve immune homeostasis in the gut. The Endocannabinoid Anandamide is an intestinal endocannabinoid which engages cannabinoid receptors (CB) on the enteric nervous system and controls appetite and energy balance. Anandamide can also engage VR1, a nonselective cation channel in neurons. The inventors of the present application have found that the Endocannabinoid Anandamide, a cannabinoid receptor ligand, can maintain immune homeostasis in the gut/pancreas and can thus be used to treat autoimmune diseases characterized by inflammation of the gut.
As used herein, a subject typically includes a mammal, more specifically a human subject. In one aspect, the subject is suffering from one or more symptoms of gut inflammation such as diarrhea, fever, fatigue, abdominal pain, abdominal cramping, blood in the stool, reduced appetite, and/or unintended weight loss. Administration of a cannabinoid receptor ligand can improve one or more of the symptoms of gut inflammation.
Also as used herein, the term gut refers to the gastrointestinal tract as well as liver, spleen, pancreas, omentum, and other organs served by the blood supply to and from the gut.
As used herein, a cannabinoid receptor ligand is a molecule that binds and stimulates or inhibits the cannabinoid receptor. While VR1 and CB2 were known to be expressed in neurons and dendritic cells, the inventors unexpectedly found that VR1 and CB2 are also expressed in the small intestinal propria. The cannabinoid receptor ligand that are useful in the methods disclosed herein include those previously identified as agonists of VR1.
Cannabinoids include Phytocannabinoids, Endocannabinoids, synthetic cannabinoids, and combinations thereof. Phytocannabinoids are cannabinoids that originate from nature and can be found in the cannabis plant. Endocannabinoids are the cannabinoids that are produced endogenously by human or animal bodies. Synthetic cannabinoids are compounds that have a cannabinoid-like structure yet are manufactured using chemical means. Depending on the method of manufacture the synthetic cannabinoid may comprise a racemic mixture of cannabinoids, in contrast to an isolated cannabinoid which will typically be a single enantiomer.
Phytocannabinoids include Tetrahydrocannabinols (such as, e.g., Delta-9-tetrahydrocannabinol (Delta-9-THC), and Delta-8-tetrahydrocannabinol (Delta-8-THC)), Cannabidiols, Cannabinols, Cannabigerols, Tetrahydrocannabivarins, Cannabidivarins, and Cannabichromenes.
Endocannabinoids include Arachidonoylethanolamine (Anandamide or AEA), 2-arachidonoyl glycerol (2-AG), 2-arachidonyl glyceryl ether (noladin ether), N-arachidonoyl-dopamine (NADA), Virodhamine (OAE), and Lysophosphatidylinositol (LPI).
Synthetic cannabinoids include Dronabinol (Marinol), Nabilone (Cesamet), Sativex, Rimonabant (SR141716), JWH-018, JWH-073, CP-55940, Dimethylheptylpyran, HU-210, HU-331, SR144528, WIN 55, 212-2, JWH-133, Levonantradol (Nantrodolum), and AM-2201.
In one embodiment, the method further comprises administering a second compound that is a vanilloid receptor 1 (VR1) agonist.
VR1 agonists include resiniferatoxin and other resiniferatoxin-like complex polycyclic compounds such as tinyatoxin, capsaicin and other capsaicin analogs such as ovanil, and other compounds that include a vanilloid moiety that mediates binding and activation of VR1. Additional VR1 agonists include capsiate, civamide, SDZ-249-665, DA-5016, arvanil, scutigeral, isovelleral, phorbol 12,13-didecanoate 20 homovanillate, phorbol 12,13-dinonanoate 20 homovanillate, and comparable substances, as well as analogs, derivatives and salts of the compounds mentioned above.
In certain embodiments, the cannabinoid receptor ligand is combined with a second active agent that is an FAAH inhibitor, an inhibitor of anandamide transporter, an inhibitor of anandamide amidase, or a combination thereof.
FAAH inhibitors are described in U.S. Pat. Nos. 8,293,724 and 8,202,893, included herein by reference for their disclosure of FAAH inhibitors. FAAH inhibitors can have the formula:
Ar—R-E,
wherein Ar is an optionally substituted aryl or heteroaryl, R is an aliphatic linker, and E is a moiety selected from trifluoromethyl ketone, boronic acid, boronic acid ester, fluorosulfone, fluorophosphonate, α-haloketone and α-haloester.
FAAH inhibitors also include N-(4-hydroxyphenyl)-arachidonylamide, palmitylsulphonylfluoride, arachidonyltrifluoromethylketone and 4-benzyloxyphenyl-n-butylcarbamate.
Inhibitors of anandamide transporters are disclosed in U.S. Pat. Nos. 7,589,220 and 8,202,893, incorporated herein by reference for their disclosure of inhibitors of anandamide transporters. Such inhibitors include compounds of the formula
wherein Z is selected from a heterocyclic ring, a substituted heterocyclic ring, a heteroaromatic ring, or a substituted heteroaromatic ring.
Additional anandamide transporter inhibitors include AM404 and OMDM-1.
Inhibitors of anandamide amidase are described in U.S. Pat. No. 6,579,900, included herein by reference in its entirety for its disclosure of inhibitors of anandamide amidase. Additional anandamide amidase inhibitors include methyl arachidonyl fluorophosphonate.
Additional active agents that may be combined with the cannabinoid receptor ligands include standard-of-care drugs that are used to treat gut inflammation, such as 5-aminosalicylic acid, TNF inhibitors, azathioprine, methotrexate, 6-mercaptopurine, steroids or probiotics, and the like.
As used herein, an effective amount of a cannabinoid receptor ligand is an amount sufficient to reduce gut inflammation in a subject and/or to reduce the symptoms associated with gut inflammation in the subject such as diarrhea, fever, fatigue, abdominal pain, abdominal cramping, blood in the stool, reduced appetite, and/or unintended weight loss. Exemplary dosage amounts of the cannabinoid receptor ligand are 5 mg/kg body weight to 100 mg/kg body weight.
As used herein, “pharmaceutical composition” means therapeutically effective amounts of the cannabinoid receptor ligand with a pharmaceutically acceptable excipient, such as diluents, preservatives, solubilizers, emulsifiers, and adjuvants. As used herein “pharmaceutically acceptable excipients” are well known to those skilled in the art.
Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavoring or coloring agents.
The active ingredient may be administered parenterally in a sterile medium, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
Pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The term “unit dosage” or “unit dose” means a predetermined amount of the active ingredient sufficient to be effective for treating an indicated activity or condition. Making each type of pharmaceutical composition includes the step of bringing the active compound into association with a carrier and one or more optional accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid or solid carrier and then, if necessary, shaping the product into the desired unit dosage form.
“Pharmaceutically acceptable salts” includes derivatives of the disclosed compounds wherein the parent compound is modified by making an acid or base salt thereof, and further refers to pharmaceutically acceptable solvates of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional salts and the quaternary ammonium salts of the parent compound formed, for example, from inorganic or organic acids. For example, conventional acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC—(CH2)n—COOH where n is 0-4, and the like. The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable.
The cannabinoid receptor ligands are used to improve the immune homeostasis in the gut of a subject suffering from an autoimmune disease characterized by inflammation of the gut. Many autoimmune diseases primarily affect the gastrointestinal tract, while other autoimmune diseases, including systemic autoimmune diseases such as lupus, result in gastrointestinal inflammation in addition to joint inflammation, for example. Exemplary autoimmune diseases that can be treated with the cannabinoid receptor ligands include autoimmune diseases primarily affecting the gastrointestinal tract such as celiac disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, inflammatory bowel disease, pernicious anemia, and primary biliary cirrhosis. Endocrinologic autoimmune diseases that have gut inflammation as a symptom include diabetes mellitus Type I, Hashimoto's thyroiditis, Grave's disease, and Addison's disease. Systemic autoimmune disorders that have gut inflammation as a symptom include systemic lupus erythematosus, Sjogren's syndrome, scleroderma, rheumatoid arthritis, and the like. While the treatment of autoimmune disease typically depends on the disease, a major goal of treatment is the reduction of inflammation. Thus, treatments that reduce gut inflammation would be helpful in treating a wide variety of autoimmune disorders.
The invention is further illustrated by the following non-limiting examples.
Mice: C57BL/6, NOD/Lt, VR1−/− (also known as TRPV1−/−), IL27RA−/− (also known as WSX-1−/−) and CX3CR1gfp/gfp, were obtained from the Jackson Laboratory (Bar Harbor, Me.). IL10-GFP reporter (Vert-X) mice were a kind gift from Dr. Roger S Thrall. All animals except NOD/Lt were on a C57BL/6 background. CX3CR1gfp/gfp were crossed with C57BL/6 mice to obtain CX3CR1gfp/+ mice. CX3CR1gfp/gfp mice were backcrossed with VR1−/− to obtain CX3CR1gfp/+ VR1−/− mice. IL27RA−/− mice were backcrossed with C57BL/6 mice to obtain IL27RA+/− mice. All animals were housed in the Center for Comparative Medicine at UConn Health. All experiments were conducted under the guidelines approved by the UConn Health Animal Care Committee.
Cell isolation: The siLP cells were isolated as per protocols known in the art. In brief, small intestines were removed from the mice, luminal contents were flushed with cold HBSS (Invitrogen, Carlsbad, Calif., USA) with 5% FBS, opened longitudinally after removing Peyer's patches and cut in to small pieces. The epithelial cells were removed by incubation with HBSS containing 1.3 mM EDTA (Invitrogen, Carlsbad, Calif., USA), 1mM HEPES (Invitrogen, Carlsbad, Calif., USA) and 0.77 mM DTT (Sigma, St. Louis, Mo., USA) at 37° C. shaking at 350 rpm. The intestinal pieces were then washed with PBS (Invitrogen, Carlsbad, Calif., USA) and digested in 100 units/ml collagenase IV (Sigma, St. Louis, Mo., USA) at 37° C. shaking at 550 rpm. Leukocytes were enriched using percoll (GE Healthcare, Piscataway, N.J., USA) gradient. The PLN, MLN and splenic cells were isolated by gently crushing the organs through 70 μm cell strainer (BD Falcon, Franklin, N.J., USA). Splenocytes were incubated with ACK lysing buffer (Invitrogen, N.Y., USA) to eliminate red blood cells for 3 mins at 37° C. and washed twice with PBS.
Flow cytometry: Fluorescently labeled antibodies used for surface staining included: CD11b (M1/70), CD11 c (N418), CX3CR1 (polyclonal), CD103 (2E7), CD64 (X54-5/7.1), MHCII (M5/114.15.2), B220 (RA3-6B2), CD3 (145-2C11), CD4 (RM4-5), CD25 (PC61) and CD62L (ME2-14) and those used for intracellular staining include: VR1 (polyclonal) IL10 (JESS-16E3), TNFα (MP6-XT22), IFNγ (XMG1.2), and IL17A (TC11-18H10.1). For detection of CB2, primary antibody against CB2 (polyclonal) was used followed by fluorescently labeled secondary antibody. All were purchased from Biolegend San Diego, Calif., USA except for TNFα (Becton Dickinson, San Jose, Calif., USA), CX3CR1 (R&D Minneapolis, Minn., USA), VR1 (Thermo Scientific, Rockford, Ill. USA), IL10 (eBioscience, San Diego Calif., USA) and CB2 (Thermo Scientific, Rockford, Ill. USA). Cells were acquired with a LSRII flow cytometer (Becton Dickinson, San Jose, Calif., USA) or sorted with a FACSAria™ machine (Becton Dickinson, San Jose, Calif., USA). Flow cytometry analysis was done with the FlowJo Version 9.7.6 (Tree Star, Ashland, Oreg,).
Intracellular staining for VR1: After surface staining and fixing, cells were incubated in permeabilization buffer (BD Biosciences, San Jose, USA). Cells were then incubated in 4% goat serum for 30 minutes to prevent non-specific binding of the secondary antibody. After washing, cells were incubated with primary antibody against VR1 (rabbit anti-mouse) or unconjugated isotype control for 30 minutes. Next the cells were washed two times and stained with fluorochrome labeled anti-rabbit secondary antibody for 30 minutes. After washing two times cells were analyzed by flow cytometry.
Intracellular cytokine staining: Cells were incubated with PMA (50 ng/ml) (Sigma Co., St. Luis, Mo., USA) plus ionomycin (1 μg/ml) (Sigma Co., St. Luis, Mo., USA) in the presence of golgi plug (BD Biosciences, San Jose, USA) for 5 hours at 37° C. followed by surface staining with fluorochrome conjugated antibodies and then intracellular staining of IL10, TNFα, IFNγ, and IL17A was performed by fixation and permeabilization according to manufacturer's protocol BD Cytofix/Cytoperm™ kit.
In vitro co-culture assays: For in vitro co-culture assays, siLP MNPs were sorted as described previously from C57BL/6 or CX3CR1gfp/+ mice. MNPs (10×104) were cocultured for 4 days with splenic naïve CD25− CD62Lhi CD4±T cells sorted from untreated C57BL/6 or IL27Ra−/− mice at 1:1 ratios in the presence of soluble αCD3 mAb (145-2C11) (0.5 μg/ml) in U-bottomed 96 well plates. The cultured cells were maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-gluatamine, 50 units/ml streptomycin, and 50 μM Beta-mercaptoethanol, all purchased from Gibco/Invitrogen (Carlsbad, Calif., USA). After 4 days, the cultured cells were washed with PBS and surface stained with CD4 and intracellular staining was performed for IL10 and IFN-γ.
Oral administration of CP, AEA and PF3845: Mice were anesthetized with intraperitoneal injection of ketamine (2 mg/kg) and xylazine (0.1 mg/kg). Vehicle control (tween 80 (5%), ethanol (85%) in PBS) or CP (10 μg) (ENZO Life Sciences, Farmingdale, N.Y., USA) or AEA (250 μg, 400 μg or 500 μg) (Cayman Chemical Co., Ann Arbor, Mich., USA) or PF3845 (5 mg/kg) (Cayman Chemical Co., Ann Arbor, Mich., USA) were gavaged in 100 μl volume using an 18-gauge feeding needle (Popper & Sons, New Hyde Park, N.Y., USA).
qPCR: Total RNA was isolated from sorted siLP MNP using the RNeasy® Micro kit Oaten, Chatsworth, Calif., USA). cDNA was made from total RNA using iSCRIPT™ cDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif.). IL-27 gene expression was measured using IL-27 TactMan® Gene Expression Assay (Life technologies, Grand Island, N.Y., USA) and CFX96™ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, Calif., USA). Gene expression was normalized to GAPDH.
Adoptive transfer: Female NOD mice were orally gavaged with CP 10 μg or vehicle at 9 and 10 weeks of age as described earlier. 2 weeks later CD4+ T cells were sorted from the PLN and spleen of the treated mice. All recipient female NOD mice were sub-lethally irradiated. Four hours after irradiation the recipient mice received 10×1010 diabetogenic splenocytes. 7×106 sorted CD4+ T cells were then transferred to naïve NOD female mice by retro-orbital injection at 11 weeks of age. Development of diabetes was monitored by measuring urine glucose using Diastix® reagent strips (Bayer, Elkhart, Ind., USA). Mice were considered diabetic after two consecutive readings of >1000 mg/dl of urine glucose.
Statistical analyses: Data were analyzed by Student's t-test (unpaired, one-tailed) using GraphPad Prism Version 6.0 (GraphPad Software, La Jolla, Calif., USA). P<0.05 was considered significant.
The expression of endocannabinoid receptors VR1 and CB2 on the major cell populations in the small intestinal lamina propria (siLP). See
VR1−/− mice were back-crossed with CX3CR1gfp/gfp reporter mice to obtain CX3CR1gfp/+ VR1−/− mice (
We orally gavaged CX3CR1gfp/+ mice with VR1 ligands CP or AEA. In case of CP (10 μg), its administration significantly increased the frequency of the regulatory CX3CR1hi Mφ but did not affect the frequency and number of the CX3CR1lo cells (
Since IL10 is a cytokine with potent tolerogenic properties in the gut, we looked at the IL10 production as a measure of tolerogenicity in the CX3CR1hi Mφ and CD103+ DCs 24 hours after treatment with CP using the IL10 GFP-reporter mice. Compared to controls, the CX3CR1hi Mφ of CP fed mice showed a significant increase in the production of IL10 (
The ability of CP to alter the ability of the siLP MNPs to induce IL10-producing regulatory T cells was tested. The CX3CR1hi Mφ and CD103+ DCs constitute the overwhelming majority of siLP MNPs, thus the total MNPs (CD11b+CD11c+ and CD11b−CD11c+ cells) (
Both the pancreatic (PLN) and mesenteric lymph nodes (MLN) drain the gut. The possibility that the absence of VR1 in addition to affecting the number of CX3CR1hi Mφ in the siLP also affects the frequency of CX3CR1+ Mφ in the gut draining lymph nodes i.e., PLN and MLN was tested. VR1−/− mice showed a significant reduction in the frequency of CX3CR1+ Mφ in the PLN (
The phenotype of the PLN CX3CR1+ Mφ was akin to that of the siLP Mφ (shown in
Given that the CX3CR1hi Mφ are critical to VR1 induced tolerance we asked if functional CX3CR1 is required for CP-elicited expansion of Tr1 cells. CX3CR1gfp/gfp mice were orally gavaged with CP or vehicle as a control and Tr1 cells were analyzed in the PLN and it was observed that in the absence of functional CX3CR1 protein, CP does not elicit Tr1 cells (
These results establish the endocannabinoid system as a major participant in maintaining tolerance in the gut. It does so through two distinct, non-overlapping and complementary, mutually re-enforcing pathways, i.e. through its profound role in maintenance/differentiation of well-known immune regulatory CX3CR1hi Mφ population, and by mediating expansion of the regulatory Tr1 T cells. Remarkably, although our enquiry began with examination of the role of VR1 in immune tolerance elicited by CP, the most established exogenous ligand of VR1, the endocannabinoid AEA, also causes similar consequences as CP. This is the first example of a neurologically active endocannabinoid playing a substantial immunological role, and suggests interesting possibilities of concordant regulation of immune tolerance and energy balance through one ligand and two receptors.
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. 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 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. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a 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”), is intended merely to better illustrate 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 as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. 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 priority to U.S. Provisional Application 62/158,730 filed on May 8, 2015, which is incorporated herein by reference in its entirety.
This invention was made with government support under grant AI095776 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62158730 | May 2015 | US |
