The invention generally relates to polycationic polymers and methods for using the same. More specifically, the invention relates to conjugated polycationic polymers and method of using polycationic polymers for treating autoimmune diseases, radiation exposure or infectious diseases.
Nucleic acids are tasked with storing the genetic information required for life, however in many disease states nucleic acids are found in excessively high amounts and contribute to disease enhancement. This is especially true in diseases where the pathological insult is primarily from the host and not from bacteria, viruses or other common pathogens. In pancreatic cancer, for example, circulating nucleic acids are more abundant when compared to non-cancerous individuals and these nucleic acids have been shown to directly enhance disease progression ultimately adding to patient morbidity.
There have been several attempts to eliminate circulating nucleic acids from these patients and our laboratory proposes to use nucleic acid scavengers (NAS) in the form of cationic polymers in this context. Currently, the interface between scavengers and reduction in aberrant inflammation is being explored because this approach is amenable to facile chemical modification and offers an alternative to current drugs.
There exists a need for deciphering the mechanisms behind polycationic polymers and their ability to ameliorate several diseases in order to translate this approach to the clinic and for novel compounds that will help in this goal. Further, these compounds may also prove to be potential therapies on their own.
Disclosed herein are conjugated polycationic polymers and methods of using the same. One aspect of the invention is a conjugated polycationic polymer, the conjugated polycationic polymer comprising a dendron, the dendron comprising a focal point, a plurality of cationic termini, and a branched cationic polymer between the focal point and the plurality of cationic termini; a detectable label; and a crosslinker, wherein the crosslinker links the detectable label and the focal point of the dendron. In some embodiments, the conjugated polycationic polymer is capable of binding a nucleic acid. In some embodiments, the conjugated polycationic polymer is capable of binding a nucleic acid-protein complex.
Another aspect of the invention is a scavenging apparatus, the scavenging apparatus comprising a plurality of conjugated polycationic polymers and a substrate, wherein the plurality of conjugated polycationic polymers are immobilized on the substrate. In some embodiments, the conjugated polycationic polymer is capable of binding a nucleic acid. In some embodiments, the conjugated polycationic polymer is capable of binding a nucleic acid-protein complex.
Another aspect of the invention is a method of scavenging a nucleic acid or negatively-charged biomolecule or complex from a solution, the method comprises contacting the solution comprising a cell-free nucleic acid or negatively-charged biomolecule or complex with a scavenging apparatus.
Another aspect of the invention is a method for the reduction of negatively-charged biomolecule or complex in a bodily fluid of a subject or a patient having an abnormally high concentration of the cell-free nucleic acid in the bodily fluid, the method comprising contacting the bodily fluid with a conjugated polycationic polymer or scavenging apparatus, wherein the contacting step reduces the concentration of the cell-free nucleic acid or negatively-charged biomolecule or complex in the bodily fluid.
Another aspect of the invention is a method for the tracking of a conjugated polycationic polymer, a cell-free nucleic acid, or negatively-charged biomolecule or complex in vitro or ex vivo, the method comprising contacting the conjugated polycationic polymer with a cell in vitro or ex vivo and determining the position of the conjugated polycationic polymer relative to a cell membrane or an organelle membrane of the cell, wherein the conjugated polycationic polymer is any of the conjugated polycationic polymers described above. In some embodiments, the conjugated polycationic polymer has been contacted with a negatively charged biomolecule to obtain a negatively charged biomolecule polymer adjunct and wherein step of determining the position of the conjugated polycationic polymer also determines the position of the negatively charged biomolecule polymer adjunct.
Another aspect of the invention is a method for the tracking of a negatively-charged biomolecule in vivo, the method comprising administering the negatively-charged biomolecule-polymer adjunct to a subject and determining the position of the conjugated polycationic polymer within the subject, where the conjugated polycationic polymer is any of the conjugated polycationic polymers described above. In some embodiments, the conjugated polycationic polymer has been contacted with a negatively charged biomolecule to obtain a negatively charged biomolecule polymer adjunct and wherein step of determining the position of the conjugated polycationic polymer also determines the position of the negatively charged biomolecule polymer adjunct.
Nucleic acid molecules were thought to be largely immunologically inert until it was discovered that the innate immune system employs pattern-recognition receptors (PRRs) to recognize various molecular patterns associated with harmful pathogens and damaged cells and to initiate inflammatory responses. In particular, various bacterial or viral derived DNA and RNA molecules were found to activate several PRRs, including at least four different Toll-like receptors (TLR3, 7, 8 and 9) and several cytoplasmic PRRs.
Given their potent immunostimulatory and proinflammatory effects, the discovery that the inappropriate activation of TLRs/PRRs is associated with a broad range of inflammatory disorders was not a surprise. More specifically, it now appears that nucleic acid-sensing TLRs play a critical role in numerous inflammatory disorders presumably because dead and dying cells release nucleic acids and nucleic acid-containing complexes into the extracellular space which induces pathogenic inflammatory responses (
Moreover, increased levels of extracellular circulating mtDNA have even been associated with increased mortality in Intensive Care Unit patients. Thus, nucleic acid-sensing TLRs and PRRs have become attractive therapeutic targets for the treatment of acute pathological inflammation as well as devastating inflammatory disorders. Unfortunately the redundancy of the TLR and PRR families as well as their ability to sense a variety of structurally different nucleic acid ligands has made it challenging to develop effective inhibitors that can broadly ameliorate the proinflammatory effects of RNA, DNA and nucleic acid-containing complexes. Moreover, because these PRRs are important for responding to infectious agents, therapeutic strategies that compromise TLR function (or their downstream effector molecules) compromise an animal's or patient's ability to combat infection. Novel anti-inflammatory agents that do not affect innate immunity toward pathogenic infection while being able to mitigate the effects of inflammation are required.
We demonstrate that polycationic polymers can inhibit the activation of nucleic acid-sensing TLRs (TLR3, 7, 8 and 9) and the inflammatory response engendered by prototypical proinflammatory nucleic acids in vitro as well as rescue animals from nucleic acid-induced fatal inflammatory shock. Moreover we show that conjugated polycationic polymers can be used to detect and sequester nucleic acids. As a result polycationic polymers may be used to treat conditions associated with elevated levels of cell-free nucleic acids, such as autoimmune diseases, infectious diseases, or acute radiation syndrome, by neutralizing the effects of proinflammatory and procoagulant nucleic acid-based DAMPs released from damaged cells while at the same time not compromising the native immune systems ability to combat infectious diseases.
Polycationic polymers, which are sometimes referred to as nucleic-acid scavenging polymers, are polymers having a plurality of cationic termini, a focal point or bridging moiety, and a branched cationic polymer between the focal point or the bridging moiety and the cationic termini. The polycationic polymers may be a dendrimer or a dendron.
Dendrimers or dendrons may be characterized by the generation number Gn. The generation number details the number of successive additions of the polymers base monomer. The generation number (Gn) may characterize the dendron's properties depending on the choice of the polymer. Properties characterizable by knowledge of the generation number and the cationic polymer include, without limitation, the number of branch points, the size of the dendron, the electronic charge, and terminal moieties. In certain embodiments, the dendron is a G2 dendron, a G3 dendron, a G4 dendron, a G5 dendron, G6 dendron, or any Gn suitable for use as a scavenger.
In some embodiments, the polycationic polymer is selected from the group consisting of a poly(β amino ester), disulfide containing poly(β amido amine) or poly(β hydroxyl amine). Preferred polymers include those in
The polycationic polymer is suitably a polycationic polymer capable of binding to a nucleic acid. Preferred polycationic polymers include biocompatible polymers (that is, polymers that do not cause significant undesired physiological reactions) that can be either biodegradable or non-biodegradable polymers or blends or copolymers thereof. PAMAM G3 was used in the examples, but other polycationic polymers are anticipated to achieve similar effects. Examples of such polymers include, but are not limited to, polycationic biodegradable polyphosphoramidates, polyamines having amine groups on either the polymer backbone or the polymer side chains, nonpeptide polyamines such as poly(aminostyrene), poly(aminoacrylate), poly(N-methyl aminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate), poly(N,N-diethylaminoacrylate), poly(aminomethacrylate), poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate), poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl aminomethacrylate), poly(ethyleneimine), polymers of quaternary amines, such as poly(N,N,N-trimethylaminoacrylate chloride), poly(methyacrylamidopropyltrimethyl ammonium chloride); natural or synthetic polysaccharides such as chitosan, cyclodextrin-containing polymers, degradable polycations such as poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA); polycationic polyurethanes, polyethers, polyesters, polyamides, polybrene, etc. Particularly preferred cationic polymers include CDP, CDP-Im, PPA-DPA, PAMAM and HDMBr. (See U.S. Pat. Nos. 9,340,591, 7,270,808, 7,166,302, 7,091,192, 7,018,609, 6,884,789, 6,509,323, 5,608,015, 5,276,088, 5,855,900, U.S. Published Appln. Nos. 2012/0183564, 20060263435, 20050256071, 200550136430, 20040109888, 20040063654, 20030157030, International Patent Publication No. WO 2014/169043, Davis et al, Current Med. Chem. 11(2) 179-197 (2004), and Comprehensive Supramolecular Chemistry vol. 3, J. L. Atwood et al, eds, Pergamon Press (1996).)
The plurality of cationic termini may be any terminal moieties that allow for the binding of negatively charged molecules. The polycationic polymer may bind nucleic acids or other negatively charged molecules to the corona of a dendrimer or dendron. Under certain conditions, the plurality of cationic termini may assist to effectively bind the nucleic acid irreversibly. Under certain under condition, the plurality of cationic termini may assist to effectively bind the nucleic acid reversibly. The plurality of cationic termini may be an ammonium terminal moiety or any other cationic termini suitable for binding to nucleic acids.
Advantageously, the binding affinity of a polycationic polymer of the invention for a nucleic acid, expressed in terms of Kd, is in the pM to mM range, preferably, less than or equal to 50 nM; expressed in terms of binding constant (K), the binding affinity is advantageously equal to or greater than 105 M−1, preferably, 105 M−1 to 108 M−1, more preferably, equal to or greater than 106M−1. Thus, the binding affinity of the sequence-independent nucleic acid-binding cationic polymers can be, for example, about 1×105 M−1, 5×105 M−1, 1×106 M−1, 5×106 M−1, 1×107 M−1, 5×107 M−1; or about 10 μM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM. “K” and “Kd” can be determined by methods known in the art, including Isothermal calorimetry (ITC), Forster Resonance Energy Transfer (FRET), surface plasmon resonance or a real time binding assay such as Biacore.
The cationic polymers bind to a wide array of different nucleic acids including ssRNA, ssDNA, dsRNA and dsDNA and of which may be presented in a complex with protein such as viral proteins, histones, HMGB1 or RIG-I. See
Conditions such as pH, presence or absence of salts, and/or temperature may affect the electronic character of the polycationic polymer and within the scope of the invention. Depending on the conditions for using the polycationic polymer, the plurality of termini or the branched polymer between a focal point or a bridging moiety and the plurality of termini may be electrically neutral. Under some conditions, the polycationic polymer has a plurality of electrically neutral termini and a branched cationic polymer between a focal point or a bridging moiety and the plurality of electrically neutral termini. Under some conditions, the polycationic polymer has a plurality of cationic termini and a branched electrically neutral polymer between a focal point or a bridging moiety and the plurality of cationic termini.
One aspect of the invention is conjugated polycationic polymers. The conjugated polycationic polymers comprise a dendron having a focal point, a plurality of cationic termini, and a branched cationic polymer between the focal point and the plurality of cationic termini, a detectable label, and a crosslink that links the detectable label and the focal point of the dendron. The conjugated polycationic polymers have the ability to bind to negatively charged molecules, such as nucleic acids or nucleic acid-protein complexes, to sequester the negatively charged molecules and/or prepare a trackable adjunct.
In some embodiments, the crosslinker is prepared by contacting a first crosslinkable moiety with a second crosslinkable moiety. The dendron may further comprise the first crosslinkable moiety and the detectable label comprises a second crosslinkable moiety, and the first crosslinkable moiety is capable of crosslinking with the second crosslinkable moiety. The first crosslinkable moiety and/or the second crosslinkable moiety may be a sulfhydryl, carbonyl, carboxyl, amine maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, carbodiimide, isothiocyanates, isocyanates, acyl azides, N-Hydroxysuccinimide ester, sulfonyl chloride, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester, or any other crosslinkable moiety.
The detectable label may be a binding label, a chromophore, an enzyme label, a bioluminescent label, a quencher, a radiolabel, or any other label suitable for a means of detection. Binding labels provide for a detectable signal via a binding event. In some embodiments, a binding label may be biotin, an antibody, an antigen, or any other label capable of providing a detectable signal via a binding event. Chromophores provide a detectable signal via the absorbance and emission of photons. In some embodiments, the chromophore is a fluorophore, a phosphor, a dye, a quantum dot, or any other chromophore capable of absorbing and emitting detectable photons. In certain embodiments, the chromophore is an Alexa Fluor such as Alexa Fluor 488 or Alexa Fluor 750. Enzyme labels provide a detectable signal via a reaction with a substrate. Bioluminescent labels provide a detectable signed via the emission of light from a protein. In certain embodiments, the bioluminescent label is a luciferase. Quenchers provide a detectable signal via the modulation of the photon emission from a chromophore. Radiolabels provided for a detectable signal via a radioactive decay.
Another aspect of the invention is a polycationic apparatus for binding and sequestering negatively charged molecules. The polycationic apparatus comprises a plurality of conjugated polycationic polymers and a substrate, wherein the plurality of conjugated cationic polymers are capable of being immobilized on the substrate. In certain embodiments, the conjugated polycationic polymers are any of the conjugated polycationic polymers described above.
In an embodiment of the apparatus, the substrate comprises a binding moiety and the detectable label binds with the binding moiety to immobilize the polycationic polymer polymer on the substrate. The binding moiety may be avidin, an antibody, or any other binding protein. When avidin is used as a binding moiety, the detectable label is an avidin-binding label. In particular embodiments when avidin is used as a binding moiety, the detectable label is biotin. When an antibody is used as a binding moiety, the detectable label is an antibody-binding label. In particular embodiments when an antibody is used as a binding moiety, the detectable label may be an antigen.
The binding moiety may also be a binding moiety that binds a protein. In particular embodiments, the binding moiety may be biotin or an antigen. When biotin is used as a binding moiety, the detectable label may be a biotin-binding label. In particular embodiments when biotin is used as a binding moiety, the detectable label is avidin. When an antigen is used as a binding moiety, the detectable label may be an antigen-binding label. In particular embodiments when an antigen is used as a binding moiety, the detectable label is an antibody.
In an alternative embodiment of the apparatus, the plurality of polycationic polymers is covalently bound to the substrate. The polycationic polymers comprise a dendron, the dendron comprising a focal point, a plurality of cationic termini, and a branched cationic polymer between the focal point and the plurality of cationic termini, and a crosslinker, wherein the crosslinker links the substrate and the focal point of the dendron. The dendron may further comprises a first crosslinkable moiety, the substrate comprises a second crosslinkable moiety, the second crosslinkable moiety capable of crosslinking with the first crosslinkable moiety; and the crosslinker is prepared by contacting the first crosslinkable moiety with the second crosslinkable moiety. In certain embodiments, the first crosslinkable moiety or the second crosslinkable moiety comprises a member selected from the group consisting of sulthydryl, carbonyl, carboxyl, amine maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, carbodiimide, isothiocyanates, isocyanates, acyl azides, N-Hydroxysuccinimide ester, sulfonyl chloride, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester.
The substrate may be any substrate suitable for binding the polycationic polymer. In certain embodiments, the substrate may be a glass, silicon, a silicon polymer, a metal, a plastic, magnetic, or an electrospun fiber. Glasses may include silica, a borosilicate, soda lime, or any other glass suitable for binding the polycationic polymer. Silicone polymers may include polydimethylsiloxane or any other silicone polymer suitable for binding the polycationic polymer. Metals may include gold, silver, platinum, or any other metal suitable for binding the polycationic polymer. Plastics may include a poly(methyl methacrylate), a poly(styrene), cyclic olefin copolymer, or any other plastic suitable for binding the polycationic polymer. Magnetic substrates may include any magnetic material suitable for binding the polycationic polymer, including, magnetic beads. The electrospun fiber may be any electrospun fiber suitable for binding the polycationic polymer, including those described in International Application Ser. No. PCT/US2015/026201 to Sullenger et al., published as WO/2015/161094 22 October 2015. Those skilled in the art will appreciate that there may be many ways to immobilize the polycationic polymer to the substrate depending on the choice of substrate.
Sequestering Negatively Charged Molecules from Solutions or Biological Samples
Another aspect of the invention is methods for scavenging negatively charged molecules, such as a nucleic acid, from a solution or a biological sample. The method comprises contacting the solution comprising a negatively charged molecule with any of the apparatuses described above. In certain embodiments, the apparatus comprises the conjugated polycationic polymers also described above. The solution may be artificially created by human intervention or a biological sample obtained from a subject or a patient. When the solution is a biological sample obtained from a subject or a patient, the solution may be blood, lymph, plasma, serum, cerebral spinal fluid, urine or any other bodily fluid. In certain embodiments, the solution or biological sample comprises cell-free nucleic acids.
In one embodiment of the invention, the conjugated polycationic polymer is bound to the substrate of the apparatus and the solution or biological sample is contacted with the bound conjugated polycationic polymer. When the solution or biological sample is contacted with the bound conjugated polycationic polymer, the cationic polymer may bind negatively charged molecules to prepare an adjunct. By forming the adjunct, the negatively charged molecules will be sequestered by the bound conjugated polycationic polymer.
In an alternative embodiment, the conjugated polycationic polymer is deposited into the solution or biological sample and the solution or sample containing the conjugated polycationic polymer is contacted with the substrate of the apparatus. By depositing the conjugated polycationic polymer into the solution or biological sample, you allow for the formation of adjuncts between the conjugated polycationic polymer and negatively charged molecules present. When the adjuncts are later contacted with the apparatus, the adjuncts may bind to the substrate through the conjugated polycationic polymers. This, in turn, sequesters the negatively charged molecules.
Another aspect of the invention includes methods for the tracking the conjugated polycationic polymer or a negatively charged biomolecule or complex, such as cell-free nucleic acid, in vitro or ex vivo. The method comprises contacting the polycationic polymer adjunct with a cell in vitro or ex vivo; and determining the position of the conjugated polycationic polymer relative to a cell membrane or an organelle membrane of the cell. In some embodiments, the conjugated polycationic polymer may be contacted with a negatively charged biomolecule or complex to prepare an adjunct that allows for simultaneous detection of the negatively charged biomolecule or complex via the adjunct. When practicing the method, it may be possible to determine that the polycationic polymer adjunct is bound to the cell membrane or the organelle membrane. When practicing the method, it may also be possible to determine that the polycationic polymer or polycationic polymer adjunct is determined is within the cell membrane or the organelle membrane. Such methods may be useful for determining whether or not the polycationic polymer and/or negatively charged molecule enter cells. If so, the methods may also be useful for determining the rate of uptake through a number of different analytical tools, including, but not limited to, flow cytometry and confocal microscopy. Such methods may also be useful for screening cell or tissue types for the ability to internalize polycationic polymers and/or prone to polycationic polymer toxicity. Further still, such methods may be able to determine if polycationic polymers localize with cellular organelles or other intracellular compartments.
Another aspect of the invention includes methods for the tracking the conjugated polycationic polymer or a negatively charged biomolecule or complex, such as a cell-free nucleic acid, in vivo. The method comprises administering the polycationic polymer to a subject and determining the position of the polycationic polymer within the subject In some embodiments, the conjugated polycationic polymer may be contacted with a negatively charged biomolecule or complex to prepare an adjunct that allows for simultaneous detection of the negatively charged biomolecule or complex via the adjunct. The administering step may comprise intravenous injection, intraperitoneal injection, subcutaneous injection, or any other suitable method of administration. Practicing the method may also allow for the determination of whether the polycationic polymer adjunct is within a tissue of the subject. This allows for the determination of the polycationic polymer localization. This further allows for the determination of pharmacokinetics, including, but not limited to rate of clearance and biological binding capacity. This method also opens up the ability to analyze which routes of administration results in more rapid degradation of the polycationic polymer.
Either of the in vitro, ex vivo, or in vivo tracking methods may use a detectable label as described above. In some embodiments, the detectable label is a chromophore. In certain embodiments, the detectable label is a fluorophore. In particular embodiments, the detectable label is an Alexa Fluor, for example Alexa Fluor 488 or Alexa Fluor 750. When the detectable label is a chromophore, the determining step may comprise exciting the detectable label and detecting the localized position of emitted photons. The position of the nucleic acid may be determined by any suitable method. Examples of methods and/or techniques for determining position, include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, confocal microscopy, flow cytometry, fluorescence-activated cell sorting, or immunohistochemistry.
Another aspect of the invention provides methods for the reduction of cell-free nucleic acid or other inflammatory mediator in a bodily fluid of a subject or a patient having an abnormally high concentration of the cell-free nucleic acid or other mediator of inflammation in the bodily fluid. The method comprises contacting the bodily fluid with a polycationic polymer or nuc scavenging apparatus, wherein the contacting step reduces the concentration of the cell-free nucleic acid, DAMPS, PAMPS or other inflammatory mediators in the bodily fluid. The polycationic polymer may be any of the conjugated polycationic polymers described above or any of the unconjugated polycationic polymers described above. The scavenging apparatus may be any of the scavenging apparati described above. In certain embodiments, the contacting step is performed within the subject or the patient. In other embodiments, the contacting step is performed outside the subject or the patient. The method may further comprise obtaining the bodily fluid from the patient and/or returning the bodily fluid to the subject or the patient. In some embodiments, the bodily fluid is blood, plasma, serum, cerebral spinal fluid, lymph, or any other bodily fluid having cell-free nucleic acids or other inflammatory mediators.
In certain embodiments, the subject or patient suffers from a condition associated with the abnormally high concentration of the cell-free nucleic acid or other inflammatory mediators. The condition may be a cancer, an effect associated with radiation therapy, an autoimmune disease, an infectious disease, or any other condition associated with abnormally high concentrations of cell-free nucleic acid or other inflammatory mediators in a bodily fluid. In particular embodiments, practicing the methods described herein may provide therapeutic benefit for the subject or patient suffering from the condition.
The polycationic polymer may be used to make pharmaceutical compositions. Pharmaceutical compositions comprising the polycationic polymers described above and a pharmaceutically acceptable carrier are provided. A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Examples of pharmaceutically acceptable carriers suitable for use in the composition include, but are not limited to, water, buffered solutions, glucose solutions, or oil-based carriers. Additional components of the compositions may suitably include, for example, excipients such as stabilizers, preservatives, diluents, emulsifiers and lubricants. Examples of pharmaceutically acceptable carriers or diluents include stabilizers such as carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine serum or skimmed milk and buffers (e.g., phosphate buffer). Especially when such stabilizers are added to the compositions, the composition is suitable for freeze-drying or spray-drying. The composition may also be emulsified.
The polycationic polymer may be administered with an addition therapeutic agent. The polycationic polymer and therapeutic agent may be administered in any order, at the same time or as part of a unitary composition. The two may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more. The polycationic polymer may be administered or used to contact a bodily fluid of the subject in conjunction with another therapy to treat the disease or condition.
An effective amount or a therapeutically effective amount as used herein means the amount of the polycationic polymer that, when administered to a subject for treating the condition is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the compositions or formulations, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.
The compositions described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, intratumoral or transmucosal absorption. Thus the compounds may be formulated as an ingestable, injectable, topical or suppository formulation. The compositions may also be delivered within a liposomal or time-release vehicle. Administration to a subject in accordance with the invention appears to exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.
It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compound or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compound of the invention and of a known agent such as tocopherol, such as by means of an appropriate conventional pharmacological or prophylactic protocol. The subject may be a human subject, a human suffering from cancer or a non-human animal subject. For example, the subject may be a domesticated animal such as a cow, pig, chicken, horse, goat, sheep, dog or cat.
The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compositions will reduce symptoms of the condition at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to pre-treatment symptoms or symptoms is left untreated. It is specifically contemplated that pharmaceutical preparations and compositions may palliate or alleviate symptoms of the disease without providing a cure, or, in some embodiments, may be used to cure the disease or disorder.
Suitable effective dosage amounts for administering the compositions may be determined by those of skill in the art, but typically range from about 1 microgram to about 50,000 micrograms per kilogram of body weight weekly, although they are typically about 50,000 micrograms or less per kilogram of body weight weekly. Large doses may be required for therapeutic effect and toxicity of the compositions is likely low. In some embodiments, the effective dosage amount ranges from about 10 to about 50,000 micrograms per kilogram of body weight weekly. In another embodiment, the effective dosage amount ranges from about 100 to about 40,000 micrograms per kilogram of body weight weekly. In another embodiment, the effective dosage amount ranges from about 500 to about 30,000 micrograms per kilogram of body weight weekly. The effective dosage amounts described herein refer to total amounts administered, that is, if more than one compound is administered, the effective dosage amounts correspond to the total amount administered. The compositions can be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.
Autoimmune disorders such as systemic lupus erythematosus (SLE) are characterized by increased production of antibodies against self-nucleic acids and their associated proteins. A number of Toll-like receptors (TLRs) that assist in recognition of these nucleic acids have been individually targeted to slow and reverse disease progression. These therapeutic strategies have shown an essential role for TLR-targeting in combating inflammatory disorders, however, their effects to date have been modest and new drugs remain to be tested.
Endosomal TLRs act as sensors of foreign RNA and DNA and elicit an innate immune response to pathogens. When tolerance is broken, these TLRs are aberrantly activated by self-nucleic acids; often associated with autoantibodies and immune complexes. This in turn leads to increased downstream activation of signaling cascades and dysregulated expression of pro-inflammatory cytokines and autoantibodies. Despite our increased understanding of TLR biology and attempts to target these particular pathways, we have been unable to develop effective ways to address the source of antigen.
Inappropriate clearance of dying cells and elevated levels of serum DNA and RNA correlates with increased autoimmune disease pathology. Studies have shown that targeting the free-circulating RNA and DNA using nucleases can be beneficial in dampening inappropriate TLR activating and improving autoimmune disease outcome.
We directly tested the hypothesis that polycationic polymers can limit pathological inflammation during the course of autoimmunity, by absorbing free-circulating DNA and RNA or nucleic acid-protein complexes. We utilized two mouse models of systemic lupus erythematosus (SLE), NZBWF1 and MRLlpr, to assess immune activation and resolution of inflammation in the presence of polycationic polymers. Both mouse strains develop spontaneous SLE, which closely mimics clinical human SLE. In addition, we further explored the hypothesis that polycationic polymers deliver a therapeutic benefit during autoimmunity without compromising the organism's ability to fight infections. To test this hypothesis we employed a PR8 influenza infection in the presence of polycationic polymers.
Here we show that polycationic polymers are capable of controlling aberrant inflammation in two separate disease models. polycationic polymers treatments resulted in improved skin inflammation and also delayed systemic lupus progression. Additionally, we demonstrated that mice treated with polycationic polymers were capable of responding to pathogenic infections such as PR8 influenza. Moreover, polycationic polymers treatment of mice during a lethal PR8 influenza infection resulted in increased survival rates. It is important to note that our studies utilized a widely used polycationic polymer: generation-3 PAMAM-G3, [NH2(CH2)2NH2]:(G=3);dendri PAMAM(NH2)32, a cationic polymer (MW6909) with a core of 1,4-diaminobutane. This molecule contains 32 surface amine groups, which allows for high affinity binding of nucleic acids, an important property that results in better polycationic polymer efficacy.
Taken together these results suggest that targeting nucleic acids with polycationic polymers could present a therapeutic strategy not only for autoimmune disorders but also for treatment of dangerous acute inflammation. We expect that the results shown for lupus will extend to other autoimmune disease wherein inflammation is associated with the pathology of the autoimmune disease, including but not limited to, psoriasis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid and associated skin diseases, cardiomyopathy, Celiac diseae, Celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, cutaneous necrotizing venulitis, discoid lupus, erythema multiforme, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic/autoimmune thrombocytopenia purpura (ITP), immunologic lung disease, immunologic renal disease, IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Ménière's disease, mixed connective tissue disease, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus-related disorders (e.g., pemphigus vulgaris), pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, spondyloarthropathies, systemic lupus erythematosis (SLE), lupus erythematosus, systemic vasculitis, takayasu arteritis, temporal arteristis/giant cell arteritis, thrombocytopenia, thyroiditis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Inflammation is a complex biological process that is necessary for clearance of pathogens. However, when acute inflammation turns chronic, it can lead to inflammatory disorders that are hard to control. Dead or dying cells release RNA and DNA into circulation. If these self-nucleic acids are not properly cleared, they can trigger activation of endosomal TLRs such as TLR7, 8 and 9. This in turn results in further downstream activation of signaling pathways and production of pro-inflammatory cytokines. In fact, multiple autoimmune disorders are characterized by elevated levels of circulating pro-inflammatory cytokines and auto-antibodies. To date, numerous studies and clinical trials have focused on addressing circulating self-RNA and DNA, TLR activation, proinflammatory cytokines and circulating auto-antibodies. Many of these drugs act on a single component or cell type of the inflammatory response, have short-term effects and do not break the TLR activation cycle. Additionally, a number of these compounds are associated with increased susceptibility to infection and decreased pathogen clearance.
The goal of our study was to break the cycle of aberrant inflammation by targeting self-nucleic acids prior to their binding of endosomal TLRs. To address our goals, we utilized a novel class of compounds, scavengers, which bind circulating nucleic acids and block TLR activation. Treatment of immune cells from both CD57/B16 wild type animals and NZBWF1 lupus prone animals with nucleic acid agonists in the presence of polycationic polymers resulted in diminished pro-inflammatory cytokine production (IL6 and TNF-a) in vitro. Cell activation through non-endosomal TLRs remained intact, thus further demonstrating the specificity of our compounds for nucleic acids. Importantly, these compounds inhibited nucleic acid-driven TLR activation in cultures of DCs derived from SLE-prone animals, suggesting that these compounds can potentially be effective in an autoimmune disease setting.
Polycationic polymers administered local and systemically in lupus prone animals improved disease outcomes in these animals. Endogenous nucleic acid-driven inflammation was diminished in the presence of polycationic polymers. In CLE models we observed reduced skin inflammation which resulted in improved disease pathology. Moreover, long-term SLE studies in the presence of polycationic polymers demonstrated the potential of these compounds to reduce levels of circulating auto-antibodies as well as decreased organ damage due to uncontrolled inflammation.
Chronic and unresolved inflammation is often associated with reduced platelet counts in patients as disease severity is increased. Here we demonstrate that treatment of lupus prone mice with polycationic polymers rescues platelet depletion thus making these compounds an important therapeutic agent, capable of acting on several different components of the inflammatory cascade as well as several different cell types.
TLR targeting has been attempted using several inhibitory compounds which bind directly to TLR7 and TLR9. Although direct inhibition of endosomal TLRs can in principle result in improved autoimmune disease outcomes, there is room for improvement. Nucleic acids are capable of activating a cohort of endosomal receptors, including but not limited to TRL7 and TLR9, thus rendering these compounds partially effective in multifaceted autoimmune disorders that rely on multiple types of receptors. Moreover, TLR7 gene mutations play an important role in disease severity and any therapies that directly target these receptors could potentially fail due to decreased mutant receptor binding. Our strategy does not rely on receptor binding, therefore, addressing a number of these concerns. We used PAMAM-G3 as the polycationic polymers in these studies due to its 32 surface amines that allow for high affinity binding of nucleic acids while lower generation PAMAM dendrimers were not as effective at inhibiting nucleic acid-mediated TLR activation in our previous in vitro studies with model TLR ligands. Additionally, generation 5 PAMAM has revealed increased toxicity in our previous and current studies at a similar dosing regimen. However our studies suggest that exploration of higher generation dendrimers with biodegradable properties or other polycationic polymers are warranted as they may further improve treatment outcomes. Moreover, a drug delivery device could prove to be an important strategy to deliver therapeutic agents slowly and uniformly and thereby increase their efficacy while eliminating any toxicity.
Lastly, our study addresses immunosuppression: a very important aspect of all therapeutic agents attempting to target aberrant inflammation. Chronic treatment with anti-inflammatory agents can lead to overall immune suppression and increased susceptibility to infections. To determine whether our therapeutic approach impacts immune responses to pathogens, we infected polycationic polymer treated animals with PR8 influenza to mimic human flu infection. We did not observe immune-suppression in treated animals, as polycationic polymers treated lupus prone mice were able to recover similarly to untreated controls. To our surprise C57B16 animals that received lethal doses of PR8 in the presence of polycationic polymer did not succumb to infection at the same rate as the control treated animals. These finding suggest that polycationic polymers may have broader application in not only controlling aberrant inflammation but in also improving the immune response to pathogens.
Thus polycationic polymers represent novel agents to potentially treat SLE as well as a wide variety of infectious diseases particularly those caused by highly pathogenic viruses such as pandemic influenza and Ebola. In the Examples we also demonstrate that administration of polycationic polymers is also capable of rescuing mice from lethal nucleic acid-induced inflammatory shock. Thus we expect that the polycationic polymers may be useful to treat viral or bacterial infections in which septic shock or large inflammatory responses are at least partially responsible for the pathology of the disease.
We have explored the use of polycationic polymerns to limit acute inflammatory shock and disseminated intravascular coagulation that are engendered following radiation exposure, specifically after 24 hours. Recent studies have demonstrated that lethal doses of ionizing radiation cause a release of both mitochondrial (mt) and nuclear DNA into the extracellular space and circulation.
As mentioned previously, extracellular DNA and RNA from damaged cells have been increasingly implicated in pathological inflammation and activation of the coagulation system, two hallmarks of Acute Radiation Syndrome. Recent studies by our group has illustrated that cationic polymers that can scavenge such extracellular nucleic acids can limit inflammation, counteract inflammatory shock and inhibit activation of the coagulation system and limit micro- and macro-vascular thrombosis. Our data demonstrates that these molecules dramatically improve survival when administered 24 hours following total body irradiation (TBI).
As used herein, the term Acute Radiation Syndrome (ARS), also referred to as radiation toxicity or radiation sickness, refers to the acute illness caused by irradiation of part, some, most or entire body by a high dose of penetrating radiation in a very short period of time. In some cases, usually a matter of minutes is all that is required to induce radiation sickness. As used herein, the term “lethal dose of radiation” refers to the dose or radiation expected to cause death to 50% of an exposed population with 30 days (LD 50/30). Typically, the LD 50/30) is in the range of from about 400 to 450 rem (4 to 5 sieverts) that is received over a very short period of time (e.g., matter of minutes).
The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. 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 facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.
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. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.
We synthesized several conjugates using a schematic similar to the generation of our biotinylated polycationic polymer (
Characterization studies on the polymers demonstrate a successful conjugation event. Shifts in excitation and emissions between the unreacted dyes and polycationic polymers are indicative of covalent bonding (
Synthesis using PAMAM G4 disulfide core as a starting material began by cleaving the disulfide bond using TCEP-HCL, resulting in a hemi-PAMAM G4 thiol. The intermediate PAM-SH was then reacted with maleimide conjugates at room temperature overnight with tri-methyl amine as a catalyst. Purification was performed by dialysis using specific molecular weight cutoffs dialysis membranes for 48 hours at 4° C. Lastly, the product was frozen and lyophilized for characterization studies and preliminary experiments. All reactions were performed in water unless otherwise specified.
Optimization of excitation and emission wavelengths was performed using SpectraMax i3 plate reader and the manufacturer's optimization software where the dyes were added to a 96-well all black plate and maximum excitations and emissions were plotted as a spectrograph heat map. See
Flow cytometry was performed with live murine macrophage (RAW) cells that were incubated with diluting concentrations of PAMSM-AF488. Cells were seeded at 1×105 cells/well in 12-well polystyrene plate overnight. Conjugated polycationic polymers were incubated with the cells for 30 min followed by extensive washing and trypsonization with PBS. Samples were then washed further before being analyzed by FACSCalibur in FL-1 (green) channel. See
Using a biotinylated cationic polymer (PAMSMB) with pancreatic cancer cells lines in vitro we are successful in scavenging pro-oncogenic damage associated molecular patterns (DAMPs) as seen by a reduction in toll-like receptors (TLRs) 3, 4, 7, and 9 activation.
Conditioned DAMP containing media is generated by exposing pancreatic cancer cell lines to radiation and allowed to incubate for three days, followed by the addition of a biotinylated cationic polymer conjugated to a streptavidin coated magnetic bead. The resultant supernatant is placed on reporter cells expressing TLR 3, 4, 7, 9 and a reduction in TLR stimulation was clearly observed in the samples that were treated with the polymer.
Inhibition of TLR activation was also seen using pancreatic cancer patient serum before and after radiation after ex-vivo scavenging. Though the difference in TLR stimulation between sera with and without polymer scavenging was not as pronounced when compared to the conditioned media samples, there was still a statistically significant difference suggesting the potential use of this biotinylated polymer as a potential therapeutic.
Using the biotinylated form of the polymer we have established a protocol to displace the DAMPs that are bound to the polymer using low-molecular weight heparin (LMWH). This method was validated by electrophoretic mobility shift assay (EMSA) and TLR stimulation.
DNA in plasmid form was bound to PAMAM-G3 via co-incubation at room temperature. Binding was verified via visualization on agarose gel. LMWH displacement was executed at 60° C. with agitation of the DNA-polymer complex. Displacement of the DNA was also verified by agarose gel visualization. This same process was repeated with TLR reporter cell assays wherein the successful binding of the DNA-polymer complex resulted in low TLR activity and successful displacement with LMWH resulted in elevated TLR activation. LMWH resulted in no activation alone.
We hypothesized that polycationic polymers might represent a useful treatment strategy for systemic lupus erythematosus (SLE) since SLE is associated anti-nucleic acid autoantibodies. Therefore we evaluated the therapeutic potential of the polycationic PAMAM-G3 in NZBW F1 mice. These animals develop an autoimmune disease resembling human SLE and are characterized by high levels of circulating auto-antibodies and pro-inflammatory cytokines First, to evaluate the potential utility of a polycationic in a localized model of lupus erythematosus, we used a well-established murine tape-stripping-induced dermal injury model, which mimics cutaneous lupus erythematosus (CLE) in humans. To determine if scavengers can bind extracellular nucleic acids and thereby limit pathology following skin injury in CLE-prone mice, NZBW F1 animals were subjected to tape stripping and subsequently treated with PAMAM-G3 subcutaneously.
We chose to employ generation-3 PAMAM (PAMAM-G3) in these studies because it binds RNA and DNA with high affinity and blocks TLR activation by artificial nucleic acid agonists in vitro and in vivo. By contrast, lower generation PAMAM molecules, with fewer than 32 surface amine groups, were not as effective as PAMAM-G3 at inhibiting CpG DNA and poly I:C RNA-mediated activation of TLRs. Higher generation PAMAM molecules (e.g. PAMAM-G5), are as efficacious as PAMAM-G3 at inhibiting nucleic acid-mediated TLR activation but they are associated with increased toxicity making it challenging to perform studies with them in lupus prone mice. To choose a therapeutic dosing regimen, we first determined the maximum tolerated dose (MTD) of PAMAM-G3 in NZBW F1 mice (100-200 mg/kg) and then administered this polycationic polymer 5-10 fold below the MTD. The CLE-prone mice were injured and then treated with PAMAM-G3 (20 mg/kg) twice per week for 14-21 days post injury. Strikingly, subcutaneous administration of PAMAM-G3 allowed CLE-prone mice to recover from skin damage much more effectively than control animals (
Previous studies in this CLE-prone mouse model have shown that skin damage leads to pronounced immune cell recruitment to the site of injury within 24 hours and that the resulting skin inflammation is dependent on signaling through nucleic acid sensing TLRs (TLR7 and TLR9). Therefore, we evaluated whether immune cell recruitment is affected by polycationic polymer treatment. NZBW F1 skin samples were obtained 24 hours post tape stripping and treatment with the polycationic polymer and the presence of immune cell infiltrates was analyzed. As shown in
Next we sought to determine if polycationic polymers can be useful for treatment of chronic inflammatory disease in a murine model of SLE. We evaluated the ability of PAMAM-G3 to reduce glomerulonephritis and circulating auto-antibody levels, hallmarks of SLE, in MRLlpr mice. As MRLlpr mice spontaneously develop SLE over a few months, we started treating 10-12 week old male MRLlpr mice twice a week with an intraperitoneal injection of PAMAM-G3 (20 mg/kg). After 10 weeks of polycationic polymer treatment, SLE-prone mice were analyzed for kidney damage, immune complex deposition in the kidney and levels of serum autoantibodies. As shown in
Since polycationic polymer treatment reduced kidney damage and glomerulonephritis, we next assessed how polycationic polymer treatment impacted complement deposition in the kidneys of MRLlpr mice. By 5 months of age, immunofluorescent staining for complement (C3c) deposits revealed that all lupus-prone mice not treated with a polycationic polymer had developed C3c deposits throughout the glomerulus (
In addition to renal dysfunction, progression of SLE in the MRCPlpr animals is characterized by increasing levels of circulating anti-nuclear and anti-DNA antibodies which further exacerbates autoimmunity in the lupus prone animals. To determine if long-term treatment with a polycationic polymer can impact the levels of anti-nuclear and anti-DNA antibodies in aging animals-prone to SLE, we treated MRCPlpr mice with PAMAM-G3 (20 mg/kg twice a week) for 2.5 months and performed ELISA and immunofluorescent assays on serum collected from the treated animals to determine autoantibody levels. The fluorescent antinuclear antibody (ANA) assay is clinically relevant and the most sensitive approach to detect serum antibodies against a variety of endogenous nuclear components in their native antigenic form. As shown in
Autoimmune disorders, including SLE are associated with decreased levels of circulating platelets. We assessed circulating platelet counts in SLE animals post polycationic polymer treatment. As shown in
Currently marketed autoimmune disease-combatting drugs and specifically lupus treatments result in severe immune suppression and an array of side effects. To determine whether polycationic polymer treatment results in immune suppression as well, we evaluated the effects of PAMAM-G3 treatment in a viral infection model in vivo using the same dosing strategy that proved effective in the lupus-prone mice (PAMAM-G3, 20 mg/kg, 2×/week). Lupus-prone animals were challenged intranasally with the mouse-adapted influenza A H1N1 strain, PR8 at a mouse lethal dose of 10% (mLD10) to determine if polycationic polymer treatment would result in increased morbidity and mortality. Mice were monitored daily and sacrificed if they lost >15% of their body weight. Polycationic polymer treatment did not increase morbidity, as monitored by weight loss, (
Our observation that polycationic polymer treatment may improve survival of lupus-prone mice when the animals are challenged at low mLDs, led us to investigate whether PAMAM-G3 might have beneficial effects on normal mice challenged with higher doses of influenza. Therefore, C57BL6/J mice were infected with a mLD50 of influenza A virus PR8 (H1N1) and treated with PAMAM-G3 at the time of viral challenge (20 mg/kg, 2×/week). Remarkably as shown in
Using the conjugated polymers provided above, we noted that the biotinylated PAMAM was able to bind to DNA both in and outside of cells. See
We evaluated the ability of the polycationic polymer PAMAM-G3 to rescue mice subject to lethal irradiation. BALB/c mice received 7.25 Gy of total body radiation. Mice were then treated by subcutaneous administration of the polycationic polymer PAMAM-G3 at a dose of 20 mg/kg or PBS vehicle 24 hours after irradiation. The treatment was repeated on day three after irradiation. Survival was monitored daily. Each group contained 10 animals. Similar experiments were obtained twice. This survival difference between PAMAM-G3 and the Vehicle treated groups is statistically significant with a P value=0.0057.
In 2009, we made the surprising observation that certain nucleic acid binding polymers can reverse nucleic acid aptamer binding to its target protein. This discovery led us to examine whether such polymers could act as NASs and inhibit the activity of proinflammatory nucleic acids that bind proteins involved in innate immunity. First, we examined whether such NASs could inhibit the activities of potent, prototypic nucleic acid molecules that are known to activate NA-sensing TLRs (TLR3, 7, 8 and 9). We discovered that certain NASs (named CDP, HDMBr, PAMAM-G3, PPA-DPA, poly L-lysine and protamine sulfate) can indeed counteract the activities of multiple nucleic acid based TLR agonists in vitro and in vivo (
C57BL/6, NZBW F1 and MRLlpr were obtained from the Jackson Laboratory (Bar Harbor, Me.). Mice were housed in a pathogen-free barrier facility at Duke University. Only male mice were used in all of our studies.
Tape Stripping: Tape stripping was performed on the dorsal area of the mice, post shaving, with standard size bandages, 20 strokes. PAMAM-G3 (20 mg/kg) (Sigma-Aldrich) was administered at the time of tape-stripping subcutaneously and every three days after injury. Animals were sacrificed at 14 days following injury. All skin samples were then harvested and fixed in 10% formalin for future histological analysis.
SLE long-term treatment: MRL-lpr male mice were injected with PAMAM-G3 (20 mg/kg) twice a week for a period of 8-10 weeks starting at 10 weeks of age. Mice were then sacrificed and blood and tissue were collected for further analysis.
PR8 infections: Mouse-adapted virus strain, influenza A/Puerto Rico/8/34 (H1N1; PR8) was obtained from Charles River. 10-week mice were anesthetized with vaporized isoflourane. Virus was administered intra-nasally in a total volume of 40 μL sterile pharmaceutical grade saline. Control mice were mock treated with pharmaceutical grade saline only. PAMAM-G3 was injected intraperitoneally at 20 mg/kg in a total volume to 200 μL of pharmaceutical grade saline. Weight loss and survival of infected mice was followed over a period of 14 days. Mice that lost 15% or more of their body weight were euthanized and recorded as dead per Duke University Institutional Animal Care and Use Committee guidelines.
B cell activation: B cells from spleens for NZBW F1 animals were purified by negative selection. Stimulation assays were performed as previously described (20)
DC culture: Murine bone marrow DCs were isolated from NZBW F1 mice and were cultured in the presence of GM-CSF (Peprotech) and IL-4 (Peprotech) as previously described (55). Stimulation assays were performed as previously described (20).
Virus microneutralization assay was performed as described previously (56) with modifications. Briefly virus and serum dilutions were performed as described and then mixed with 100 μl of freshly trypsinized MDCK-London cells containing 1.5×104 cells in 96-well cell culture treated plates. Negative controls consisted of cells alone, while positive controls contained virally infected cells. Plates were incubated for 20 hours before fixation with acetone. Endogenous biotin in wells was blocked with PBS containing 0.1% avidin (Life Technologies) for 15 minutes followed by washes and any bound avidin was blocked PBS containing 0.01% biotin (Sigma Aldrich) for 15 minutes. Plates were analyzed for positive infection via ELISA. Mouse monoclonal biotinylated anti-NP antibodies MAB8257B and MAB8258B (Millipore), dilution 1:6,000, and HRP-streptavidin conjugate (BD Biosciences), dilution 1:4,000 were used in the ELISA. Color was developed using OPD substrate (Sigma-Aldrich) in citrate buffer (Sigma-Aldrich), and optical density was measured at 490 nm in plate reader (Molecular Devices,). The highest serum dilution that generated >50% specific signal was determined to be the neutralization titer. 50% specific signal=(OD490 virus control—OD490 cell control)/2+OD490 cell control.
Skin lesion scoring was conducted in a blinded fashion by a trained veterinarian pathologist as previously described (28, 29). Briefly, skin samples were collected, fixed in 10% formalin, paraffin embedded (FFPE) and further processed for H+E staining. Epidermal thickness, degree of ulceration, intraepithelial inflammation, dermal inflammation and panniculus inflammation were assessed and graded on a scale from 0 to 3:0-normal skin architecture, 1-mild inflammation with slight epidermal hyperplasia, 2-moderate inflammation with noticeable epidermal hyperplasia-3-severe inflammation with marked epidermal hyperplasia. All parameters were scored separately and summed to reach a total disease score.
For kidney disease, kidneys were collected and further processed as FFPE samples for H+E staining or snap frozen in OCT for immunofluorescence staining. Glomerulonephritis scoring was done as previously described in a blinded fashion by a trained veterinarian pathologist (7). Briefly, kidneys were scored for glomerulonephritis on a scale of 1 to 4: 1-normal, 2-mild, 3-moderate and 4-severe. This scoring takes into account glomerular changes, interstitial changes and severity of lymphoplasmatic infiltration into the kidney.
Slides coated with Crithidia luciliae (Scimedx) were rehydrated with PBS for 30 minutes. Samples were then blocked for two hours (PBS, 0.1% Tween-20, 5% goat serum and 1% rat anti-mouse CD16/CD32). Serum samples were subsequently added to the slides at various dilutions (1:40-1:360). Serum Ab levels were detected using secondary antibody goat anti-mouse IgG FITC. Kidneys from treated animals were processed as described above. 5 μm sections were then stained with anti-complement antibody. All slides were mounted and images were acquired using a Zeiss Axiovert 500 confocal immunofluorescent microscope. Images were analyzed for staining intensity using image J.
Monoclonal Abs included: B220, CD45.2, CD4, CD8, IFN□, CD3, CD11c and GL7 (eBioscience). Single cell suspensions of cultured cells were counted and 106 cells were suspended in FACS buffer (PBS plus 2% FBS) and stained with various antibody combinations. In addition skin samples collected at 24-post injury were enzymatically digested with 0.28 u/ml Liberase 3 (Roche) for 30 minutes at 37° C. then treated with DNAse, filtered and stained with various antibodies. Flow cytometry was performed on a Gallios flow cytometer and FACSCanto. All data was analyzed with FlowJo software.
Statistical significance was determined with two-tailed Student's t test or analysis of variance (ANOVA). Long-rank Mantel-Cox test was performed on all survival curve graphs. All p values less than 0.05 were considered significant.
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No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 62/385,664, filed Sep. 9, 2016; U.S. Provisional Application No. 62/301,034, filed Feb. 29, 2016, and U.S. Provisional Application No. 62/250,700, filed Nov. 4, 2015, all of which are incorporated herein by reference in their entirety.
This invention was made with Government support under Federal Grant Nos. AI067798 and AI093960 awarded by the NIH. The Federal Government has certain rights to this invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2016/060652 | 11/4/2016 | WO | 00 |
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62250700 | Nov 2015 | US | |
62301034 | Feb 2016 | US | |
62385664 | Sep 2016 | US |