WO2008068480A2 - Method for identifying substrates of transporter proteins expressed in the lung - Google Patents

Abstract

The present invention relates to an assay method for identifying an agent that is taken up and retained intracellularly in lung tissue comprising the steps of: (a) contacting a test sample comprising a lung transporter protein with an agent that acts on an intracellular target in the lung; (b) identifying an agent that is a substrate for the lung transporter protein; and (c) measuring the retention time of the agent in the lung tissue, wherein a retention time of at least 0.5 hours is indicative of an agent that is retained in the lung tissue.

Description

METHOD

FIELD

The present invention relates to lung transporter proteins. In particular, the present invention relates to the use of lung transporter proteins in assay methods for identifying an agent, acting on an intracellular target, that is taken up and retained intracellular^ in the lung tissue.

BACKGROUND

Transporters localized on the plasma membrane recognise the structures of substances to be transported such that specific substances are selectively transported. Transporters may also recognise foreign substances, such as drugs, and transport them into cells. It is believed that most drugs permeate through the plasma membrane fundamentally by simple diffusion depending on their physicochemical properties - such as molecular size, hydrophobicity, and hydrogen-binding capacity. In the case of ionic drugs, it is believed that only molecules in the non-dissociated form can permeate through the plasma membrane according to the pH partition hypothesis. However, it has become evident that a number of drugs penetrate through the cell membrane by a specific mechanism other than simple diffusion. Therefore, active or facilitated cell uptake transporter systems exist in many organs that require efficient exchange of intracellular and extracellular substances.

Since various transporters are thought to be distributed in organs and cells based on their physiological roles, their distribution and functions may be specific to organs. Therefore, transporters are expected to be important to impart an organ specificity to pharmacokinetics. In other words, an organ-specific drug delivery system can be constructed utilizing transporters. For this purpose, it is necessary to identify various transporters at the molecular level and analyze their properties in detail. Horvath et a (2006) accepted for publication in The American Journal of Respiratory and Critical Care Medicine provide evidence for a facilitated cell uptake mechanism for cationic drugs in the human airway epithelia, accomplished by apically polarized expression of the pH dependent organic cation transporters OCTNl and OCTN2. In vitro functional studies on primary airway epithelial cells isolated from donor lungs indicated that cationic drug cell uptake is mainly associated with OCTN2. These findings also suggested that the OCTNs carry commonly used cationic bronchodilators - such as formoterol and albuterol but not the more lipohilic bronchdilator salmeterol. However, the mechanism of action of these drugs in the lung is mediated by binding to extracellular receptors in the lung and is consequently not directly dependent on cell uptake.

It is important to understand how drugs, acting on intracellular targets in the lung are taken up by cells and absorbed into the blood since effective airway cell uptake and slow absorption into the blood of these drugs is a prerequisite for good local pharmacological effects during a suitable time period. The most common inhaled drugs with an intracellular target are the glucocorticosteroids used in the therapy of inflammatory diseases of the lung. However, glucocorticosteroids are lipophilic compounds that easily penetrate into cells by passive diffusion and their cell uptake is consequently not dependent on transporter mediated cell uptake. For hydrophilic drugs carrying a net positive or negative charge at physiological pH, transporter mediated cell uptake in the lung tissue may be of high importance for reaching an intracellular target in the lung. Furthermore, the intracellular residence time of such drugs, affecting the duration of action, may be determined by the inherent permeability of the drug and/or whether the drug is a substrate for drug efflux/export transporters, catalysing an active/facilitated transport of the drug from the interior of the cells into the systemic circulation (blood). Consequently, the conventional modifications to enhance hydrophobicity of drugs to increase their intracellular availability may no longer be necessary. SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the surprising finding that transporter mechanisms in the lung not only control the cell uptake of agents and thus concentration in the lung tissue but may also (eg. in combination with passive permeability properties of an agent) control the intracellular retention time in the lung.

The present invention is also based on the finding that the lung tissue not only contains cationic uptake transporter proteins, but also anionic uptake and efflux/export transporter proteins.

This knowledge may provide new important strategies for the discovery of lung selective compounds with an intracellular target that can be used in the prevention and/or treatment (eg. local prevention and/or treatment) of lung diseases.

SUMMARY ASPECTS OF THE PRESENT INVENTION

In a first aspect, there is provided an assay method for identifying an agent that is retained intracellularly in lung tissue comprising the steps of: (a) contacting a test sample comprising a lung transporter protein with an agent that acts on an intracellular target in the lung; (b) identifying an agent that is a substrate for the lung transporter protein; and (c) measuring the retention time of the agent in the lung tissue, wherein a retention time of at least 0.5 hours is indicative of an agent that is retained in the lung tissue.

In a second aspect, there is provided an assay method for identifying an agent that modulates the activity of a lung transport protein comprising the steps of: (a) identifying an agent that is a substrate for the lung transport protein; and (b) measuring the activity of the lung transport protein in the presence and absence of the agent; wherein a difference in the activity of the lung transport protein in the sample as compared to a control is indicative that said agent modulates the activity of the lung transporter protein; and wherein said lung transporter protein is a monocarboxylic acid transporter protein, and/or a nucleoside transporter, and/or an amino acid transporter, and/or an oligo-peptide transporter, and/or a bile acid transporter, and/or OAT4, OATP-D, OATP-H, OCTN3, MATEl, MATE2, NATl, DAT, 5HTT, OSTalpha, OSTbeta and/or OCTl, OCT2, OCT3, CT2, OCTNl, and\or OCTN2.

In a third aspect, there is provided an assay method for identifying an agent that is a substrate for a lung transporter protein comprising the steps of: (a) contacting a sample comprising a lung transporter protein with an agent; (b) identifying an agent that is a substrate for the lung transport protein; and (c) measuring the uptake of the agent by the lung transporter protein in the presence and absence of the agent; wherein a difference in the uptake of said agent by the lung transporter protein as compared to a control is indicative that said agent is a substrate for a lung transporter protein; and wherein said lung transporter protein is a monocarboxylic acid transporter protein, and/or a nucleoside transporter, and/or an amino acid transporter, and/or an oligo-peptide transporter, and/or a bile acid transporter, and/or 0AT4, 0CTN3, MATEl, MATE2, OSTalpha, OSTbeta, and/or OCTl, OCT2, OCT3, CT2, OCTNl, and\or OCTN2.

In a fourth aspect, there is provided a process comprising the steps of: (i) performing the assay method according to the preceding aspects; (ii) identifying an agent; and (iii) preparing a quantity of that agent.

In a fifth aspect, there is provided a process comprising the steps of: (i) performing the assay according to the preceding aspects; (ii) identifying an agent capable of being transported by a lung transporter protein; (iii) modifying said agent; and (iv) preparing a pharmaceutical composition comprising said modified agent.

In a sixth aspect, there is provided a pharmaceutical composition comprising an agent identified by the assay method according to the preceding aspects or the process according to the preceding aspects admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof. In a seventh aspect, there is provided a process of preparing a pharmaceutical composition comprising admixing an agent identified by the assay method according to any of the preceding aspects or the process according to any of the preceding aspects with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.

In an eighth aspect, there is provided a method of treating a disease in a human or animal which method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an agent identified by the assay method of any of the preceding aspects or the process of any of the preceding aspects, wherein the agent is capable of modulating the disease and wherein said composition is optionally admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof.

In a ninth aspect, there is provided an agent identifiable, preferably, identified by the assay method of any of the preceding aspects.

In a tenth aspect, there is provided an agent identifiable, preferably, identified by the assay method according to any of any of the preceding aspects for use in the treatment and/or prevention of disease.

In an eleventh aspect, there is provided an agent identifiable, preferably, identified by the assay method of any of the preceding aspects in the manufacture of a composition for the treatment of a disease.

In a twelfth aspect, there is provided an assay method, a process, a pharmaceutical composition, a method, an agent or a use substantially as described herein with reference to the accompanying Figures. SUMMARY EMBODIMENTS OF THE PRESENT INVENTION

In one embodiment, a retention time of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 25 hours, or at least 30 hours is indicative of an agent that is retained in the lung tissue.

In one embodiment, the lung transporter protein is a cationic lung transporter protein.

In one embodiment, the cationic lung transporter protein is selected from the group consisting of OCTl, CT2, OCT2, OCT3, OCTNl, 0CTN2 and OCTN3 or mixtures thereof comprising at least two cationic lung transporter proteins.

In one embodiment, the lung transporter protein is a monocarboxylic acid transporter protein.

In one embodiment, the monocarboxylic acid transporter protein is selected from the group consisting of MCTl, MCT3, MCT4 and MCT5 or mixtures thereof comprising at least two monocarboxylic acid transporter proteins.

In one embodiment the lung transporter protein is a neurotransmitter transporter selected from the group consisting of NATl, DAT and 5HTT.

In one embodiment, the lung transporter protein is a nucleoside transporter.

In one embodiment, the nucleoside transporter is selected from the group consisting of CNTl, CNT2, CNT3, ENTl, ENT2, ENT3 and ENT4 or mixtures thereof comprising at least two nucleoside transporters.

In one embodiment, the lung transporter protein is an amino acid transporter. In one embodiment, the amino acid transporter is selected from the group consisting of PATl, PAT2, PAT3, PAT4, NBAT, RBAT, 4F2HC, CATl, CAT2, CAT3, CAT4, LATl, LAT2, LAT3, y+LAT-2, LPI, y+LATl, y+LAT-1 and LPI-PCl or mixtures thereof comprising at least two amino acid transporters.

In one embodiment, the lung transporter protein is an oligo-peptide transporter.

In one embodiment, oligo-peptide transporter is PEPTl and/or PEPT2.

In one embodiment, the lung transporter protein is a bile acid transporter.

In one embodiment, the bile acid transporter is NTCP and/or IBAT.

In one embodiment, the lung transporter protein is selected from the group consisting of OAT4, 0CTN3, MATEl, MATE2, OSTalpha, OSTbeta, NATl, DAT, 5HTT, OATP-D, and/or OATP-H

In one embodiment, the disappearance of the agent from the lung is used as a measure of the retention time of the agent in the lung tissue.

In one embodiment, the retention time is measured in a test animal.

In one embodiment, the agent is administered to the test animal via intratracheal instillation or inhalation (eg. by nebulisation or dry powder inhalation).

In one embodiment, the process comprises the step of: iv) preparing a pharmaceutical composition comprising that agent.

FIGURE LEGENDS

Figure 1 Uptake of compound A into HEK293 cells transfected with the human OCTl or empty control vector, (a) Time course of compound A (2.5 uM) uptake in hOCTl and empty vector transfected HEK293 cells; (b) Concentration dependence of compound A uptake in hOCTl and empty vector transfected HEK293 cells (1 minutes incubation time).

Figure 2

Uptake of compounds A-G into HEK cells transfected with the human OCTl, OCT2 and OCT3 or empty control vector (pTREX) during 4 min of incubation at a compound concentration of 2.5 uM

Figure 3

Amount of compounds H-K in the lung (expressed as percent of dose) after dosing to the rat lung in vivo. The passive permeability of the compounds are stated in the Table (expressed as PAMPA or Caco2 A-B values)

Figure 4

Relative human transporter mRNA expression of lung epithelial brushings from (top to bottom): (a) 1 healthy representative individual; (b) 1 healthy representative smoker; and (c) 1 representative COPD patient as measured by TaqMan analysis.

Figure 5

Concentration dependent inhibition of TNFα-induced release of IL-8 by compound A. The IC50 was measured in HEK293 cells transfected with hOCTl and compared to the IC50 in empty vector transfected cells.

LUNG TRANSPORTER PROTEIN

As described herein, agents that are substrates for lung transporter proteins may be identified such that the cell uptake in the lung tissue and absorption into blood, can be modulated. Based on the detailed analysis of the substrate specificity of the lung transporter proteins, agents can be designed and optimised so a suitable efficacy and duration of efficacy in the lung can be obtained.

It may be efficacious to administer an agent that is intracellularly retained in the lung since the duration of the effect of the agent in the lung tissue may be longer. In one embodiment, the effect of the agent in the lung is increased or decreased in time. Accordingly, identifying any lung transporter proteins that accepts the agent as substrate and then defining the residence time of the agent in the lung, makes it possible to identify agents that are not only substrates for the protein but which have a defined residence time in the lungs. Thus based on the detailed analysis of the retention time and transporter specificity, agents can be designed and optimised so the absorption of the drugs from the intracellular compartment of the lungs into the blood can be controlled.

The intracellular residence time of an agent may also be controlled by the passive permeability of the agent once it has been taken up intracellularly by transporter proteins.

The transporter protein is a transporter protein that is expressed in the lung tissue - such as lung epithelium, endothelium or other cells in the lung. Thus, by way of example, the lung transporter protein may be a transporter protein whose mRNA is expressed in lung epithelial brushings. The lung transporter proteins include those having the activity to transport substrates not only from the extracellular to the intracellular space of the lung but also from the intracellular to the extracellular space of the lung, including efflux/export transport from the lung into the blood. In one embodiment, the lung transporter protein is a protein that is expressed in healthy individuals, healthy smokers and/or patients suffering from COPD, asthma, CF and/or other lung diseases.

In one embodiment, the transporter protein is a cationic uptake transporter protein. The cationic uptake transporter protein may transfer a variety of organic cations - such as TEA, carnitine, noradrenalin, adrenalin, guanidine, acetylcholine quinidine, verapamil, pyrilamine, acyclovir, amantadine, desipramine, cimetidine. Specific examples of cationic uptake transporter proteins include, but are not limited to OCTl, CT2, 0CT2, OCT3, OCTNl, OCTN2 and OCTN3. Accordingly, the cationic lung transporter protein is selected from the group consisting of OCTl, CT2, OCT2, OCT3, OCTNl, 0CTN2 and OCTN3 or mixtures thereof comprising at least two cationic lung transporter proteins.

In another embodiment, the lung transporter protein is a monocarboxylic acid transporter protein. Examples of monocarboxylic acid transporters include, but are not limited to MCTl, MCT3, MCT4 and MCT5. In one embodiment, the monocarboxylic acid transporter protein is selected from the group consisting of MCTl, MCT3, MCT4 and MCT5 or mixtures thereof comprising at least two monocarboxylic acid transporter proteins.

In another embodiment, the lung transporter protein is a nucleoside transporter. Examples of nucleoside transporters include, but are not limited to CNTl, CNT2, CNT3, ENTl, ENT2, ENT3 and ENT4. In one embodiment, the nucleoside transporter is selected from the group consisting of CNTl, CNT2, CNT3, ENTl₅ ENT2, ENT3 and ENT4 or mixtures thereof comprising at least two nucleoside transporters.

In another embodiment, the lung transporter protein is an amino acid transporter. Examples of amino acid transporters include, but are not limited to PATl, PAT2, PAT3, PAT4, NBAT, RBAT, 4F2HC, CATl, CAT2, CAT3, CAT4, LATl, LAT2, LAT3, y+LAT-2, LPI, y+LATl, y+LAT-1 and LPI-PCl. In one embodiment, the amino acid transporter is selected from the group consisting of PATl, PAT2, PAT3, PAT4, NBAT, RBAT, 4F2HC, CATl, CAT2, CAT3, CAT4, LATl, LAT2, LAT3, y+LAT-2, LPI, y+LATl, y+LAT-1 and LPI-PCl or mixtures thereof comprising at least two nucleoside transporters.

In another embodiment, the lung transporter protein is an oligo-peptide transporter. Examples of oligo-peptide transporters include, but are not limited to PEPTl and/or PEPT2. In another embodiment, the lung transporter protein is a bile acid transporter. Examples of bile acid transporters include, but are not limited to NTCP and/or IBAT.

In another embodiment, the lung transporter protein is OAT4, 0CTN3, MATEl, MATE2, OSTalpha and/or OSTbeta. In one embodiment, the lung transporter protein is selected from the group consisting of OAT4, OCTN3, MATEl, MATE2, OSTalpha, OSTbeta, NATl, DAT, 5HTT, OATP-D, and/or OATP-H .

Examples of lung transporter proteins are provided in Table 1.

Transporter proteins may be prepared as recombinant proteins using recombination techniques or natural proteins. Recombinant proteins can be prepared, for example, by culturing cells transfected with DNA encoding the transporter protein. DNAs may be cDNA, genomic DNAs, and/or synthetic DNAs. The DNAs can be used for producing proteins as recombinant proteins. That is, it is possible to prepare proteins as recombinant proteins by inserting DNAs encoding proteins into an appropriate expression vector, culturing transformants obtained by transfecting suitable cells with the vector, and purifying the proteins thus expressed. Cells to be used for producing recombinant proteins include, for example, mammalian cells - such as COS cells, CHO cells, NIH3T3 cells, etc., insect cells - such as Sf9 cells, yeast cells, E. coli, and so on. Vectors used for expression of recombinant proteins vary depending on the host cells, including, for example, pcDNA3 (Invitrogen), Gateway expression system (Invitrogen), pEF-BOS (Nucleic Acids Res., 1990, 18(7), p5322), etc. for mammalian cells, "BAC-to-BAC baculovirus expression system" (GIBCO BRL), etc. for insect cells, "Pichia Expression Kit" (Invitrogen), etc. for yeast cells, pGEX-5. times.- 1 (Pharmacia), "QIAexpress system" (Qiagen), etc. for E. coli. Host cells can be transformed with vectors, for example, by the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (Boehringer Mannheim), the electroporation method, or the calcium chloride method. Recombinant proteins can be purified from recombinants thus obtained using standard methods that are known in the art. Suitably, binding to one or more transporter proteins is measured using cells, or membrane vesicles and the like, that express transporter proteins since transporter proteins are typically dependent on a membrane environment. Cells transfected with DNA encoding a transporter protein may be prepared using methods that are well known in the art. Transfected cells include, for example, mammalian cells - such as COS cells, CHO cells, HEK 293 cells, MDCK cells, NIH3T3 cells, etc., insect cells - such as Sf9 cells, yeast cells, E. coli, and so on. Typically, the recombinant transporter protein will be expressed in the membrane of the transfected cell.

Membrane vesicles naturally expressing one or more transporter proteins may also be prepared using methods that are well known in the art.

ASSAY METHODS

In one broad aspect, there is provided methods of screening compounds to identify agents, acting on intracellular targets in the lung tissue and that are taken up by transporters into lung cells and are retained therein e.g. for at least 0.5 hours.

As used herein the "activity of a lung transporter protein" includes the uptake of a substrate (e.g. an agent) by the lung transporter protein into the lung tissue cells and/or the export of a substrate (e.g. an agent) by the lung transporter protein out of the lung tissue cells.

Accordingly, in one aspect, there is provided an assay method for identifying an agent that is retained intracellularly in lung tissue comprising the steps of: (a) contacting a test sample comprising a lung transporter protein with an agent that acts on an intracellular target in the lung; (b) identifying an agent that is a substrate for the lung transporter protein; and (c) measuring the retention time of the agent in the lung tissue, wherein a retention time of at least 0.5 hours is indicative of an agent that is retained in the lung tissue. There is also provided a method of identifying agents that modulate the activity of the lung transporter protein, the method comprising measuring the activity of the lung transporter protein in the presence of an agent or after the addition of an agent that is a substrate for the lung transporter protein in: (a) a cell line into which has been incorporated recombinant DNA comprising any of the DNA sequences or an allelic variation thereof coding for a lung transporter protein; (b) a cell population or cell line genetically-modified with a lung transporter protein DNA that naturally selectively expresses the lung transporter protein; (c) a membrane vesicle (e.g. an isolated membrane vesicle) which naturally expresses DNA comprising any of the DNA sequences or an allelic variation thereof coding for a lung transporter protein.

The screening method may measure the binding of an agent to the lung transporter protein, or to cells or membranes bearing the lung transporter protein, or a fusion protein thereof by means of a label directly or indirectly associated with the agent. Alternatively, the screening method may involve competition with a labelled competitor. Further, these screening methods may test whether the agent results in a signal generated by modulation of the lung transporter protein, using detection systems appropriate to the cells bearing the protein.

For example, a cell or membrane preparation expressing a lung transporter protein may be contacted with an agent. The ability of the agent to generate a response following interaction with the protein is then measured.

By way of further example, screening of peptide libraries or organic libraries made by combinatorial chemistry with a recombinantly expressed lung transporter protein or a variant, homologue, fragment or derivative thereof or cell lines or membrane vesicles expressing the lung transporter protein or a variant, homologue, fragment or derivative thereof may be useful for identification of therapeutic agents that are substrates for the lung transport protein and function by modulating the activity of the lung transporter protein. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art. For example, nucleotide sequences encoding the lung transporter protein may be expressed in a cell line, which can be used for screening.

A lung transporter polypeptide, its immunogenic fragments or oligopeptides thereof can be used for screening therapeutic compounds in any of a variety of drug screening techniques. The polypeptide employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between the lung transporter protein and the agent being tested may be measured.

Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the lung transporter protein and is based upon the method described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984. In summary, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the lung transporter protein fragments and washed. A bound lung transporter protein is then detected - such as by appropriately adapting methods well known in the art. A purified lung transporter protein can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.

The assay method may be a high throughput screen (HTS). In this regard, the teachings of WO 84/03564 may be adapted for the lung transporter protein.

In one embodiment, the uptake by the lung transporter protein is measured using cells from or derived from lung tissue - such as primary airway epithelial cells - that are exposed to the fluorophore 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP⁺). Cell fluorescence can be quantified using ASP⁺-specific excitation (465-495 nm) and emission (595-615 nm) filters as described in Am J Respir Cell MoI Biol 2001; 25:500- 506 and Am J Respir Cell MoI Biol 2002;27:746-751. Suitably, the activity of the lung transporter protein can also be measured in this way.

In another embodiment, the uptake by the lung transporter protein is measured using cells transfected with specific lung transporters - such as HEK 293 cells transfected with OCT transporters - that are exposed to the fluorophore 4-[4-(dimethylamino)-styryl]-iV- methylpyridinium (ASP⁺). Cell fluorescence can be quantified using ASP⁺-specific excitation (465-495 nm) and emission (595-615 nm) filters. This method is amenable to high throughput screening.

In another embodiment, the uptake by the lung transporter protein is measured indirectly by using cells transfected with specific lung transporters and then measuring the biological effect of the drug. For example, uptake of an IKK2 inhibitor can be shown by measuring TNFα-induced IL-8 release in cells transfected with a lung transporter protein, e.g. OCTl.

As described herein, it has been surprisingly observed that the transport mechanisms in the lung not only control the uptake and thus concentration of agents in the lung cells but, in combination with passive permeability, also control the intracellular retention time thereof in the lung or a tissue thereof. Thus, in another broad aspect, there is provided a method of screening compounds to identify agents that more effectively reach their intracellular targets and/or that are retained in the lungs.

In one embodiment, there is provided an assay method for identifying an agent that is retained in the lung comprising the steps of: (a) contacting a test sample comprising a lung transporter protein with an agent; (b) identifying an agent that is a substrate for the lung transporter protein; and (c) measuring the retention time of the agent in the lung, wherein a retention time of at least 0.5 hours is indicative of an agent that is retained in the lung. Methods for measuring the retention time of an agent in the lung are described herein below. In one embodiment, the disappearance of the agent from the lung is used as a measure of the retention time of the agent in the lung tissue.

In a further embodiment, there is also provided a method of identifying an agent that modulates the retention time of an agent that is a substrate for a lung transporter protein, the method comprising contacting the lung transporter protein or the nucleotide sequence (or a variant, homologue, fragment or derivative thereof) coding for same with the agent and then measuring the retention time of the agent in the lung.

Thus, in certain embodiments, the lung transporter protein or a variant, homologue, fragment or derivative thereof and/or a cell line that expresses the lung transporter protein thereof may be used to screen for agents - such as antibodies or peptides - that are retained in the lung, thereby identifying a therapeutic agent that is taken up by the lung transporter protein and is retained therein.

INTRACELLULAR TARGET

As described herein, the agent acts on an intracellular target in the lung. In other words, the agent interacts with (eg. binds to directly or indirectly) a target that is expressed, produced or resides in the lung tissue (eg. inside cells of the lung tissue).

Examples of intracellular targets include, but are not limited to, kinases - such as IKK2, p38 kinase, EGF receptor tyrosine kinase, protein kinase C isoforms, MAP kinases, phosphatidylinositol and phosphatidylinositolphosphate kinases; phosphodiesterases - such as PDE4 (including PDE4D); intracellular receptors - such as retinoic acid receptors and steroid receptors; and other targets which are partly or completely localised to the intracellular space e.g. epithelial sodium channel EnaC and NFKB.

RETENTION TIME As used herein, the term "retention time" refers to the time taken for half of the agent that is administered to be eliminated from the lung.

The retention time of an agent in the lung may be measured using various methods that are known in the art.

In one embodiment, the retention time in the lungs is measured using a test animal. As used herein, the term "test animal" refers to any animal useful in the methods of the present invention. The test animal is typically a mammal- such as an adult mammal. In one embodiment, the test animal is a rat, hamster, rabbit, guinea pig or mouse.

The agent under test may be administered via various routes - such as inhalation. Administration via inhalation may be performed either using, for example, dry powder inhalation or nebulisation of a solution/suspension into an inhalation chamber in which the animals are exposed either by nose only or whole body to the aerosol. .

Intratracheal instillation (i.t.) is another dosing technique can be used. Briefly, the animals are lightly anaesthetised. A cannula, connected to a syringe, is inserted in the trachea normally at the bifurcation and the agent is injected into the trachea.

Time points are typically chosen to cover agents that are absorbed rapidly or slowly into the systemic circulation.

In one embodiment, the retention time of the agent in the lung is measured for at least about 0.5 hr, at least about 0.75hr, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 25 hours, and/or at least about 30 hours.

In another embodiment, the retention time is at least 4 hours for an agent that is administered twice daily in humans. At each sampling occasion, the animals are killed by cervical dislocation and the lung is dissected out with all non-relevant tissue removed and the subjected to LC-MS/MS analysis.

The results from the LC-MS/MS analysis of the lung tissue is given as the amount of compound present in the whole lung (nmol/lung). The lung disappearance half life (lung T¹A) can be calculated and is considered of importance for evaluation of dosing interval needed to maintain effective therapy and can also be used as a measure of lung retention time.

Optionally, BAL fluid may be collected for analysis of compound concentration.

The intracellular residence time of an agent may be controlled by a low passive permeability and/or by transporter mediated efflux/export.

DIFFERENCE

In one embodiment, a difference in the activity of the lung transporter protein in the sample as compared to a control is used as an indicator that said agent modulates the activity of the lung transporter protein. The difference may be an increase or a decrease in the activity of the lung transporter protein. Suitably, an increase in the activity of the lung transporter protein is indicative that the agent increases or enhances the activity of the lung transporter protein. Suitably, a decrease in the activity of the lung transporter protein is indicative that the agent decreases or diminishes the activity of the lung transporter protein.

The control may be positive or a negative control. Thus, by way of example, the positive control may be an agent that is known to increase or decrease the activity of the lung transporter protein. By way of further example, the negative control may be an agent that is known not to increase or decrease the activity of the lung transporter protein. In another embodiment, a difference in the uptake of said agent by the lung transporter protein as compared to a control is indicative that said agent is a substrate for a lung transporter protein. Suitably, the difference is an increase in the uptake of said agent by the lung transporter protein as compared to a control.

The control in terms of transfected cells may be the corresponding empty vector transfected cells. The passive permeability may also be determined in the empty vector cells.

The control may also be a positive or a negative control. Thus, by way of example, the positive control may be an agent that is a known substrate for the lung transporter protein. By way of further example, the negative control may be an agent that is known not to be a substrate for the lung transporter protein.

TEST SAMPLE

The test sample may be or may be derived from a biological sample.

Biological samples may comprise any lung tissue or lung cell preparation in which at least one lung transporter protein is present, and may vary in nature depending on the particular lung transporter protein to be tested.

Biological samples may be provided by obtaining a lung biopsy specimen, a lung tissue explant, a lung organ culture or any other tissue or cell preparation from a subject or a biological source which comprises at least one lung transporter protein.

The biological sample may be may be derived from airway epithelia - such as human airway epithelia. The subject or the biological source of the test sample may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortal izable cell lines, somatic cell hybrids, differentiated or differentiatable cell lines, transformed cell lines and the like.

In one specific embodiment, the test sample is a cultured cell — such as a cultured lung cell or ALI (air liquid interface) lung epithelial cell culture.

In another specific embodiment, the test sample is a cell line (eg. a HEK 293 cell or a MDCK cell) transfected with a lung transporter protein,

AGENT

As used herein, the term "agent" may be a single entity or it may be a combination of entities.

In one embodiment, the agent is a "hydrophilic" compound. Advantageously, the hydrophilic compounds identified by the methods described herein enables rapid and efficient intracellular uptake in the lung tissue with a suitable intracellular residence time.

In another embodiment, the agent is a "hydrophilic" compound carrying a positive or negative charge at physiological pH.

In another embodiment, the agent is a "hydrophilic" compound carrying a positive or negative charge at physiological pH that acts on at least one intracellular target in the lung and is retained therein.

The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial. The agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule. The agent may even be an antibody.

The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.

By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesiser or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof, cell-free expression e.g. in rabbit reticulocyte lysate, wheat germ or E, coli S30 extracts.

Typically, the agent will be an organic compound. Typically the organic compounds will comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. For some applications, preferably the agent comprises at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. For some applications, the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group.

The agent may contain halo groups. Here, "halo" includes fluoro, chloro, bromo or iodo.

The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.

The agent of the present invention may be capable of displaying other therapeutic properties.

The agent may be used in combination with one or more other pharmaceutically active agents.

The agent may be or may be derived or derivable from an agent that is conventionally administered by inhalation and may include or be based on salts of cromoglycic acid, e.g. sodium cromoglycate; salts of nedocromil, e.g. nedocromil sodium; inhaled steroids - such as beclomethasone dipropionate, tipredane, budesonide and fluticasone; anticholinergic agents - such as ipratropium bromide; bronchodilators, e.g. salmeterol, salbutamol, reproterol, terbutaline, isoprenaline and fenoterol, and salts thereof; enzymes; vitamins and antihistamines.

The agent may be or may be derived or derivable from a drug that is used to treat asthma (both chronic and acute) - such as steroid inhalers (eg. beclomethasone diproprionate, budesonide, flunisolide, fluticasone proprionate, triamcinolone and acetonide); cromolyn sodium and nedocromil, long acting beta-2 agonists (such as salmeterol, formoterol and albuterol), methylxanthines (such as theophylline and mepyramine-theophylline acetate), leukotriene modifiers (such as zafirlukast, zileuton, montekulast and pranlukast), quick relief beta-2 agonists (such as albuterol, bitolterol, pirbuterol, terbutaline and bambuterol), anticholinergics (such as ipatropium bromide), systemic corticosteroids (such as methylprednisolone, prednisolone, prednisone and deflazacort) and experimental agents (including monoclonal antibodies directed against intracellular adhesion molecules or IgE), thromboxane A2 synthetase inhibitors (OKY-046), thromboxane prostanoid receptor antagonists (S- 1452), other eicosanoid modifiers (alprostadil vs. PGEl, dinoprostone vs. PGE2, epoprostenol vs. prostacyclin and PGI2 analogues (e.g. PG 12 beraprost), seratrodast (e.g.AA-2414), ozagrel (OKY-046)), phosphodiesterase 4 isoenzyme inhibitors, thromboxane A2 synthetase inhibitors (e.g. azelastine), ditec (low dose disodium cromoglycate and fenoterol), platelet activating factor receptor antagonists (Y-24180), antihistamines, anti-thromboxane A2 (SWR-00151), antibradykinins (such as icatibant), agents that inhibit activated eosinophils and T-cell recruitment (e.g. ketotifen), IL- 13 blockers (such as soluble IL- 13 receptor fragments), IL-4 blockers (such as soluble IL-4 receptor fragments), ligands that bind and block the activity of IL- 13 or IL-4, and xanthine derivatives (such as pentoxify line and A802715).

If combinations of active agents are administered, then they may be administered simultaneously, separately or sequentially.

MODULATING

The term "modulating the retention time" may refer to preventing, suppressing, alleviating, restoring, increasing, extending, or elevating or otherwise affecting the retention time of an agent in the lung.

In one embodiment, the term "modulating the retention time" may refer to increasing, elevating or extending the retention time of an agent in the lung. According to this embodiment, the duration of the effect of the agent in the lung tissue will be increased, elevated or extended.

The retention time of an agent in the lung may be modulated by affecting the degree of binding or interaction between the lung transporter protein on the inside of the cell and the agent. By way of example, the retention time in the lung tissue may be extended if the agent binds to or interacts less readily with the lung transporter protein on the inside of the cell and is exported out of the cell via the lung transporter protein more slowly. By way of further example, the retention time may be decreased in the lung if the agent binds to or interacts more readily with the lung transporter protein on the inside of the cell and is therefore exported out of the cell more rapidly. Alternatively, the intracellular retention time in the lung tissue may be extended if the agent binds with the lung transporter on the outside of the cell and is then transported to the inside of the cell from which no further active/facilitated transport into the blood exist. In this case, the further transport into the blood will be solely dependent on the passive permeability of the agent.

The term "'modulating the activity of a lung transporter protein" may refer to preventing, suppressing, alleviating, restoring, increasing, elevating or otherwise affecting the activity of a lung transporter protein. Suitably, the activity of the lung transporter protein is modulated by an agent that is a substrate for said protein.

DISEASE

Modulating the intracellular retention time of an agent in the lung may be used in the treatment and/or prevention of disturbances of airway diseases - such as obstructive lung disease or obstructive airway disease (OAD) - in order to extend the duration of the effect of the agent.

"Obstructive lung disease" or "obstructive airway disease" (OAD) are terms used to describe a complex of chronic and acute conditions that have in common airflow limitation or airflow obstruction. OADs includes asthma, emphysema, chronic bronchitis, and chronic bronchiolitis. The sites of airway obstruction in OADs vary from the upper airways to the most peripheral bronchioles. The exact cause of most diseases of the airways is not well understood. The definition of airway diseases add to the confusion. Chronic bronchitis is defined clinically by the chronic presence of cough and sputum production. Emphysema, on the other hand, is defined anatomically, on the basis of the breakdown of lung tissue and the enlargement of the alveolar sacs. OADs all have airway narrowing as a disease parameter and they also share inflammation as a component of the disease process.

In one embodiment, the disease is asthma, which is a chronic lung disease characterised by coughing, chest tightness, shortness of breath, and wheezing due to a reversible obstruction of airflow resulting from inflammation and hyper-responsiveness of the airways. An asthma attack is a dangerous overreaction by the immune systems, the lungs pump out mucus and inflammatory molecules, clogging and swelling constricted airways; in severe cases, all airflow is cut off and the attack may be fatal. In sensitized individuals, inhalation of allergens may produce inflammation of the airway lining, and precipitate a flare-up of asthma. Asthma may also occur as a result of other inflammatory stimuli - such as respiratory tract infections. Individuals who have become sensitized to specific foods may have severely and possibly life-threatening reactions after ingestion of these substances. Asthma, once thought of as a "simple" hypersensitivity reaction, is now known to be a complex condition with a probable spectrum of causes and contributing factors, with airway inflammation as its central attribute.

In another embodiment, the disease is cystic fibrosis.

PHARMACEUTICALS

There is also provided a pharmaceutical composition comprising a therapeutically effective amount of an agent and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Accordingly, even though the agents described herein can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.

Normally, the agents will be administered as a dry powder by inhalation or as suspension/solution by nebulization to the lung It is also possible to administer the agents in sustained release formulations.

Thus, there is also provided a method of treating an individual in need of same comprising administering to said individual an effective amount of the pharmaceutical composition.

Typically, the pharmaceutical compositions - which may be for human or animal usage - will comprise any one or more of a pharmaceutically acceptable diluent, carrier, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. As indicated above, the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Such molecules may provide the basis for treatment of various abnormal conditions of the lung. For example, such molecules may be used to increase the activity and/or expression of the lung transporter protein, thereby increasing the interaction between the lung transporter protein and its substrate(s) in conditions where it would be preferable to increase lung transporter protein activity. By way of further example, such molecules may be used to decrease the activity and/or expression of the lung transporter protein, thereby decreasing the interaction between the lung transporter protein and its substrate(s) in conditions where it would be preferable to decrease lung transporter protein activity. By way of further example, the agent may be retained in the lung, thereby extending the duration of effect of the agent.

The pharmaceutical composition could be for veterinary (i.e. animal) usage or for human usage.

Thus, there is also provided pharmaceutical compositions comprising effective amounts of one or more agents in admixture with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant (including combinations thereof) - such as a stabilising compound, saline, buffered saline, dextrose, and/or water.

The pharmaceutical compositions obtained may be useful for preventing and/or treating one or more of the diseases described above.

PRODRUG

It will be appreciated by those skilled in the art that the agent may be derived from a prodrug. Examples of prodrugs include certain protected group(s) which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form an agent that is pharmacologically active.

It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, may be placed on appropriate functional itis of the agents. Such prodrugs are also included within the scope of the invention.

PHARMACEUTICALLY ACTIVE SALT

The agent may be administered as a pharmaceutically acceptable salt. Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

CHEMICAL SYNTHESIS METHODS

The agent may be prepared by chemical synthesis techniques. It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example, as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by PJ.Kocienski, in "Protecting Groups", Georg Thieme Verlag (1994).

It is possible during some of the reactions that any stereocentres present could, under certain conditions, be racemised, for example, if a base is used in a reaction with a substrate having an having an optical centre comprising a base-sensitive group. This is possible during e.g. a giianylation step. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well-known in the art.

The compounds and salts may be separated and purified by conventional methods.

Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomer^ mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base.

The agent or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesise the agent in whole or in part. For example, if the agent comprises a peptide, then the peptide can be synthesised by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra). Synthesis of peptide agents (or variants, homologues, derivatives, fragments or mimetics thereof) can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the agent, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent.

CHEMICAL DERIVATIVE

The term "derivative" or "derivatised" as used herein includes chemical modification of an agent.

Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.

CHEMICAL MODIFICATION

The agent may be a modified agent - such as, but not limited to, a chemically modified agent.

The chemical modification of an agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction.

In one aspect, the agent may act as a model (for example, a template) for the development of other compounds.

ADMINISTRATION The components may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the components are is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

Advantageously compositions for topical administration to the lung include dry powder compositions (eg. by inhalation) and spray compositions (eg. as suspension/solution by nebulization).

Dry powder compositions for topical delivery to the lung may, for example, be presented in capsules and cartridges for use in an inhaler. Formulations generally contain a powder mix for inhalation of the agent and a suitable powder base - such as lactose or starch. Alternatively, the agent may be presented without excipients. Packaging of the formulation may be suitable for unit dose or multi-dose delivery. In the case of multi- dose delivery, the formulation can be pre-metered or metered in use.

Pharmaceutical formulations may be non-pressurised and adapted to be administered as a dry powder topically to the lung via the buccal cavity.

Spray compositions may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs - such as a metered dose inhaler, with the use of a suitable liquefied propellant. Aerosol compositions may be either a suspension or a solution and generally contain the agent and a suitable propellant - such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, especially 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n- propane or a mixture thereof. The aerosol composition may optionally contain additional formulation excipients well known in the art such as surfactants e.g. oleic acid or lecithin and cosolvents e.g. ethanol. One example formulation is excipient free and consists essentially of (e.g. consists of) the agent (optionally in combination with another therapeutically active ingredient) and a propellant - such as 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane and/or mixtures thereof. Another example formulation comprises the agent, a propellant selected from 1,1,1,2-tetrafluoroethane, 1,1, 1,2,3,3, 3-heptafluoro-n-piOpane and/or mixtures thereof and a suspending agent which is soluble in the propellant e.g. an oligolactic acid or derivative thereof

Medicaments for administration by inhalation may have have a controlled particle size. The optimum particle size for inhalation into the bronchial system is usually about 1-10 μm, preferably 2.5 μm. Particles having a size above 20 μm are generally too large when inhaled to reach the small airways. To achieve these particle sizes the particles of thr agent may be size reduced by conventional means e.g. by micronisation. The desired fraction may be separated out by air classification or sieving. When an excipient - such as lactose - is employed, generally, the particle size of the excipient will be much greater than the inhaled medicament. When the excipient is lactose it will typically be present as milled lactose.

Formulations for administration topically to the nose include pressurised aerosol formulations and aqueous formulations administered to the nose by pressurised pump. Formulations may be non-pressurised and adapted to be administered topically to the nasal cavity. The formulation may contain water as the diluent or carrier for this purpose. Aqueous formulations for administration to the lung or nose may be provided with conventional excipients - such as buffering agents, tonicity modifying agents and the like. Aqueous formulations may also be administered to the nose by nebulisation.

The components can also be administered in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

If the pharmaceutical is a tablet, then the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for administration (delivery) may include, but are not limited to, one or more of oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation optionally in combination with chitosan microspheres to ensure adhesion), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, trandermal (by ultrasound and iontophoresis), intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural and sublingual.

The term "administered" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, heipes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.

DOSE LEVELS

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular ^■ condition, and the individual undergoing therapy.

FORMULATION

The component(s) may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.

GENE THERAPY

The present invention encompasses gene therapy whereby a nucleotide sequence (or a variant, homologue, fragment or derivative thereof) coding for the lung transporter protein is regulated in vivo. For example, regulation of expression may be accomplished by administering compounds that bind to the nucleotide coding sequence, or control regions associated with the nucleotide coding sequence for the lung transporter protein, or its corresponding RNA transcript to modify the rate of transcription or translation.

By way of example, a nucleotide sequence encoding a lung transporter protein, may be under the control of an expression regulatory element - such as a promoter or a promoter and enhancer. Suitably, the enhancer and/or promoter is active in at least the lung, such that the nucleotide sequence coding for the lung transporter protein is preferentially expressed. The enhancer element or other elements conferring regulated expression may be present in multiple copies. Likewise, or in addition, the enhancer and/or promoter may be preferentially active in one or more specific cell types of the lung,

The level of expression of the nucleotide sequence coding for the lung transporter protein, may be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (that is, deletion analysis).

Expression of the lung transporter protein may also be controlled using a drug-inducible promoter system - such as the tetracycline operon - which will ensure up to 10,000-fold induction of the protein only when it is required, by taking a course of tetracycline.

RIBOZYMES

Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Accordingly, the agent may be an engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of the RNA sequences encoding the lung transporter protein.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide sequence inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridisation with complementary oligonucleotides using ribonuclease protection assays.

Both antisense RNA and DNA molecules and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesising oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

SiRNA

A nucleic acid may be contacted with a gene encoding a lung transporter protein to modulate the activity and/or expression of the protein, for example, at the level of transcription, transcript stability, translation or post-translational stability. Accordingly, the agent may be an antisense nucleic acid sequence or an siRNA. The inhibition of gene expression using antisense technology is well known. For example, antisense constructs are described in detail in US 6,100,090 (Monia et al), and Neckers et al., 1992, CrU Rev Oncog 3(1-2): 175-231.

Post-transcriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA) is a conserved cellular defence mechanism for controlling the expression of foreign genes. It is thought that the random integration of elements such as transposons or viruses causes the expression of dsRNA, which activates sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi). The mechanism of RNAi involves the processing of long dsRNAs into duplexes of 21-25 nucleotide (nt) RNAs. These products are called small interfering or silencing RNAs (siRNAs) which are the sequence-specific mediators of mRNA degradation. In differentiated mammalian cells dsRNA >30bp has been found to activate the interferon response leading to shut-down of protein synthesis and nonspecific mRNA degradation (Stark et al 1998). However this response can be bypassed by using 21nt siRNA duplexes (Elbashir et al 2001, Hutvagner et al 2001) allowing gene function to be analysed in cultured mammalian cells.

The siRNA may comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or modification of one or more nucleotides.

Such alterations can include the addition of non-nucleotide material - such as modified nucleotides - to, for example, the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant or even more resistant to nuclease digestion.

Typically, the siRNA will be in the form of isolated siRNA comprising short double- stranded RNA from about 17 nucleotides to about 29 nucleotides in length - such as approximately 19-25 contiguous nucleotides in length - that are targeted to a target mRNA. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is identical to a target sequence contained within the target mRNA.

A target sequence on the target mRNA encoding the lung transporter protein may be selected from a given sequence - such as a cDNA sequence - corresponding to the target mRNA, using various methods in the art. For example, the rational design of siRNAs is described in Nat Biotechnol. (2004) 22(3):326-30. siRNAs can be designed based on the following guidelines. Firstly, a sequence of around 21 nucleotides in the target mRNA is identified that begins with an AA dinucleotide. Each AA is recorded and the 3' adjacent nucleotides are identified as potential siRNA target sites. This is based on the observation by Elbashir et al. (EMBO J (2001) 20: 6877-6888, Nature (2001) 41 1 : 494- 498. 2 and Genes & Dev. (2001) 15: 188-200) that siRNAs with 3' overhanging UU dinucleotides are the most effective. However, siRNAs with other 3' terminal dinucleotide overhangs have been shown to effectively induce RNAi. Preferably, target sites from among the sequences identified above are then further selected using one or more the following criteria: (i) selecting siRNAs with 30-50% GC content; (ii) avoid stretches of > 4 T's or A's in the target sequence; (iii) select siRNA target sites at different positions along the length of the gene sequence; and (iv) eliminate any target sequences with more than 16-17 contiguous base pairs of homology to other coding sequences.

If the selected siRNA sequences does not function for silencing, the following steps may be used. A search may be conducted for sequencing errors in the gene and possible polymorphisms. Studies on the specificity of target recognition by siRNA indicate that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation. A second and/or third target may also be selected and the corresponding siRNA prepared and tested. Although siRNA silencing is highly effective by selecting a single target in the mRNA, it may be desirable to design and employ two independent siRNA duplexes to control the specificity of the silencing effect.

siRNA may be obtained using a number of techniques known to those of skill in the art. For example, the siRNA may be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesiser. The siRNA may be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. A full-length double-stranded transcript of the lung transporter protein may also be generated by in vitro transcription and annealing of the two strands. The the double-stranded RNA can then be cleaved with a nuclease - such as ribonuclease III or Dicer - into siRNAs for transfection in to cells.

siRNA may be recombinantly produced using methods known in the art. For example, siRNA may be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. The recombinant plasmids may also comprise inducible or regulatable promoters.

VARIANTS/HOMOLOGUES/DER1VATIVES/FRAGMENTS

The present invention encompasses the use of variants, homologues, derivatives and fragments thereof of the sequences described herein.

The term "variant" is used to mean a naturally occurring polypeptide or nucleotide sequences which differs from a wild-type sequence.

The term "fragment¹" indicates that a polypeptide or nucleotide sequence comprises a fraction of a wild-type sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The sequence may also comprise other elements of sequence, for example, it may be a fusion protein with another protein. Suitably, the sequence comprises at least 50%, at least 65%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the wild-type sequence.

Suitably, the fragment retains 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% activity of the wild-type polypeptide or nucleotide sequence.

The fragment may be a functional fragment. By a "functional fragment" of a molecule is understood as a fragment retaining or possessing substantially the same biological activity as the intact molecule. In all instances, a functional fragment of a molecule retains at least 10% and at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the intact molecule.

The term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term "homology" can be equated with "identity".

In the present context, a homologous sequence is taken to include an amino acid sequence, which may be at least 75, 85 or 90 % identical, preferably at least 95%, 96%, 97%, 98 % or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity {i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. MoI. Biol., 403-410), the GENEWORKS suite of comparison tools and CLUSTAL. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8).

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix - such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

Should Gap Penalties be used when determining sequence identity, then suitably the following parameters are used:

For polypeptide sequence comparison the following settings may be used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Suitably, the degree of identity with regard to an amino acid sequence is determined over at least 5 contiguous amino acids, determined over at least 10 contiguous amino acids, over at least 15 contiguous amino acids, over at least 20 contiguous amino acids, over at least 30 contiguous amino acids, over at least 40 contiguous amino acids, over at least 50 contiguous amino acids, or over at least 60 contiguous amino acids.

The sequences may also have deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example, according to the Table below. Amino acids in the same block in the second column and suitably in the same line in the third column may be substituted for each other:

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution - such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids - such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha* and alpha- disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids - such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid", 7- amino heptanoic acid*, L-methionine sulfone*^*, L-norleucine*, L-norvaline*, p-nitro-L- phenylalanine*, L-hydroxyproline , L-thioproline*, methyl derivatives of phenylalanine (Phe) - such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)^#, L-Tyr (methyl)*, L- Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L- diaminopropionic acid ^# and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups - such as methyl, ethyl or propyl groups - in addition to amino acid spacers - such as glycine or β- alanine residues. A further form of variation involves the presence of one or more amino acid residues in peptoid form will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the α-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example, Simon RJ et al, PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of nucleotide sequences.

GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1

Data obtained with compound A (intracellular IKK2 inhibitor)

The efficacy of compound A in the lung after local administration is driven entirely by local intracellular concentrations in the lung. Efficacy in the rat LPS model (measured as inhibition of neutrophil influx into bronchial-alveolar fluid) can be observed already at low dosages (3 ug/kg) after intratracheal instillation (i.t.) to the lung. In sharp contrast to this, no efficacy in the lung can be observed after systemic administration (by i.p.) up to 10 mg/kg despite high systemic blood exposure. Apparently, the drug is not available to the specific intracellular site of action in the lung after systemic administration but is so after local administration.

After /./. administration of 1 mg/kg to the rat, the disappearance TV. of the compound from the lung was approximately 1 h lasting for 8-10 h. At later time points, when the concentration in lung had dropped below 10 umol/L, the rate of disappearance from the lung was slower (T'/i: 5-6 h). At much lower doses by i.t administration (0.050 and 0.025 mg/kg), the lung levels were measured at 8 and 12 h after dosing and were surprisingly high suggesting a saturable PK mechanism in the lung. Indeed after dosing of 0.030 mg/kg to the lung by dry powder inhalation (DPI), the initial disappearance T!/_ was rapid but already at 30-60 min after dosing when the lung concentrations again dropped below 10 μM the subsequent T¹A approached 5-6 h and remained so during the whole experimental period (for 30h).

These results show that there exists a saturatable uptake/binding mechanism for compound A in the lung.

Since the compound is a highly hydrophilic base, we investigated the possibility if it was a substrate for the polyspecific organic cation transport proteins (OCTs). These membrane bound proteins are present in several tissues like the liver, kidney etc and their function is to facilitate transport of endogenous hydrophilic bases in and out of cells (1). Recently, it has been shown that they are present in the air luminal membrane of ciliated epithelial cells in rat and human lung (2). There are 7 subtypes of the protein (OCTl, OCT2, OCT3, OCTNl, OCTN2, OCTN3 and CT2 (OCT6)), distinguishing in tissue and cell localisation. The compound was incubated with HEK293 cells transfected with hOCTl. Compound A was rapidly taken up by HEK cells expressing hOCTl but only very slowly by HEK cells transfected with an empty vector (Figure 1).

The cell uptake at different concentrations showed that compound A is a high affinity substrate for this cell uptake transporter protein with a Km of approximately 3 μM.

On the basis of this information, we propose a hypothesis for efficacy and duration of compound A in the lung. A saturable uptake mechanism of compound A into lung epithelial cells appears to exist that possibly is facilitated by polyspecific organic cation transport proteins (OCTs). This mechanism may target the epithelial cells in the lung. The low permeability of the compound is another important property which helps retain the compound in the lung and ensures that compound taken up intracellullary does not escape too rapidly into the systemic circulation. Accordingly, it can also be hypothesised that at high lung concentrations (> 10 μM) of the compound, the lung cell uptake transporter system is saturated and as a consequence, the compound is rather quickly (T/4 Ih) escaping the lung into the blood through pores/tight junctions in the lung epithelial layer. At low lung concentrations (< 10 μM), on the other hand, the compound is efficiently taken up into the epithelial cells by cation transporters. The further transport into the blood from the intracellular compartment is a slower process (T 14 6h) probably due to the low passive permeability of the compound.

As mentioned above, there is a pure local effect with compound A and the blood levels do not seem to contribute to the effect. This may be explained by a preferential expression of transporter proteins at the air luminal membrane of the lung epithelial cells.

Example 2

Data obtained with other IKK2 inhibitors

The results obtained with compound A led us to speculate if other IKK2 inhibitor compounds are taken up intracellularly by similar mechanisms. We therefore incubated some IKK2 inhibitors with OCTl, OCT2 and OCT3 transfected cells and measured the cell uptake in comparison with control cells (empty vector). From Figure 2 it can be seen that compounds A-F are all substrates for OCTl. Compound C also seems to be a substrate for OCT2 and OCT3. In contrast to the other compounds, Compound G is a more lipophilic compound with a high passive permeability and is not a substrate for cation cell uptake transporters. The results suggest that it might be possible to design compounds that are good substrates for lung OCTs in combination with a low passive permeability in order to target specific cells in the lung with a long duration of effect. Example 3

Permeability properties

The passive permeability and/or transporter catalysed efflux from the intracellular compartment into the blood might be one way of controlling the intracellular residence time of compounds taken up into lung cells by transporters. A low passive permeability seems furthermore to be a requirement for compounds transported by cationic cell uptake transporters. The influence of permeability for the lung disappearance TV₂ in rat lung in vivo after i.t. administration of different IKK2 inhibitors is shown in Figure 3. As can be seen, compounds (I, J and K) with a higher passive permeability (higher PAMPA or Caco2 values) have a shorter lung disappearance TV. than e.g compound H which is a low passive permeability compound.

The results strongly indicate that the passive permeability is a component (in addition to drug transporter mechanisms) that controls the intracellular residence time in lung.

Example 4

Lung transporters

The importance of transporters for absorption, tissue distribution and elimination of compounds has been known for some years but is still a rapidly growing research area within DMPK. The main focus has been on transporters of importance for the blood-brain barrier, their presence in the liver and kidney for the elimination of drugs into bile and urine and their presence in the GI-tract as determinants for drug absorption/efflux. Little is known about transporters in the lung. Data shown above indicates that transporters in the lung might be of fundamental importance for local uptake intracellular^ as well as for local intracellular residence time in the lung of inhaled drugs. In an attempt to find out which transporter proteins that are present in the human lung, we undertook TaqMan analysis of lung epithelial brushings from 1 normal individual, 1 healthy smoker and 1 COPD patients (Figure 3).

As can be seen in Figure 3 , mRNA for a number of transporters were found in human lung. Among the cation transporters, the highest expression was found for OCTNl and 0CTN2 and among the anion transporters, mRNA for OATP-D and OATP-H was found at the highest levels. In addition, mRNA for a number of ABC efflux transporters as well as for OST alpha was found. Although, the mRNA expression levels of OCTl were low, it cannot be excluded that a significant cellular compound uptake may occur via this transporter. Significant protein levels of OCT 1-3 have previously been detected at the luminal side of airway epithelial cells (2). However, due to higher expression, OCTNl and 0CTN2 may be of quantitatively greater importance in facilitating lung cell uptake of hydrophilic basic compounds in the lung.

Example 5

Fhiorometric organic cation transporter inhibition assay

Introduction

This fluorescence based inhibition assay using ASP⁺ (4-(4-(dimethylamino)styryl)-N- methylpyridiniumiodide) as fluorescent substrate was developed for investigation of compound interactions with organic cation transporters found in lung. ASP⁺ is a substrate of OCTs, it is not cell toxic and at physiological pH this compound is permanently charged with a very low permeability across membranes. ASP⁺ is strongly fluorescent when taken up into cells, but virtually non-fluorescent in aqueous buffer solutions [1, 2, 3]. Adherent hOCTl transfected HEK 293-cells in 96-well plates are incubated with the test compounds and the ASP⁺ uptake in the cells is monitored online by spectrofluorometry. The initial rate of ASP⁺ uptake in the presence of test compound is compared to uptake in absence of inhibitor. 100 μM Verapamil (or other potent OCT inhibitor may be used as a positive control and shows almost 100% inhibition. The assay may either be used as a spot test, measuring % inhibition at two concentrations (10 and 100 μM), or for the IC₅₀ determination with eight different concentrations of test compound. It displays a useful tool for identification of hOCT inhibitors and may thereby also provide an indication of potential substrates. We have also demonstrated that this assay may be used with HEK293 cells transfected with other organic cation transporters e.g. OCT2, OCT3, under the same conditions, to identify inhibitors of other organic cation transporters.

Method

• HEK293 cells transfected with organic cation transporters e.g. hOCTl are seeded in Poly-D-Lysine coated 96-well plates (black/ clear) at a density of 4* 1Oe⁵ cells/ ml for 2 days growing and 3* 1Oe⁵ cells/ ml for 3 days growing.

• Approx. 24 hours before running the assay the medium is changed for protein expression stimulation by 10 mM sodium butyrate if needed to achieve higher transporter expression [4, 5].

• Cells should be 90-100 % confluent at the time of the assay.

• Prewarm waterbath, thermomixer,fluorescence reader, substrate solution (4 uM ASP⁺ solution in assay HBSS pH 7.4) and assay buffer (HBSS pH 7.4) for washing the cells at 37⁰C.

• Prepare master plate of test compound/s containing either 2-3 (spot test) or 8 (IC₅₀ determination) concentrations of each test compound.

• Prime injection pump of the fluorescence reader with prewarmed ASP⁺ solution and set layouts and test conditions.

• Prewarm test compound plate for 5-10 min. at the thermomixer.

• Wash the plated cells twice with 200 μl per well prewarmed assay buffer. Avoid damaging the cell monolayer.

• Then pipet very carefully 100 μl each well prewarmed inhibitor solution from the master plate to the cells. Insert plate into the reader and meassure a baseline to check if test compound is fluorescent itself. • Start injection (100 μl/ well) of the ASP⁺ solution (4 μM results in 2 μM) by the automatic injection pump.The uptake is monitored online by spectrofluorometryCalculate the %inhibition or IC₅₀ based on the initial rate of ASP+ uptake.

References

[I] A. Stachon et al. (1996) Cell. Physiol. Biochem. 6: 72-81 [2] A. Stachon et al. (1997) Cell. Physiol. Biochem. 7: 264-274 [3] T. Mehrens et al. (1999) J Am. Soc. Nephrol. 11 : 1216-1224 [4] J. Grunberg et al. (2003) Biotechn. 34 (5): 968-972 [5] Y. Cui et al. (2001) MoI. Pharmacol. 60 (5): 934-943

Example 6

Transporter substrate assay

Introduction

This Example describes how to culture HEK 293-cells (human embryonic kidney 293s cells) stably transfected with drug transporters and how to identify and characterise substrates of lung drug transporters in these cells. The description applies to cells transfected with different drug transporters expressed in lung - such as human OCTl, and BCRP, . However a similar cell culture and assay protocol may also be used for HEK 293-cells transfected with other lung transporters of the OATP, OCT, OCTN, OAT and ABC families. Briefly, compounds are incubated with test compound for a designated time. The uptake of compound is stopped by washing cells and thereafter adding lysing solution. The amount of compound in the cell lysate is measured by LC-MS/MS or, if compound is radiolabeled, by scintillation counting. The assay may be used for initial uptake/efflux rate determinations as well as concentration dependence (Km and Vmax) investigations. Empty vector transfected cells are used as a control and also to estimate the passive permeability of compounds. This example is focussing on a procedure optimised for organic cation transporters using non-labelled compound. The conditions are similar for other transporters although the time of incubation is different depending on that the time course is different depending on the transporter type.

Cell culture protocol

• The transfected HEK 293-cells are subcultured twice a week at 70-80% confluency. The medium used is DHI supplemented with 10% FBS, 2 inM L- glutamine, 1.8 mM CaC12 and 500 μg/mL geneticin

• The cells are passaged by washing with prewarmed D-PBS^""and treating cells with trypsine/ EDTA for 1 minute. Detach the cells and collect cells in a centrifuge tube with warm medium.

• Centrifuge 4 min at 115xg (800 rpm) at room temperature.

• Discard supernatant from cells, resuspend cell pellet gently in 10 ml prewarmed medium and count the cells.

• Seed 1x10° per T75-flask for 3-4 days growing.

Uptake assay protocol

• Seed cells (HEK293 cells transfected with transporter and empty vector transfected cells as a control) in Poly-D-Lysine coated 12-well dishes at a density of 3xlθe⁵ cells/mL for 3 days growing. At the time of the assay the cells should be 90-100% confluent.

• Approx. 24 hours before running the assay, sodium butyrate is added to the medium if needed to increase transporter protein expression.

• Wash the plated cells twice with approximately 500 μl HBSS/well. Use preheated (37⁰C) buffer and keep cell plates on plate warmer. Pipette and remove buffer carefully to avoid damaging the cell monolayer.

• Preincubate cell plates with 500 μl HBSS for 5 min.

• Remove buffer and add compound diluted in HBSS. The volume should be 500 μl/well. Routinely compounds are incubated at 2.5 uM. Include a reference substance as a positive control.

• Incubate for selected time intervals, routinely an incubation time of 4 min is used. For Km and Vmax determinations 1 min is preferred to ensure initial linear rate. • Remove solution and add approximately 500 μl ice cold HBSS.

• Wash cells twice in ice cold HBSS.

• Remove buffer and make sure all is completely removed since removal of excess substrate is critical for good results.

• Add 500 μl lysing solution (methanol/1% HAc containing internal standard) to each well. The internal standard compensates for the loss in volume during incubation and sample preparation.

• Transfer the content of each well to a 96-deep well plate and centrifuge 1500 g for 10 minutes.

• Transfer the supernatant to a new 96-deep well plate, dilute each sample with water (1 :3).

• Analyse with LC-MS/MS using standard curve and internal standard.

• Calculate uptake rate and compare compound uptake rates between transporter and empty vector transfected cells to evaluate the transporter-mediated component of the uptake. Calculate Km and Vmax to evaluate the affinity and maximal uptake rate.

Example 7

Lung PK studies in small animals

Measurements of compound concentrations in lung after local administration is of fundamental importance in projects where the aim is to target the lung. For inhaled compounds, the blood concentrations do not necessarily mirror the concentration in lung since the whole idea behind local administration is to create a non-equilibrium condition between lung and blood. Preferably, animal experimental methodology should be used. Materials & Methods

Animals

Rats and mice are the most common animals used in these studies. Guinea pigs and hamsters can also be used. The choice of animal species should be guided by the species used in the animal PD studies or disease model studies so that the results can be compared to biological effect data.

Dosing methodology

Inhalation can be performed either by using dry powder inhalation or nebulisation of a drug solution/suspension into an inhalation chamber in which the animals are exposed either nose only or whole body to the aerosol

Intratracheal instillation (i.t.) is another dosing technique used in these experiments.

Briefly, the animals are lightly anaesthetised by isoflurane. A special cannula, connected to a syringe, is inserted in the trachea normally at the bifurcation and max 1 mL/kg is injected into the trachea.

Formulations for i.t. instillation

In general, a solution of the test compound should be used as first choice. By using a solution, the dissolution step of the compound in lung can be avoided and data obtained from the study is more easily interpreted. However, in many cases, the solubility of the compound is too low and dosing of a suspension is therefore the only choice.

Time points for collection of lung and blood

When the study is planned, it's usually not known if the compound is absorbed rapidly or slowly into the systemic circulation. Therefore, time points should be chosen to cover for both scenarios. The following suggested time points are sufficient for a full profile: 5, 15, 30 min and 1.5, 4, 8, 24 and 3Oh. Animal experiment

At each dosing occasion, the syringe + tracheal cannula is weighed before and after administration. The animals are also weighed before each administration.

At the sampling occasion, the animal(s) is anaesthetized with isoflurane, pentobarbital or with other relevant anaesthezia. Vena jugularis or arteria carotis is cut with scissors to drain as much blood as possible from the lungs. The lung is then carefully dissected out with all non-relevant tissue removed.

Calculations

The results from the LC_MS/MS analysis is given as concentration of compound in the whole lung (nmol/L) The lung disappearance half life (lung TV2) can be calculated and is considered of importance for evaluation of dosing interval needed to maintain effective therapy and can also be used as a measure of lung retention time.

Example 8

Compound screening for cellular pharmacological activity using HEK293 cells transfected with hOCTl

Some IKK2 inhibitors with low passive permeability, known to be potent in biochemical cell free assays and in an in vivo pharmacology model did not show any potent effect in different cell models e.g. PBMC and HeLa cells. To evaluate whether this was due to the lack of uptake transporters in PBMC and HeLa cells consequently resulting in no cellular uptake and thus no interaction with the intracellular target, a pharmacological screening model using HEK293 cells transfected with hOCTl was developed. HEK293 cells transfected with hOCTl or empty vector as control were stimulated with TNFα and IL-8 levels were measured 24 hours after the stimuli. For key compounds selected from the biochemical assay screen, the IC50 of TNFα-induced IL-8 release dropped significantly in hOCTl -transfected cells compared to empty vector transfected cells. An example is shown in figure 5 in which the IC50 for compound A decreased around 60-fold in hOCTl transfected cells compared to empty vector cells. With other compounds an even larger decrease was observed. To follow up on this change in IC50, the intracellular concentration of compound A λvas estimated from analysing amount of the compound in cell lysates. When HEK293 cells transfected with hOCTl were incubated with 25 nM compound A in the medium a 10-fold increase in the intracellular concentration was observed after 1 hour of incubation compared to when compound A was incubated with HEK293 cells with empty vector. This indicates that hOCTl is transporting compound A into the cells and by concentrating the compound intracellularly, IKK2 is exposed to a higher concentration of the compound than expected from the concentration added to the medium.

For several other compounds, the correlation of potency between the biochemical assay, cellular assay and in vivo pharmacology model improved significantly when using HEK293 cells transfected by hOCTl. A cell-based pharmacology screening model using the HEK293 cells transfected with an uptake transporter and empty vector transfected cells as control was therefore developed.

Materials & Methods

HEK293 cells transfected with an organic cation transporter e.g. hOCTl are seeded in poly-D-lysine coated 96-well plates at a density of 3-4x105 cells/mL and grown for 2-3 days. Empty vector transfected cells are cultured in the same way and and used as controls.

TNFα (10 ng/mL) is added to the cells either alone or together with an IKK2 inhibitor at different concentrations.

After 24 hours of incubation, the plates are centrifuged at 4°C and medium collected.

IL-8 levels are measured in the medium using FMAT. TABLE 1: Accession numbers for the lung transport proteins

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All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biology (eg. molecular biology), physiology and/or pharmacology or related fields are intended to be within the scope of the following claims.

Claims

1. An assay method for identifying an agent that is taken up and retained intracellularly in lung tissue comprising the steps of:

(a) contacting a test sample comprising a lung transporter protein with an agent that acts on an intracellular target in the lung;

(b) identifying an agent that is a substrate for the lung transporter protein; and

(c) measuring the retention time of the agent in the lung tissue,

wherein a retention time of at least 0.5 hours is indicative of an agent that is retained in the lung tissue.

2. The assay method according to claim 1, wherein a retention time of at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 25 hours, or at least 30 hours is indicative of an agent that is retained in the lung tissue.

3. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is a cationic lung transporter protein.

4. The method according to claim 3, wherein the cationic lung transporter protein is selected from the group consisting of OCTl, CT2, OCT2, OCT3, OCTNl, 0CTN2 and 0CTN3 or mixtures thereof comprising at least two cationic lung transporter proteins.

5. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is a monocarboxylic acid transporter protein.

6. The assay method according to claim 5, wherein the monocarboxylic acid transporter protein is selected from the group consisting of MCTl, MCT3, MCT4 and MCT5 or mixtures thereof comprising at least two monocarboxylic acid transporter proteins.

7. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is a nucleoside transporter.

8. The assay method according to claim 7, wherein nucleoside transporter is selected from the group consisting of CNTl, CNT2, CNT3, ENTl, ENT2, ENT3 and ENT4 or mixtures thereof comprising at least two nucleoside transporters.

9. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is an amino acid transporter.

10. The assay method according to claim 9, wherein the amino acid transporter is selected from the group consisting of PATl, PAT2, PAT3, PAT4, NBAT, RBAT, 4F2HC, CATl, CAT2, CAT3, CAT4, LATl, LAT2, LAT3, y+LAT-2, LPI, y+LATl, y+LAT-1 and LPI-PCl or mixtures thereof comprising at least two amino acid transporters.

1 1. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is an oligo-peptide transporter.

12. The assay method according to claim 11, wherein the oligo-peptide transporter is PEPTl and/or PEPT2.

13. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is a bile acid transporter.

14. The assay method according to claim 13, wherein the bile acid transporter is NTCP and/or IBAT.

15. The assay method according to claim 1 or claim 2, wherein the lung transporter protein is selected from the group consisting of 0AT4, 0CTN3, MATEl, MATE2, OSTalpha ,OSTbeta, NATl, DAT, 5HTT, OATP-D, and/or OATP-H.

16. The assay method according to any of the preceding claims, wherein the disappearance of the agent from the lung is used as a measure of the retention time of the agent in the lung tissue.

17. The assay method according to any of the preceding claims, wherein the retention time is measured in a test animal.

18. The assay method according to any of the preceding claims, wherein the agent is administered to the test animal via intratracheal instillation or inhalation..

19. An assay method for identifying an agent that modulates the activity of a lung transport protein comprising the steps of:

(a) identifying an agent that is a substrate for the lung transport protein; and

(b) measuring the activity of the lung transport protein in the presence and absence of the agent;

wherein a difference in the activity of the lung transport protein in the sample as compared to a control is indicative that said agent modulates the activity of the lung transporter protein; and

wherein said lung transporter protein is a monocarboxylic acid transporter protein, and/or a nucleoside transporter, and/or an amino acid transporter, and/or an oligo-peptide transporter, and/or a bile acid transporter, and/or 0AT4, OCTN3, MATEl, MATE2, OSTalpha, OSTbeta, OCTl, OCT2, 0CT3, CT2, OCTNl, 0CTN2, 0CTN3, NATl, DAT, 5HTT, OATP-D, and/or OATP-H.

20. An assay method for identifying an agent that is a substrate for a lung transporter protein comprising the steps of:

(a) contacting a sample comprising a lung transporter protein with an agent; and

(b) identifying an agent that is a substrate for the lung transport protein; and

(c) measuring the uptake of the agent by the lung transporter protein in the presence and absence of the agent;

wherein a difference in the uptake of said agent by the lung transporter protein as compared to a control is indicative that said agent is a substrate for a lung transporter protein; and

wherein said lung transporter protein is a monocarboxylic acid transporter protein, and/or a nucleoside transporter, and/or an amino acid transporter, and/or an oligo-peptide transporter, and/or a bile acid transporter, and/or OAT4, OCTN3, MATEl, MATE2, OSTalpha, OSTbeta, OCTl, OCT2, 0CT3, CT2, OCTNl, 0CTN2, OCTN3, NATl, DAT, 5HTT, OATP-D, and/or OATP-H.

21. A process comprising the steps of:

i) performing the assay method according to any of claims 1-20;

ii) identifying an agent; and

iii) preparing a quantity of that agent.

22. A process according to claim 21, comprising the step of:

iv) preparing a pharmaceutical composition comprising that agent.

23. A process comprising the steps of:

i) performing the assay according to any one of claims 1 to 20;

ii) identifying an agent capable of modulating the retention time of the lung transporter protein;

iii) modifying said agent; and

iv) preparing a pharmaceutical composition comprising said modified agent.

24. A pharmaceutical composition comprising an agent identified by the assay method of any one of claims 1 to 20 or the process of any one of claims 21 to 23 admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof.

25. A process of preparing a pharmaceutical composition comprising admixing an agent identified by the assay method of any one of claims 1 to 20 or the process of any one of claims 21 to 23 with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.

26. A method of treating a disease in a human or animal which method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an agent identified by the assay method of any one of claims 1 to 20 or the process of any one of claims 21 to 23, wherein the agent is capable of modulating the disease and wherein said composition is optionally admixed with a pharmaceutically acceptable carrier, diluent excipient or adjuvant and/or combinations thereof.

27. An agent identifiable, preferably, identified by the assay method according to any one of claims 1 to 20.

28. An agent identifiable, preferably, identified by the assay method according to any one of claims 1 to 20 for use in the treatment and/or prevention of disease.

29. Use of an agent identifiable, preferably, identified by the assay method according to any one of claims 1 to 20 in the manufacture of a composition for the treatment of a disease.

30. An assay method, a process, a pharmaceutical composition, a method, an agent or a use substantially as described herein with reference to the accompanying Figures.