WO2011035140A1 - Methods and compositions for delivery of contrast moieties to the lungs - Google Patents

Abstract

Disclosed are imaging compositions formulated for inhalation comprising a conjugate of a pulmonary surface active agent and a contrast agent. The surface active agent has an affinity for the human alveolar/gas interface and comprises at least a portion of a mammalian lung surfactant or a mimic thereof. The disclosure also provides a method of imaging respiratory tissue in a mammal suffering from or at risk of suffering from a lung disease comprising administering to the subject a conjugate comprising a contrast agent for imaging lung tissue and a surface active agent by inhalation in an amount effective to permit respiratory tissue imaging.

Description

METHODS AND COMPOSITIONS FOR DELIVERY OF CONTRAST MOIETIES

TO THE LUNGS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 61/243,721, filed September 18, 2009, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] There are many reasons why improved lung imaging could be beneficial in the diagnosis, prognosis, and management of lung diseases and disorders. For example, the ability to detect the exact location in the lung tissue of a bronchioalveolar or squamous cell carcinoma, adenocarcinoma, or other solid tumor, particularly when it is in its early stage of development, could have significant benefit in the management of these typically fatal diseases. In pulmonary disorders including chronic obstructive pulmonary disease (COPD), chronic bronchitis, and emphysema, there is a chronic obstruction of air flow in and out of the lungs or a change in lung morphology. The obstruction that manifests in these disorders is often permanent and progresses over time. Exacerbations, which are an acute worsening of respiratory function, result in increased morbidity and mortality. The ability to image the fine structure of the affected tissue with improved resolution may aid the management of such diseases.

[0003] Imaging of respiratory tissues in patients suffering from pulmonary disorders is often needed for performing diagnosis and monitoring of patients. Such can be accomplished, for example, by ultrasonic imaging, X-ray imaging, computed tomography, magnetic resonance imaging (MRI), optical imaging, and radionuclide imaging such as Positron Emission

Tomography (PET) and Single Photon Emission Computed Tomography (SPECT). Contrast agents have been used in conventional imaging approaches when the inherent or native signal in vivo is absent or poor. A contrast agent serves to provide a stronger, more easily identifiable signal to an otherwise poorly detectable tissue site.

[0004] Up to this time, many of the known contrast agents have been introduced into patients by injections, which is invasive and can be uncomfortable. There are well-known challenges of delivering any medicament to the deep lungs by inhalation. In addition, lung imaging is particularly challenging due to the predominance of air in the lungs and potential artifacts created by cardiac and respiratory motions. A further obstacle with the delivery of contrast agents to the lungs, whether the agent is delivered orally, parenterally, or by inhalation, is achieving meaningful residence times, given the rapid clearance of xenobiotics by the lung. Thus, there remains an unmet need for effective lung imaging.

SUMMARY

[0005] This disclosure describes methods and compositions for delivering contrast moieties to the lungs. It is now appreciated that one problem associated with imaging respiratory tissues is the difficulty in obtaining sufficient residence times of contrast moieties (e.g. , contrast agents) in the lungs. The lungs are very adept at clearing foreign matter, such that contrast moieties may be cleared from the lung before the desired image is achieved. Also, attempts at enhancing imaging of lung tissue suffer from excessive background signal which presents itself as noise. Accordingly, pulmonary tissue imaging could be improved if a contrast agent with preferential affinity for lung tissue could be developed, and a means to enable distribution throughout the lungs could be found.

[0006] Pulmonary surfactants are secreted by Type II pneumocytes in the lungs of all mammals to reduce surface tension within the alveoli, thereby preventing alveolar collapse during expiration. Pulmonary surfactant comprises a complex of lipids and proteins, spread across the alveolar surface, and are maintained in the lung for extended periods. In accordance with this invention, the residence time of contrast moieties in the lung and its distribution there within are enhanced by linking the contrast moiety to a surfactant protein, truncated

polypeptide, or a mimic thereof and/or a surfactant lipid. Administering contrast moiety linked to a surfactant protein or polypeptide and/or a surfactant lipid as a conjugate provides increased duration (dwell time) of the agent in the lung and better and more thorough distribution of the agent within the vast lung surface area including distal alveoli. This results in several advantages: substantially fewer and/or smaller doses of the contrast agent are possible;

administration becomes rapid and convenient by inhalation; there may be better patient compliance; and the contrast agent is better localized to the lung tissue resulting in decreased systemic toxicity and higher localized lung concentrations for enhanced efficacy. [0007] In one aspect, the invention provides an imaging composition, formulated for inhalation, comprising a surface active agent that has an affinity for the human alveolar/gas interface. The surface active agent comprises at least a portion of a mammalian lung surfactant protein or polypeptide or mimic thereof that is substantially non-immunogenic to humans or tolerably immunogenic. In some embodiments, the surface active agent may additionally comprise a mammalian lung surfactant lipid. This surface active agent is associated with a contrast moiety, and preferably bonded to the contrast moiety, covalently or by chelation, for example. The class of such compositions as described herein is referred to simply as "imaging conjugates."

[0008] The composition also may comprise a targeting moiety, which binds to an extracellular or cell surface-bound target or other target accessible to the pulmonary/gas interface within the lung. The extracellular or cell-surface target may be, by way of example, scar tissue, a colony of infectious microorganisms, or a receptor such as an EGF receptor, a TNF receptor, a VEGF receptor, or a P2X or P2Y purinergic receptor. Such compositions are referred to herein as "targeted imaging conjugates," and comprise a subgroup of the imaging conjugates. The targeting moiety may improve the contrast of the tissue bearing the target. Targeting moieties may include a wide variety of ligands known to bind preferentially to an extracellular or cell surface bound target (e.g. , a cell surface receptor or its ligand) such as agonists, antagonists, inhibitors, or specific binders such as antibodies and their engineered derivatives such as Fab, Fab', F(ab')₂, Fv, and SFv fragments. A targeting moiety may be attached to a surface active agent or to a contrast moiety.

[0009] The compositions can be administered to a subject suffering from lung disease, or at risk of contracting lung disease, including, but not limited to, emphysema, tuberculosis, cystic fibrosis, asthma, COPD, and lung cancer, in order to image the respiratory tissues or portions thereof.

[0010] In one embodiment, the surface active agent comprises a human lung surfactant protein or polypeptide or a non-human mammalian lung surfactant polypeptide or a fraction thereof. The compositions may also include lipid components in admixture with the conjugate, e.g. , dipalmitoylphosphatidylcholine (DPPC). Exemplary non-human mammalian lung surfactants include bovine, porcine, or ovine lung surfactants or a fraction thereof. The agent may comprise or be derived from a mammalian lung surfactant harvested from the lungs of a human or non-human mammal. [0011] In another embodiment, the surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide, an allelic variant thereof, or a synthetic mimic thereof. The agent may comprise a natural surfactant polypeptide, such as SP-A, SP-B, SP-C, SP-D, portions thereof, or mixtures thereof. The agent may comprise a mixture of SP-A, SP-B, SP-C, SP-D or portions thereof. Exemplary peptides include at least about a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid fragment of a natural surfactant polypeptide which retains surface active property, e.g., spread throughout the lungs mimicking the preference for the air/lung tissue interface of the naturally occurring surfactant polypeptides. The surface active agent may comprise at least a portion or optionally the entirety of SP-B. Exemplary SP-B

polypeptides include at least about a 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid fragment of SP-B. An SP-B peptide may be a truncated amino-terminal peptide or a truncated carboxy- terminal peptide. An exemplary SP-B peptide may be a 25-amino acid amino terminal peptide. Synthetic 25-mer SP-B has been shown to retain a high percentage of the surfactant activity of the intact full length protein.

[0012] In another embodiment, the surface active agent comprises a synthetically produced peptide. A peptidomimetic may comprise at least one deletion or amino acid substitution mutant of a mammalian or human lung surfactant polypeptide.

[0013] In another embodiment, the surface active agent may comprise a surfactant polypeptide that is produced synthetically, e.g., recombinantly or by peptide synthesis technology. A recombinant mammalian lung surfactant polypeptide, such as SP-A, SP-B, SP- C, SP-D, or a portion thereof may be produced by expressing the DNA coding for SP-A, SP-B, SP-C, SP-D, or a portion thereof in a prokaryotic or eukaryotic expression system.

Recombinant surfactant polypeptides may be the same or differ from mammalian lung surfactant polypeptides. A recombinant polypeptide may comprise at least one deletion or amino acid substitution mutant of a mammalian, preferably a human lung surfactant polypeptide. Synthetic mimics of lung surfactant peptides may be used, for example, a 21- amino acid peptide with structural similarities to pulmonary surfactant protein B available from Discovery Labs and known in the art as "KL4". See U.S. Pat. Nos. 5,164,369, 5,260,273 and 5,407,914, incorporated herein by reference. Other amphipathic SP-B or SP-C polypeptide mimetics may be used. [0014] In another embodiment, the surface active agent comprises a surfactant lipid. In some embodiments, the surface active agent comprises both a surfactant polypeptide and a lipid. Exemplary surfactant lipids include phospholipids (e.g., dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, and phosphatidylglycerols), neutral lipids, and cholesterol.

[0015] The surface active agent and/or targeting moiety (if used) may be bonded to a contrast moiety. The surface active agent preferably is covalently bonded or chelated to a contrast moiety. The contrast moiety may be linked to either a surfactant protein or surfactant lipid or both. In certain embodiments, the contrast moiety may be bonded to an amino- or carboxy-terminal amino acid or an internal amino acid of the surfactant polypeptide. In some embodiments, the contrast moiety may be covalently bonded to a surfactant lipid through an amide or ester bond. In instances where the contrast moiety contains a carboxylic acid, a surfactant lipid bearing an amino group (or the surfactant lipid is modified to contain an amino group) is reacted with the contrast moiety in the presence of a peptide coupling reagent. In other embodiments, the contrast moiety may be covalently bonded to a surfactant lipid through an amide or ester bond, or via an ether, amino, or amido-alkylene linkage. It is also

contemplated that the contrast moiety may be bonded to a targeting moiety.

[0016] In certain embodiments, more than one contrast moiety may be bound to the surface active agent or the targeting moiety (if used). For example, a single contrast moiety may be bound to the surface active agent and mixed with at least one other contrast moiety bound to the surface active agent. When more than one contrast moiety is bound to the surface active agent, the contrast moieties may be bound to surfactant proteins, polypeptides, or mimics thereof, and/or surfactant lipids. It is also contemplated that at least one contrast moiety may be bound to the surface active agent and at least one contrast moiety may be bound to the target moiety.

[0017] In one embodiment, the contrast moiety is extended with an amino acid or mimetic linker, such as a glycine linker, to create an unnatural amino acid that can be used in automated peptide synthesis. The extended molecule (e.g. , the contrast moiety plus the amino acid linker) can then be attached to the surface active agent, for example, through an amino- or hydroxyl- group.

[0018] The contrast moiety is carried by the surface active agent and may spread throughout the lungs induced by the polypeptides surfactant activity. Targeted imaging conjugates bind to an extracellular or cell- surface bound target that is accessible to the pulmonary/gas interface, and may permit resolution of a structure bearing the target within a background of surfactant spread throughout the lung. The contrast moieties may include, but are not limited to, X-ray contrast agents, MRI contrast agents, optical contrast agents, atomic particle emitters, and ultrasonic contrast agents. In certain embodiments, more than one contrast moieties may be bonded to a surface active agent (and/or targeting moiety, if one is used) and administered in combination. When more than one contrast moiety is bonded to a surface active agent (and/or targeting moiety, if one is used), the contrast moiety may be the same contrast moiety, a member of the same class of contrast moieties, or a member of a different class of contrast moieties. It is contemplated that some contrast moieties may be better suited for bonding to the surface active agent where others may be better suited for bonding to the targeting moiety (e.g. , it is preferable to bind ultrasonic contrast agents to a targeting moiety.)

[0019] The imaging composition is delivered to the lungs of a human patient by an inhalation device. Exemplary inhalation devices include fixed dose inhalers, metered dose inhalers, and nebulizers.

[0020] In another aspect, the invention provides a method for imaging respiratory tissues in a mammal, the method comprising administering the imaging composition to the mammal by inhalation or pulmonary instillation in an amount effective to spread throughout at least a portion of the alveolar surface area of the lungs of the mammal, and detecting and displaying the position of the contrast moiety to indicate the presence or position of a tissue of interest. In certain embodiments, a mammal requiring imaging of respiratory tissue may be suffering from lung inflammation, lung disease, or a lung disorder. The method may also comprise administering an imaging conjugate comprising a contrast moiety bonded to a surface active agent and optionally a targeting moiety, where the surface active agent is characterized by an affinity for the human alveolar/gas interface, and wherein the surface active agent comprises at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof that is substantially non-immunogenic or tolerably immunogenic to humans. The imaging conjugate is administered to the subject by inhalation or instillation in an amount effective to permit determination of the presence or position of all or a portion of the mammalian lung tissue or of a cell or tissue bearing a target in the lung. [0021] The administration serves to target the contrast moiety to the lungs of a subject in need thereof. The compositions of matter and the methods of the invention have the dual advantage that they reduce the systemic bioavailability and distribution of the contrast moiety relative to inhalation administration of an unconjugated contrast moiety, and this provides an advantage in dealing with off target toxicities. The compositions of matter and the methods of the invention also facilitate distribution and increase the residence time of the contrast moiety in the lung relative to inhalation administration of an unconjugated contrast moiety, and therefore very significantly improve lung bioavailability. The result can be a reformulation or redesign of a known contrast moiety that has unacceptable toxicity and/or poor lung tissue discriminating power, thereby to provide enhanced contrast of respiratory tissues and substructures therein to produce a clinically valuable novel contrast moiety.

[0022] In one embodiment, the administration of a contrast moiety-surface active agent conjugate reduces the dosing frequency relative to administration of an unconjugated contrast moiety. The administration step may be a single dose administered prior to imaging the mammal's respiratory tissue. Alternatively, the administration step may be repeated at multiple doses prior to imaging of respiratory tissues (e.g., at least two, three, four or more doses) administered over a 30 minute, 1 hour, two hour, three hour or longer time period. In some embodiments, the administration step may be repeated once daily, every other day, every three days, every four days, every five days, weekly, biweekly, monthly, bimonthly, quarterly, semi- annually, or annually to obtain images of respiratory tissue. The administration step may be conducted using an inhaler, an aerosol, particulates with or without propellants, metered dosages, or a nebulizer.

[0023] In certain embodiments, the mammal in need of respiratory tissue imaging may be suffering from lung inflammation or disease or is at risk of suffering from a lung disease. The subject in need of respiratory tissue imaging may be suffering from emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory distress disorder (RDS), pneumonia, tuberculosis or other bacterial infection, cystic fibrosis, and/or lung cancer.

[0024] In certain embodiments, it is advantageous to produce data sets derived from imaging healthy lung tissue to develop a data filter that can be used to remove or minimize the effect of background and to improve interpretation of corresponding images of diseased lung. In other embodiments, data generated from imaging lung tissue characterized by different diseases (e.g. , emphysema or tuberculosis), or varying degrees of disease severity or progression may be used as standards or "fingerprints" against which the nature of or severity of disease in a presenting patient can be assessed.

BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1A shows the nucleic acid sequence that encodes human surfactant protein A (SEQ ID NO: 1). Figure IB shows the amino acid sequence for human surfactant protein A (SEQ ID NO: 2).

[0026] Figure 2A shows the nucleic acid sequence that encodes human surfactant protein B (SEQ ID NO: 3). Figure 2B shows the amino acid sequence for human surfactant protein B (SEQ ID NO: 4). Figure 2C shows the amino acid sequence for mature human surfactant protein B (SEQ ID NO: 5).

[0027] Figure 3A shows the nucleic acid sequence that encodes human surfactant protein C (SEQ ID NO: 6). Figure 3B shows the amino acid sequence for human surfactant protein C (SEQ ID NO: 7). Figure 3C shows the amino acid sequence for mature human surfactant protein C (SEQ ID NO: 8).

[0028] Figure 4A shows the nucleic acid sequence that encodes human surfactant protein D (SEQ ID NO: 9). Figure 4B shows the amino acid sequence for human surfactant protein D (SEQ ID NO: 10). Figure 4C shows the amino acid sequence for mature human surfactant protein D (SEQ ID NO: 11).

[0029] Figure 5A shows the nucleic acid sequence that encodes bovine surfactant protein A (SEQ ID NO: 12). Figure 5B shows the amino acid sequence for bovine surfactant protein A (SEQ ID NO: 13).

[0030] Figure 6A shows the nucleic acid sequence that encodes bovine surfactant protein B (SEQ ID NO: 14). Figure 6B shows the amino acid sequence for bovine surfactant protein B (SEQ ID NO: 15).

[0031] Figure 7A shows the nucleic acid sequence that encodes bovine surfactant protein C (SEQ ID NO: 16). Figure 7B shows the amino acid sequence for bovine surfactant protein C (SEQ ID NO: 17). [0032] Figure 8A shows the nucleic acid sequence that encodes bovine surfactant protein D (SEQ ID NO: 18). Figure 8B shows the amino acid sequence for bovine surfactant protein D (SEQ ID NO: 19).

[0033] Figure 9A shows the nucleic acid sequence that encodes porcine surfactant protein A (SEQ ID NO: 20). Figure 9B shows the amino acid sequence for porcine surfactant protein A (SEQ ID NO: 21).

[0034] Figure 10A shows the nucleic acid sequence that encodes a partial porcine surfactant protein B (SEQ ID NO: 22). Figure 10B shows a partial amino acid sequence for porcine surfactant protein B (SEQ ID NO: 23).

[0035] Figure 11 A shows the nucleic acid sequence that encodes porcine surfactant protein C (SEQ ID NO: 24). Figure 1 IB shows the amino acid sequence for porcine surfactant protein C (SEQ ID NO: 25).

[0036] Figure 12A shows the nucleic acid sequence that encodes porcine surfactant protein D (SEQ ID NO: 26). Figure 12B shows the amino acid sequence for porcine surfactant protein D (SEQ ID NO: 27).

[0037] Figure 13A shows the nucleic acid sequence that encodes ovine surfactant protein A (SEQ ID NO: 28). Figure 13B shows the amino acid sequence for ovine surfactant protein A (SEQ ID NO: 29).

[0038] Figure 14A shows the nucleic acid sequence that encodes ovine surfactant protein B (SEQ ID NO: 30). Figure 14B shows the amino acid sequence for ovine surfactant protein B (SEQ ID NO: 31).

[0039] Figure 15A shows the nucleic acid sequence that encodes ovine surfactant protein C (SEQ ID NO: 32). Figure 15B shows the amino acid sequence for ovine surfactant protein C (SEQ ID NO: 33).

[0040] Figure 16A shows the nucleic acid sequence that encodes a partial ovine surfactant protein D (SEQ ID NO: 34). Figure 16B shows a partial amino acid sequence for ovine surfactant protein D (SEQ ID NO: 35).

[0041] Figure 17 shows photographs (A-D) of lung histology sections from four mice instilled with saline.

[0042] Figure 18 shows photographs (A-D) of lung histology sections from four mice with HNE (human neutrophil elastase)-induced emphysema. DETAILED DESCRIPTION

Surfactant Proteins

[0043] The surface active agent component of the constructs of the invention comprises at least a portion of a mammalian lung surfactant polypeptide that is substantially non- immunogenic to humans. The polypeptide or portion thereof may be a mammalian lung surfactant moiety or a synthetic mimic thereof. Exemplary surfactant polypeptides may be animal-derived, recombinant, synthetic (made in a peptide synthesizer), analogs, or peptide mimetics.

[0044] Natural lung surfactant proteins include SP-A, SP-B, SP-C, SP-D, or portions thereof, alone or in combination with lipids (U.S. Pat. No. 5,302,581). In some embodiments, the surface active agent comprises the full length surfactant polypeptide. In other

embodiments, the surface active agent comprises a portion of a surfactant polypeptide. For example, human SP-B is a 79 amino acid residue polypeptide, however, the N-terminal 25 amino acid residues of SP-B possess therapeutic effects comparable to the whole peptide (Kurutz and Lee, Biochem., 41, 9627-36 (2002)). Exemplary peptides of natural lung surfactant proteins may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Exemplary peptides of human SP-B are shown in Table 1.

[0045] In one embodiment, the surface active agent comprises human lung surfactant obtained by lung lavage of human cadavers at autopsy or by lung lavage of consenting adults.

[0046] In certain embodiments, the surface active agent comprises a non-human mammalian lung surfactant or a fraction thereof. Exemplary non-human surfactants include bovine, porcine or ovine lung surfactants or a fraction thereof. The non-human surfactant may be harvested from the lungs of a non-human mammal using techniques that are well known in the art. For example, porcine surfactant may be obtained from newborn and/or adult pigs harvesting the broncho alveolar lavage (BAL) of the lungs with saline as described in Bernhard et ah, Am. J. Respir. Cell Mol. Biol. 17:41-50 (1997), which is incorporated herein by reference. Harvested BAL fluid is centrifuged to remove cells and then the cell-free BAL fluid is further centrifuged to generate a raw surfactant pellet. CUROSURF®, a natural porcine lung surfactant consisting of polar lipids (mainly phospholipids), SP-B, and SP-C may be used. Ovine surfactant may be obtained from whole lung lavages of adult sheep as described by

Brackenbury et al., Am. J. Respir Cir. Care Med. 163: 1135- 1142 (2001), which is incorporated herein by reference. The harvested alveolar lavage is centrifuged to remove cellular debris, followed by further centrifugation to obtain a pellet corresponding to a surfactant aggregate pellet. Bovine surfactant may also be obtained from the lung lavages of adult cows as described by Panda et al. (J Colloid Interface Sci., 311:551-5 (2007)), which is incorporated herein by reference. Alveofact®, Infasurf®, and Survanta®, natural bovine surfactant extracts containing phospholipids, neutral lipids, SP-B and SP-C polypeptides may also be used.

[0047] Proteins and polypeptides derived from or having characteristics similar to those human lung surfactant may also be used. For example, SP-B may be isolated from bovine surfactant using differential organic extraction, column chromatography, and/or preparative SDS-PAGE as described by Beers et ah, Am. J. Physiol Lung Cell Mol. Physiol. 262:L773- L778 (1992), which is incorporated herein by reference.

[0048] The mammalian lung surfactant polypeptides or portion thereof can also be recombinantly produced. Recombinant SP-A, SP-B, SP-C, SP-D, or a portion thereof is obtainable by expression of a DNA sequence coding for SP-A, SP-B, SP-C, SP-D, or a portion thereof in a suitable prokaryotic or eukaryotic expression system using various known techniques. Recombinant vectors, which are readily adapted to include a isolated nucleic acid encoding a surfactant polypeptide or a portion thereof, host cells containing the recombinant vectors, and methods of making such vectors and host cells as well as using them for the production of the encoded polypeptides by recombinant techniques are well-known. The nucleic acids encoding a surfactant polypeptide or a portion thereof may be provided in an expression vector comprising a nucleotide sequence encoding a surfactant polypeptide that is operably linked to at least one regulatory sequence. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. The vector copy number, the ability to control that copy number, and the expression of any other protein encoded by the vector, such as antibiotic markers, should be considered. The subject nucleic acids may be used to cause expression and over-expression of a kinase or phosphatase polypeptide in cells propagated in culture, e.g., to produce proteins or polypeptides, including fusion proteins or polypeptides.

[0049] Host cells may be transfected with a recombinant gene in order to express a surfactant polypeptide or portion thereof. The host cell may be any prokaryotic or eukaryotic cell. For example, a polypeptide may be expressed in bacterial cells, such as E. coli, insect cells (baculo virus), yeast, or mammalian cells. In those instances when the host cell is human, it may or may not be in a live subject. Other suitable host cells are known to those skilled in the art. Additionally, the host cell may be supplemented with tRNA molecules not typically found in the host so as to optimize expression of the polypeptide. Other methods suitable for maximizing expression of the polypeptide will be known to those in the art.

[0050] Methods of producing polypeptides are well-known in the art. For example, a host cell transfected with an expression vector encoding a surfactant polypeptide or portion thereof may be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained cytoplasmically. Cells are then harvested, lysed, and the protein is isolated from the cell lysates.

[0051] A cell culture includes host cells, media, and other by-products. Suitable media for cell culture are well known in the art. The polypeptide may be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, gel filtration chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite

chromatography, lectin chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of a polypeptide of the invention, and high performance liquid chromatography (HPLC) is employed for purification. Thus, a nucleotide sequence encoding all or a selected portion of a surfactant polypeptide may be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the sequence into a polynucleotide construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures. Similar procedures, or modifications thereof, may be employed to prepare recombinant polypeptides of the invention by microbial means or tissue-culture technology.

[0052] Expression vehicles for production of a recombinant protein include plasmids and other vectors. For instance, suitable vectors for the expression of a polypeptide of the invention include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX- derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. [0053] In certain embodiments, mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-I), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant protein by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the [beta]-gal containing pBlueBac III).

[0054] In another embodiment, protein production may be achieved using in vitro translation systems. In vitro translation systems are, generally, a translation system which is a cell-free extract containing at least the minimum elements necessary for translation of an RNA molecule into a protein. An in vitro translation system typically comprises at least ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex, comprising the cap-binding protein (CBP) and eukaryotic initiation factor 4F (eIF4F). A variety of in vitro translation systems are well known in the art and include commercially available kits. Examples of in vitro translation systems include eukaryotic lysates, such as rabbit reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect cell lysates and wheat germ extracts. Lysates are commercially available from manufacturers such as Promega Corp., Madison, WI; Stratagene, La Jolla, CA; Amersham, Arlington Heights, IU.; and GIBCO/BRL, Grand Island, NY. In vitro translation systems typically comprise macromolecules, such as enzymes, translation, initiation and elongation factors, chemical reagents, and ribosomes. In addition, an in vitro transcription system may be used. Such systems typically comprise at least an RNA polymerase holoenzyme,

ribonucleotides and any necessary transcription initiation, elongation and termination factors. In vitro transcription and translation may be coupled in a one-pot reaction to produce proteins from one or more isolated DNAs. When expression of a carboxy terminal fragment of a polypeptide is desired, e.g., a truncation mutant, it may be necessary to add a start codon (ATG) to the oligonucleotide fragment containing the desired sequence to be expressed. It is well-known in the art that a methionine at the N-terminal position may be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat et al, (1987) J Bacteriol. 169:751-757) and Salmonella typhimurium and its in vitro activity has been demonstrated on recombinant proteins (Miller et al., (1987) PNAS USA 54:2718-1722). Therefore, removal of an N-terminal methionine, if desired, may be achieved either in vivo by expressing such recombinant polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al).

[0055] Polypeptides of the invention may also be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes provide for certain advantages in their use. Conservative substitutions are those in which one amino acid residue is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite binding activity.

[0056] Polypeptides of the invention may also be truncated relative to the full-length mature polypeptide. Polypeptides may be truncated at either the amino-terminus, carboxy- terminus, or both termini. Polypeptides may be truncated by at least one amino acid, or at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 amino acids.

[0057] A mammalian lung surfactant polypeptide or a portion thereof can be synthesized from amino acids by techniques that are known to those skilled in the polypeptide art. A summary of the many techniques available may be found in J. M. Steward and J. D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969, and J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983 for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis.

[0058] In general, these methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group (e.g., lysine).

[0059] Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final

polypeptide. That polypeptide is then washed by dissolving in a lower aliphatic alcohol, and dried. The dried surfactant polypeptide can be further purified by known techniques, if desired.

[0060] In certain embodiments, commonly used methods such as t-BOC or f-MOC protection of alpha-amino groups can be used. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C-terminus of the peptide (See, Coligan et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can be synthesized, for example, by the well known solid phase peptide synthesis methods described in Merrifield, /. Am. Chem. Soc. 85: 2149, 1962, and Stewart & Young, 1969, Solid Phase Peptides Synthesis, pp. 27-62, using a copoly(styrene- divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0°C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.

[0061] In one embodiment, recombinant and/or synthetic SP-B peptides contain amino acids 2, 4, 6, and 9 of SEQ ID NO:5. Prolines 2, 4, and 6 and tryptophan 9 of SEQ ID NO:5 may constitute essential structural motifs for protein function. In some embodiments, SP-B peptides may be substituted at any amino acid residue other than tryptophan 9 amino acid (relative to SEQ ID NO:5).

[0062] A lung surfactant polypeptide mimic is generally a polypeptide that is engineered to mimic the essential attributes of human surfactant protein. An exemplary mimetic peptide mimics SP-B. One example of a SP-B mimic is KL4, a 21 amino acid residue peptide comprising the sequence KLLLLKLLLLKLLLLKLLLLK (SEQ ID NO: 94). This SP-B mimetic protein is also known as Lucinactant (Surfaxin®, Discovery Laboratories).

Surfactant Lipids

[0063] In certain embodiments, a surface active agent for use in the invention comprises a surfactant protein, a portion thereof, or mixtures thereof, which associates with natural surfactant lipids in vivo. In other embodiments, a surface active agent for use in the invention comprises a a lipid-protein complex.

[0064] Surface active agent for use in the invention disclosed herein may comprise one or more lipids. Natural mammalian lung surfactant is a complex of phospholipids, neutral phospholipids, and proteins. Over 90% of the surfactant is lipids; around half of which is dipalmitoylphosphatidylcholine (DPPC). Phosphatidylcholine molecules form a large portion of the lipid in surfactant and have saturated acyl chains. Phosphatidylglycerol (PG) is also present, it has unsaturated fatty acid chains that fluidize the lipid monolayer at the interface. Neutral lipids and cholesterol are also present. If the compositions of the invention are formulated together with a lipid, use of such naturally occurring molecules are preferred. [0065] In some embodiments, the optionally included surface active agent can comprise, for example, from as little as about 0.05 to 95% weight percent lipid, so long as the resulting composition has surfactant activity. By weight percent is meant the percentage of a compound by weight in a composition by weight. Thus, a composition having 50 weight percent lipid contains, for example, 50 grams lipids per 100 grams total composition. A surface active agent may contain 0.1 to 50 weight percent lipid, although higher concentrations of lipid can be used. Surface active agents containing both phospholipid and a surfactant polypeptide or portion thereof can contain, therefore, 0.1, 1, 10, 50, 80, to almost 100 weight percent lipid and about 50, 20, 10, to less than 1 weight percent surfactant polypeptide. Alternatively, surface active agents may contain the reverse ratios of lipid to surfactant polypeptide.

[0066] The term "lipid" as used herein refers to a naturally occurring, synthetic or semisynthetic (e.g., modified natural) compound which is generally amphipathic. The lipids typically comprise a hydrophilic component and a hydrophobic component. Exemplary lipids include, but are not limited to, phospholipids, fatty acids, fatty alcohols, neutral fats, phosphatides, oils, glycolipids, aliphatic alcohols, waxes, terpenes and steroids. The phrase semi- synthetic (or modified natural) denotes a natural compound that has been chemically modified in some fashion.

[0067] Examples of phospholipids include native and/or synthetic phospholipids.

Phospholipids that can be used include, but are not limited to, phosphatidylcholines (saturated and unsaturated), phospatidylglycerols, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids, phosphatidylinositols, sphingolipids, diacylglycerides, cardiolipin, ceramides, cerebrosides and the like. Exemplary phospholipids include, but are not limited to, dipalmitoyl phosphatidylcholine (DPPC), dilauryl phosphatidylcholine (DLPC) (C12:0), dimyristoyl phosphatidylcholine (DMPC) (C14:0), distearoyl phosphatidylcholine (DSPC), diphytanoyl phosphatidylcholine, nonadecanoyl phosphatidylcholine, arachidoyl

phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) (CI 8: 1), dipalmitoleoyl phosphatidylcholine (C16: l), linoleoyl phosphatidylcholine (C18:2), myristoyl palmitoyl phosphatidylcholine (MPPC), steroyl myristoyl phosphatidylcholine (SMPC), steroyl palmitoyl phosphatidylcholine (SPPC), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyl palmitooleoyl phosphatidylcholine (PPoPC), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylglycerol (DOPG), palmitoyloleoyl phosphatidylglycerol

(POPG), dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dimyristoylphosphatidylserine (DMPS),

distearoylphosphatidylserine (DSPS), palmitoyloleoyl phosphatidylserine (POPS), soybean lecithin, egg yolk lecithin, sphingomyelin, phosphatidylinositols, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidic acids, and egg phosphatidylcholine (EPC).

[0068] Examples of fatty acids and fatty alcohols include, but are not limited to, sterols, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, dipamlitic acid, and the like. Exemplary fatty acids include palmitic acid. Exosurf®, a mix of DPPC, cetyl alcohol, tyloxapol, and sodium chloride may be used as synthetic lung surfactant.

[0069] Examples of fatty acid esters include, but are not limited to, methyl palmitate, ethyl palmitate, isopropyl palmitate, cholesteryl palmitate, palmityl palmitate sodium palmitate, potassium palmitate, tripalmitin, and the like.

[0070] Surfactant polypeptide and surfactant lipids interact by hydrostatic interactions. Charged amino acids interact with the lipid polar head groups and hydrophobic amino acids interact with phospholipid acyl side chains. For example, SP-B and SP-C are proteins having significant hydrophobic character. Both SP-B and SP-C preferentially bind anionic lipids, such as phosphatidylglycerol (PG), and not DPPC. SP-A and SP-D are more hydrophilic proteins and interact with a broad range of amphipathic lipids, including glycerophospholipids, sphingophospholipids, glycosphingolipids, lipid A, and lipoglycans. SP-A binds DPPC. By way of example, hydrostatic interactions are observed with the SP-B mimetic, KL4, and lipids in natural surfactant or lipids comprised in the surface active agent. For example, the lysine residues in the KL4 peptide interact with the charge head groups of DPPC and the hydrophobic leucine resides interact with the phospholipid acyl side chains of phosphatidylglycerol.

[0071] In certain embodiments, an imaging composition as disclosed herein comprises a surface active agent comprising a portion of a mammalian lung surfactant polypeptide or mimic thereof and does not additionally comprise a lipid or a mixture of lipids. Imaging compositions administered by inhalation comprising surface active agents comprising only a portion of a mammalian lung surfactant polypeptide or mimic thereof can interact with natural surfactant in the lungs through hydrostatic interactions. For example, recombinant SP-B can interact with natural surfactant in the lungs by binding anionic phospholipids, such as phosphatidylglycerol. [0072] In other embodiments, an imaging composition as disclosed herein comprises a surface active agent comprising both a portion of a mammalian lung surfactant polypeptide or a mimic thereof and at least one lipid. To facilitate absorption of imaging compositions comprising both a polypeptide or mimic thereof and at least one lipid into natural surfactant in the lungs, phopholipid monolayers mimicking those found in natural surfactant can be used. Exemplary lipid mixtures include dipalmitoylphosphatidylcholine and

palmitoyloleoylphosphatidylglycerol, for example at a 7:3 w/w ratio. The mammalian lung surfactant polypeptide can be inserted into the phosphoplipid monolayer and the protein/lipid mix can be absorbed into the natural surfactant at the alveolar/gas interface in the lungs following inhalation.

Contrast Moieties

[0073] Imaging of respiratory tissues can be obtained, for example, by ultrasonic imaging, X-ray imaging (or x-radiation), computed tomography, magnetic resonance imaging (MRI), optical imaging (or optical radiation), and radionuclide imaging such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT).

[0074] The term "contrast moieties" is used interchangeably herein with the term "contrast agents" to refer to substances that enhance the contrast of structures or fluids within the body in medical imaging. Contrast moieties may include, but are not limited to X-ray contrast agents, MRI contrast agents, optical contrast agents, atomic particle emitters (e.g., radionuclides) and ultrasonic contrast agents.

[0075] Exemplary of X-ray contrast agents useful for radiography and computed tomography typically are iodine or Barium compounds such as inorganic barium salts or soluble iodine containing compounds, which may be ionic or non-ionic. Exemplary iodinated contrast agents include, but are not limited to, Diatrizoate, Metrizoate, Ioxaglate, Iopamidol, lohexol, loxilan, lopromide, and lodixanol. X-ray contrast agents comprised of metal chelates (U.S. Pat. No. 5,417,959) and polychelates comprised of a plurality of metal ions (U.S. Pat. No. 5,679,810) may be used. In addition, multinuclear cluster complexes may be used as X-ray contrast agents (U.S. Pat. Nos. 5,804,161, WO91/14460, and WO 92/17215).

[0076] MRI contrast agents may be paramagnetic, superparamagnetic or ferromagnetic nonoparticles or microparticles. Exemplary MRI contrast agents include metal chelates, in which the metals are selected from the group consisting of lanthanide series members of atomic number 57-70, and transition metal members having an atomic number selected from the group consisting of 21-29, 42 and 44. Most commonly used MRI contrast agents are chelates of Gadolinium, Iron, or Manganese. Exemplary MRI contrast agents include, but are not limited to, Gadolinium chelates such as Gadodiamide, Gadobenate Dimeglumine, Gadopentetate Dimeglumine, Gadoteridol, Gadofosveset Trisodium, and Gadoversetamide; Iron oxide particles such as Ferucarbotran, Ferumoxtran-10, and Ferumoxides; and Manganese salts or chelates such as Mangafodipir Trisodium. Additional examples of paramagnetic metal chelate MRI contrast agents include, but are not limited to, Gd (DTPA) ^~:gadolinium (III)- diethylenetriamine-N,N,N',N",N"-pentaacetate; Gd(DTPA)-BMA:gadolinium(III)- diethylenetriamine-N,N,N',N",N"-pentaacetate-bis(methylamide); Dy(DTPA)^2"

:Dysprosium(III)-diethylenetriamine-N,N,N',N",N"-pentaacetate; GD(DOTA)^":

gadolinium(III)-l,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetate; Mn(CDTA)^2":

manganese(II)-trans— 1 ,2-cyclohexylenedinitrilotetraacetate; Mn(NOTA) ^~:manganese(II)- 1 ,4,7- triazacyclonoane-N,N',N"-triacetate; MN(EDTA) ^":manganese(II)-ethylenediaminetetracetate; Mn(HEDT A)^" : managnese(II) -hydroxylethylethylenediaminetriacetate ; Fe(EHPG) : Iron(III) - N,N'-ethylenebis(2-hydroxyphenylglycine)ethylenediamine ; Fe(HBED):Iron(III)-N,N'-bis(2- hyroxybenzyl)ethylenediaminediacetate, and the like.

[0077] Ultrasound contrast agents may be gas-filled microbubbles, which differ in their shell makeup and gas core makeup. Exemplary gas-filled microbubbles include, but are not limited to, gas-filled microbubbles encapsulated with denatured albumin as described in U.S. Pat. Nos. 4,572,203, 4,718,433, 4,774,958, and 4,844,882; gas-filled microbubbles

encapsulated by liposomes as described in U.S. Pat. Nos. 5,088,499 and 5,123,414; gas-filled, free microbubbles as described in U.S. Pat. Nos. 5,393,524, and 5,409,688; and saccharide containing solid particles that are mixed with a diluent to produce an ultrasound contrast agent as described in U.S. Pat. Nos. 4,442,843 and 4,681,119. Additional examples of ultrasound contrast agents include Perflutren Protein-Type A Microspheres Injectable Suspension

(OPTISON®, GE Healthcare), Galactose-Palmitic Acid Microbubbles (Levovist®, Schering AG), Perflutren Lipid Microspheres (DEFINITY®, Bristol Myers Medical Imaging), and Perflexane Lipid Microspheres (IMAGENT®, Photogen Inc.). Inhalable gaseous ultrasound contrast agents have been described in WO 93/06869 and U.S. Pat. Nos. 5,406,950 and

6,013,243. [0078] Atomic particle emitters useful for radionuclide based imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) are also contemplated. Typical PET diagnostic agents are radiolabeled with one or more atoms that exhibit positron emission (such as certain isotopes of carbon, nitrogen, oxygen, fluorine, or rubidium, including 11 C, 13 N, 15 O, 18 F, and 82 Rb). For example, radiolabeled amino acids may be used as metabolic tracers for PET imaging which include, but are not limited to, [ⁿC]a-aminoisobutyric acid (AIB), L-[^UC] methionine (Met), L-[¹⁸F]fluoro-a- methyl tyrosine,

0-(2-[ 18 F]fluoroethyl)tyrosine and trans- l-amino-3-[ 18 F]fluorocyclobutyl-l-carboxylic acid (FACBC). In addition, radionuclides may be incorporated either into compounds normally used by the body such as glucose (or glucose analogues), water or ammonia, or into molecules that bind to receptors or other sites of drug action. SPECT diagnostic agents are labeled with gamma-emitting atoms, such as ^99mTc, ⁶⁷Ga, ^mIn and ¹²³I. Exemplary SPECT agents include, but are not limited to, ^99mTc-tetrofosmin (Myoview, GE healthcare), ^99mTc-sestamibi

(Cardiolite, Bristol-Myers Squibb), and ^99mTc-HMPAO (hexamethylpropylene amine oxime).

[0079] Optical contrast agents can provide either positive or negative optical contrast.

Exemplary optical contrast agents include, but are not limited to, near infrared optical imaging dyes, cyanines and other optical dyes, isosulfan blue or other absorbing contrast agents, indocyanine green, porphyrins or other fluorophores, methyl red or other biologically responsive dyes, colored or fluorescent proteins and other gene products, quantum dots and other spectroscopically distinct physical constructs, and contrast-filled micelles. Optical contrast agents may comprise suitable optical dyes, such as EMR-absorbing and voltage- sensitive dyes which are safe for in vivo administration. Such dyes may include cyanines, merocyanines, oxonols, styryl dyes, and the like. Optical contrast agents such as optical dyes have been previously described (e.g., U.S. Pat. No. 5,494,031 and U.S. Pat. No. 4,805,623). More recently, new optical dyes have been reported that may have application to real-time optical localization and targeting (U.S. Pat. No. 5,672,333, U.S. Pat. No. 5,698,397, WO 97/36619, and WO 98/48838). Colored or fluorescent proteins such as the luciferase protein or the green fluorescent protein family may also be employed.

[0080] In certain embodiment, a single contrast moiety is bonded to a surface active agent or to a targeting moiety. In other embodiments, at least two contrast moieties may be bonded to a surface active agent and/or a targeting moiety (if one is used). When more than one contrast moiety is bonded to the a surface active agent (or targeting moiety, if one is used), the contrast moiety may be the same contrast moiety, a member of the same class of contrast moieties, or a member of different classes of contrast moieties.

Targeting Moieties

[0081] In certain embodiments, a targeting moiety may be covalently attached to a surfactant polypeptide or a contrast moiety to form a targeted imaging conjugate. Targeting moieties contemplated herein bind preferentially to an extracellular or cell surface bound target on a cell or a tissue accessible to the pulmonary gas interface of the lung, and thereby permit determination of the presence or position of a cell or a tissue bearing said target (e.g., an extracellular or cell surface bound target, e.g., a cell surface receptor or its ligand). Targeting moieties may include agonists, antagonists, inhibitors or other specific binders such as an antibody or an engineered derivative thereof, such as Fab, Fab', F(ab')₂, Fv, and SFv fragments.

[0082] Exemplary biological markers for cancer may include, but are not limited to, epidermal growth factor receptor (EGFR), transforming growth factor β receptor (TGF R), vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), ephrin receptor (EphR), estrogen receptors (ER), nicotinic acetylcholine receptors (nAChR), and other receptor tyrosine kinases (RTKs) known to those skilled in the cancer art. Endogenous ligands for these receptors include, for example, EGF, TGF , VEGF, IGF, PDGF, FGF, ephrin, estrogen, nicotine, and acetylcholine.

[0083] Exemplary cell surface receptors for cells infected with Mycobacterium tuberculosis include, but are not limited to, DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), complement receptors (CRs) such as CR1, CR3, and CR4, mannose receptor (MR), surfactor protein A (Sp-A) receptor, CD 14, scavenger receptors, IL-12 receptor, Fey receptors, and the P2X or P2Y purinergic receptors such as the P2X7 receptors. Known ligands for these receptors include, for example, complement factors such as C3b, mannose, SP-A, lipopolysaccharides, polyanionic macromolecules and particles, IL-12, IgG, ATP and ATP analogues. [0084] Exemplary cell surface receptors known to mark scar tissue include, but are not limited to, nerve growth factor receptor (NGFR), transforming growth factor β receptor (TGFPR), and insulin-like growth factor receptor (IGFR). Ligands for these receptors include, for example, NGF, TGF , and IGF. Linkage

[0085] Many strategies can be employed to link a contrast moiety, a targeting moiety, and a surface active agent to one another for use in the invention. In an exemplary embodiment, a contrast moiety is associated with a surface active agent, e.g., by covalent bonding or chelation to form an imaging conjugate. For example, one or more contrast moieties can be attached to the surface active agent either directly or using a linker that preserves the biological activity of the contrast moiety and retains significant dwell time of the surface active agent at the lung/air interface.

[0086] In some embodiments, a targeting moiety is attached to the surface active agent and/or the contrast agent to form a targeted imaging conjugate. For example, a targeting moiety can be attached to a contrast moiety and/or a surface active agent either directly using a linker that preserved the targeting ability of the targeting moiety, biological activity of the contrast moiety, and retains significant dwell time of the surface active agent at the lung/air interface.

[0087] In each case, at least one additional residue can be added at the amino- or carboxy- terminus or at an internal amino acid residue of a surfactant polypeptide of the type disclosed herein to generate a linker for bonding a contrast moiety. In an exemplary embodiment, SP-A, SP-B, SP-C, SP-D, or portions thereof, may be extended by at least one amino acid to create an unnatural amino acid or short amino acid sequence, e.g., four to eight amino acids long, by automated peptide synthesis. Alternatively, the native sequence of the human or animal form of these protein domains beyond these regions displaying the surfactant activity may be included as a natural linker.

[0088] A contrast moiety may be conjugated to the C-terminal or N-terminal amino acid of the surface active agent by bonding with the carboxyl group of the C-terminal amino acid or the amino group of the N-terminal amino acid. In certain embodiments, a contrast moiety is conjugated to the N-terminal amino acid of a surfactant polypeptide (e.g., SP-B). A targeting moiety may be optionally conjugated to the surfactant polypeptide at the C-terminus or another intermediate position in the polypeptide so not to interfere with the contrast moiety. In another embodiment, a contrast moiety may be conjugated to the C-terminal amino acid of a surfactant polypeptide (e.g. , SP-B). A targeting moiety may be optionally conjugated to the surfactant polypeptide at the N-terminus or another intermediate position in the polypeptide so not to interfere with the contrast moiety. In yet another embodiment, the contrast moiety is conjugated at an internal amino acid of a surfactant polypeptide (e.g., SP-B). The targeting moiety may be optionally conjugated to the N-terminal or C-terminal or another internal position in the surfactant polypeptide.

[0089] A contrast moiety may be conjugated to a surfactant lipid, e.g. , 1,2- dimyristoylphosphatidylethanolamine. For example, the contrast moiety may be covalently bonded to a surfactant lipid through an amide or ester bond. In instances where the contrast moiety contains a carboxylic acid, a surfactant lipid bearing an amino group (or the surfactant lipid is modified to contain an amino group) is reacted with the contrast moiety in the presence of a peptide coupling reagent, e.g. , HATU. In other embodiments, the contrast moiety may be covalently bonded to a surfactant lipid through an amido-alkylene linker, or via an ether or amino linkage. The amino linkage may be constructed by, for example, installing a 1- bromopropyl group onto the contrast agent, and then reacting with an amino-containing surfactant lipid.

[0090] Alternatively, a contrast moiety may be conjugated to the targeting moiety in a targeted imaging conjugate. For example, a targeting moiety may be conjugated to the C- terminal or N-terminal amino acid of the surface active agent by bonding with the carboxyl group of the C-terminal amino acid or the amino group of the N-terminal amino acid. In some embodiments, a targeting moiety is conjugated to an internal amino acid of a surfactant polypeptide (e.g. , SP-B). The contrast moiety is then conjugated to the targeting moiety at a position to maintain the targeting function of the targeting moiety, the dwell time of the surfactant polypeptide, and the biological activity of the contrast moiety. The contrast moiety may be conjugated to the N-terminus, C-terminus, or another intermediate position of the targeting moiety.

[0091] The contrast moiety may be bonded directly to the amino acid or via a linker to the surface active agent and/or targeting moiety. Representative covalent linkages include an ester, an amide, urea, carbamate, sulfonamide, ether, thioether, disubstituted amino, or a trisubstituted amine. (March, Advanced Organic Chemistry, 4th Ed., John Wiley & Sons, 1992.) Other linkage types could also be used. One strategy is to synthesize a derivative of the contrast moiety as may be necessary in specific cases to create a selectively reactive chemical group in a region of the molecule chemically separate from its active region at locations suggested by structure function analysis studies.

[0092] One type of covalent linker comprises amino acid residues. Such linkers may comprise at least one residue or can be 40 or more residues, more often 1 to 10 residues, and most often 1 to 5 or 5-10 amino acid residues in length. The linker is usually a small, water- soluble, neutral polar or non-polar amino acid or unstructured peptide. Typical amino acid residues used for linking are glycine, tyrosine, cysteine, lysine, glutamic acid, and aspartic acid, or the like. One linker frequently used where linked moieties each are intended to retain their independent function is a glycine rich sequence comprising between one to five glycine residues. Another linker frequently used in similar contexts where linked moieties each are intended to retain their independent function is Glycine and Serine rich synthetic sequences such as Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser, or [Gly Gly Gly Gly Ser]n where n is one, two, or three.

[0093] In another embodiment, a contrast moiety is covalently linked to a surfactant polypeptide and/or targeting moiety through a phenylalanine linker attached either to the C- terminus of the surfactant polypeptide or the C-terminus of the targeting moiety. The phenylalanine linker preferably does not significantly alter the physical properties of the surfactant polypeptide or the targeting moiety. A contrast agent, e.g., fluorine- 18 for use in positron emission tomography, can be covalently attached to the phenyl group of the phenylalanine linker. In other embodiments, the contrast agent is covalently linked to an amino group of the surfactant moiety and/or targeting moiety through an amide linkage. The amide linkage can be formed by, for example, reacting a surfactant polypeptide and/or targeting moiety bearing a primary amino group with a contrast agent bearing a carboxylic acid group in the present of a peptide coupling agent, such as HATU.

[0094] Other strategies for attaching an contrast moiety and/or a targeting moiety to a surface active agent are contemplated including by chelation, ionic attraction, or non-covalent association such as hydrophobic-hydrophobic interaction. [0095] Further, one of skill in the art will appreciate, based on the teachings herein, that minor structural changes can be made to various contrast moieties and/or targeting moieties to make them more suitable for attachment to the surfactant active agent or one another. For example, an amino group or amide group of a contrast agent or targeting moiety may be reacted with methyl 3-chloro-3-oxopropanoate or methyl 3-chloropropanoate to install a linking group. The methyl ester of the linking group could then be converted to a carboxylic acid or acid chloride and reacted with an amino group of a surfactant active agent in order to form a conjugate. In other embodiments, an ester group of a contrast agent or targeting moiety may be converted to a carboxylic acid in order to facilitate amide coupling with a surfactant active agent or one another. These and other function group manipulations will be appreciated by one of skill in the art. See, for example, J. March, Advanced Organic Chemistry, McGraw Hill Book Company, New York, (1992, 4^th edition).

[0096] Once a contrast moiety and surfactant active agent have been selected, a library of fusion constructs may then be created, comprising individual species exploiting different points of attachment on the chemical structure of the contrast moiety and on the peptide, different length linkers, different linker chemistries, different length surfactant peptides, etc., all with a view to improving the binding constant of the contrast moiety to its target, improving activity, reducing immunogenicity, or for other purposes. The desired combination of different surfactant polypeptide domains, linkers, and attachment points can be generated, for example, by brute force construction of a desired number of candidate constructs. A library of such constructs may be generated using standard molecular biology protocols. As noted, the contrast moiety may be attached at either the N-terminal or the C-terminal, or at an

intermediate location. The size/length, and amino acid sequence of the mammalian lung surfactant polypeptide or a mimic thereof may be varied. Nucleic acids encoding the various mammalian lung surfactant polypeptide or a mimic thereof can be recombinantly fused and cloned in suitable expression vectors, under the control of operatively linked promoters and transcription regulators. The construct may also be post translationally modified as may be necessary or desirable in specific instances, e.g., glycosylated or pegylated.

[0097] The resulting library can be screened for the ability to permit imaging of respiratory tissues in experimental animals. Conventional imaging approaches can be utilized to gauge the efficacy of the contrast moiety to enhance lung imaging in vivo. For example, MRI imaging can be utilized to assess the whether the contrast moiety can enhance and improve the visibility of respiratory structures.

[0098] Once one or a group of surfactant active agents are optimized for safety, distribution and dwell time within the lungs, by appropriate screening or otherwise, its properties may be further enhanced by one or more rounds of mutagenesis and additional selection/screening according to art known methods. This will lead to a functionally optimized structure, which can be used repeatedly to enhance the efficacy within the lungs of a wide variety of different contrast moiety classes and individual species for the imaging of respiratory tissues.

Methods of Administration

[0099] Compositions of the invention are delivered to the lungs by inhalation. Inhalation devices, such as inhalers (including dry powder inhaler and metered dose inhalers) and nebulizers (also known as atomizers) may be used to deliver the disclosed compositions to the lungs. Exemplary dry powder inhalers can be obtained from Inhale Therapeutic Systems as described in U.S. Pat. Nos. 5,458,135; 5,740,794; 5,785,049, which are herein incorporated by reference. Dry powder inhalers can also be obtained from 3M as described in U.S. Pat.

6,029,661, which is incorporated herein by reference.

[0100] The compositions disclosed herein may also be administered using a metered dose inhaler (MDI) containing a solution or suspension of contrast moiety in a pharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon (CFC) or fluorocarbon, as described in U.S. Pat. No. 5,320,094 and U.S. Pat. No. 5,672,581, both incorporated herein by reference.

Metered dose inhalers are designed to deliver a fixed unit dosage of medicament per actuation or "puff", for example in the range of 10 to 5000 microgram medicament per puff. Exemplary metered dose inhibitors can be obtained from 3M as described in U.S. Pat. Nos. 5,224,183; 5,290,534; 5,511,540; 6,454,140; and 6,615,826, which are incorporated herein by reference . Metered dose inhalers may also be CFC-free. Imaging compositions to be used with an inhaler may be in the form of aerosolized solid particles or droplets of liquid or suspension.

[0101] Alternatively, the compositions described herein may be dissolved or suspended in a solvent, e.g., water or saline, and administered by nebulization. Exemplary nebulizers for delivering an aerosolized solution include the AERx™ (Aradigm), the Ultravent®

(Mallinkrodt), the Pari LC Plus™ or the Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, and the Acorn II® (Marquest Medical Products). Imaging Formulation

[0102] Imaging compositions disclosed herein can be formulated into a solution and/or a suspension of particles in a carrier appropriate for inhalation into the respiratory tract and the lungs. Such carriers are also well known to the ordinary artisan familiar with inhalants for the delivery of fine droplets and insufflations for the delivery of inhalable fine particles, on the order of, for example, from about 0.5 to 1 micron, and preferably from about 0.5 to about 0.7 micron, comprised of powders, mists or aerosols, into the respiratory tract as described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Ed. By Arthur Osol, which is incorporated herein by reference.

[0103] In one embodiment, imaging compositions for inhalation administration can be administered as powders. The powdered contrast agent or composition is normally located within a container such as a hard gelatin capsule or a blister package, or a multi-dose devise. The capsule or blister is ruptured or broached within in an inhaler device, thereby enabling the powder to be inhaled. Generally, the mean particle size of the contrast moiety used for inhalation is between 1 and 10 micron with the size range between 2 and 5 microns being particularly suitable for penetrating the peripheral airways of the lungs. Such particle size ranges are commonly achieved by micronisation or spray drying.

[0104] A powdered imaging composition is often administered as a composition comprising a blend or mixture of the contrast moiety with an inert carrier. Usually the inert carrier has a mean particle size substantially larger than that of the contrast moiety. This provides, among other advantages, an improvement in the flow properties and dispensing accuracy of the composition.

[0105] Commonly described carrier materials for contrast moiety, include calcium carbonate and sugars, for example sucrose, mannitol or dextrose or, more particularly, lactose, which are pharmaceutically acceptable and pose no problems of toxicity, since any residues imbibed during dosing are well tolerated upon digestion or may be easily eliminated by disillusion (e.g., in the case of the sugars) or mucocilliary clearance from the lung.

[0106] The composition in the capsule or blister is frequently about 25 mgs. This weight probably represents the maximum quantity of powder that may be comfortably inhaled without undue side effects, such as coughing, and also corresponds to the minimum quantity that is usually dispensed by filling machines. [0107] In certain embodiments, compositions formulated for powder inhalation comprise a carrier present at a concentration of about 95.0 to 99.99%. More particularly, from 97.0 to 99.9%, especially from 98.0 to 99.8%, by weight. Processes for preparing such powders, by the application or adaptation of known methods, also constitute features of the invention.

[0108] In other embodiments, the imaging composition may be formulated as an aerosol formulation using methods well known in the art. One widely used method for dispensing such an aerosol formulation involves making a suspension formulation of the contrast moiety as a finely divided powder in a liquefied propellant gas. Alternatively a solution formulation can be prepared where the contrast moiety is dissolved in a propellant system, perhaps containing solubilizers and co-solvents to aid dissolution of the contrast agent. Pressurized metered dose inhalers (pMDI) are normally used to dispense such formulations to a patient. Propellants may include chlorofluorocarbon (CFC), fluorocarban (FC), or hydrofluroalkane (HFA) propellants.

Imaging Methods

[0109] The disclosure also provides methods for imaging respiratory tissue in a mammal, the method comprising administering the imaging composition by inhalation or pulmonary instillation in an amount effective to spread throughout at least a portion of the alveolar surface area of the lungs of the mammal, and detecting and displaying the position of the contrast moiety to indicate the presence or position of a tissue of interest. In some embodiments, detection and displaying the position of the contrast moiety in lung tissue may be used to assess or evaluate alveolar wall morphology. For example, high density alveolar wall structure is characteristic of healthy lung tissue whereas lung tissue with large vacuoles and deteriorated alveolar wall structure is characteristic of lung disorders, e.g., emphysema (compare Figures 17 and 18).

[0110] In certain embodiments, a mammal requiring imaging of respiratory tissue may be suffering from lung inflammation, a lung disease, or a lung disorder. The method comprises administering to the subject an imaging conjugate comprising a contrast moiety bonded to a surface active agent, which has an affinity for the human alveolar/gas interface and which comprises at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof that is substantially non-immunogenic to humans. In some embodiments, a targeted imaging conjugate is administered to the subject by inhalation in an amount effective to permit determination of the presence or position of a cellular tissue bearing a target in the lung. The mammal may be a human, monkey, chimpanzee, horse, dog, cat, cow, sheep, pig, rat or mouse. In exemplary embodiments, the mammal is a human.

[0111] The mammal in need of respiratory tissue imaging may be suffering from lung inflammation or is suffering from or at risk of suffering from lung disease or lung disorder. Exemplary lung diseases or disorders that may require imaging include, but are not limited to, emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory distress disorder (RDS), pneumonia, tuberculosis or other bacterial infection, cystic fibrosis, and/or lung cancer. Dosage

[0112] The administration of imaging conjugates or targeted imaging conjugates may reduce the dosing frequency relative to administration of an unconjugated contrast agent. The administration step may be a single dose administered prior to imaging the mammal's respiratory tissue. Alternatively, the administration step may be repeated at multiple doses prior to imaging of respiratory tissues (e.g. , at least two, three, four or more doses)

administered over a 30 minute, one hour, two hour, three hour or longer time period. In some embodiments, the administration step may be repeated once daily, every other day, every three days, every four days, every five days, weekly, biweekly, monthly, bimonthly, quarterly, semiannually, or annually to obtain images of respiratory tissue.

EXEMPLIFICATION

[0113] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way.

Example 1: Conjugation of a PET Scanning Contrast Agent to a SP-B Peptide

[0114] An exemplary contrast agent having Formula 1 can be prepared according to the procedures described below. The procedure involves first attaching a protected tributyltin

18 18 phenylalanine to a SP-B peptide, and then installing F. It is contemplated that the F-labeled construct can be used as a PET scanning contrast agent.

H₂N^— FPIPLPYCWLCRALIKRIQAMIPKG

1

[0115] It is further contemplated that, in certain embodiments, Formula 1 may be modified to include a chemical targeting moiety that has affinity for a particular protein or other biological target. For example, it is contemplated that covalent attachment of an ADAM inhibitor to the 18 F-labeled construct of Formula 1 would provide a compound useful for the early detection of cancer. In certain embodiments, the ADAM inhibitor is covalently attached to an amino group (such as the N-terminal amino group of Formula 1) either directly or through a linker. It is also contemplated that covalent attachment of an adenosine triphosphate (or an analog thereof) to the 18 F-labeled construct of Formula 1 would provide a compound useful for the early detection of tuberculosis.

Detailed Synthetic Procedure

[0116] Step 1: Protected para-iodo-phenylalanine compound 2 shown in Scheme 1 is reacted with hexabutyl distannane in the presence of a palladium catalyst at elevated temperature to provide tributylstannyl phenylalanine 3, according to procedures described by Langer, O. Bioorg. Med. Chem., 9(3):677-94 (2001).

SCHEME 1

2 3

[0117] Step 2: Tributylstannyl phenylalanine 3 is treated with peptide coupling reagent HATU (i.e., 2-(lH-7-azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate methanaminium) in the presence of N,N-diisopropylethylamine (DIPEA) and peptide 4 in order to covalently attach the peptide to the derivatized phenylalanine. The reaction product is then treated with piperidine to remove the Fmoc protecting group, thereby affording tributylstannyl- peptide conjugate 5. Additional aspects of the synthetic procedures amenable to the above preparation are described by Gordon, L.M., Protein Sci., 5(8): 1662-75 (1996).

SCHEME 2

[0118] Step 3: Tributylstannyl-peptide conjugate 5 is treated with a fluorinating reagent, e.g., [F 18 ]F₂, followed by addition of hydrobromic acid to provide compound 1. Detailed procedures for installing the F 18 label are described by Eskola, O., Nucl. Med. Biol., 31(1): 103- 10 (2004).

SCHEME 3

H2N-

FPIPLPYCWLCRALIKRIQAMIPKG H2N CRALIKRIQAMIPKG

5 1

Example 2: Conjugation of a MRI Contrast Agent to a SP-B Peptide

[0119] An exemplary peptide conjugate having Formula 4 can be prepared according to the procedures described below in Scheme 4. The procedure involves subjecting triamine 1 to hydrogenation conditions to remove the benzyl protecting group and provide carboxylic acid 2. This carboxylic acid is treated with peptide coupling reagent HATU and peptide 3 to form a synthetic intermediate peptide conjugate (not shown in Scheme 4). Amenable reaction conditions for the peptide coupling include those based on Nguyen, L., Trends Cell Biol., 15(5):269-76 (2005). This synthetic intermediate is treated with a mixture of trifluoracetic acid (TFA), water, and triisopropylsilane, followed by reaction with piperidine to remove the protecting groups and afford the peptide conjugate having Formula 4. The Gd MRI contrast agent is formed by adding GdCl₃ (1.1 eq.) to peptide conjugate 4 at room temperature in water at pH 7, according to literature procedures described in Vaccaro, M., Langmuir, 22(15):6635- 43 (2006). Excess, uncomplexed Gd³⁺ is removed by basifying to pH 10 with sodium hydroxide and subjecting the resulting mixture to centrifugation. The triamine 1 starting material used in this reaction sequence can be prepared based on procedures described by Anelli, P.L., Bioconjugate Chem., 10: 137-140 (1999).

SCHEME 4

[0120] It is further contemplated that, in certain embodiments, the peptide conjugate of Formula 4 may be modified to include a chemical targeting moiety that has affinity for a particular protein or other biological target. For example, it is contemplated that covalent attachment of an ADAM inhibitor to the peptide conjugate of Formula 4 would provide a compound useful for the early detection of cancer, particularly lung cancer. In certain embodiments, an ADAM inhibitor is covalently attached to an amino group (such as the N- terminal amino group of Formula 1) either directly or through a linker. It is also contemplated that covalent attachment of an adenosine triphosphate (or an analog thereof) to the peptide conjugate of Formula 4 would provide a compound useful for the early detection of

tuberculosis. Example 3: Conjugation of a Targeting Moiety to a SP-B Peptide

[0121] It is contemplated that various targeting moieties known in the art can be conjugated to a SP-B peptide. Representative examples of targeting moieties include marimastat and other compounds known to block ADAMs that shed EGF ligands from lung cancer cells. Exemplary synthetic procedures are described in detail below.

[0122] Part i: An exemplary peptide conjugate having Formula 3 can be prepared according to the procedures described below in Scheme 5. The procedure involves reacting marimastat (1) with peptide 2 in the presence of peptide coupling agent EDCl and N- methylmorpholine (NMM) to provide peptide conjugate 3. Marimastat is known to block ADAMs that shed EGF ligands from lung cancer cells, and it is contemplated that the marimastat-peptide conjugate has the ability to block ADAMs that shed EGF ligands from lung cancer cells.

IQAMIPKG

3

[0123] Part 2: The exemplary peptide conjugate having Formula 3 can be prepared according to the procedures described below in Scheme 6. The procedure involves attaching a small molecule inhibitor of ADAM family of proteins to a SP-B Peptide via a malic acid linker. Specifically, malonic acid is first reacted with peptide coupling reagent EDCl in the presence of N-methylmorpholine in dichloromethane (DCM), and then carboxamide 1 is added to the reaction mixture, followed by addition of peptide 2 to form the peptide conjugate of Formula 3. The small molecule inhibitor 1 is understood to block ADAMs that shed EGF ligands from lung cancer cells, and it is contemplated that the peptide conjugate having Formula 3 has the ability to block ADAMs that shed EGF ligands from lung cancer cells.

SCHEME 6

3. H₂N^— FPI PLPYCWLCRALIKRIQAMI PKG

2

Example 4: Conjugation of Adenosine Triphosphate to a SP-B Peptide

[0124] An exemplary peptide conjugate incorporating an adenosine triphosphate moiety, as represented by Formula 4, can be prepared according to the procedures presented in Scheme 7. The procedure involves reacting carboxylic acid 1 with peptide 2 in the presence of peptide coupling reagent EDCI and N-methylmorpholine to form an intermediate peptide conjugate (not shown). This peptide conjugate is reacted with piperidine to remove the Fmoc protecting group and provide peptide conjugate 3. Reaction of the sodium salt of adenosine triphosphate (ATP) with peptide conjugate 3 in the presence of CMD-CDI (i.e., N-cyclohexyl- V'-(2- morpholinoethyl)carbodiimide methyl-p-toluene sulfonate) at pH 7 covalently links the ATP moiety to the peptide conjugate to provide the desired peptide conjugate 4. It is contemplated that peptide conjugate 4 can be used to treat cystic fibrosis and/or tuberculosis. Further, it is contemplated that this synthetic procedure would be amenable to preparing peptide conjugates that incorporate an analog of ATP. Additional procedures for coupling reactions using carboxylic acid 1 are provided in Jayachandran, R., Cell, 130(l):37-50 (2007). Procedures for preparing peptide 2 are provided in Gordon, L.M., Protein Sci., 5(8): 1662-75 (1996) and Nam, N.H., Bioorg. Med. Chem., 12(22):5753-66 (2004). SCHEME 7

AMIPKG

2. Piperidine, DMF

3

4

Example 5: Conjugation of a PET Scanning Contrast Agent to a Surfactant Lipid

[0125] It is contemplated that a PET Scanning Contrast Agent can be covalently bound to a phospholipid surfactant via an amide bond. For example, it is contemplated that reaction of a phospholipid, e.g., 1,2-dimyristoylphosphatidylethanolamine 1, with tributylstannyl

phenylalanine 2 in the presence of peptide coupling reagent HATU (i.e., 2-(lH-7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyl uronium hexafluorophosphate methanaminium) and N,N-diisopropylethylamine (DIPEA) will provide conjugate 3. The reaction product is then treated with piperidine to remove the Fmoc protecting group, thereby affording tributylstannyl- phospholipid conjugate 4. Additional aspects of the synthetic procedures amenable to the above preparation are described by Gordon, L.M., Protein Sci., 5(8): 1662-75 (1996).

[0126] To convert conjugate 4 to a form suitable for use in PET scanning, conjugate 4 is treated with a fluorinating reagent, e.g., [F 18 ]F₂, followed by addition of hydrobromic acid to provide compound 5. Detailed procedures for installing the F 18 label are described by Eskola, O., Nucl. Med. Biol., 31(1): 103-10 (2004).

Example 6: Conjugation of a MRI Contrast Agent to a Surfactant Lipid

[0127] It is contemplated that a MRI Contrast Agent can be covalently bound to a phospholipid surfactant via an amide bond or ester bond. For example, it is contemplated that reaction of 1,2-dimyristoylphosphatidylethanolamine 1 with carboxylic acid 2 from Example 2 in the presence of peptide coupling reagent HATU (i.e., 2-(lH-7-azabenzotriazol-l-yl)-l, 1,3,3- tetramethyl uronium hexafluorophosphate methanaminium) and N,N-diisopropylethylamine (DIPEA), followed by acidic saponification of the ester groups will provide conjugate 3. It is contemplated that a Gd MRI contrast agent can be formed by adding GdCl₃ (1.1 eq.) to conjugate 3 at room temperature in water at pH 7, according to literature procedures described in Vaccaro, M., Langmuir, 22(15):6635-43 (2006).

[0128] Similarly, reaction of carboxylic acid 2 with oxalyl chloride to generate an acid chloride intermediate, followed by reaction with distearoyl phosphatidylglycerol could be used to covalently bond distearoyl phosphatidylglycerol with carboxylic acid 2 via an ester bond.

SCHEME 10

myristoyl tBu0₂C C0₂tBu

Example 7: Conjugation of a Targeting Moiety to a Surfactant Lipid

[0129] It is contemplated that a targeting moiety, such as a peptide targeting moiety, can be covalently bound to a fatty acid surfactant via an amide bond. For example, it is contemplated that reaction of a fatty acid surfactant, e.g., lauric acid 2, with targeting peptide 1 in the presence of peptide coupling reagent HATU (i.e., 2-(lH-7-azabenzotriazol-l-yl)-l, 1,3,3- tetramethyl uronium hexafluorophosphate methanaminium) and N,N-diisopropylethylamine (DIPEA) will provide conjugate 3, as illustrated in Scheme 11. SCHEME 11

Targeting Peptide NH₂ + H0₂

1 2

INCORPORATION BY REFERENCE

[0130] This application is related to International Patent Application No.

PCT/US2008/065776, filed, June 4, 2008, and U.S. Patent Application No. 61/121,405, filed December 10, 2008; the complete disclosure of each application is incorporated herein by reference. The entire disclosure of each of the patent documents and scientific articles referred to herein is also incorporated by reference for all purposes.

EQUIVALENTS

[0131] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0132] What is claimed is:

Claims

CLAIMS 1. An imaging composition formulated for inhalation, comprising:

a surface active agent characterized by an affinity for the human alveolar/gas interface and comprising at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof; and, bonded to said agent,

a contrast moiety who's position in the lungs is detectable from outside the body to enable production of an image of at least a portion of the respiratory tissues of a mammal, the detection being effected by means of detection of x-radiation, optical radiation, magnetic resonance, or atomic particle emission.

2. An imaging composition comprising a contrast moiety who's position in the body is detectable outside the body by means of detection of x-radiation, optical radiation, magnetic resonance, or atomic particle emission to enable production of an image of at least a portion of the tissues of a mammal, wherein the improvement comprises:

a surface active agent bonded to said contrast agent characterized by an affinity for the human alveolar/gas interface and comprising at least a portion of a mammalian lung surfactant polypeptide or a mimic thereof, thereby to adapt said composition for imaging of mammalian respiratory tissues.

3. The composition of claim 1 or 2 wherein said surfactant polypeptide comprises a human lung surfactant, a non human mammalian lung surfactant such as a porcine or bovine lung surfactant, a peptidomimetic having surface active properties comprising a deletion or amino acid substitution mutant of a mammalian lung surfactant polypeptide, or a fraction thereof.

4. The composition of claim 1 or 2 wherein the surfactant polypeptide comprises a synthetic or recombinantly produced portion of the polypeptide component of a mammalian lung surfactant moiety.

5. The composition of claim 1 or 2 wherein the surfactant polypeptide comprises at least a portion of SP-A, SP-B, SP-C, SP-D, or a mixture thereof.

6. The composition of claim 1 or 2 wherein the surfactant polypeptide comprises at least a portion of SP-B such as the 25 amino acid fragment from the N-terminus of SP-B.

7. The composition of claim 1 or 2 further comprising a mammalian lung surfactant lipid.

8. The composition of claim 1 or 2 wherein said surfactant polypeptide comprises or is derived from a mammalian lung surfactant harvested from the lungs of a mammal.

9. The composition of claim 1 or 2 wherein said surfactant polypeptide comprises a peptide selected from the group consisting of SEQ ID Nos. 36 through 93.

10. The composition of any of the preceding claims, wherein the contrast moiety is selected from the group consisting of barium salts and iodine containing X-ray contrast agents.

11. The composition of any of the preceding claims, wherein the contrast moiety is selected from the group consisting of paramagnetic, superparamagnetic and ferromagnetic metal nonoparticles or chelates for use as MRI contrast agents.

12. The composition of any of the preceding claims, wherein the contrast moiety is selected from the group consisting of near infrared optical imaging dyes, cyanines and other optical dyes, isosulfan blue or other absorbing contrast agents, indocyanine green, porphyrins or other fluorophores, methyl red or other biologically responsive dyes, colored or fluorescent proteins and other gene products, quantum dots and other spectroscopically distinct physical constructs, and contrast- filled micelles.

13. The composition of any of the preceding claims, wherein the contrast moiety utilizes positron-emitting radionuclide or gamma-emitting radionuclide.

14. The composition of any of the preceding claims wherein more than one contrast moiety is bonded to the surfactant polypeptide.

15. The composition of any of the preceding claims further comprising a targeting moiety attached to said surfactant polypeptide or said contrast moiety which targeting moiety binds preferentially to an extracellular or cell surface-bound target on a cell or tissue accessible to the pulmonary/gas interface of the lung thereby to permit determination of the presence or position of a cell or tissue bearing said target.

16. The composition of claim 15 wherein the targeting moiety binds preferentially to a cancer cell.

17. The composition of claim 16 wherein the cancer cell comprising a receptor selected from the group consisting of epidermal growth factor receptor (EGFR), transforming growth factor β receptor (TGF R), vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), ephrin receptor (EphR), estrogen receptor (ER), and nicotinic acetylcholine receptor (nAChR).

18. The composition of claim 15 wherein the targeting moiety binds preferentially to an infectious microorganism.

19. The composition of claim 18 wherein the infectious microorganism comprising a receptor selected from the group consisting of DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), complement receptor CR1, complement receptor CR3, and complement CR4, mannose receptor (MR), Surfactor Protein A (Sp-A) receptor, CD 14, scavenger receptor, IL-12 receptor, Fey receptor, P2X purinergic receptor, P2X7 receptor, and P2Y purinergic receptor.

20. The composition of claim 15 wherein the targeting moiety binds preferentially to a scar tissue.

21. The composition of claim 20 wherein the scar tissue comprises a receptor selected from the group consisting of nerve growth factor receptor (NGFR), transforming growth factor β receptor (TGF R), and insulin-like growth factor receptor (IGFR).

22. The composition of any of the preceding claims disposed in an inhalation device for use by a human patient.

23. A method of imaging respiratory tissue in a mammal, the method comprising administering the imaging composition of any of claims 1-22 to the mammal by inhalation or pulmonary instillation in an amount effective to spread throughout at least a portion of the alveolar surface area of the lungs of the mammal, and detecting and displaying the position of the contrast moiety to indicate the presence or position of a tissue of interest.

24. Use of a composition of any of claims 1-22 for the preparation of a medicament for use as a contrast agent for imaging tissue in a mammalian respiratory tract.

25. The composition of any of claims 1-22 wherein the surface active agent is covalently attached to the contrast moiety by a linker.

26. The composition of claim 15 wherein the surface active agent is covalently attached to the targeting moiety by a linker.

27. The composition of claim 25 or 26 wherein the linker comprises an amide, ester, urea, carbamate, ether, thioether, disubstituted amine, or trisubstituted amine.

28. The composition of any of claims 1-22 wherein the contrast moiety is covalently attached to the N-terminus of a mammalian lung surfactant polypeptide.

29. The composition of anyone of claims 1-22 wherein the contrast moiety is covalently attached to the C-terminus of a mammalian lung surfactant polypeptide.

30. The composition of any of claims 25 or 26 wherein the linker comprises one or more glycine residues.