WO2008003682A1

WO2008003682A1 - Non invasive method for assessing mucus clearance

Info

Publication number: WO2008003682A1
Application number: PCT/EP2007/056652
Authority: WO
Inventors: Nicolau Beckmann; Rainer Kneuer; Philipp Schmidt
Original assignee: Novartis Ag
Priority date: 2006-07-06
Filing date: 2007-07-02
Publication date: 2008-01-10

Abstract

The present invention relates to the field of imaging. More specifically, the invention provides a probe for use in a method for assessing mucus clearance, comprising a polysaccharide polymer capable of binding to mucus, coupled to at least one imaging contrast agent. In a preferred embodiment, the probe is made of aminodextran and coupled to MRI contrast agent and/or a near infra-red fluorochrome, such as Gadolinium chelating group complex and/or Cy5.5, respectively.

Description

NON INVASIVE METHOD FOR ASSESSING MUCUS CLEARANCE

The present invention relates to the field of optical molecular imaging or magnetic resonance imaging. More specifically, the invention provides a probe for use in a method for assessing mucus clearance, comprising a polysaccharide polymer capable of binding to mucus, coupled to at least one imaging contrast agent. In a preferred embodiment, the probe is made of aminodextran and coupled to a magnetic resonance imaging (MRI) contrast agent and/or near infrared fluorochrome, such as Gadolinium chelating group complex and/or Cy5.5 respectively.

Airway mucus hypersecretion is a feature of several lung diseases like asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. It is indicative of poor asthma control and contributes to morbidity and mortality (Rogers 2004). Excess mucus not only obstructs airways but also contributes to airway hyperresponsiveness. Furthermore, asthma might have a specific mucus hypersecretory phenotype. Goblet cell hyperplasia and submucosal gland hypertrophy are shared with COPD (Rogers 2005) which comprises multiple components including pulmonary inflammation, airway remodelling and mucociliary dysfunction. The latter features contribute to the development of chronic, progressive airflow limitation. The mucociliary dysfunction component of COPD is due to mucus hypersecretion coupled with a decrease in mucus transport, and represents an important pathophysiological feature requiring appropriate treatment. There is currently huge research interest in identifying targets involved in inducing mucus abnormalities, which should lead to the rational design of anti-hypersecretory drugs for treatment of airway mucus hypersecretion.

Mucociliary clearance is an important mechanism for removing inhaled particles, secretions and cellular debris from the respiratory tract. In humans and large animals, assessment of mucociliary clearance is most commonly performed using inhaled radiolabeled aerosols and scintigraphy (Foster and Wagner 2001 ; Morgan et al 2004). In small animals, the clearance of fluorescent polysterene microspheres from the lung is determined by terminal serial bronchoalveolar lavage fluid (BAL) analysis (Coote et al 2004). Evidently, using radioactive materials or performing terminal experiments has its limitations and is not satisfactory, especially for use in human. Therefore, there is a need to have a noninvasive method to measure mucocilliary clearance at high spatial resolution in mammals.

Dextran is an oligosaccharide being considered for use in the treatment of cystic fibrosis (CF) because of the therapeutic potential it has demonstrated in animal and in vitro experiments. It has been shown that dextran exhibits significant mucolytic activity in vitro in CF sputum (Feng et al 1998), while enhanced mucociliary clearance rates have been observed when aerosolized dextran was delivered to dogs, suggesting that dextran reduces cross-linkage bonding in the mucus, thus leading to reduced mucous viscoelastic modulus (Feng et al 1999).

The present invention is based, at least in part, on the discovery that the combination of a polysaccharide polymer, such as dextran or derivatives, and a contrast agent allows for the efficient and effective detection of mucus clearance in mammals by optical imaging or MRI. This contrast agent can thus be used to characterize molecular targets in respiratory diseases and in pathways involved in physiological and pharmacological modulation of mucus clearance.

DETAILED DESCRIPTION OF THE INVENTION

The instant application thus provides an imaging probe for use in a method for assessing mucus clearance, comprising a polysaccharide polymer capable of binding to mucus, coupled to at least one imaging contrast agent.

As used herein "mucus clearance" or "clearability" refers to the ability of the mucus to be cleared from the respiratory tract. This can include without limitation, mucociliary clearance or cough.

Any biocompatible (physiologically compatible) polymer known in the art may be employed in the imaging probe or methods of the subject invention. Preferably, the polymer chosen is one which is made of dextran or derivatives thereof, such as aminodextran. The polymer can be of either synthetic or natural origin. The polymer may also be of varying molecular weights, such as high molecular weight (equal to or greater than 70,000 weight average molecular weight) or low molecular weight (less than 70,000 weight average molecular weight). Preferably, when Dextran is used, the polymers are low molecular weight polymers, more preferably having a molecular weight of about 70,000 or less, and most preferably of about 10,000 or less. For example, aminodextran with an average molecular weight of 10,000 Da is used.

In one specific embodiment, one imaging contrast coupled to the polymer is a magnetic resonance imaging (MRI) contrast agent. This includes any agent that is physiologically tolerable and capable of providing enhanced contrast for MRI. MRI agents typically have the capability of altering the response of a tissue to magnetic fields. Said MRI contrast agent can include a paramagnetic or a superparamagnetic element. Any type of paramagnetic agent known in the Art and which can be used for the methods of the invention is suitable. In one embodiment, said paramagnetic element is Gadolinium (III). For example, one can use a gadolinium-chelating group complex, such as bifunctional derivatives of gadolinium- diethylenetriamine penta-acetic acid (Gd-DTPA), gadoterate meglumine (Gd-DOTA), or a lanthanum chelating group complex. Examples of superparamagnetic agents include a metal oxide, such as Fe, Co, Ni, Cu, Zn, As, Se, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Po, or At oxide. Contrast agents such as those described in US4,687,658; 5,314,680 and 4,976,950 can be used for preparing the imaging probes of the invention. As used herein, the term "bifunctional derivatives" means that the molecule comprises at least one function to chelate the paramagnetic element and an other reactive group for conjugation of the chelate to the polysaccharide polymer.

In another embodiment, one imaging contrast agent is a fluorochrome. As used herein, a "fluorochrome" includes fluorochromes that fluoresce in the near-infrared region (in the range of 650-1100nm), e.g., after excitation in the far-red range of visible light wavelengths. Specific examples include the cyanine dyes, such as Cy5™, Cy5.5™ and Cy7™ (Amersham Biosciences, Piscataway, NJ), ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750 (Molecular Probes, Eugene, OR), IRDye38™, IRDyeδO™, IRDyeδO™ (LiCor, Lincoln, NE), NIR-1 and IC5-OSu (Dojindo, Kumamoto, Japan); FAR-Blue, FAR-Green Onem and FAR-Green Two (Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American Dye Source, Montreal, Canada), Atto680 (Atto-Tec, Siegen, Germany), DY-680, DY-700, DY-730, DY-750, DY-782 (Dyomics, Jena, Germany), EVOBIue (Evotec, Hamburg, Germany) and indocyanine green and its analogs and derivatives (Akorn et al.) and LaJoIIa Blue™ (Diatron). One of skill in the art would appreciate that a large number of fluorochromes with different chemical and optical properties can be used to produce the probe of the invention. In one specific embodiment, said fluorochrome is selected among the group consisting of the following: Cy5.5, Cy7, Cy 7.5, Alexa Fluor 680, Alexa Fluor 750.

The contrast agents are coupled to the polymer capable of binding to mucus, such as dextran. As used herein, the term "coupling" means "connected by any attractive force between atoms to allow the combined aggregate to function as a unit". This includes, but not limited to, chemical bonds such as covalent bonds (e.g., polar or non-polar), non covalent bonds such as ionic bonds, metallic bonds and bridge bonds, and hydrophobic and van-der- Waals interactions. In a preferred embodiment, the polysaccharide polymers is covalently bound with a contrast agent according to known coupling methods such as those described by Hermanson, 1996.

According to a preferred embodiment, said probe essentially consists of a polysaccharide polymer covalently bound to cyanine dyes, such as Cy5.5, and an MRI contrast agent, such as Gd-DOTA.

The probes of the invention are typically suspended in a suitable delivery vehicle, such as sterile saline. The vehicle may also contain stabilizing agents, carriers, excipients, stabilizers, emulsifiers, and the like, as is recognized in the art.

In a specific embodiment, the probe is formulated as a dry powder appropriate for in vivo inhalation. Preferably, the size of the dry powder particles has an average median aerodynamic diameter (MMAD) of less than 10μm, more preferably less than 5μm, and most preferably less than 2μm. More preferably, the probe formulated as a dry powder comprises a polymer made of dextran or derivatives thereof, such as aminodextran, having a low molecular weight of 10,000 Da or less.

Another aspect of the invention pertains to the use of dextran polymers or a probe as defined above, for imaging mucus secretion in an animal. Said animal is preferably a mammal, a non-human mammal or a human. In one specific embodiment, when the contrast agent is a near-infrared fluorochrome, considering the depth of penetration for near-infrared light in tissue being several centimeters, small animals are preferably used in the method. In one specific embodiment, said small animal is a non-human mammal, for example a rodent, and more preferably a small rodent selected among the group of rats, mice, rabbits, guinea pigs and hamsters.

In yet another aspect, the invention provides a kit comprising the probe as above-defined and instructions for the performance of an assay for imaging mucus secretion, more specifically, the kit is for use in a method for imaging mucus secretion in a mammal as defined hereafter.

Another aspect of the invention is directed to a non-invasive method for imaging mucus secretion in a mammalian subject in vivo, comprising: a. administering to the subject an MRI composition comprising a mucus binding polysaccharide polymer coupled to an MRI contrast agent, b. obtaining a magnetic resonance image of the lung or any mucus secreting regions of interest, wherein said image represents mucus secretion in the region of interest. Said MRI composition can be for example any of the probe defined above.

The invention further pertains to a non-invasive method for imaging mucus secretion in a mammalian subject in vivo, comprising a. administering to the subject an optical imaging composition comprising a mucus binding polysaccharide polymer coupled to an optically active molecule; b. Illuminating the subject with light source in a region of interest; and, c. visually monitoring the presence of the optical imaging composition in the region of interest, thereby obtaining an image, wherein said image represents mucus secretion in the region of interest.

Said optical imaging composition can be a fluorescent dye, a bioluminescent dye or a near infrared fluorescent dye.

In any of the imaging methods of the invention, the imaging probe can be administered by known manner. For example, it can be administered to the respiratory tract or lung by inhalation by using a nebulizer and/or an endotracheal tube. Dispensing devices can include dry powder inhalers or dry powder insuflators when the composition is formulated as a dry powder.

The amount of probe administered to the animal will depend on different parameters, including the species, the weight of the animal and the composition of the probe.

The invention also relates to a method of identifying a compound that modulates mucus clearance in a mammalian subject, said method comprising: a. administering a candidate compound to a test subject; b. assessing the rate of mucus clearance in said test subject by the imaging method defined above; and, c. comparing the rate of mucus clearance between the test animal and a control animal to which no or a reference candidate compound has been administered to, d. wherein any significant difference in mucus clearance between the control and test animals is indicative that said candidate compound is a compound capable of modulating mucus clearance in said animal.

As used herein, the term "a compound that modulates mucus clearance" refers to any compound that alters mucus clearance either by improving mucus clearability (ie, by mucociliary clearance or cough) or by inhibiting mucus secretetion (e.g. small molecule inhibitors of CXCR2). Such compounds which assist clearance are traditionally referred to as "mucolytic" agents, or "mucoactive" agents.

To identify a test compound that is capable of modulating mucus clearance in the method above, one would measure or determine the rate of mucus clearance in the absence of the administration of the candidate substance (the control animal). In a specific embodiment, the control animal can be the same animal as the test animal, the rate of mucus clearance being first assessed before the administration of the compound to the animal and then assessed after or with the administration of the compound. A test compound, which increases the rate of mucus clearance relative to that observed in its absence, is indicative of a candidate substance being a mucoactive agent with ability to stimulate or increase the rate of mucus clearance. Conversely, the test compound may be identified as one which slows the rate of mucus secretion. Such compounds may be useful in the treatment or amelioration of disorders which manifest defect in the mucus clearance, including cystic fibrosis, chronic bronchitis, bronchiectasis and bronchial asthma.

The test compound may be screened among peptides, polypeptides, proteins, antibodies or nucleic acids, or any other synthetic or natural products. Compounds selected among those being structurally related to other known modulators of mucus clearance are preferred. More specifically, the compounds may be screened among those that are structurally related to existing mucoactive agents.

Other features and advantages of the invention will become apparent from the following examples.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Labeling of AminodextraniO with Cy5.5 and Gd-pBn-DOTA Figure 2: Axial sections through the thorax of a single rat in approximately the same position and acquired at various time points following i.t. challenge with LPS (1 mg/kg). The black and white arrows indicate the two components of the MRI signal detected in the lung. The animal respired freely during image acquisitions and neither respiratory nor cardiac triggering was applied.

Figure 3: Intra-tracheal administration of the imaging probe 1 in solution (0.2 ml.) led to prominent increase in the intensity of edematous signals induced by LPS, which persisted 24 h after contrast agent delivery. The presence of the contrast agent predominantly in the left lobe, where the inflammatory response had been detected by MRI, was confirmed by NIRF. The NIRF image was acquired from isolated lungs harvested from the animal immediately after the MRI session.

Figure 4: Electron microscopy of milled probe 1.

Figure 5: Prominent signal increases in the lung of an LPS-challenged rat, induced by the imaging probe 2 administered i.t. as a powder.

Figure 6: Confocal microscopy (mucus: blue; nuclei: red) and PAS/Alcian blue (mucus: violet; nuclei: blue) stained sections of lungs from LPS-challenged rats, (a) Confocal microscopy (left) demonstrates that the imaging probe 2 formulated as dry powder bound specifically to mucus, (b) the imaging probe 2 also marked mucus present in goblet cells, (c) No mucus was labeled when vehicle was administered, (d) The number of goblet cells was the same in LPS-challenged rats treated with the imaging probe of example 2 (left) or vehicle (right).

EXAMPLES 1. Example 1 : synthesis and characterization of an imaging probe of the invention

The general synthesis reaction is shown in figure 1. Probe 1:

AminodextraniO (425 mg, 0.042 mmol; MW: 10.000 Da; Molecular Probes) was dissolved over a period of 2 h (swelling) in NaHCO₃ (0.1 M, 15 ml) and adjusted to pH 9 by Na₂CO₃ (0.1 M). After adding Cy5.5-NHS (1.1 eq., 46.9 mg, 0.046 mmol) dissolved in NaHCO₃ (0.1 M, 0.5 ml) the resulting mixture was stirred for 15 min at room temperature. pSCN-Bn-DOTA (6 eq., 177 mg, 0.253 mmol; Macrocyclics) was dissolved in NaHCO₃ (0.1 M, 5 ml) adjusted to pH 9 by Na₂CO₃ (0.1 M) and added to the reaction mixture that was stirred additional 18h at room temperature. Part of the reaction mixture (0.2 ml) was purified by size exclusion chromatography (sephadex, bidest. water). After lyophilization the labeling ratio of the DOTA- ligand was determined by ¹H-NMR. The adduct was purified by ultrafiltration (MWCO 5.000, PES, 5 x NaHCOs, 0.1 M) until the filtrate appeared colorless. The buffer was exchanged (2 x citrate buffer, 0.1 M, PH 8) and after dissolving Gadolinium chloride hexahydrate (5 eq., 78.4 mg, 0.211 mmol; Sigma) in citrate buffer (0.1 M) and adjusting to pH 8 by NaOH (1 M) the reaction mixture was stirred for 72h at room temperature. Purification was carried out by ultrafiltration (MWCO 5.000, PES, 5 x citrate buffer, 0.1 M; pH 8, 5 x bidest. water) until the filtrate was free of unbound gadolinium ions (xylenol orange test at pH 6). Elemental analysis showed labeling of 4.49% Gd/mol: 4 x Gd per molecule. Cy5.5 ratio was determined by an UV-absorbance concentration assay at wavelength maximum of the dye: 0.75 x Cy5.5 per molecule.

Probe 2:

Aminodextran70 (500 mg, 7.14 μmol; MW: 70.000 Da; Molecular Probes) was dissolved over a period of 2 h (swelling) in NaHCO₃ (0.1 M, 15 ml) and adjusted to pH 9 by Na₂CO₃ (0.1 M). After adding Cy5.5-NHS (1.1 eq., 7.9 mg, 7.81 μmol) dissolved in NaHCO₃ (0.1 M, 0.5 ml) the resulting mixture was stirred for 15 min at room temperature. pSCN-Bn-DOTA (36 eq., 178 mg, 0.256 mmol; Macrocyclics) was dissolved in NaHCO₃ (0.1 M, 5 ml) adjusted to pH 9 by Na₂CO₃ (0.1 M) and added to the reaction mixture that was stirred additional 18h at room temperature. Part of the reaction mixture (0.2 ml) was purified by size exclusion chromatography (sephadex, bidest. water). After lyophilization the labeling ratio of the DOTA- ligand was determined by ¹H-NMR. The adduct was purified by ultrafiltration (MWCO 50.000, PES, 5 x NaHCO₃, 0.1 M) until the filtrate appeared colorless. The buffer was exchanged (2 x citrate buffer, 0.1 M, PH 8) and after dissolving Gadolinium chloride hexahydrate (30 eq., 79.2 mg, 0.213 mmol; Sigma) in citrate buffer (0.1 M) and adjusting to pH 8 by NaOH (1 M) the reaction mixture was stirred for 72h at room temperature. Purification was carried out by ultrafiltration (MWCO 50.000, PES, 5 x citrate buffer, 0.1 M, pH 8; 5 x bidest. water) until the filtrate was free of unbound gadolinium ions (xylenol orange test at pH 6). Elemental analysis showed labeling of 4.42% Gd/mol: 24 x Gd per molecule. Cy5.5 ratio was determined by an UV-absorbance concentration assay at wavelength maximum of the dye: 0.55 Cy5.5 per molecule. 2. Example 2: powder formulation

Probe 1 (1.34 g) was ground to a fine powder using a ball mill (dismembrator) at 2000 rpm and zirconia grinding balls (diameter 3 mm, 20 g) in n-hexane for 5 x 1 h . Partly compressed aggregates / agglomerates of irregular shaped particles were obtained (diameter up to 80 μm; diameter of irregular shaped particles < 1 - app. 7 μm; > 90 % < 5 μm) as shown by electron microscopy. These particles were small enough to be applied by the syringe jet. Probe 2 could not be milled in homogenous sized particles and stuck to the syringe tube.

3. Method of imaging mucus secretion in an animal

Animals

Male BN rats (Iffa-Credo, L'Arbresle, France) weighing approximately 250 g were used in this study.

Maintenance Conditions

Time of acclimatization was of seven days. Rats were housed under standard conditions (temperature 20-24°C, relative humidity minimum 40%, light/dark cycle 12 hrs) and fed a standard diet (Nafag Nr. 890) and water supply ad libitum.

Statement on Animal Welfare

Animal handling, care and experimental use were in line with the Swiss Federal Law for animal protection (animal license BS No. 1989).

LPS Challenge

Animals were anesthetised with 4% isoflurane (Abbott, Cham, Switzerland) and LPS from Salmonella typhosa (Sigma, Dorset, UK; 1 mg/kg dissolved in 0.2 ml saline) was administered intra-tracheally (i.t.) and the animals allowed to recover.

MRI

Measurements were carried out with a Biospec 47/40 spectrometer (Bruker, Karlsruhe, Germany) operating at 4.7 T. A gradient-echo sequence with repetition time 5.6 ms, echo time 2.7 ms, band width 100 kHz, flip angle of the excitation pulse approximately 15°, field of view 6x6 cm², matrix size 256x128 and slice thickness 1.5 mm was used throughout the study. A single slice image was obtained by computing the 2DFT of the averaged signal from 60 individual image acquisitions and interpolating the data set to 256x256 pixels. There was an interval of 530 ms between individual image acquisitions, resulting in a total acquisition time of 75 s for a single slice. A birdcage resonator of 7 cm diameter was used for excitation and detection. During MRI measurements, rats were anesthetised with 2 % isoflurane in a mixture of O₂/N₂O (1 :2), administered via a face mask, and placed in supine position. The body temperature of the animals was maintained at 37 °C by a flow of warm air.

Histology

Challenged rats were killed by an overdose of pentobarbital (250 mg/kg i.p.). After being removed from the thorax, lungs were immersed in 10% neutral formalin buffer for 72 h. After fixation, transverse sections were cut through the median part of the left, the right apical, the median and the caudal lobes, dehydrated through increasing graded series of ethylic alcohol, and processed into paraffin wax overnight. Three serial slices of 3 μm thickness were cut from each section. Following stainings were then applied: (i) hematoxylin/eosin to assess general morphology; (H) PAS/Alcian blue reaction to detect mucus and goblet cells. One slice was left unstained for the detection of Cy5.5 by confocal microscopy.

Near-Infrared Fluorescence (NIRF) Imaging

Since the aminodextran contrast agent was also labeled by Cy5.5, NIRF images were acquired from isolated lungs, harvested immediately after an MRI session. For fluorescence excitation, three laser diodes operating at 660 nm with a power of 10 mW/cm² were used. The fluorescent light emitted from the lung samples was detected by a charge-coupled device (CCD) camera (Hamamatsu Photonics, Schϋpfen, Switzerland) equipped with a focusing lens system (macro lens 60 mm, 1 :2.8, Nikon). The CCD features low noise and low dark signal enabling low light level detection as well as long integration times. The matrix size of the images was 532x256 pixels. A hard filter was used for selecting the wavelength at detection (700 nm). Data acquisition (i.e. integration) times ranged from 0.5 to 2.0 s depending on the intensity of fluorescence. The experiment was controlled by a PC using the Siemens SYNGO^® software.

Results • In vivo Imaging

Figure 2 shows representative transverse sections through the thoracic region of a BN rat before and at various times following i.t. exposure to LPS (1 mg/kg). Clear signal changes were present in the lung within a few hours after application of endotoxin. The signals in response to LPS had two components. One (black arrows) was characterized by a diffuse signal and was particularly prominent until about 48 h after LPS challenge. A second component (white arrows), characterized by an irregular appearance and much weaker signal intensity, was present in the first hours after LPS challenge but predominated at the later time points. It was not possible to differentiate the individual components as they overlapped. Previous studies had shown that the edematous signals induced by LPS were predominantly due to secreted mucus (Beckmann et al 2002; Tigani et al 2002).

At 24 h following LPS, a prominent increase of the intensity of MRI edematous signals was observed after i.t. administration of the imaging probe of Example 1 in solution (0.2 mL) (figure 3). The increased signal was detected 24 h after contrast agent. At this time point, NIRF confirmed the presence of the contrast agent in the isolated lung from the same animal.

As the vehicle (0.9% NaCI) itself showed MRI signal enhancement in the rat lung, an aminodextran contrast agent in powder form was developed (see paragraph 2 above) to avoid this interference. The contrast agent would be injected with a dry powder insuflator. Probe 2 bearing a 70.000 g/Mol dextran backbone could not be milled in homogenous sized particles and was stuck to the syringe tube. Probe 1 (10.000 g/Mol) led to particles that were small enough to be applied by the insuflator (Figure 4). Figure 5 shows how the imaging probe of the invention led to a prominent increase in the intensity of edematous signals immediately after its i.t. administration. Histology revealed that the contrast agent bound specifically to mucin, secreted or present in goblet cells (Figure 6).

Discussion

The present results suggest that, in LPS-challenged BN rats, the imaging probes of the invention binds specifically to mucus. Careful histological examination revealed that not only secreted mucus, but also mucin encountered in goblet cells was labeled by the contrast agent. This may indicate that the time course of MRI signals in the lungs of LPS-challenged animals following administration of the contrast agent does not reflect only mucus clearance, but also the resolution of mucus plugging. It is also conceivable that the imaging probe is only slowly cleared from the lung, thus contributing to enhance the signal from continuously secreted mucus. Indeed, upon inhalation, particles initially deposited on airway surfaces are subsequently cleared by various clearance mechanisms. Depending on their initial site of deposition, a fast- and a slow-clearance phase have been observed in particle retention experiments. The fast-clearance phase has traditionally been interpreted as tracheobronchial (TB) clearance, whereas the slow-clearance phase is commonly attributed to mechanical clearance from the alveolar region (ICRP 1994). While there is general agreement that mucociliary transport is the principal clearance mechanism in the TB region during the first 24 h of exposure, experimental data from bolus inhalation studies postulated the existence of a slow bronchial clearance phase (Stahlhofen et al 1990). However, recent calculations of mucociliary clearance in the human TB tree suggested that these findings might be partly explained by a delayed clearance from peripheral bronchiolar airways in an asymmetric lung structure (Asgharian et al 2001 ).

Tracheal mucociliary clearance velocities in rats display a wide range of values, presumably because of differences in measurement techniques. Values found in the literature are 8.1 mm/min (Giordano and Morrow 1972), 5.1 mm/min (Patrick and Stirling 1977), and 1.9 mm/min (Felicetti et al 1981 ). Anesthesia may influence (increase) mucociliary transport. These velocities suggest that with the exemplified imaging probe, a delayed clearance is observed with MRI.

Being doubly labeled, the imaging probe provides the opportunity to perform NIRF acquisitions in vivo as well.

REFERENCES

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Claims

1. An imaging probe for use in a method for assessing mucus clearance, comprising a polysaccharide polymer capable of binding to mucus, coupled to at least one imaging contrast agent.

2. The probe according to Claim 1 , wherein said polysaccharide polymer is made of dextran or derivatives thereof, such as aminodextran.

3. The probe according to Claim 1 or 2, wherein at least one imaging contrast is a magnetic resonance imaging (MRI) contrast agent.

4. The probe according to any of Claims 1-3, wherein said MRI contrast agent includes a paramagnetic or a superparamagnetic element.

5. The probe according to Claim 4, wherein said paramagnetic element is Gadolinium (III).

6. The probe according to Claim 5, wherein said MRI contrast agent is Gadolinium chelating group complex

7. The probe according to any one of Claims 1-6, wherein one imaging contrast agent is a fluorochrome that has absorption and emission maximum between 650 and 1 100 nm.

8. The probe according to Claim 7, wherein said fluorochrome is selected among the group consisting of the following: Cy5.5, Cy7, Cy 7.5, Alexa Fluor 680, Alexa Fluor 750.

9. The probe according to any of Claims 1-8, wherein said probe essentially consists of a polysaccharide polymer covalently bound to cyanine dyes, such as Cy5.5, and an MRI contrast agent, such as Gd-DOTA.

10. The probe according to any of Claims 1-9, wherein said probe is formulated as a dry powder appropriate for in vivo administration.

11. The probe according any of Claims 1-10, wherein said probe has an average molecular weight comprised between 500.000 and 3.000, more preferably between 70.000 and 3.000 , most preferably between 10.000 and 3.000.

12. A non-invasive method for imaging mucus secretion in a mammalian subject in vivo, comprising: a. administering to the subject a magnetic resonance imaging composition comprising a mucus binding polysaccharide polymer coupled to a MRI contrast agent, by inhalation or injection into the trachea; and, b. obtaining a magnetic resonance image of the lung or any mucus secreting regions of interest, wherein said image represents mucus secretion in the region of interest.

13. The method according to Claim 12, wherein said magnetic resonance imaging composition is a probe according to any one of Claims 3 to 6 and 9.

14. A non-invasive method for imaging mucus secretion in a mammalian subject in vivo, comprising a. administering to the subject an optical imaging composition comprising a mucus binding polysaccharide polymer coupled to an optically active molecule; b. illuminating the subject with light source; and, c. visually monitoring the presence of the optical imaging composition in the subject, thereby obtaining an image, wherein said image represents mucus secretion in the region of interest.

15. The method according to Claim 14, wherein said optically active molecule is a fluorescent dye, a bioluminescent dye or a near infrared fluorescent dye.

16. The method according to any of Claims 12-14, wherein said mammalian subject is a non-human mammal, for example a rodent, and more preferably a small rodent selected among the group of rats, mice, rabbits, guinea pigs and hamsters.

17. A method of identifying a compound that modulates mucus clearance in a mammalian subject, said method comprising: a. administering a candidate compound to a test subject; b. assessing the rate of mucus clearance in said test subject by the method according to Claim 14; and, c. comparing the rate of mucus clearance between the test animal and a control animal to which no or a reference candidate compound has been administered to, wherein any significant difference in mucus clearance between the control and test animals is indicative that said candidate compound is a compound capable of modulating mucus clearance in said animal.

18. The use of a dextran polymers or a probe according any of Claims 1-1 1 , for assessing mucus clearance in a mammalian subject.

19. The use according to Claim 18, wherein said animal is non-human mammal, for example a rodent, and more preferably a small rodent selected among the group of rats, mice, rabbits, guinea pigs and hamsters.

20. A kit comprising the probe according to any of Claims 1-11 and instructions for the performance of an in vivo assay for determining mucus clearance in a mammalian subject.