Patent Application
20110014292
The present invention relates generally to a method of prophylactically or therapeutically treating antigen-induced airway tissue inflammation and agents for use therein. More particularly, the present invention provides a method of prophylactically or therapeutically treating allergic airway inflammation and agents for use therein via the administration of the method of the present invention is useful, inter alia, in the treatment and/or prophylaxis of conditions characterised by antigen-induced airway tissue inflammation.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Inflammation of the lung tissue, despite being a normal part of the immune response, is nevertheless a potentially serious condition which, where very severe or even mild but chronic, can lead to significant and sometimes irreversible damage to the lung tissue. Still further, the onset of inflammation can be localised to one lung or it may spread to both.
The lung inflammatory process is characterised by inflammatory changes in large and small airways leading to damage of the alveoli and capillaries. In chronic inflammation, the repair of the epithelium is impaired resulting in mucus hypersecretion, airway narrowing and fibrosis and destruction of the parenchyma. The intensity and cellular characteristics of chronic airway inflammation varies as the disease progresses. Once inflammatory cells are activated, they release mediators which damage lung structures. These include a wide range of potent proteases (Shapiro, 1998), oxidants, and toxic peptides. Activation may further lead to the release of chemotactic peptides that perpetuate inflammation and tissue damage (Senior and Griffin, 1980, J Clin Invest, 66).
More specifically, the airways and lung parenchyma are found to exhibit high numbers of macrophages, T-lymphocytes (predominantly CD8+T cells), and neutrophils. Leukotriene B4 (LTB4), interleukin 8 (IL-8), and tumor necrosis factor-α (TNF-α) are the major inflammatory mediators involved in this process. The induction of this inflammatory response ultimately leads to:
(ii) Vascular damage
(iii) Airway narrowing or fibrosis; and
(iv) Elastin destruction.
In contrast to other types of injury and repair processes, the inflammation and tissue remodelling (fibrosis) observed in airway inflammation is often irreversible and may therefore exist through the life of the afflicted individual.
Pulmonary inflammation can be caused by a wide variety of factors including:
Although inflammation is a normal and necessary part of an effective immune response, it can nevertheless quickly become very damaging if left unchecked or if induced in response to an innocuous antigen. The disease conditions which are characteristically associated with pulmonary inflammation include asthma, chronic obstructive pulmonary disease, cystic fibrosis, lung fibrosis, acute lung injury and ARDS.
In terms of airway hyperresponsiveness, despite increasing evidence that airborne particulate matter detrimentally affects lung function, much remains to be determined about the exact component(s) of particulate matter which are responsible, with roles attributed to particulates (fine and ultrafine), ozone and nitrogen dioxide (Brunekreef et al. 2002. Lancet 360:1233-1242; Heinrich et al. 2004, Curr Opin Allergy Clin Immunol 4:341-348; Gauderman et al. 2007, Lancet 369:571-577.). The particle concentration in ambient air is dominated by ultrafine particles (<100 nm in diameter) (Brunekreef et al. 2002, supra; Oberdorster, G. 2001, Int Arch Occup Environ Health 74:1-8). Ultrafine particles are postulated to contribute disproportionately to the morbidity and mortality associated with particle inhalation (Oberdorster 2001, supra; Bernstein et al. 2004, J Allergy Clin Immunol 114:1116-1123). Interest in the health effects of nanoparticles in the lung is also being driven by their increasing use in industrial and pharmaceutical applications. The evidence suggests that ultrafine particulate matter may play a disproportionately large role in asthma exacerbations (Peters et al. 1997, Am J Respir Crit Care Med 155:1376-1383; von Klot et al. 2002, Eur Respir J 20:691-702), and that nanoparticles of a variety of compositions instilled into the lung induce inflammation/toxicity (Brown et al. 2001, Toxicol Appl Pharmacol 175:191-199; Kaewamatawong et al. 2005, Toxicol Pathol 33:743-749; Renwick et al. 2004, Occup Environ Med 61:442-447; de Haar et al. 2005, Toxicol Sci 87:409-418). It is now clearly recognized that dendritic cells play a critical role in establishment and maintenance of pulmonary immune responses (Lambrecht and Hammad, 2003, Nat Rev Immunol 3:994-1003; Vermaelen and Pauwels, 2005, Am J Respir Crit Care Med 172:530-551). However, knowledge of the potential effects of interaction between nanoparticles with pulmonary antigen presenting cells/dendritic cells is scarce. Accordingly, there is a need to better understand the immunological responses of airway tissue to ultrafine particles in order to facilitate the development of effective therapeutic and prophylactic treatment regimes.
Particles are readily taken up by peripheral dendritic cells in vitro and in vivo, with ultrafine beads 40-100 nm showing preferential uptake (Fifis et al. 2004, J Immunol 173:3148-3154; Foged et al. 2005, Int J Pharm 298:315-322). Recent findings show that peripheral dendritic cells are sensitive to particles in the 40-50 nm size range, and when conjugated to antigen these nanobeads induce potent immune responses in small (Fifes et al. 2004, supra) and large animals (Scheerlinck et al. 2006, Vaccine 24:1124-1131). In addition, human lung-derived alveolar macrophages avidly internalize such nanobeads in vitro (Pouniotis et al. 2004, Clin Exp Immunol 143:363-372), thereby facilitating the onset of an immunological response. In work leading up to the present invention, however, it has been determined that in contrast to expectations based on published literature, inert nanobead instillation profoundly inhibits development of key parameters of antigen-induced airway inflammation. These findings present a new model for preventing or treating airway inflammation due to induction of a lung state resistant to the normal inflammatory triggers encountered upon antigen sensitization and challenge.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
One aspect there is provided a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
In another aspect there is provided a method of therapy or prophylaxis of antigen-induced lung tissue inflammation in a mammal said method comprising contacting said lung tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
In yet another aspect there is provided a method of therapy or prophylaxis of allergen induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
In still another aspect there is provided a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an inert 35 nm-55 nm particle.
In a further aspect there is provided a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an inert particle of 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm or 55 nm.
Yet another aspect is directed to the use of an ultrafine particle in the manufacture of a medicament for the treatment or prophylaxis of antigen-induced airway tissue inflammation in a mammal wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
The present invention is predicated, in part, on the determination that whereas ultrafine particles are known to induce airway inflammation, in particular allergic airway inflammation, a subgroup of such particles can in fact facilitate the induction or maintenance of normal airway tissue homeostasis. Accordingly, this finding has facilitated the development of methods of prophylactically or therapeutically treating conditions characterised by antigen induced airway inflammation, which conditions have, to date, often been treated with corticosteroids, in order to reduce inflammatory symptomology. This is generally recognised as a necessary but nevertheless undesirable treatment regime due to the side-effects associated with prolonged administration of corticosteroids. Accordingly, in addition to providing an effective means of achieving an immunological outcome which has not been attainable to date, the method of the present invention is also very simple to routinely perform and avoids the use of treatment regimes associated with unwanted side effects, such as corticosteroid treatment.
Accordingly, in one aspect there is provided a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
By reference to “airway tissue” is meant the tissue of the passages which run from the mouth and nose, including the mouth and nose, into the lungs, together with the alveoli. The largest of the passages which runs from the oral and nasal cavities is the trachea (also known as the “windpipe”). In the chest, the trachea divides into two smaller passages termed the bronchi, each of these being further characterised by three regions termed the primary bronchus, secondary bronchus and tertiary bronchus. Each bronchus enters one lung and divides further into narrower passages termed the bronchioles. The terminal bronchiole supplies the alveoli. This network of passages is often colloquially termed the “bronchial tree”. Without limiting the present invention in any way, the predominant cell types in the pseudostratified columnar tracheal and bronchial epithelia include basal, intermediate, goblet, and ciliated cells. The simple columnar epithelia of bronchioles contain two main cell types termed Clara and ciliated cells. The most distal and functionally specialised epithelia of the lung include the gas exchanging air spaces; squamous type I pneumocytes and cuboidal type II pneumocytes.
In one embodiment, said airway tissue is lung tissue.
According to this embodiment there is provided a method of therapy or prophylaxis of antigen-induced lung tissue inflammation in a mammal said method comprising contacting said lung tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
Without limiting the present invention to any one theory or mode of action, the inflammatory response is a complex response characterised by a series of physiological and/or immunological events which are induced to occur by the release of a cytokine cascade in response to any one of a variety of stimuli including, but not limited to, tissue injury, infection, an immune response (such as to a pathogen or an innocuous agent—as occurs with allergies), or disease (such as tumour formation or an autoimmune response).
The physiological events which characterise inflammation include:
(i) vasodilation
(ii) increased vascular permeability
(iii) cellular infiltration
(iv) changes to the biosynthetic, metabolic and catabolic profiles of affected organs
(v) activation of the cells of the immune system.
It should therefore be understood that reference to an “inflammatory response” is a reference to any one or more of the physiological and/or immunological events or phases that are induced to occur in the context of inflammation and, specifically, in response to the signals generated by the cytokine cascade which directs the inflammatory response.
For example IL-1, TNFα and IL-6 are well known for their functions as pro-inflammatory mediators. It should also be understood that an inflammatory response within the context of the present invention essentially includes a reference to a partial response, such as a response which has only just commenced, or to any specific phase or event of a response (such as the phases and events detailed in points (i)-(v), above, or any other effect related to inflammation including, but not limited to, the production of acute phase proteins—including complement components and fever). Reference to a “chronic” inflammatory response should be understood as a reference to a response which is not acute. More specifically, it is of a prolonged duration, such as weeks, months or even indefinitely. An “acute” inflammatory response, however, is a reference to the immediate and early response to tissue injury such as physical, chemical or microbial insult. An acute inflammatory response is usually complete within a short duration, typically hours to a few days.
Without limiting the present invention to any one theory or mode of action, in certain circumstances the acute inflammatory process, characterized by neutrophil infiltration and oedema, gives way to a predominance of mononuclear phagocytes and lymphocytes. This is thought to occur to some degree with the normal healing process but becomes exaggerated and chronic when there is ineffective elimination of foreign materials as occurs in certain infections (e.g. tuberculosis) or following introduction of foreign bodies (e.g. cigarette smoke) or deposition of crystals (e.g. urate crystals). Chronic inflammation is often associated with fusion of mononuclear cells to form multinucleated gigant cells, which eventually become a granuloma. Chronic inflammation is also seen under conditions of delayed hypersensitivity.
In terms of the present invention, it has been determined that antigen induced airway tissue inflammation can be prophylactically or therapeutically treated by administering an ultrafine particle which can induce or maintain normal airway tissue homeostasis. By “antigen” is meant any proteinaceous or non-proteinaceous molecule which is capable of inducing an immune response in the airway tissue, this inherently involving the onset of airway tissue inflammation. Examples of such antigens include, but are not limited to, pathogens (such as viral, bacterial or parasitic), tobacco related particles, environmental particles, plant derived particles (such as pollens), chemical or other synthetic form of pollutant (such as airborne pollutants present in smog), other airborne particles (such as dust related allergens) or organism derived particles (such as house dust mite faeces). It should be appreciated that the subject allergen may be one which is generally expected to function as a foreign, immunogenic molecule, such as a chemical molecule found in pollutants, or it may be one which is innocuous, such as grass pollen. The nature of the immune response which is generated may take any form. For example, immune responsiveness to some innocuous allergens often takes the form of a delayed type hypersensitivity reaction while immune responsiveness to other classes antigens may take the form of a hypersensitivity response which is not delayed or it may take the form of another class of immune response which is not regarded as a typical hypersensitivity response, such as a cell mediated response to a virus.
Accordingly, in one embodiment there is provided a method of therapy or prophylaxis of allergen induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
In another embodiment there is provided a method of therapy or prophylaxis of pathogen induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
Examples of such pathogens includes, but is not limited to, respiratory syncytial virus, rhinovirus, influenza virus, cytomegalovirus and parainfluenza virus.
Without limiting the present invention to any one theory or mode of action, the administration of ultrafine particles in accordance with the method of the invention is characterised by extensive redistribution of dendritic cell subsets across lung compartments, particularly in the airways. This state is characterized by low effector T cell responses (both Th1 and Th2), but maintenance of normal (baseline) CD4+CD25+Foxp3+Treg frequencies during local allergen challenge. Accordingly, reference to “homeostasis” in accordance with the method of the present invention should be understood as a reference to the maintenance of an airway tissue physiological state which is a non-inflammatory state. To this end, it should be understood that said homeostasis is intended as a reference only to inflammatory-related homeostasis of the airway tissue and not to the homeostasis of other physiological factors, unrelated to inflammation, such as pulmonary related homeostasis, surfactant-related homeostasis and the like.
The ultrafine particles of the present invention are preferably inert. By “inert” is meant that the particles are substantially devoid of toxic contaminants.
As detailed hereinbefore, it has been determined that a subpopulation of ultrafine particles can induce or maintain non-inflammatory airway tissue homeostasis. An “ultrafine” particle should be understood as a particle of less than 100 nm. As exemplified herein, this subpopulation includes inert 30 nm-70 nm ultrafine particles. Still more preferably, said particles are from 35 nm-65 nm and yet more particularly 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm or 64 nm. Most particularly, said particles are 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm or 55 nm.
The present invention therefore more particularly provides a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an inert 35 nm-55 nm particle.
Yet more particularly there is provided a method of therapy or prophylaxis of antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an inert particle of 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm or 55 nm.
It should be understood that with respect to the subject ultrafine particle there is no particular limitation on the shape or surface morphology that the ultrafine particles may take. Generally, the particles will be spherical or spheroidal in shape. For avoidance of any doubt, reference to the “size” of the particles is intended to be that of the largest dimension provided by a cross section of a given particle. Thus, in the case of spherical particles the size is the diameter of the sphere, as measured to the outer perimeter of the sphere.
The particles may be in the form of primary particles, or in the form of an aggregation of primary particles. Generally, the particles will be in the form of primary particles.
The structure of the particle may be homogeneous or heterogeneous in terms of composition and also in terms of the physical state of the constituent components that form the composition. For example, the structure of the particles may be formed from one or more components that are in a solid state. The particles may also have a core-shell type structure in which the outer shell is formed from one or more components that are in a solid state and the inner core is formed from one or more components that are in a liquid state. Having said this, it will be appreciated that in order to function in accordance with the invention, the particles will at least have an outer surface or shell that is formed from one or more components that are in a solid state. Generally, the particles will be formed from one or more components that are in a solid state.
As used herein, reference to a component of the particles being in a “solid” or “liquid” state is meant that the component has that physical state at a temperature of no less than that which would be experienced by the particle when in vivo (i.e. generally at a temperature of no less than about 37° C.).
The particles may be formed from any suitable material provided that it does not promote a toxic response when used in accordance with the invention. In other words, at the very least the outer surface of the particles that makes contact with lung tissue will be formed from, or coated/grafted with, an inert material. Examples of suitable materials that the particles may be formed from or coated with include, but are not limited to, polymer, inorganic material such as ceramic and glass, metal or an organic material, such as glycine.
The particles are preferably made from a polymeric material. The polymeric material may or may not be biodegradable. In the context of the present invention, by a polymeric material being “biodegradable” is meant that the physical structure of the polymeric material is degraded in vivo such that the polymer can ultimately be excreted from the host. Degradation of the polymeric material may occur via physical or chemical pathway. Where a biodegradable polymer is to be used, its degradation products should not be toxic to the host. Examples of polymeric materials from which the particles may be formed include, but are not limited to, polystyrene, polyacrylates, polymethacrylates, polyolefins such as polypropylene and polyethylene, polyfluorocarbons such as Teflon, polyurethanes, polyamides, polycarbonates and polyesters. Suitable biodegradable polymers include, but are not limited to, biodegradable polyurethanes, biodegradable polyesters and biodegradable polycarbonates. The outer surface of the particles may be provided with functional groups that can be used to alter the surface characteristics of the particles. For example, the functional groups may be used to provide a charge at the particle surface or they may be used as a reaction site to tether or graft a surface modifying agent to the particle. Such functional groups may include, but are not limited to, amine groups, carboxyl groups, hydroxyl groups and sulfate groups. Examples of surface modifying agents that may be tethered or grafted at the surface of the particles include, for example, amino acids, such as glycine. Techniques for tethering or grafting surface modifying agents to the surface of a substrate such as a particle are generally well known in the art.
The particles may also comprise one or more therapeutic agents such as a pharmaceutically active compound. Upon being positioned within the lung, such particles may be designed to release the agent into the host in a controlled manner. For example, the agent might be dispersed throughout the polymeric matrix of a polymer particle and diffuse from polymer matrix in a desired manner into the lung.
Particles suitable for use in accordance with the invention may be prepared using known techniques. The particles may also be obtained commercially. For example, suitable particles may be purchased from Polysciences Inc. Warrington, Pa., USA under the Tradename Polybead®.
In yet another aspect there is provided a method of therapy or prophylaxis of a condition characterised by antigen-induced airway tissue inflammation in a mammal said method comprising contacting said airway tissue with an effective amount of an ultrafine particle wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
Preferably, said airway tissue is lung tissue. Still more preferably, said antigen is an allergen. Yet more preferably said ultrafine particle is an inert particle of 30 nm-70 nm, more preferably 35 nm-65 nm and most preferably about 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm or 55 nm.
The term “mammal” as used herein includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes). Preferably, the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.
The method of the present invention is useful as a therapeutic or a prophylactic treatment. By prophylactic treatment is envisaged the administration of said ultrafine particles in individuals who have not yet developed antigen-induced airway tissue inflammation but may, for example, be at risk of developing such a condition. In this regard, and without limiting the present invention in any way, it has been demonstrated that where said ultrafine particles are administered to non-inflamed airway tissue, this tissue is able to maintain its non-inflammatory homeostasis in the face of subsequent antigen challenge. In the context of a therapeutic treatment regime, the method of the present invention can reduce the level of inflammation, thereby inducing a shift back towards normal non-inflammatory homeostasis. Accordingly, it should be understood that reference to maintaining or reducing non-inflammatory airway tissue homeostasis is a reference to not just entirely preventing the onset of airway inflammation or eliminating pre-existing inflammation but also to at least partially reducing said inflammation or, in the context of the prophylactic aspects of this invention, reducing the extent or severity of the onset of an airway inflammatory state.
Accordingly, reference herein to “treatment” and “prophylaxis” is to be considered in its broadest context. The term “treatment” does not necessarily imply that a subject is treated until total recovery. This is a particularly significant point in relation to the present invention since in the context of disease conditions in which airway tissue inflammation is one of a range of symptoms, other symptoms may not be alleviated by this method. In this situation, the method of the invention is “treating” the disease condition in terms of reducing or eliminating the occurrence of a highly undesirable symptom but may not eliminate other symptoms unrelated to inflammation which may nevertheless be induced by the antigen. For example a toxic antigen, such as a pollutant, may nevertheless exert other systemic outcomes associated with the toxicity of the antigen itself. Similarly, “prophylaxis” does not necessarily mean that the subject will not develop some symptomology. However, the method of the present invention may slow or reduce the onset or degree of inflammation. The term “prophylaxis” may therefore be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the severity of an existing condition. To this end, the nanoparticles of the present invention may therefore be administered as a pretreatment to the onset of the condition in issue. In this context, for example, the nanobeads may be administered prior to vaccination with the antigen (such as a pathogen or allergen) after vaccination with the antigen or subsequently to infection.
Yet another aspect is directed to the use of an ultrafine particle in the manufacture of a medicament for the treatment or prophylaxis of antigen-induced airway tissue inflammation in a mammal wherein said ultrafine particle induces or maintains non-inflammatory airway tissue homeostasis.
Preferably, said airway tissue is lung tissue. Still more preferably, said antigen is an allergen. Yet more preferably said ultrafine particle is an inert particle of 30 nm-60 nm, more preferably 35 nm-65 nm and most preferably about 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm or 55 nm.
There are a wide range of well characterised conditions which are associated with antigen induced airway inflammation. For example, allergens induce airway hypersensitivity, such as Type I hypersensitivity, and pathogens cause infection, such as viral infection, bacterial infection or parasitic infection. The inhalation of other types of particulate matter, such as the particles present in tobacco smoke, smog or other pollution can induce inflammation associated with one or more of a range of conditions such as asthma, emphysema, COPD, acute respiratory distress syndrome, pneumonia, acute lung injury, lung fibrosis and bronchiectasis.
The present invention is preferably achieved by administering to said mammal an effective amount of a modulatory agent as hereinbefore defined. To this end, an “effective amount” means an amount necessary to at least partly attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of the particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The present invention further contemplates a combination of therapies, such as the administration of the modulatory agent together with other proteinaceous or non-proteinaceous molecules which may facilitate the desired therapeutic or prophylactic outcome. For example, in the context of the therapeutic treatment of asthma, one may seek to maintain ongoing anti-inflammatory therapies until such time as the method of the present invention has become effective.
Administration of the ultrafine particles of the present invention hereinbefore described, in the form of a pharmaceutical composition, may be performed by any convenient means. The particles of the pharmaceutical composition is contemplated to exhibit therapeutic or prophylactic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Dosage regimens may be adjusted to provide the optimum response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
The modulatory agent may be administered in any convenient or suitable manner although respiratory routes are preferred. For example, one may administer by inhalation or insufflation of powders or aerosols (including by nebulizer); intratracheal or intranasal.
For inhalation, the composition of the invention can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. See, e.g., Patton (1998) Biotechniques 16:141-143; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.), Aradigm (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale Therapeutic Systems (San Carlos, Calif.), and the like. For example, the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, e.g., air jet nebulizers.
In accordance with these methods, the composition defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By “coadministered” is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject particles may be coadministered together with anti-inflammatory or other relevant drugs in the context of asthma treatment. By “sequential” administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules, These molecules may be administered in any order.
Yet another aspect of the present invention is directed to the use of an ultrafine particle for the therapeutic or prophylactic treatment of antigen-induced airway tissue inflammation in a mammal.
The present invention is further described by reference to the following non-limiting examples.
Female BALB/c mice aged 7-8 weeks were obtained from Laboratory Animal Services (Adelaide, South Australia) and housed in the Alfred Medical Research and Education Precinct animal facility. Numbers of mice per group are indicated in the Figure legends. All experimental protocols were approved by the precinct Animal Ethics Committee.
Mock bead conjugation was performed as follows. Polybead carboxylate microspheres (0.0471 μm; Polysciences Inc. Warrington, Pa. USA #15913) were added to a glass tube at 1% solids (@ 1.46×1014 particles/ml) and sonicated for 5 minutes. MES buffer (2-[N-Morpholino]ethanesulfonic acid; MP Biomedicals Irvine, Calif. USA #195309) was added to 50 mM and the pH adjusted to 6. EDAC (N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride; Sigma-Aldrich, Castle Hill NSW #E1769) was added to 4 mg/ml, and pH adjusted to 6.5. The beads were mixed at room temperature for 2 hours. Glycine (Sigma-Aldrich #G7126) was added to 7 mg/ml, and mixed for 30 minutes. The beads were dialysed overnight against PBS at 4° C., and sonicated in a water bath sonicator prior to use. In certain experiments mice received FITC-labelled particles (20 μg in 50 μl saline) or saline as control; pilot experiments indicated that the effects of unlabeled versus FITC-labelled beads were indistinguishable (data not shown). Beads prepared under GLP conditions with endotoxin below the level of detectability of the LAL assay similarly gave indistinguishable results from beads prepared under normal laboratory conditions (data not shown). On days −36 and −34 mice were anaesthetised and nanobeads (20 μg) or saline delivered intratracheally (50 μl). On days −24 and −12 mice were sensitized intraperitoneally with saline or OVA (50 μg; Sigma-Aldrich) adsorbed to aluminium hydroxide. Mice were challenged intratracheally with saline or OVA (25 μg) on days 0, 2, 5 and 7 as described previously (Hardy et al. 2003, Am J Respir Crit Care Med 167:1393-1399). In some experiments 25 μg FITC-conjugated OVA (Molecular Probes, Eugene, Oreg., USA #023020) was used for the final (4th) challenge. Mice were killed 24 hours after the final challenge (
Methods were as described previously (Hardy et al. 2003, supra). Blood was collected from the inferior vena-cava and serum collected. LN suspensions were prepared by gently grinding through a 70 μm cell strainer (BD Falcon, San Jose, Calif., USA #352350). BAL was performed with 0.4 ml 1% FCS in PBS and 3 further lavages of 0.3 ml. Viable LN and BAL cells were counted in a hemocytometer. For differentials, BAL cytospots were Giemsa-stained (Merck, Kilsyth Victoria) and ≧200 cells identified by morphological criteria.
For flow cytometric analysis we used an enzymatic tissue digestion protocol as described (Vremec et al. 1992, J Exp Med 176:47-58) with modifications. The right ventricle was perfused with 5 ml Ca2+/Mg2+-free HBSS (Invitrogen, Mt. Waverly Victoria #14175095) with 0.01 M EDTA, pH 7.2. The trachea including major bronchi as they entered the lung were dissected free from the lung, and draining LN, trachea and lung tissue separately minced with a scalpel blade on ice. Tissue fragments were pelleted by centrifugation (10 minutes, 350 g at 4° C.). LN, trachea and lung were digested in collagenase type III (1 mg/ml; Worthington, Lakewood, N.J. USA) and DNase type I (0.025 mg/ml; Roche Diagnostics, Sydney NSW #1284932) at 25° C. in the dark mixing continuously; after 1 hour fresh collagenase/DNase solution was added and digestion continued for 1 hour. The reaction was stopped by adding one 10th volume of EDTA and 3% FCS and mixing for 5 minutes. The cell suspension mixed with a pipette to break up clumps and filtered through a 70 μm cell strainer (BD Falcon), red cells lysed, and washed twice in staining buffer [3% FCS, 3% pooled normal mouse serum, 5 mM EDTA (pH 7.2) and 0.1% Na-Azide in Ca2+/Mg2+-free HBSS]. Viable tracheal, lung, and LN leukocytes were counted in a hemocytometer.
Non-specific FcR binding was blocked by incubating cells in CD16/CD32 block (BD Biosciences, San Jose, Calif., USA) and 3% pooled normal mouse serum in EDTA-containing staining buffer (see above). Cells were stained on ice for 20 minutes with combinations of the following antibodies/conjugates diluted in staining buffer (all BD unless noted): CD11b-PE, CD11c-APC, CD40-biotin, CD80-biotin, CD86-biotin, class II MHC (I-Ad)-biotin, DEC-205-PE (Cedarlane, Hornby, Ontario, Canada), F4/80-PE (Caltag, Burlingame, Calif. USA), and streptavidin-PerCP. Appropriate isotype control antibodies were used. Cells were protected from light at all times. Approximately 1×106 events were acquired on a FACSCalibur™ (BD), and analyzed on FlowJo (Tree Star, Ashland, Oreg. USA).
One million cells/well were plated in a 96 well V-bottom plate and blocked with FACS staining buffer for 30 min on ice. Surface staining was performed with CD4-PerCP and CD25-PE or isotype control (IgG2b-PE) diluted in staining buffer for 30 min on ice, followed by two washes in staining buffer. Cells were incubated in 100 μl/well fixation/permeablisation buffer (eBiosciences #88-8118) for 30 min on ice and washed once with permeablisation buffer. Cells were intracellularly stained with Foxp3-APC antibody or isotype control (rat IgG2a-APC) diluted in permeablisation buffer+2 μl normal mouse serum for 30 min on ice, washed once and resuspended in 1% paraformaldehyde.
IL-4, IL-5, and IL-13 ELISPOT were performed as described previously (Hardy et al. 2003, Clin Exp Allergy 36:941-950). IFNγ ELISPOT was performed using AN18 capture and R4-6A2 biotinylated detection antibodies (Mabtech, Mossman, NSW Australia; #3321-3-1000 and #3321-6-1000) and hydrophobic membrane plates (Millipore #MAIPS4510).
OVA-specific IgE was detected as described previously (Hardy et al. 2003, supra). Briefly, ELISA plates were coated with OVA (10 μg/ml) and incubated with IgG-depleted serum diluted 1:5, followed by anti-mouse IgE-biotin and streptavidin-peroxidase. Absorbance was read at 490 nm; results are expressed as raw OD readings minus background (no serum added).
BAL fluid was acid activated prior to detection of TGF-β according to the manufacturer's instructions (R&D Systems #DY1679). The limit of detection was 8 pg/ml. For IL-10 analysis, IL-10 capture and detection antibodies (#551215 and #554465, respectively, BD) were used according to the manufacturer's instructions. BAL fluid was used neat, 1:2 and 1:4. Detection was performed with streptavidin-HRP (Amersham Biosciences, #RPN1231) and reaction product developed with 3,3′,5,5;-TetraMethylBenzidine (Zymed, Calif. USA, #00-2023). The reaction was stopped with an equal volume of 1M HCl and plates read at 450 nm.
Formalin-fixed paraffin embedded lung sections were stained with periodic acid-Schiff (PAS) reagent. The numbers of PAS-positive cells per small bronchiole (approx. basement membrane circumference 0.5 mm) were counted. Six to ten airways were counted per mouse.
Statistics were analysed using SPSS 12.0.1 software. The Mann-Whitney U test or paired Student's t-Test were used as appropriate, with differences considered statistically significant at p<0.05.
To test the hypothesis that ultrafine particle instillation in the lung promotes susceptibility to allergic lung inflammation 47 nm nanobeads were instilled intratracheally into the mouse lung 12 days prior to sensitization with OVA/alum, and mice were subsequently (36 days later) challenged intratracheally with OVA, or saline as control (
Antigen-specific Th2 cells play a critical role in development of allergic airway inflammation. We asked whether the induction of local Th2 immunity (in lung draining LN) would be affected by nanobead instillation. The frequency of OVA-stimulated draining LN cells producing the Th2 cytokines IL-4, IL-5 and IL-13 in the saline/OVA/OVA group were all at ≧78 cells per 106 cells (
The frequency of cells producing IFN-γ in both the beads/OVA/OVA and saline/OVA/OVA groups was comparable and low at <20 per 106 cells (data not shown). These results show that nanobeads instilled into the airways of allergen sensitized mice impair the induction of a Th2-biased response in the draining LN and that this is not as a consequence of immune deviation and associated increased IFN-γ production.
An alternative potential mechanism by which nanobeads could prevent the induction of airway inflammation by decreasing Th2 immunity would be by a direct effect on APC in the lung, particularly DC. To address this possibility we instilled naïve mice with FITC-labelled nanobeads to determine their short and long-term effects on lung APC and included for the analysis a range of general APC and DC subset markers. We used saline instillation as control. Lung DC are not a homogeneous population with DC from trachea (representing airways) and parenchyma having distinct functional and phenotypic properties (Huh et al. 2003, J Exp Med 198:19-30; von Garnier et al. 2005, J Immunol 175:1609-1618). We therefore determined separately the consequences of nanobead instillation on leukocyte numbers and phenotype from trachea, lung parenchyma and draining LN. The main findings were that bead instillation caused a marked reduction in frequency of CD11c+MCHII+DC in tracheal digests particularly at d1 following instillation, and this effect persisted to d7 (
Nanobead Instillation Increases Proportions of CD11bhi DC
Consistent with others (von Garnier et al. 2005, supra; Wikstrom and Stumbles 2007, Immunol Cell Biol 85:182-188), we found a population of CD11c+ cells which express CD11b at high density. Particle instillation increased the proportion of lung CD11c+CD11bhi cells from 2-3% of total cells in normal mice to 9% by d3 and d7 post-instillation (
Nanobead-induced changes in the composition of the DC ‘milieu’ across different pulmonary immune compartments may result in differential migration or maturational status. By d3 lung parenchymal CD11c+CD11bhi cells had increased CD40, CD80 and CD86 mean fluorescence intensity 2-4 fold, and this decreased slightly by d7 (
We hypothesized that the above noted re-distribution of APC across lung compartments in turn results in different and/or less APC taking up intratracheally instilled allergen explaining the downstream lack pulmonary Th2 immunity. To determine whether particles alter APC migration during acute allergic airway inflammation mice received particles prior to systemic OVA sensitization, and were challenged with OVA intratracheally and FITC-labelled OVA at the 4th challenge thereby permitting tracking of pulmonary APC which have endocytosed FITC-labelled allergen (Vermaelen et al. 2001, J Exp Med 193:51-60; Vermaelen et al. 2003, Am J Respir Cell Mol Biol 29:405-409) (
Nanobead Instillation Prevents Increases in CD11bhi and MHCIIhi Allergen-Laden Cells in the Lung but not the Draining LN
Dramatic differences between DC subpopulations in the airways could be an independent local effect, or may also be reflected in differential DC distribution in the lung and draining LN. The proportions of FITC+CD11c+ cells in the lung were decreased in the saline/OVA/OVA group (5.7±1%) compared to the control no inflammation group (9±1.5%, saline/saline/OVA, p<0.05), and nanobead instillation partially prevented this (7±1%, beads/OVA/OVA). The low frequency of CD11bhi FITC+CD11c+ cells in the ‘no inflammation’ groups (10±2% and 17±4%, saline/saline/OVA and beads/saline/OVA, respectively) was markedly increased by airway inflammation (51±2%, saline/OVA/OVA, p<0.001) and this was partially prevented by nanobead instillation (35±5%, p<0.05,
Rapid redistribution of APC and specifically CD11c+ DC from lung and trachea to the LN results in a novel homeostatic condition resistant to induction of allergic airway inflammation, with dramatically reduced allergen uptake locally in the airway/trachea by CD11c+ DC. Limited effector T cell stimulation is observed downstream, in particular an absence of the expected Th2 immunity elicited locally even in the presence of peripheral sensitization (as evidenced by IgE induction in the bead/OVA/saline group in
Although this novel mechanism explains all the findings, we used the same particle treatment and allergen sensitisation/challenge protocol to explore the possibility that bead treatment could promote regulatory cytokines and/or regulatory T cells (Treg) in the lung or lung-draining LN. Nanobead treatment induced modest decreases in BAL fluid concentrations of the immunomodulatory cytokine TGF-β(saline/OVA/OVA versus beads/OVA/OVA, p<0.05), while nanobeads instilled into mice without airway inflammation caused slightly increased TGF-β concentrations (p<0.01,
Female BALB/c mice aged 7-8 weeks were obtained from Laboratory Animal Services (Adelaide, South Australia) and housed in the Alfred Medical Research and Education Precinct animal facility. All experimental protocols were approved by the precinct Animal Ethics Committee.
BGP was purchased from Greer Laboratories Inc. (Lenoir, N.C., USA) as dry, non-defatted pollen, and 1 g of pollen extracted in 5 ml of 1 mM NH4HCO3 overnight at 4° C. on a rotating wheel. After centrifugation, the supernatant was dialyzed against PBS overnight, filtered through a 0.2-μm filter, and the protein content determined using the Bio-Rad Microassay (Bio-Rad, USA).
Polybead carboxylate microspheres (0.05 μm and 0.45 μm; Polysciences Inc. Warrington, Pa. USA #15913 and #09836, respectively) were glycine-coated as described previously (Fifis et al. 2004, J Immunol 173:3148-3154). In certain experiments mice received FITC-labelled particles (0.04 μm and 0.5 μm, Invitrogen-Molecular Probes, Carlsbad Calif., #F8795 and #F8813, respectively); pilot experiments indicated that the effects of unlabelled versus FITC-labelled particles were indistinguishable (data not shown). To determine particle effects on allergic airway inflammation, mice received saline (control) or particles (20 μg/50 μl) intratracheally (i.t.) (Hardy et al. 2003, supra) on d0 and d2. Mice were sensitised i.p. with saline or OVA (50 μg; Sigma-Aldrich) adsorbed to aluminium hydroxide on d12 and d22. Mice were challenged i.t. with saline or OVA (25 μg) on d32, d34, d37 and d39 as described previously (Hardy et al. 2003, supra). In certain experiments mice received FITC-labelled OVA for the final (4th) challenge, or were sensitised and challenged with BGP. Mice were killed 24 hours after the final challenge.
Collection and preparation of blood, lung-draining LN, and BAL were as described previously (Hardy et al. 2003, supra). Viable LN and BAL cells were counted in a haemocytometer. For differentials, BAL cytospots were Giemsa-stained (Merck, Kilsyth Victoria) and ≧200 cells identified by morphological criteria. Tissue digestion was performed as described previously (Vremec et al. 1992, supra) with modifications. The right ventricle was perfused with 5 ml Ca2+/Mg2+-free HBSS (Invitrogen #14175095) with 0.01 M EDTA, pH 7.2. Lung-draining LN were minced with a scalpel blade, while lung tissue was chopped with a tissue chopper (Mickle Laboratory Engineering Co. Ltd, Gomshall, Surrey, UK). Tissue fragments were digested in collagenase type III (1 mg/ml; Worthington, Lakewood, N.J., USA) and DNase type I (0.025 mg/ml; Roche Diagnostics, Sydney NSW #1284932) at 25° C. mixing continuously for 45 minutes (LN) or 1 hour (lung). The reaction was stopped by adding one 10th volume of EDTA and 3% FCS and mixing for 5 minutes. The cell suspension was filtered through a 70 □m cell strainer (BD Falcon), red cells lysed, and washed in staining buffer [3% FCS, 3% pooled normal mouse serum, 5 mM EDTA (pH 7.2) and 0.1% Na-Azide in Ca2+/Mg2+-free HBSS]. Viable cells were counted in a haemocytometer.
Non-specific FcR binding was blocked by incubating cells in CD16/CD32 block (BD Biosciences, San Jose, Calif., USA). Cells (0.5-1×106) were stained on ice for 20 minutes with combinations of the following antibodies/conjugates (all BD unless noted): CD11b-PE, CD11c-APC, CD40-biotin, MHCII-PE and MHCII-biotin (AMS 32.1) and streptavidin-PerCP. Appropriate isotype control antibodies were used. All dilutions were in staining buffer (see above). Acquisition was on a FACSCalibur™ (BD), and analysis performed on FlowJo (Tree Star, Ashland, Oreg., USA).
IL-4, IL-5, and IL-13 ELISPOT were performed as described previously (Hardy et al. 2006, supra). IFN-γ ELISPOT was performed using AN18 capture and R4-6A2 biotinylated detection antibodies (Mabtech, Mossman, NSW Australia; #3321-3-1000 and #3321-6-1000) and hydrophobic membrane plates (Millipore #MAIPS4510).
OVA-specific IgE was detected as described previously (Hardy et al. 2003, supra).
Formalin-fixed paraffin embedded lung sections were stained with periodic acid-Schiff (PAS) reagent. The number of PAS-positive cells per small bronchiole (approx. basement membrane circumference 0.5 mm) were counted. Six to ten airways were counted per mouse.
Statistics were analysed using SPSS 15.0.1 software. Data were analysed for normality, and log-transformed as necessary prior to analysis by independent samples t-Test, ANOVA or two-way ANOVA with Tukey's HSD post-hoc analysis, as appropriate. Differences were considered statistically significant at p Group sizes are indicated in the Figure legends. All values are mean±s.e.m.
Previous findings suggesting that ultrafine particles have superior ability to promote acute airway inflammation relative to fine particles were based on analyses performed hours post-particle instillation ((Brown et al. 2001, supra; Kaewamatawong et al. 2005, supra; Renwick et al. 2004, supra). However, the long-term effects of ultrafine particles on lung immune cells, particularly particles devoid of potentially toxic (Borm et al. 2006, Part Fibre Toxicol 3:11; Oberdorster et al. 2005, Environ Health Perspect 113:823-839) or pro-inflammatory chemicals (e.g. transition metals (Wilson et al. 2002, Toxicol. Appl. Pharmacol. 184:172-179) are unknown. The long-term consequences of pulmonary exposure to chemically inert glycine-coated 50 nm (nanoparticles) and 500 nm (microparticles) polystyrene particles (Fifis 2004, supra; Mottram et al. 2007, Molecule. Pharm. 4:73-84) were tested in a well-established model of acute allergic airway inflammation (Hardy et al. 2003, supra; Hardy et al. 2006, supra). In the following investigations, particles were instilled prior to allergen sensitisation/challenge, and not coadministered with allergen, in contrast to other studies (Alessandrini et al. 2006, J. Allergy. Clin. Immunol. 26:2706-2713; deHaar et al. 2006, Clin. Exp. Allergy 36:1469-1479). Mice received 50 or 500 nm particles intratracheally 10-12 days prior to systemic allergen sensitisation, with allergen challenges commencing d32 (
To investigate further 50 nm particle pre-treatment affected pulmonary Th2 immunity by interfering with peripheral antigen sensitisation, prior to the local challenge with antigen, the effect of 50 nm particles in mice which were sensitised but not challenged was investigated. Allergen-specific IgE was induced at comparable concentration in ovalbumin (OVA)-sensitised mice, whether pre-treated with 50 nm particles or not (0.11±0.36 OD units versus 0.08±0.002, saline versus 50 nm, respectively). Furthermore, in the absence of allergen challenge, 50 nm particles did not prevent the induction of Th2 cytokine producing cells in the lung-draining lymph node (LN) (nano/OVA/sal) (
Strikingly, 50 nm particle instillation prior to sensitisation/challenge (nano/OVA/OVA) caused a generalised inhibition of key Th2 cytokine production (IL-4, IL-5 and IL-13) in the lung-draining LN (
It was also confirmed that nanoparticle protective effects were not unique to our OVA-induced model of allergic airway inflammation in the BALB/c (H-2b) strain. Nanoparticle pre-treatment of C57BL/6 (H-2d) mice markedly inhibited eosinophilic airway inflammation and Th2 cytokine production in the draining LN (
Pulmonary dendritic cells (DC) play a critical role in the generation of allergic immune responses (Lambrecht 2003, supra). To study whether 50 nm particle treatment affected allergen uptake by DC, allergen-sensitised mice were challenged with FITC-labelled allergen to allow the subsequent identification of allergen-laden DC (OVA-FITC+CD11c+ cells)28 (
Regulatory CD4+CD25+T cells expressing high levels of Foxp3 (Treg) play a central role in the regulation of allergic asthma and downregulation of Th2 immunity (Hawrylowicz & O'Garra 2005, Nat. Rev. Immunol. 5:271-2831; Kearley et al. 2005 supra; Strickland et al. 2006, supra). The frequency of lung CD4+CD25+Foxp3+ cells in naive mice was found to be increased 2-fold by 50 nm but not 500 nm particles at d31 post-instillation (0.6±0.1% versus 1.2±0.2%, saline versus 50 nm, respectively, p=0.031). Furthermore, in mice with allergic airway inflammation the frequency of draining LN CD4+CD25+Foxp3+ cells was decreased by one third (sal/sal/OVA versus sal/OVA/OVA,
Collectively these findings demonstrate that inert nanoparticles have the capacity to generate a lung state resistant to allergic airway inflammation. Importantly, this feature is strictly size-dependent, with nanoparticles but not microparticles being able to turn off Th2 immunity, characterised by a decrease in stimulatory DC in the lung and an increased proportion of lung and draining LN residing Treg. Since 50 nm, but not 500 nm particles prevented the full spectrum of innate and adaptive inflammatory responses elicited upon allergen challenge in atopic (sensitised) animals, size clearly pays a critical role in enabling this novel biological activity.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007905630 | Oct 2007 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2008/001528 | 10/15/2008 | WO | 00 | 9/27/2010 |
