Patent Application
20140163085
The present invention relates to oligonucleotides useful in the treatment of inflammation and respiratory diseases, and specifically to oligonucleotide inhibitors of phosphodiesterase expression.
The alveolar and airway epithelium is recognized as a dynamic barrier that plays an important role in regulating inflammatory and metabolic responses to oxidative stress, sepsis, endotoxemia, and other critical illnesses in the lung. The respiratory epithelium, in particular, is a primary target of inflammatory conditions/infections at the epithelial-blood interface, and is itself capable of amplifying an inflammatory signal by recruiting inflammatory cells and producing inflammatory mediators.
Chronic Obstructive Pulmonary Disease (COPD) is one example of an inflammatory airway and alveolar disease where persistent upregulation of inflammation is thought to play a role. Inflammation in COPD is characterized by increased infiltration of neutrophils, CD8 positive lymphocytes, and macrophages into the airways. Neutrophils and macrophages play an important role in the pathogenesis of airway inflammation in COPD because of their ability to release a number of mediators including elastase, metalloproteases, and oxygen radicals that promote tissue inflammation and damage. It has been suggested that inflammatory cell accumulation in the airways of patients with COPD is driven by increased release of pro-inflammatory cytokines and of chemokines that attract the inflammatory cells into the airways, activate them and maintain their presence. The cells that are present also release enzymes (like metalloproteases) and oxygen radicals which have a negative effect on tissue and perpetuate the disease. A vast array of pro-inflammatory cytokines and chemokines has been shown to be increased within the lungs of patients with COPD. Among them, an important role is played by tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8), which are increased in the airways of patients with COPD.
Other examples of respiratory diseases where inflammation seems to play a role include: asthma, eosinophilic cough, bronchitis, cystic fibrosis, pulmonary hypertension, acute respiratory distress syndrome (ARDS), acute and chronic rejection of lung allograft, sarcoidosis, pulmonary fibrosis, rhinitis and sinusitis.
Asthma is defined by airway inflammation, reversible obstruction and airway hyperresponsiveness. In this disease the inflammatory cells that are involved are predominantly eosinophils, T lymphocytes and mast cells, although neutrophils and macrophages may also be important. A vast array of cytokines and chemokines have been shown to be increased in the airways and play a role in the pathophysiology of this disease by promoting inflammation, obstruction and hyperresponsiveness.
Eosinophilic cough is characterized by chronic cough and the presence of inflammatory cells, mostly eosinophils, within the airways of patients in the absence of airway obstruction or hyperresponsiveness. Several cytokines and chemokines are increased in this disease, although they are mostly eosinophil directed. Eosinophils are recruited and activated within the airways and potentially release enzymes and oxygen radicals that play a role in the perpetuation of inflammation and cough.
Acute bronchitis is an acute disease that occurs during an infection or irritating event for example by pollution, dust, gas or chemicals, of the lower airways. Chronic bronchitis is defined by the presence of cough and phlegm production on most days for at least 3 months of the year, for 2 years. One can also find during acute or chronic bronchitis within the airways inflammatory cells, mostly neutrophils, with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and mucus production that occur during these diseases.
Lung transplantation is performed in patients with end stage lung disease. Acute and more importantly chronic allograft rejection occur when the inflammatory cells of our body, lymphocytes, do not recognize the donor organ as “self”. Inflammatory cells are recruited by chemokines and cytokines and release a vast array of enzymes that lead to tissue destruction and in the case of chronic rejection a disease called bronchiolitis obliterans.
Sarcoidosis is a disease of unknown cause where chronic non-caseating granulomas occur within tissue. The lung is the organ most commonly affected. Lung bronchoalveolar lavage shows an increase in mostly lymphocytes, macrophages and sometimes neutrophils and eosinophils. These cells are also recruited and activated by cytokines and chemokines and are thought to be involved in the pathogenesis of the disease.
Pulmonary fibrosis is a disease of lung tissue characterized by progressive and chronic fibrosis (scarring) which will lead to chronic respiratory insufficiency. Different types and causes of pulmonary fibrosis exist but all are characterized by inflammatory cell influx and persistence, activation and proliferation of fibroblasts with collagen deposition in lung tissue. These events seem related to the release of cytokines and chemokines within lung tissue.
Acute rhinitis is an acute disease that occurs during an infection or irritating event, for example, by pollution, dust, gas or chemicals, of the nose or upper airways. Chronic rhinitis is defined by the presence of a constant chronic runny nose, nasal congestion, sneezing and pruritis. One can also find within the upper airways during acute or chronic rhinitis inflammatory cells with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and mucus production that occur during these diseases.
Acute sinusitis is an acute, usually infectious disease of the sinuses characterized by nasal congestion, runny, purulent phlegm, headache or sinus pain, with or without fever. Chronic sinusitis is defined by the persistence for more than 6 months of the symptoms of acute sinusitis. One can also find during acute or chronic sinusitis within the upper airways and sinuses inflammatory cells with a broad array of chemokines and cytokines. These mediators are thought to play a role in the inflammation, symptoms and phlegm production that occur during these diseases.
There is a growing body of evidence suggesting an intimate link between inflammation and neoplastic diseases. The tumor microenvironment is shaped by cells entering it, and their functions reflect the local conditions. Successive changes occurring at the tumor site during tumor progression resemble chronic inflammation. This chronic inflammatory reaction seems to be largely orchestrated by the tumor, and it seems to promote tumor survival. It has become evident that early and persistent inflammatory responses observed in or around developing neoplasms regulates many aspects of tumour development (matrix remodelling, angiogenesis, malignant potential) by providing diverse mediators implicated in maintaining tissue homeostasis, e.g., soluble growth and survival factors, matrix remodelling enzymes, reactive oxygen species and other bioactive molecules.
As described above, these inflammatory respiratory diseases or diseases in which inflammation plays a critical role are all characterized by the presence of mediators that recruit and activate different inflammatory cells which release enzymes or oxygen radicals causing symptoms, the persistence of inflammation and when chronic, destruction or disruption of normal tissue.
A therapeutic approach that would decrease pro-inflammatory cytokine and chemokine release by a vast array of cells while having a reduced effect on the release of anti-inflammatory mediators or enzymes may have an advantage over current therapies for inflammatory respiratory diseases or any other systemic inflammatory disease.
The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers participating in signaling transduction pathways and mechanisms. Both cAMP and cGMP are formed from their respective triphosphates (ATP and GTP) by the catalytic activity of adenylyl (adenylate) or guanylyl (guanylate) cyclase. Inactivation of cAMP/cGMP is achieved by hydrolytic cleavage of the 3′-phosphodiester bond catalyzed by the cyclic-nucleotide-dependent phosphodiesterases (PDEs), resulting in the formation of the corresponding, inactive 5′-monophosphate. It has been shown that the inflammatory response and its progression is exquisitely sensitive to modulations in the steady-state levels of cyclic nucleotides, where target cells for their effects extend beyond immune cells to include accessory cells, such as airway smooth muscle, epithelial and endothelial cells, and neurons. The cyclic nucleotide PDEs are a large, growing multigene family, comprising at least 11 families of PDE enzymes. The profile of selective and nonselective PDE inhibitors in vitro and in vivo, therefore, suggests a potential therapeutic utility as antidepressants, antiproliferative, immunomodulatory, tocolytics, inotropes/chronotropes, and cytoprotective agents. Use of antisense oligonucleotides (“AONs”) directed against PDE are described in co-owned WO 05/030787 and WO 07/134,451.
For potential clinical uses, oligonucleotides should exhibit stability against degradation by serum and cellular nucleases, show low non-specific binding to serum and cell proteins, exhibit enhanced recognition of the target mRNA sequence, demonstrate cell-membrane permeability and elicited cellular nucleases when complexed with complementary mRNA. It is well documented that oligonucleotides containing natural sugars (D-ribose and D-2-deoxyribose) and phosphodiester (PO) linkages are rapidly degraded by serum and intracellular nucleases, which limit their utility as effective therapeutic agents. Chemical strategic modifications have been described for oligonucleotides in order to improve their stability and efficacy as therapeutic agents. The main chemical changes included, modification of the sugar moiety, the base moiety, and/or modification or replacement of the internucleotide phosphodiester linkage. To date the most widely studied analogues are the phosphorothioate (PS) oligodeoxynucleotides, in which one of the non-bridging oxygen atoms in the phosphodiester backbone is replaced with a sulfur.
For example, some PO linkages within immunostimulatory sequences such as CpG-containing oligonucleotides have been replaced with PS linkages to increase the stability of the oligonucleotide. However, generally PO linkages within the CpG motif are required and therefore a mixed PO/PS backbone is needed for maximal immunostimulatory activity of the CpG-containing oligonucleotides.
There are examples of antisense oligonucleotides with PO/PS linkages and incorporation of FANA modifications. However, such modifications can reduce the activity of the antisense oligonucleotide, as described in co-owned WO09/137,912.
Other modifications to oligonucleotides include the replacement of adenosine residues with 2-amino-2′-deoxyadenosine (DAP), as described in co-owned WO 03/004511.
It would be desirable to have improved oligonucleotides directed against PDEs for use in treating inflammation and respiratory diseases.
In an aspect, there is provided an oligonucleotide directed against a target Phosphodiesterase (PDE), wherein the oligonucleotide is capable of hybridizing to at least a portion of a nucleic acid sequence encoding the PDE under stringent conditions, and wherein,
In a further aspect, there is provided a pharmaceutical composition comprising one oligonucleotide described herein and a pharmaceutically acceptable carrier.
In a further aspect, there is provided the pharmaceutical composition described herein for the treatment of inflammation.
In a further aspect, there is provided the pharmaceutical composition described herein for treatment of respiratory disease.
In a further aspect, there is provided a method of treating respiratory disease in a subject comprising administering the pharmaceutical composition described herein.
In a further aspect, there is provided the use of the pharmaceutical composition described herein in the preparation of a medicament for the treatment of respiratory disease.
In a further aspect, there is provided the use of the pharmaceutical composition described herein for the treatment of respiratory disease.
In a further aspect, there is provided an oligonucleotide comprising the base sequence of any one of SEQ ID NOs. 1-72.
In a further aspect, there is provided an oligonucleotide consisting of any one of SEQ ID NOs. 1-127.
Table 1 identifies AON sequences with specificity for the human phosphodiesterase (PDE) isoform 7A in accordance with the present invention.
Table 2 identifies AON sequences with specificity for the human phosphodiesterase (PDE) isoforms 4B and 4D in accordance with the present invention.
Table 3 illustrates the homology between human AON sequences and the sequences from different animal species (mouse, rat and monkey).
Table 4 identifies the AON sequences with specificity for the human phosphodiesterase (PDE) isoforms 4B, 4D and 7A containing modified chemistry in accordance with the present invention.
Table 5 identifies the oligonucleotide sequences used as control in accordance with the present invention.
Table 6 identifies AON sequences with specificity for the mouse phosphodiesterase (PDE) isoforms in accordance with the present invention.
Table 7 describes the real-time PCR primers used to quantify target genes and inflammatory markers in in vitro and in vivo models.
Table 8 describes the influx of inflammatory cells in bronchoalveolar lavages and the histopathological changes following administration of the AON sequences to the lungs of mice.
Table 9 identifies AON sequences used in in vivo toxicology studies in accordance with the present invention.
Table 10 identifies the AON sequences with specificity for the human (3-chain and CCR3 containing modified chemistry in accordance with the present invention.
There is described herein oligonucleotides directed against phosphodiesterase (PDE) isoforms in order to downregulate the expression thereof. The oligonucleotides have a mixed phosphodiester/phosphorothioate backbone (P2M) and at least one 2-amino-2′-deoxyadenosine (DAP) nucleotide. It has been surprisingly found that the P2M and DAP modifications exhibit synergy in improving anti-PDE oligonucleotides by increasing their potency, efficacy or stability and/or decreasing their toxicity. For example, the oligonucleotides described herein preferably exhibit unexpectedly higher efficacy/toxicity ratios.
Accordingly, in an aspect, there is provided an oligonucleotide directed against a target Phosphodiesterase (PDE), wherein the oligonucleotide is capable of hybridizing to at least a portion of a nucleic acid sequence encoding the PDE under stringent conditions, and wherein,
Preferably, the target PDE is selected from the group consisting of PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE7A1, PDE7A2, PDE7A3 and PDE7B, preferably PDE7A, PDE4B and PDE4D.
In some embodiments, ratio of phosphorothioate to phosphodiester bonds is between 30:70 and 70:30, preferably 40:60 and 60:40, 45:55 and 55:45 and 50:50.
In some embodiments, the oligonucleotide is at least 80% complementary to the target PDE, preferably 100%.
The oligonucleotide preferably exhibits a efficacy/toxicity ratio greater than 0.25.
In some embodiments, the oligonucleotide is between 15-25 nucleotides in length, preferably between 18-22 nucleotides in length.
In one embodiment, the oligonucleotide has a base sequence selected from the group consisting of SEQ ID NOs. 1-72, wherein at least one adenosine is replaced with DAP, and is preferably SEQ ID NOs. 1, 33, 36, 41 and 42.
In a preferably embodiment, the oligonucleotide comprises, preferably consists of, any one of SEQ ID NOs. 81, 86, 90, 92, 95, 97, 99, 101, 103, 105, 114, 119, 121, 123, 125 and 127.
In a further aspect, there is provided a pharmaceutical composition comprising one oligonucleotide described herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical compositions comprises at least two of the oligonucleotides described herein, preferably directed against PDE7A and PDE4B, or PDE7A and PDE4D respectively. In a preferably embodiment, the oligonucleotide directed against PDE4B or PDE4D also downregulates the other.
In a further aspect, there is provided the pharmaceutical composition described herein for the treatment of inflammation.
In a further aspect, there is provided the pharmaceutical composition described herein for treatment of respiratory disease, preferably at least one of chronic obstructive pulmonary disease, asthma, eosinophilic cough, bronchitis, acute and chronic rejection of lung allograft, sarcoidosis, pulmonary fibrosis, rhinitis, sinusitis, viral infection or a neoplastic disease.
In a further aspect, there is provided a method of treating respiratory disease in a subject comprising administering the pharmaceutical composition described herein.
In a further aspect, there is provided the use of the pharmaceutical composition described herein in the preparation of a medicament for the treatment of respiratory disease.
In a further aspect, there is provided the use of the pharmaceutical composition described herein for the treatment of respiratory disease.
In a further aspect, there is provided an oligonucleotide comprising the base sequence of any one of SEQ ID NOs. 1-72, preferably wherein at least one adenosine is replaced with DAP.
In a further aspect, there is provided an oligonucleotide consisting of any one of SEQ ID NOs. 1-127.
The terms “nucleic acid” and “nucleic acid molecule” as used interchangeably herein, refer to a molecule comprised of nucleotides, i.e., ribonucleotides, deoxyribonucleotides, or both. The term includes monomers and polymers of ribonucleotides and deoxyribonucleotides, with the ribonucleotide and/or deoxyribonucleotides being connected together, in the case of the polymers, via 5′ to 3′ linkages. However, linkages may include any of the linkages known in the nucleic acid synthesis art including, for example, nucleic acids comprising 5′ to 2′ linkages. The nucleotides used in the nucleic acid molecule may be naturally occurring or may be synthetically produced analogues that are capable of forming base-pair relationships with naturally occurring base pairs.
“Bases” includes any one of the natively found purine and pyrimidine bases, adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U), but also any modified or analogous forms thereof. Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogues, aza and deaza purine analogues, and other heterocyclic base analogues, wherein one or more of the ring atoms and/or functional groups of the purine and pyrimidine rings have been substituted by heteroatoms, e.g., carbon, fluorine, nitrogen, oxygen, sulfur, and the like. Preferably, such bases include, but are not limited to, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine. Bases may also include, but are not limited to, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N4-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E)-5-(2-bromovinyl)uracil, N6-methyladenine, 2-chloroadenine, 2-fluoroadenine, 2-chloroadenine, N6-cyclopropyl-2,6-diaminopurine, nicotinamide, 2-aminopurine, 1,2,4-triazole-3-carboxamide.
The term “nucleic acid backbone” or “internucleoside linkage” as used herein refers to the structure of the chemical moiety linking nucleotides in a molecule. This may include structures formed from any and all means of chemically linking nucleotides.
The term “oligonucleotide” as used herein refers to a nucleic acid molecule from about 2 to about 100 nucleotides, and in increasing preferability, 2 to 80 nucleotides, 4 to 35 nucleotides, 10 to 30 nucleotides, 15 to 25 nucleotides, and 18 to 22 nucleotides.
Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (for example, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions involves a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of highly stringent conditions involves the use of higher temperatures in which the washes are identical to those above except the temperature of the final two 30 min. washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of very highly stringent conditions involves the use of two final washes in 0.1×SSC, 0.1% SDS at 65° C.
The terms “treatment”, “treating”, “therapy” and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or amelioration of an adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a subject as previously defined, particularly a human, and includes:
The term “pharmaceutically acceptable” as it is used herein with respect to carriers, excipients, surfactants and compositions refers to substances which are acceptable for use in the treatment of a subject patient that are not toxic or otherwise unacceptable for administration by any of the routes herein described.
The formulations of the present invention are preferably administered directly to the site of action and, thus, preferably are topical, including but not limited to, oral, intrabuccal, intrapulmonary, rectal, intrauterine, intratumor, nasal, intrathecal, inhalable, transdermal, intradermal, intracavitary, iontophoretic, ocular, vaginal, intraarticular, otical, transmucosal, rectal, slow release or enteric coating formulations. Without limiting any of the foregoing, formulations of the present invention may also be intracranial, intramuscular, subcutaneous, intravascular, intraglandular, intraorgan, intralymphatic, intraperitoneal, intravenous, and implantable. The carriers used in the formulations may be, for example, solid and/or liquid carriers.
Reference may be made to “Remington's Pharmaceutical Sciences”, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, for other carriers that would be suitable for combination with the present oligonucleotide compounds to render compositions/formulations suitable for administration to treat respiratory disease.
Optionally, the presently described oligonucleotides may be formulated with a variety of physiological carrier molecules. The presently described oligonucleotides may also be complexed with molecules that enhance their ability to enter the target cells. Examples of such molecules include, but are not limited to, carbohydrates, polyamines, amino acids, peptides, lipids, and molecules vital to cell growth. For example, the oligonucleotides may be combined with a lipid, the resulting oligonucleotide/lipid emulsion, or liposomal suspension may, inter alia, effectively increase the in vivo half-life of the oligonucleotide.
The pharmaceutical compositions provided herein may comprise oligonucleotide compounds described above and one or more pharmaceutically acceptable surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the oligonucleotides of the invention have been previously described in U.S. Application Publication No. 2003/0087845, the contents of which are incorporated herein with respect to surfactants. The application states that suitable surfactants “ . . . include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamelar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomeric and polymeric, poly (vinyl amine) with dextran and/or alkanoyl side chains, Brij 35™, Triton X-100™ and synthetic surfactants ALEC™, Exosurf™, Survan™ and Atovaquone™, among others. These surfactants may be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound additions to the 5′ and/or 3′ ends of the AONs.
The oligonucleotide component of the present compositions may be contained in a pharmaceutical formulation within a lipid particle or vesicle, such as a liposome or microcrystal. As described in U.S. Pat. No. 6,025,339, the lipid particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as the oligonucleotide is contained therein. Positively charged lipids such as N-[1-(2,3-dioleoyloxi) propyl]-N,N,N-trimethyl-ammoniumethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. No. 4,880,635 to Janoff et al.; U.S. Pat. No. 4,906,477 to Kurono et al.; U.S. Pat. No. 4,911,928 to Wallach; U.S. Pat. No. 4,917,951 to Wallach; U.S. Pat. No. 4,920,016 to Allen et al.; U.S. Pat. No. 4,921,757 to Wheatley et al.; etc.
The composition of the invention may be administered by any means that transports the oligonucleotide compound to the desired site, such as for example, the lung. The oligonucleotide compounds disclosed herein may be administered to the lungs of a patient by any suitable means, but are preferably administered by inhalation of an aerosol comprised of respirable particles that comprise the oligonucleotide compound.
The oligonucleotides may be formulated to be administered in a dry powder inhaler, metered dose inhaler, nebulizer, soft mist inhaler and by any other suitable device having the capacity to deliver oligonucleotides to the lungs via inhalation route.
The composition of the present invention may be administered into the respiratory system as a formulation including particles of respirable size, e.g. particles of a size sufficiently small to pass through the nose, mouth and larynx upon inhalation and through the bronchi and alveoli of the lungs. In general, respirable particles range from about 0.5 to 10 microns in size. Particles of non-respirable size that are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably thus minimized. For nasal administration, a particle size in the range of 10-500 μM is preferred to ensure retention in the nasal cavity.
A solid particulate composition comprising the oligonucleotide compound may optionally contain a dispersant that serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which may be blended with the antisense compound in any suitable ratio, e.g., a 1 to 1 ratio by weight.
Liquid pharmaceutical compositions of active compound (the oligonucleotide compound(s)) for producing an aerosol may be prepared by combining the oligonucleotide compound with a suitable vehicle, such as sterile pyrogen free water or phosphate buffered saline.
The aerosols of liquid particles comprising the oligonucleotide compound may be produced by any suitable means, such as with a nebulizer. Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active oligonucleotide ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, anti-bacterials, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants.
The aerosols of solid particles comprising the active oligonucleotide compound(s) and a pharmaceutically acceptable surfactant may likewise be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles that are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. The active oligonucleotide ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 150 μL, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or hydrofluoroalkanes and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents.
The aerosol, whether formed from solid or liquid particles, may be produced by the aerosol generator at a rate of from about 1 to 150 litres per minute.
The present invention will be more readily understood by referring to the examples that are given to illustrate the following invention rather than to limit its scope.
A549 cells (human lung carcinoma; ATCC; #CCL-185) were grown in Ham's F12 media containing 10% FBS, 100 U/mL Penicillin and 100 μg/mL Streptomycin. Flp-In T-Rex 293 cells (human transformed embryonic kidney; Invitrogen; #R780-07) were cultivated in DMEM containing 10% FBS, penicillin 100 U/mL and streptomycin 100 μg/mL. NHBE primary cells (normal human bronchial epithelial; Cederlane; #CC-2540) were cultivated in BEBM media (500 mL) supplemented with 2 ml BPE, 0.5 mL Hydrocortisone, 0.5 mL hEGF, 0.5 mL Epinephrine, 0.5 mL Transferrin, 0.5 mL Insulin, 0.5 mL Retinoic Acid, 0.5 mL Triiodothyronine (Cederlane, cat#CC-3170). CYNOM-K1 cells (cynomolgus monkey skin embryonic; ECACC; #90071809) were cultivated in EMEM (EBSS) media containing 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids, 100 U/mL Penicillin and 100 μg/mL Streptomycin. L2 cells (rat lung epithelial; ATCC; #CRL-149) were cultivated in F12K media containing 10% FBS, penicillin 100 U/mL and streptomycin 100 μg/mL. NIH3T3 cells (mouse fibroblast; ATCC; #CRL-1658) were cultivated in DMEM media containing 10% BCS, penicillin 100 U/mL and streptomycin 100 μg/mL. RAW264.7 cells (Leukaemic monocyte macrophage; ATCC; #TIB-71) were cultivated in DMEM media containing 10% FBS, 1 mM sodium pyruvate, penicillin 100 U/mL and streptomycin 100 μg/mL. Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers. PBMC were isolated by Ficoll-Hypaque density gradient centrifugation of EDTA K3 blood from normal donors. PBMC were plated at 0.2×106 cells/200 μL in 48-well plates (for suspension cells) in AIM-V culture media. TF-1 cells (erythroleukemia) were maintained in RPMI-1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 10% non-inactivated FBS and 2 ng/mL rhGM-CSF.
Phosphorothioate-DNA (PS-DNA) AON (Sigma Genosys), Phosphorothioate-FANA (PS-FANA) AON (Topigen, Montreal or UcDNA, Calgary), DAP, P2M and P2M-DAP AON (Biospring, Germany), 2′-OMe AON (Biospring, Germany) or Lock nucleic acid (LNA) AON (Exiqon, USA) were resuspended in sterile water and diluted in Opti-MEM for in vitro transfection or diluted in PBS for in vivo studies.
A549 cells were seeded at 0.25×106 cells/mL (200 μL per well in 48-well plates) in HAM-F12 medium supplemented with 10% serum without antibiotics then incubated overnight at 37° C. prior to transfection with AON/Lipofectamine complexes (ratio of 1 μg AON:1 μL Lipofectamine 2000). Flp-In T-Rex 293 cells were seeded at 0.9×106 cells/mL (200 μL per well in 48-well plates) in DMEM medium supplemented with 10% serum without antibiotics then incubated overnight at 37° C. prior to transfection as described above. NHBE cells were seeded at 0.5×106 cells/mL (200 μL per well in 48-well plates) in BEBM complete medium without antibiotics for 24 h prior to transfection as described. L2 cells were seeded at 1.75×106 cells/mL (400 μL per well in 24-well plates) in F12K medium supplemented with 10% serum without antibiotics then incubated overnight at 37° C. and transfected as described above. CYNOM-K1 cells were seeded at 0.25×106 cells/mL (200 μL per well in 48-well plates) in EMEM medium supplemented with 10% serum without antibiotics then incubated overnight at 37° C. prior to transfection with AON/Lipofectamine complexes (ratio of 1 μg AON:2 μL Lipofectamine 2000). NIH 3T3 cells were seeded at 0.25×106 cells/mL (200 μL per well in 48-well plates) in DMEM medium supplemented with 10% serum without antibiotics then incubated overnight at 37° C. prior to transfection with AON/Lipofectamine complexes (ratio of 1 μg AON:3 μL Lipofectamine 2000). The day before the transfections, TF-1 cells were diluted as to reach a density between 0.6 and 0.8×106 cells/mL the following day. The day of transfection, cells were plated in 12 well-plates at a density of 0.5×106 cells/well in 400 μL growth media without antibiotics and then transfected with AONs mixed with the Lipofectamine 2000 transfection reagent according to a ratio of 1 μg nucleic acid:1 μl lipid. The complexes were incubated for 20 min. and added to the cells for 24 h treatment at 37° C., 5% CO2.
RNA was extracted from cell pellets according to the RNAeasy mini kit protocol using the QiaVac 24 manifold from Qiagen and treated with DNase according to Qiagen's procedure. RNA was quantified using the RiboGreen reagent according to the manufacturer's protocol (Invitrogen).
Preparation of first-strand cDNA was performed using the Superscript First-Strand Synthesis System for RT-PCR kit (Invitrogen), in a total reaction volume of 20 μL. 1 μg of RNA was first denatured at 65° C. for 5 min., with 0.5 mM of each dNTPs, 0.5 μg of oligo (dT)18, 2 pmol of PDE4B gene specific reverse primer (5′-tctttgtctccctgctgga-3′) and chilled on ice for at least 1 min. The mixture was incubated at 42° C. for 2 min. and a second pre-mix containing 1× First-Strand Buffer, 10 mM DTT, and 40 units of SuperScript II RT was added. Reactions were incubated at 42° C. for 10 min., at 50° C. for 1 h and inactivated by heating at 70° C. for 15 min.
PCR reaction mixtures were performed with 3 □L of cDNA reaction in a total volume of 20 μL in presence of 0.4 mM of each PCR primer and 4 μL of LC FastStart DNA Master SYBR Green 1 PLUS. Step 1 (Denaturation): 95° C., 10 min (slope 20° C./sec); Step 2 (Cycles×40): 95° C., (slope 20° C./sec); 57° C. or 59° C., 5 sec. (slope 20° C./sec) 72° C., 10 sec. (slope 20° C./sec); Step 3 (Melting curve):95° C., (slope 20° C./sec); 70° C., 30 sec. (slope 20° C./sec); 95° C., 0 sec. (slope 0.1° C./sec); Step 4 (Cool): 40° C., 30 sec (slope 20° C./sec)
PCR primer sequences used for each gene are described in Table 6. Quantification of PCR products was performed using the RelQuant program (Roche).
Cells were harvested and lysed for 30 min at 55° C. in 1× lysis mixture from the Quantigene® 2.0 kit (Affymetrix). Cell lysates were hybridized overnight at 55° C. in Quantigene® 2.0 capture plates in the presence of specific probe sets and mRNA signal was amplified (at 55° C.) and detected (at 50° C.). mRNA expression was linearly quantified by luminescence signal using the Luminoskan.
For β-chain or CCR3 protein measurements, TF-1 were harvested 24 h post-transfection, washed twice with PBS, and incubated with eotaxin-biotin or IL-3-biotin for 1 h at 4° C. Avidin-FITC was then added and cells incubated for 30 min at 4° C. Cells were washed twice with PBS and fixed in 4% paraformaldehyde before flow cytometry analysis.
NHBE cells were stimulated for 4 h with 10 ng/mL cytomix (TNF-α+IL-1β+IFN-γ) at the end of the transfection period. A549 cells were stimulated for 6 h with 10 ng/mL IL-1β at the end of the transfection period. The mRNA expression of inflammatory markers was analyzed by real-time PCR. The protein secretion of was measured in cells supernatant by multiplex ELISA (SearchLight service, Aushon), with normalization to the cell viability as measured by alamarBlue assay.
RAW264.7 cells were seeded at 0.13×106 cells/mL in complete media without antibiotics in 24-well plates and incubated in the presence of AON sequences or control CpG immunostimulatory sequences for 24 h. PBMC were seeded at 1×106 cells/mL with AIM-V media without antibiotics in 48-well plates and treated as described above. Following treatment, culture supernatants were collected and protein content assayed by ELISA following the manufacturer's directions.
CD-1 male mice (25-30 g, Charles River) were acclimated to the animal facility (Mispro, Montreal, Canada) for approximately one week prior to the beginning of treatment. Mice were anesthesized with isoflurane (or ketamine/xylazine for t=0 h time point) and intubated. Aerosols of AONs (0.2 mg/kg) were administered into the trachea using a microsprayer (Penn-Century). Immediately after AON delivery (time=0 h) or at various time-points following delivery (time=2 h, 8 h, 16 h, 24 h, 48 h, 72 h and 144 h), mice were sacrificed using ketamine/xylazine. The lungs were removed, snap frozen in liquid nitrogen and stored at −80° C. until preparation of tissue homogenates. Lung tissues were homogenized in lysis buffer, centrifuged, and the resulting supernatants were snap frozen. A Hybridation-Enzyme-Linked-Immunosorbent-Assay (H-ELISA) method was used to perform quantification of each AON using specific probe sets. Standard curves were prepared for each AON using mouse lung tissue homogenate as a matrix.
For all the animal studies the housing and care of mice (male C57Bl/6, Charles Rivers Laboratory) used in this study were provided according to protocols approved by Mispro Biotech's Institutional Animal Care and Use Committee, in conformity with Canadian Council on Animal Care (CCAC) guidelines. On days 1 to 5 and 8 to 12, groups of mice were anesthesized with isoflurane and intubated. Aerosols of vehicle (endotoxin-free PBS) or AONs or administered into the trachea using a microsprayer (Penn-Century). Bronchoalveolar lavages (BAL) were performed 24 h after the last treatment and differential cell counts were determined on at least 300 cells using standard morphological criteria. Lung tissues were harvested, formalin fixed and paraffin sections prepared. Histopathological evaluations were performed on Hematoxylin & Eosin stained slides.
The rat studies were performed at ITR Laboratories Canada (Bale d'Urfe, QC) in compliance with GLP regulations. Briefly, Male and female Sprague-Dawley rats received 14 consecutive doses of vehicle or 5 mg/kg of mg/kg of TOP004/TOP005 or TOP006/TOP007 (in a 1:1 w/w ratio in saline) administered daily as aerosols using a inhalation exposure system. The animals were examined 1-2 times daily for clinical symptoms including a qualitative assessment of food consumption, and body weight was measured weekly. Electrocardiographic (ECG) activity was recorded and ophthalmic examinations were conducted for animals pre-study and on Day 14.
One day after the last dose (Day 15), the rats (8/sex/group) were euthanized. Terminal procedures included complete gross necropsy examination, collection and preservation of approximately 40 tissues, and measurement of the weights of all major organs. Respiratory tract tissues (nasal cavity, nasopharynx, larynx, pharynx, trachea, bronchi, lungs including carina and bronchial lymph nodes) from all animals were examined by light microscopy, and all collected tissues was examined for all high dose and control group animals.
On day 1, groups of mice were anesthesized with isoflurane and intubated. Aerosols of vehicle (endotoxin-free PBS), AONs or controls were administered into the trachea using a microsprayer (Penn-Century). On day 1, other groups of mice were treated by gavage with vehicle (0.5% methylcellulose) or roflumilast (Rasayan). One hour after drug treatment, mice were exposed to saline or LPS (0.04 mg/mL) for 15 min in a plexiglass chamber. BAL were performed 3 h post-challenge and differential cell counts were determined on at least 300 cells using standard morphological criteria.
On days 1, 3, 6 and 8, or just 6 and 8 groups of mice were anesthesized with ketamine and xylazine (60 mg/kg and 12 mg/kg, i.p.) and intubated. Aerosols of vehicle (endotoxin-free PBS), AONs or controls were administered into the trachea using a microsprayer (Penn-Century). On days 6 to 9, other groups of mice were treated by gavage with vehicle (0.5% methylcellulose) or roflumilast (Rasayan). On days 6 to 9 (3 h after drug treatment), mice were nose-only exposed to the smoke of two 2R4f reference cigarettes (University of Kentucky) per day. Cigarette smoke was delivered to mice using a nose-only exposure system (Proto-Werx), at a rate of 1 puff (20 ml) per min. BAL were performed 18-24 h after the last smoke exposure and differential cell counts were determined on at least 300 cells using standard morphological criteria.
The sequences of the AON directed against the phosphodiesterase (PDE) isoform 7A are presented in Table 1. The potency of some selected phosphorothioate (PS) AON sequences (PS-DNA) is demonstrated in
The sequences of the AON directed against the PDE isoforms 4D and 4B are presented in Table 2. The potency of some selected PS-DNA AON sequences is demonstrated in
Some selected AON sequences targeting the human PDE genes were designed to maximize homology with the PDE gene sequence from other species, specifically the mouse, rat and monkey sequences. Table 3 describes the sequence homology and number of mismatch of selected AON sequences across these different species.
This example relates to the enhanced efficacy of specific AONs against various mRNA targets when P2M and DAP modifications are incorporated into the chemistry of the AON. Tables 4a-4-d and 10a-10b describe the compositions of AON modified with DAP residues and P2M linkages.
All adenosine bases of selected PS AON sequences were replaced with 2-amino-2′-deoxyadenosine bases (PS-DAP) and efficacy compared to unmodified PS-DNA AON sequences for their efficacy at reducing target mRNA expression in Flp-In T-Rex 293 cells transfected for 24 h. As shown in
In
The DAP base modification was incorporated into AON sequences containing P2M linkage modifications.
In
In
In
In
This example relates to the prolonged efficacy of P2M-DAP-containing AONs. P2M modifications are expected to reduce the stability of the AON due to the lower content of PS linkages, rendering it less resistant to nucleosidase digestion, which would shorten its activity in vitro and in vivo. The half-life of P2M-DAP-containing AONs was assessed in lung tissue. P2M-DAP-containing AONs were administered to mice via intra-tracheal aerosol and AON content measured at different time-points following administration.
In
Surprisingly, despite a relatively short half-life, the DAP base and P2M linkage modifications of the AON sequences have sustained inhibitory activity up to 72 h in vitro (
This example relates to efficacy of P2M-DAP-containing AON on the biological function of different cell types, specifically the secretion of cytokines and chemokines. Cytokines and chemokines are important mediators of cell activation and recruitment while metalloproteases (MMP) are important modulators of tissue remodelling. Lung epithelial cells can play a role in the pathophysiology of inflammatory respiratory diseases through the secretion of chemokines that lead to the recruitment of immune cells, such as neutrophils and monocytes/macrophages, cytokines and metalloproteases. Interleukin-8 (IL-8/CXCL8) and monocyte chemoattractant protein-1 (MCP-1/CCL2) levels are increased in COPD patients (Szilasi M et al. Pathology Oncology Research. 2006, 12:52-60). The levels of IL-8 and MCP-1 may be involved in neutrophil and macrophage recruitment respectively. Similarly, MMPs may contribute to the pathogenesis of COPD (Baraldo et al., Chest 2007, 132:1733-40). MMP-2 and MMP-12 expression is increased in COPD patients.
In
In
This example relates to the relative reduction of the immune and inflammatory response of P2M-DAP modified AONs in vitro as well as in vivo following chronic dosing administration to mice.
The response of unmodified PS-DNA and DAP-modified AON sequences was also assessed in vivo following administration by inhalation of 14 consecutive doses of 5 mg/kg to rats (saline was used as control). Lung tissue histolopathology was assessed 24 h following the last AON treatment.
The response of P2M-DAP-modified AON sequences intra-tracheal administration of 10 doses of 2.5 mg/kg to mice (PBS was used as control). Differential cell counts of bronchoalveolar lavage (BAL) and lung tissue histolopathology were assessed 24 h following the last AON treatment. Table 8 summarizes the influx of inflammatory cells in BAL and the histopathological assessment of the selected P2M-DAP and unmodified AON sequences (PS-DNA). As shown in
Table 8 also summarizes the efficacy/toxicity benefit ratio calculated for the selected P2M-DAP-containing AON sequences. This ratio takes into account the activity (referred to efficacy index or as example here the maximal target knockdown obtained at a given concentration) of the AON vs its relative toxicity (here referred to lung toxicity index or the sum of all the findings and their severity grade). Results in Table 8 show very low ratio values for the unmodified AON sequences (PS-DNA) and higher ratio values (>0.25) for the P2M-DAP-containing AON sequences.
This example relates to the efficacy of AON targeting the PDE4B/4D and PDE7A when P2M-DAP modifications are incorporated into the chemistry of the AON in an in vivo model of acute lung inflammation in mice. Tables 4c, 4d and 6 describe the compositions of AONs targeting the human or mouse PDE4B/4D and PDE7A and modifications with P2M-DAP.
The activity of P2M-DAP-modified AONs targeting the PDE4B/4D and PDE7A was demonstrated in an in vivo model of LPS-induced acute lung inflammation model in mice. In this model of inflammation, mice are exposed to LPS resulting in a marked influx of neutrophils in the lungs of the animals (
This example relates to the efficacy of AON targeting the PDE4B/4D and PDE7A when P2M-DAP modifications are incorporated into the chemistry of the AON in an in vivo model of cigarette smoke-induced lung inflammation in mice. Tables 4c, 4d and 6 describe the compositions of AONs targeting the human or mouse PDE4B/4D and PDE7A and modifications with P2M-DAP.
The activity of P2M-DAP-modified AONs targeting the PDE4B/4D and PDE7A was also demonstrated in an in vivo model of cigarette smoke-induced lung inflammation model in mice. In this model of inflammation, mice are exposed to cigarette smoke for 4 consecutive days resulting in a marked influx of neutrophils in the lungs of animals (
The long acting nature of the P2M-DAP-modified AONs was again demonstrated, as they were administered every 2 days compared to roflumilast, which was administered daily.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references cited herein are incorporated by reference.
aTarget site on PDE7A1 mRNA sequence (accession # NM_002603)
bPercentage of PDE7A mRNA expression in Flp-In T-Rex 293 cells (260 nM)
aTarget site on PDE4B1 (accession #NM_001037341) or PDE4D1 (accession # NM_001104631) mRNA sequence
bPercentage of PDE4B or PDE4D mRNA expression in Flp-In T-Rex 293 cells (260 nM)
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aPercentage of phosphorothioate linkage in AON sequence.
cPercentage of difference compared to PDE mRNA inhibition in Flp-In T-Rex 293 cells treated with 236 nM PS-DNA.
x
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aPercentage of phosphorothioate linkage in AON sequence.
aPercentage of phosphorothioate linkage in AON sequence.
aPercentage of phosphorothioate linkage in AON sequence.
aPercentage of phosphorothioate linkage in AON sequence.
aPercentage of phosphorothioate linkage in AON sequence.
This application claims priority from U.S. Provisional Patent Application No. 61/446,144 filed on Feb. 24, 2011.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2012/000169 | 2/23/2012 | WO | 00 | 2/12/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61446144 | Feb 2011 | US |
