Patent Application
20120141549
Not applicable.
The present invention relates to methods and compositions for inhibiting inflammatory processes (such as asthma) in the lungs, wherein oligonucleotides targeting IgE receptors and NFkappaB are administered via an aerosolized microsphere formulation.
Allergic asthma is a complex inflammatory disease of the lungs characterized by variable airflow obstruction, airway hyperresponsiveness (AHR) and airway inflammation (1-7). The inflammatory process consists of a chronic infiltration by mast cells, basophils, eosinophils, dendritic cells and B/T-lymphocytes, each playing a distinct role as part of a network of local inflammation (2, 4, 6-12).
The key biomarker of allergic asthma is the IgE antibody, now considered to be the critical “gatekeeper” of the human allergic response (4, 10, 13-23). Mast cells and basophils are the main cellular modulators of IgE bioactivity and largely responsible for the generation of the autacoids that amplify the asthmatic phenotype in lungs (1, 6, 7, 10, 24-30). These autacoids affect not only the recruitment and function of other immune cells in the vicinity, but also influence the patency and remodelling of the environment (extracellular matrix, smooth muscle, blood vessels) given the close anatomic disposition of lung mast cells with airway, smooth muscle and vasculature (1, 6, 7, 10, 24-30).
Although the interaction of IgE with its specific allergen is important, the relationship between IgE and its receptors on cells is critical in allergic asthma. The hallmark of an allergic response is immediate hypersensitivity where IgE binds two cell surface receptors (FcERI and FcERII/CD23), although immediate hypersensitivity is mediated mainly via FcERI (also known as the high affinity IgE receptor) (1-3, 13, 14, 19-24, 31-43). Crosslinking of IgE-bound FcERI, especially on mast cells, leads to the early phase of the allergic response, involving mast cell degranulation and the synthesis of lipid mediators, cytokines and chemokines (1-3, 13, 14, 19-24, 31-43). These proteins will then initiate the late phase which peaks a few hours later and involves the recruitment and activation of other inflammatory cells at sites sensitive to allergen.
Allergens also activate IgE-sensitised antigen-presenting cells, like dendritic cells, which in turn promote IgE production by B-cells to replenish the IgE consumed in the allergic reaction, thereby maintaining mast cell and antigen presenting cell sensitisation (1-3, 6, 7, 11-14, 19-24, 27, 31-51). FcERI is highly expressed on tissue mast cells, quite likely as a result of IgE-mediated upregulated FcERI expression (1-3, 6, 7, 11-14, 19-24, 27, 31-51). FcERI is expressed as an abg2 tetramer on mast cells and basophils and as an ag2 trimer on human but not mouse monocytes, eosinophils and smooth muscle cells (13, 14, 20, 23-25, 28, 52-54).
IgE also binds to a low affinity receptor termed FcERII, and identified as CD23 (35-37, 39, 40, 55, 56). A C-type lectin, CD23 is constitutively expressed on B-cells, airway smooth muscle cells, dendritic cells, lung alveolar macrophages, neutrophils and eosinophils (35-37, 39, 40, 55, 56). IgE acts as a positive regulator of CD23 cell surface expression, just as is the case for FcERI and so does IL-4 (35-37, 39, 40, 55, 56). CD23 activation results in IL-1b, IL-6 and TNF-alpha production by macrophages and dendritic cells and IgE binding on B-cells and dendritic cells enhances the uptake, processing and presentation of allergen epitopes to allergen-specific T-cells and B-cells (35-37, 39, 40, 55, 56). Degranulation and production of autacoids is dependent on immediate signaling of FcERI, however, late signaling events require transcriptional activation and maintenance of gene expression of autacoids and enzymes involved in their biosynthesis (1, 11, 25-27, 57).
NF-kB is a critical regulator of gene transcription in inflammation and is responsive to FcERI signaling in mast cells and dendritic cells (11, 17, 44, 46, 57-59). NF-kB consists of a number of homodimeric and heterodimeric proteins that belong to the Rel family and collectively transactivate proinflammatory genes in a wide variety of immune cells including mast cells, basophils, dendritic cells and lymphocytes (60-64). NF-kB signaling is controlled by a complex series of kinase networks at multiple levels in response to extracellular receptor and non-receptor signals (60-64). Pharmacologic inhibitors of NF-kB and upstream kinases are currently at various stages of development for inflammatory disease including allergy (17, 44, 46, 47, 57, 58, 60-68). Many of the proinflammatory cytokines and enzymes involved in autacoid synthesis expressed in activated mast cells, basophils and dendritic cells are regulated by NF-kB (17, 44, 46, 47, 57, 58, 60-68) and are downregulated in response to NF-kB inhibitors.
Given the role of the IgE molecule in the asthmatic inflammatory response, reducing its levels or inhibiting its actions on mast cells, basophils and dendritic cells could have a major impact on asthma prevention and therapy of active disease. A humanised monoclonal antibody directed against IgE is now in clinical use (Omalizumab; Xolair) and an anti-CD23 antibody (IDEC-152) is also in development after exhibiting a good safety profile (3, 5, 9, 19, 22, 34, 36-38, 42, 43, 55). These therapies, attractive as they are, do not completely eliminate IgE levels. “Escaped” IgE can therefore still activate acute processes in mast cells, basophils and dendritic cells. In alternate approaches, antisense DNA technology has been used to downregulate FcERI and NF-kB (44, 69-72) or other molecules involved in allergic asthma (Respirable AntiSense OligoNucleotides; “RASONs”; 73-78), but successful application of these methodologies has been impeded by the short half-life of ODNs and the need for an effective concentration of molecules to accumulate in lung target tissue.
The present invention relates to methods and compositions for inhibiting inflammatory processes (such as asthma) in the lungs, wherein oligonucleotides (“ONs”) targeting IgE receptors and NFkappaB are administered via an aerosolized microsphere formulation. It is based, at least in part, on the discoveries that (i) when this mixture of ONs is administered to mast cell and dendritic cell lines in vitro, the production of proinflammatory cytokines is considerably attenuated, and (ii) microspheres were found to reach a large population of lung cells following intratracheal instillation.
For purposes of clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) therapeutic ONs;
(ii) microsphere formulations; and
(iii) methods of treatment.
ONs that may be used according to the invention may target the high-affinity IgE receptor FcERI, the low-affinity IgE receptor CD23, or the p65 subunit of NfkappaB. For human therapeutic applications, preferably the human mRNA sequence is the basis for the target, but the invention also provides for use of non-human sequences that may be used either in animal model systems for confirmation of therapeutic effects and for dosage adjustment, for example, but not limited to, the murine sequence or a related rodent sequence, or for the treatment of non-human animals suffering from airway inflammation.
FcERI occurs on cells as a tetramer composed of an alpha, beta, and two disulfide-linked gamma chains. Genes encoding any of these subunits may be targets according to the invention. As one non-limiting example, the target may be the human alpha subunit for example, and not by way of limitation, having at least a portion of the sequence as set forth in SEQ ID NO: 5. As another non-limiting example, the target may be the murine alpha subunit for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 6. As another non-limiting example, the target may be the human beta subunit for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 7. As another non-limiting example, the target may be the murine beta subunit for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 8 or a sequence which is at least 95 percent homologous thereto. As another non-limiting example, the target may be the human gamma subunit for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 9. As another non-limiting example, the target may be the murine gamma subunit for example, and not by way of limitation, having a sequence as set forth in SEQ ID NO: 10.
In further non-limiting embodiments of the invention, an ON may target CD23. As one non-limiting example, the target may be human CD23 for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO 11: As another non-limiting example, the target may be murine CD23 for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 12.
In further non-limiting embodiments of the invention, an ON may target NFkappaB. As one non-limiting example, the target may be human NFkappaB for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 13. As another non-limiting example, the target may be murine NfkappaB for example, and not by way of limitation, having at least a portion of a sequence as set forth in SEQ ID NO: 14. In further non-limiting embodiments of the invention, an ON may target a binding sequence of the NF-KappaB transcription factor where the ON is a decoy for the CONSENSUS BINDING SEQUENCE OF NF-kappaB and its homologues (i.e. RelB and all other transcription factors binding to the NF-kappaB consensus sequence (SEQ. ID 5′-CTGGGGACTTTCCAT-3′ (SEQ ID NO:1)); for example but not by way of limitation the ON may comprise (5′-CTGGGGACTTTCCAT-3′ (SEQ ID NO:1)), for example in a double stranded duplex where a first strand comprises an ON 1=5′-CTGGGGACTTTCCAT-3′ (SEQ ID NO:1) and a second strand comprises ON2=5′ ATGGAAAGTCCCCAG 3′ (SEQ ID NO:2).
An ON according to the invention may be entirely or predominantly comprised of deoxyribonucleotides (an “ODN”) or entirely or predominantly comprised of ribonucleotides (an “ORN”), and may comprise one or more modified nucleotide, for example, nucleotides joined via a phosphorothioate bond. An ON may be an antisense ON, a catalytic ON, or an RNAi or siRNA ON, as are known in the art. An ON may be single stranded or double stranded or have single stranded as well as double stranded portions. In preferred non-limiting embodiments, the ON is an ODN.
An “antisense ON” refers to an ON that is complementary to at least a portion of a primary transcript or mRNA and that inhibits the expression of a target gene (see U.S. Pat. No. 5,107,065). The complementarity of an antisense ON may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. An antisense ON may be between about 10 and 100 nucleotides, or between about 10 and 50 nucleotides, or between about 15 and 100 nucleotides, or between about 15 and 50 nucleotides, or between about 10 and 35 nucleotides, or between about 15 and 35 nucleotides.
A catalytic ON is an ON that has enzyme catalytic activity and comprises a portion homologous to its target gene. For example, but not by way of limitation, a catalytic RNA may be referred to as a “ribozyme” and may catalyze hydrolysis and/or aminotransferase activity.
siRNA typically comprises a polynucleotide sequence identical or homologous (e.g., at least 90 percent or at least 95 percent or at least 98 percent homologous) to a portion of a target gene linked directly, or indirectly, to a polynucleotide sequence complementary to the sequence of the target gene (or fragment thereof) (see, for example, Hunter, 1999, Curr. Biol. 9:R440-442; Hamilton el al., 1999, Science 286:950-952; Ding, 2000, Curr. Opin. Biotechnol. 11:152-156). In a nonlimiting example, a RNAi or siRNA molecule may be between about 6 and 50 nucleotides. In particular nonlimiting examples, the RNAi or siRNA molecule is between about 10 and 35 nucleotides, or between about 10 and 25, or between about 15 and 25 nucleotides. An siRNA may optionally comprise a polynucleotide linker sequence of sufficient length to allow for the two polynucleotide sequences to fold over and hybridize to each other; however, a linker sequence is not necessary. The linker sequence is typically designed to separate the antisense and sense strands of siRNA sufficiently as to limit the effects of steric hindrance and allow for the formation of dsRNA molecules and preferably does not hybridize with sequences within the hybridizing portions of the siRNA molecule.
Ribozymes, antisense polynucleotides, and siRNA molecules may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation) may be used to transcribe the RNA strand {or strands); the promoters may be known inducible promoters such as baculovirus. Inhibition may be targeted by specific transcription in an organ, tissue, or cell type. The RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
The ON may be chemically or enzymatically synthesized by manual or automated reactions. The ON may be synthesized by a cellular nucleic acid polymerase or a bacteriophage polymerase (e.g, T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the ON may be purified prior to use. For example, an ON can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. The ON may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
Homology may be determined using standard techniques, for example using software known in the art such as BLAST or PASTA.
Specific, non-limiting examples of ON that may be used according to the invention include: homologous FcERI-alpha (human/mouse from 3′ end of sequence): gtg tee aca gca aac aga ate (SEQ ID NO:3); homologous (mouse/human) CD23 (also from 3′ end): gca gaa ggc gte gtt cca {SEQ ID NO:15); mouse FcERI-alpha: cca gtg acc ate gcc ect g (SEQ ID NO:16); mFcERIa control: cag ggg cga tgg tea ctg g (SEQ ID NO:17); hFcERlalpha-1: aca gta gag tag ggg att cca tgg cag gag cca tct tct tea tgg act cc (SEQ ID NO:18); hFcERlalpha-2: tte aag gag ace tta ggt ttc tga ggg act get aac acg cca tet gga ge (SEQ ID NO:19); and hFcERII (CD23): tet ctg aat att gae ctt cct cca tgg egg tee tgc ttg gat tet ccc ga (SEQ ID NO:20).
Activity of an ON according to the invention may be determined by an in vitro assay, for example in a cell-free system or cell culture, to test the ability of the ON to inhibit the expression of its target protein. As specific, non-limiting examples, validation of inhibitory activity may be performed using rodent and human mast cell and basophil cell lines such as LAD2, P815, Ku812 and MC9 by FACS ascertainment of surface levels of target and by steady state quantitative RT-PCR and/or Western blot analysis. Dendritic cells lines may also be used in the testing. As one potential criterion, an ON may be selected for use or further study based on its demonstrating, in cell culture, relatively less , off-target knockdown activity using genome-wide expression array technology on cell line transcripts (88-91). In addition or alternatively, the activity of the ON may be assessed by determining its affect on autacoid and cytokine production in culture supernatants, for example using ELISA/Luminex 24 hours following FcERI crosslinking using IgE:DNP-HSA exposure, or by ascertaining nuclear p65 NF-kB levels by standard mobility shift assay (49, 51, 68, 69, 79, 92-97).
Additionally, activity of an ON may be confirmed using an in vivo assay. For in vivo testing an ON may preferably be comprised in a microsphere, as discussed below. As a specific, non-limiting example, ON, preferably comprised in microspheres, may be administered to Balb/c mice by aerosol into the respiratory tract using a Bennet nebuliser. The mice may be placed inside a Plexiglas chamber and may be administered 10-200 micrograms of dry ON-containing powder per mouse, where the average diameter of each ON-containing particle is preferably about 1-2 microns and the flow rate may be from 10-20 liters/min for 20-60 minutes. Comparisons may be made to mice exposed to formulated “scrambled” oligonucleotide sequences and, where microspheres are used, to “empty” microspheres. At various times after treatment, mice in the treatment and non-treatment groups may be euthanized and the effectiveness of ON at inhibiting expression and/or functionality of target may be assessed, for example by measuring the levels of FcERI alpha, beta and gamma chain, CD23 and p65 NF-kB steady state mRNA in bronchiolar lavage fluid cells and/or by FACS measurement of cell surface FcERI a chain and CD23 in parallel cells. Efficacy may also be assessed in bioassays in vitro, where bronchiolar lavage fluid cells isolated from treated mice may be exposed to FcERI (FcERI crosslinking by IgE-dinitrophenyl-HSA exposure) and NF-kB activation (exposure to LPS) followed by cytokine and autacoid measurement in culture supernatant along with ascertainment of the degree of FcERI-dependent intracellular signaling (phospho-Syk, phospho-Lyn by Western blot) (12, 49-51, 68, 69, 79, 92-106).
Effectiveness of an ON may also be assessed in a model for asthma, for example but not limited to the ovalbumin-induced and the house dust mite allergen-triggered models of acute and chronic allergic asthma (reviewed in 107, 108). Specifically, airway function may be measured using the flow interrupter method on the basis of the response of total respiratory system resistance to saline and increasing doses of intravenous methacholine as well as whole body plethysmography to calculate enhanced pause response to methacholine (12, 50, 98-106). In addition to these physiologic measurements of airway function, lung histology may be assessed and morphometry may be performed to determine extracellular matrix (and collagen) deposition and remodeling, airway epithelium mucus content, smooth muscle cell proliferation, density and morphometry of goblet cell hyperplasia.
The present invention provides for microspheres that carry one or more ON, as described above, and for formulations comprising said microspheres.
In certain embodiments, microspheres carry one or more species of ON that targets the high-affinity IgE receptor FcERI.
In certain embodiments, microspheres carry one or more species of ON that targets the low-affinity IgE receptor CD23.
In certain embodiments, microspheres carry one or more species of ON that targets the p65 subunit of NfkappaB.
According to certain embodiments of the invention, a plurality of (two or more) different ONs as described above are comprised in a microsphere formulation. In such embodiments, an individual microsphere may carry only one species of ON, and a therapeutic formulation may comprise a mixture of microspheres bearing different ONs, or, alternatively, an individual microsphere may itself carry a mixture of ONs, or, alternatively, a combination of microspheres some carrying one species of ON and others carrying more than one species of ON may be used.
A microsphere is a particle having a diameter of between about 10 nanometers and 10 micrometers or between about 100 nanometers and 1 micrometer or between about 200 nm-2 micrometers or between about 200-400 nanometers.
ON-carrying microspheres may be prepared by methods known in the art. In particular, non-limiting embodiments, the microspheres may be prepared using a method as set forth in Kovacs et al., 2009, J. Biomaterials Science 20:1307-1320 or Zheng et al., 2006, J. Biomater. Sci. Polymer Edn. 17(12):1389-1403, both incorporated by reference in their entireties herein.
In particular, non-limiting embodiments of the invention, polycation-coated poly(D,L-lactide-co-glycolide) (“PLGA”) microspheres may be used as carriers of ONs into the lungs. The PLGA microspheres preferably have an average hydrodynamic size between 300-500 nm, such that they may be rapidly internalized by phagocytic cells and are sufficiently small to pass through conducting airways to reach the deep lung, yet with density high enough to prevent them from being exhaled (for example, a density of between about 0.5-1 g/cm3. In particular non-limiting embodiments, the polycation may be a peptide. Said cationic peptide may be of a length between about 8 and 25 amino acids and may preferably comprise between about 10 and 20 cationic amino acid residues such as histidine, ornithine or ariginine to facilitate ODN uptake and to promote ODN escape from endosomes or phagosomes upon internalization (80). In a specific, non-limiting embodiment, the cationic peptide comprises Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-Orn-His-His-His-His-His-His (SEQ ID NO:4).
In one specific, non-limiting embodiment, the microsphere may be a PLGA-Ni particle prepared using a double emulsion (w/o/w) solvent evaporation method. 90 mg PLGA and 0.6 mg 1,2-dioleoyl-m-glycero-3-((N(5-amino-1-carboxypentyl)-imino-diacetic acid]-succinyl) nickel salt)) (“DOGS-NTA-Ni”) may be dissolved in 3 ml methylenechloride. To this organic phase 200 μL double distilled water may be added and the mixture sonicated (20 W) for 2 minutes. This primary emulsion may be added to 20 ml double distilled water and the resulting mixture may be allowed to evaporate for four fours to remove the methylenechloride. Particles may be washed twice in double distilled water and recovered by centrifugation. The resulting solids may be confirmed using Fourier transform infrared spectroscopy to contain DOGS-NYA-Ni. ON (preferably ODN)loading may be accomplished by first coating the particles (30 μL from 4.5 mg/ml stock) with O10H6 (ornithine (10)-histidine (6); 60 μg), SEQ ID NO:4 followed by addition of one or more type of ON (0.06 nmoles) in 300 μl double distilled water. Equilibration of each component may be achieved using gentle shaking for 30 minutes at room temperature.
In another specific, non-limiting embodiment of the invention, PLGA/O10H6 microspheres may be prepared using a double emulsion (w/o/w) solvent evaporation technique (Jain, 2000, Biomaterials 21:2475). 5 μg O10H6 may be added to 200 μL of an aqueous solution containing 40 μg of one or more type of ON (preferably an ODN) and gently shaken for 0.5 hours at room temperature. This aqueous solution may be incorporated into a methylene chloride solution of PLGA (3% w/v) by sonication. A Fisher model 100 sonic microprobe may be used to introduce 24 W of energy (over 2 minutes) to form this water-in-oil emulsion. The primary emulsion may be added drop-wise into 20 ml of an aqueous solution of PVA (0.5% w/v) to form a water-in-oil-in-water emulsion. The resulting double emulsion may be stirred for about 4 hours in a chemical fume hood to allow the methylene chloride to evaporate. Particles may be recovered by ultracentrifugation and washed twice with double distilled water to remove excess PVA and un-trapped ON.
ON-carrying microspheres may optionally be freeze-dried, stored, and reconstituted in distilled water. Addition of sucrose (2% w/v) prior to freeze-drying may improve recovery.
The formulation comprising ON-carrying microspheres may be a suspension, an emulsion, or a dry powder.
Formulations to be used therapeutically may further comprise a suitable pharmaceutical carrier , optionally a preservative, and optionally one or more additional active agent, for example, but not limited to, a bronchodilator such as theophylline, albuterol, salmeterol, formoterol, a leukotriene modulator such as Montelukast, Zafirlukast, or Zileuton, and/or an anti-inflammatory agent such as fluticasone, budesonide, mometasone, beclomethasone, or ciclesonide and/or a mast cell stabilizer such as cromolyn or nedocromil, or an antibiotic, in therapeutically beneficial concentrations. The formulation may optionally comprise a propellant and/or additional excipients as appropriate.
In certain non-limiting embodiments, the present invention provides for a method of treating an inflammatory lung disorder comprising administering, to a subject in need of such treatment, an effective amount of microspheres carrying a mixture of ONs comprising at least one ON that inhibits expression of an IgE receptor and at least one ON that inhibits expression of NFkappaB. In specific, non-limiting embodiments of the invention, the following combinations of ON may be used:
In certain specific non-limiting embodiments, the present invention provides for a method for reducing an inflammatory response in the lungs, for example in the context of treating asthma, comprising administering, to a subject in need of such treatment, an effective amount of microspheres carrying an ON that targets FcERI alpha. For example, but not by way of limitiation, reduction in the inflammatory response may be measured as a decrease in a cytokine selected from the group consisting of GM-CSF, IFNg, IL-9, IL-15, IL-17, KC, and TNFa.
The subject may be a human subject or a non-human subject such as a non-human primate, a rodent such as a mouse or rat, a dog, a cat, a horse, etc.
Disorders which may be treated according to the invention include but are not limited to asthma (e.g. bronchial asthma) and other IgE-related inflammatory airway disorders.
A formulation comprising an effective amount of ON-bearing microspheres as set forth above may be administered via the airway of a subject in need of such treatment. Administration to the lungs may be performed using devices known in the art, including but not limited to a nebulizer, a metered dose inhaler, or a dry powder inhaler. As an alternative to administration via the mouth, the formulation may be administered through the nose. In a specific, non-limiting embodiment of the invention, the flow rate of delivery of microspheres into the lungs is between about 10-20 L/minute for between about 20-60.
An effective amount of ON means an amount that results is an amelioration of pulmonary performance, for example, but not limited to, a subjective reduction of respiratory distress, a reduced cough, an increase in the forced expiratory volume, and/or an increase in the forced expiratory volume over 1 second/ forced vital capacity ratio, where the increase may be, for example and not by way of limitation, by at least 5 percent, or by at least 10 percent, or by at least 15 percent or by at least 20 percent or by at least 25 percent.
In specific, non-limiting embodiments of the invention, the amount of ON administered per kilogram of a subject may be between about 1-100 or between about 10-50 or between about 1-10.
In non-limiting embodiments of the invention, ON-bearing microspheres may be administered either as needed, or once a day, or at least once a day, or twice a day, or three times a day, or four times a day, or once every other day, or twice a week, or once a week, or once every two weeks, or once a month. Treatment can be on a one-time basis, using a regimen as described in the previous sentence for a limited period of time, continuously, or using a regimen interrupted by a period of non-treatment.
Microspheres made with poly-(d,l-lactide-co-glycolide), or PLGA, SEQ ID NO. 4 loaded with ODN were prepared by a double emulsion solvent evaporation method. 90 mg PLGA was dissolved in 3 ml MeCl2. To this organic phase 200 μL deionized water was added and sonicated (20 W) for 2 min. This primary emulsion was added to 20 ml of deionized water containing 0.8% polyvinyl alcohol and the resulting mixture was allowed to evaporate for 4 h to remove MeCl2. Particles were washed twice in deionized water and recovered by centrifugation. Particles coated with SEQID NO. 4 were loaded with ODN.
The resulting microsphere had an average diameter of 280 nm with ρ˜1 g/cm3.
Experiments were then performed to evaluate the effect of FcERI ODN-loaded microspheres in a murine model for asthma induced by ovalbumin (“OVA”) [107, 108]. Briefly, six to twelve week old Balb/c mice were sensitized on day 0 by intraperitoneal injection of 50 ug chick ovalbumin (OVA) and 2 mg alum in 200 microliters PBS. On Day 6 mice were pretreated with 30 microliters (about 50 micrograms) of particle suspension (particles were loaded with antisense FcERIa oligonucleotides at a ratio of 18.5 micrograms DNA per mg particles). The mice were placed under deep anaesthesia by inhalation of 3% isoflurane for about 5 minutes. Mice were held by the scruff of the neck in an upright supine position and 15 microliters of the particle suspension was pipetted on to each nares. Inhalation of the particle suspension was verified by audible glottal clicking as the fluid passed into the trachea. On Days 7, 8, 9, and 10 mice were again treated with 50 micrograms of AS-FcERIa particles. Following treatment, the mice were returned to their cages and allowed to recover. Thirty minutes following treatment with particle suspension, mice were challenged intranasally with 50 micrograms ovalbumin. The procedure is identical to intranasal delivery of particle suspension. Mice received 25 microliters of a 1 microgram/microliter solution of ovalbumin in PBS applied to each nares. On day 10, three hours following the final ovalbumin challenge, bronchoalveolar lavage was performed to recover intrapulmonary exudate and leukocytes.
In a first series of studies, the amount of histamine present in BALF was compared in mice either with or without OVA-induced asthma as early as 24 hours following the last OVA challenge, treated or untreated with FcERIa ODN microspheres. As can be seen in
In another series of experiments, the BALF fluid of mice with or without OVA-induced asthma, either treated with FcER1 antisense ODN-loaded microspheres, PBS, or empty microspheres, was tested for the concentrations of various cytokines, including granulocyte colony-stimulating factor (“G-CSF”), granulocyte macrophage colony-stimulating factor (“GM-CSF”), interferon gamma (“IFNg”), interleukin-1beta (“IL-1b”), interleukin-2 (“IL-2”), interleukin-4 (“IL-4”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”), interleukin-9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12 (p70) (“IL-12(p70)”), interleukin-13 (“IL-13”), interleukin-15 (“IL-15”), interleukin-17 (“IL-17”), interferon-gamma-induced protein 10 (“IP-10”), keratinocyte-derived cytokine (“KC”), monocyte chemotactic protein-1 (“MCP-1”), macrophage inflammatory protein-1a (“MIP-1a”), the cytokine known as RANTES (for regulated upon activation, normal T cell expressed and secreted), and tumor necrosis factor alpha (“TNFa”). The results are shown in
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| Number | Date | Country | |
|---|---|---|---|
| 61406409 | Oct 2010 | US |
