Title of Invention

"A METHOD FOR PRODUCING A POWDER TO BE INHALED FOR TREATING OR PREVENTING MICROBIAL INFECTIOUS DISEASE OF THE RESPIRATORY ORGANS"

Abstract A method for producing a powder to be inhaled for treating or preventing microbial infectious disease of the respiratory organs, comprising the steps of preparing an aqueous solution containing a collectin family member protein, a sugar, NaCl and CaCl2 and spray-drying said aqueous solution.
Full Text Technical Field
[1] The present invention relates to a composition for treating or preventing microbial
infectious diseases comprising at least one member protein of the collectin family or its variant. This composition is suitable for inhalation via the respiratory system or direct delivery to epidermal cells at the site of infection. The present invention also relates to a method for producing said composition in a spray-dried form as to allow direct delivery to the sites of infection.
[2]
Background Art
[ 3] Mannose-binding lectin (MBL) is a human blood protein known to be involved in
innate immunity against microbial infections. MBL recognizes the specific gly-cosylation patterns of the proteins on the cell surface of the infecting microorganism and binds them to suppress the microbial infection according to three pathways described below: In the first pathway, MBL binds the microbial glycosylated protein to form a complex, and then it activates MBL associated serine proteases (MASPs). The MASPs, in turn, cleave C4 and C2, leading to an activation of the complement system. In the second pathway, MBL, bound to the glycosylated microbial cell surface proteins, serves as an opsonin and directs phagocytosis by neutrophils and macrophages. The same bound MBL can also neutralize the infectivity of the microbes in the third pathway, blocking their proliferation. Thus the most important initial step in the MBL's defense against microbial infections is the recognition of and binding to the microbes.
[4]
[51 MBL, a member of the collectin family, shares a common structure consisting of a
collagen domain and a carbohydrate recognition domain with other members. The MBL monomer has a molecular weight of 32 kDa. It has a C-type carbohydrate recognition domain (CRD) at the C-terminus, a cysteine-rich region at the N-terminus, and a collagen domain in between. Three MBL monomers associate to form a triple helical complex through their collagen domains. The helical complex, in turn, forms an intermolecular disulfide bond via a cysteine in the N-terminal region to yield a hexameric complex. This triple helix is a structural feature shared by all the collectin family members including surfactant proteins A (SP-A) and D (SP-D), collectin-liver 1

(CL-L1), and collectin-placenta 1 (CL-P1). Thus these member proteins belonging to the collectin family all share similar physicochemical properties. These collectin family proteins have been known to share another characteristic of playing an important role in the pre-immune defense against microbial infections in sera and the pulmonary surface as well (Hans-Jurgen et al, Protein Science,3:1143, 1994). Besides the ones listed above, other family members include CL-43, a bovine conglutinin, and human conglutinin homolog.
[6]
[7] MBL is known to be capable of binding various microorganisms. MBL most often
binds viruses with outer coats (viral envelopes). Representative examples include: influenza virus (Hartshorn, K. L. et al, J. Clin. Invest., 91:1414, 1993; Kase, T. et al., Immunol, 97:385, 1999), human immunodeficiency virus (Ezekowitz, R. A. et al, J. Exp. Med., 169:185, 1989; Haurum, J. C. et al, AIDS, 7:1307, 1993), herpes virus (Fischer, C. B. et al, Scan. J. Immunol, 39:439, 1994) and SARS corona virus (Ksiazek, T. G. et al, N. Eng. J. Med., 348:1953, 2003; Peiris, J. S. M. et al, Lancet, 361:1319, 2003). Rhinoviruses, responsible for the common cold, are expected to be good binders to MBL as well. Among bacteria, Staphylococcus aureus (Neth, O. et al, Infect. Immunol, 68:688, 2000) and Hemophilus influenzae (Van E. et al, Clin. Exp. Immunol, 97:411, 1994) are reported to be good binders, whereas among fungi, Candida albicans (Tabona, P. et al, Immunol, 85:153, 1995) is known.
[8]
[9] Among the microbes that bind MBL, influenza virus, rhinovirus, SARS corona
virus and influenza virus of animal origin cause symptoms mainly through the infection of the respiratory epithelia, whereas S. aureus and H. influenzae infect lungs. S. aureus can infect external wounds and C. albicans is involved in vaginitis. Since treating MBL in a solution phase is not suitable for curing these respiratory diseases and external wounds, special formulations are required. Although there have been many studies on MBL, the extent to which MBL is involved in the defense against the infections in epithelia and external wounds mentioned above is poorly understood. The only relevant study is one reporting a detection of MBL in saliva and breast milk (Tregoat, V. et al, J. Clin. Lab. Anal, 16(6): 304, 2002).
[10]
[11] Upon influenza infection, the virus colonizes the epithelial cells that line the
surfaces of respiratory organs. Once the virus has amplified itself inside the infected cell, new virus cells attack neighboring epithelial cells. Thus, it is possible to disrupt contact between an epithelial cell and the virus using MBL which is capable of recognizing the glycosylated microbial surface proteins and binds them. For infections in cells exposed to the surface, the mechanism that involves physical blocking through


binding can be chosen for defense from the three pathways mentioned above. The opsonin pathway can be employed for defense when macrophages emerge on the surface. In fact, it was observed in cultured cells that physical neutralization by MBL binding was sufficient for blocking microbial infection of neighboring cells. For example, MBL added to the culture medium prevented viral infection upon SARS corona virus inoculation of cultured cells (Korean patent gazette No. 1020040106194). Similar prevention of infection by MBL has been observed for influenza virus as well. (Wakamiya, N. et al, Biochem. Biophys. Res. Commun., 187:1270, 1992; Hartley, C. A. et al, J. Virol, 66:4358, 1992; Patrick, C. R. etal, J. Virol, 71:8204, 1997; Kase, T. et al, Immunol, 97:385, 1999)
[12]
[131 In order to treat external wounds and respiratory infections with MBL, it is
desirable in many ways to formulate the protein into a powder. A powder formulation is capable of an effective delivery to sites of infection, delivers an optimal amount of MBL, lessens side effects thanks to its topical application and reduces the amount used.
[14]
[15] Freeze-drying and spray-drying are used in general to formulate protein drugs into
powders. Freeze-drying is most widely employed nowadays. Freeze-drying is suitable for heat-sensitive proteins, but it is not suitable for producing a uniform powder with a diameter of a few micrometers, a form that can be readily inhaled. Freeze-drying also tends to concentrate proteins between the ice crystals while cooling. This concentration brings about a rapid change in the pH and ionic strength surrounding the protein to cause denaturation and precipitation (Schwartz, P. L. et al, Endocrinology, 92(6): 1795, 1973; Koseki, T. etal, J. Biochem., 107:389, 1990). In contrast, spray-drying involves spraying a continuous stream of a liquid sample to form microscopically dispersed droplets, while instantaneously drying them with hot air at the same time. Spray-drying has been in use for formulating various drugs. Spray-drying has the advantage of producing powders whose particle sizes are suitable for delivering drugs to respiratory tracts and lungs. Spray-drying also consumes less energy so that time and cost can be saved at the production line (Broadhead, J. et al, Drug Dev. Ind. Pharm., 18(11&12):1169, 1992). Since proteins in general are not stable against heat, there are not many cases of protein drug formulations spray-dried with hot air. There are attempts, however, to apply spray-drying as follows: oxyhemoglobin (Labrude, P. et al, J. Pharm. Sci., 78(3):223, 1989), human growth hormone (Mumenthaler, M. et al, Pharm. Res., 11(1):12, 1994; Bosquillon, C, J. Cont. Rel, 96:233, 2004), tissue plasminogen activator (t-PA, Mumenthaler. M. et al, Pharm. Res., 11(1): 12, 1994), DNase (Chan, H. K. et al, Pharm. Res., 14(4):431, 1997), parathyroid hormone

(Codrons, V. et al., J. Pharm. Sci., 92(5):938, 2003) and humanized monoclonal antibody (anti-IgE, Maa, Y. F. etai, Biotechnol Bioeng., 60(3):301, 1998).
[16]
[17] Especially when producing spray-dried protein compositions for respiratory ap-
plications, it is desirable to formulate proteins into powders sized 5 Q or less. The particle size, shape and water content of such powders are important factors in terms of treatment efficacy (Hickey, A. J. et al., Pharm. Tech., 18:58, 1994). The major determinants of the physical characteristics of such powders are mechanical conditions such as the feed velocities for hot air and the protein solution, temperature and spray pressure (Maa, Y. F. et al, Pharm. Res., 15(5):768, 1998)as well as the identities and concentrations of the additives to the protein solution such as salts, sugars and excipient proteins (Andya, J. D. et al, Pharm. Res., 16(3):350, 1999; Maa, Y. F. et al, Pharm. Dev. Tech., 2(3):213, 1997). Also in the powder formulation of recombinant human MBL by spray-drying, the important considerations are that spray-dried MBL powder should maintain its ability to activate complement through the specific binding to the glycosylated, MBL-binding proteins in the presence of serine proteases when dissolved and that such ability is not lost during long-term storage.
[18]
[19] However, there have been no studies reported on spray-drying methods for
producing human recombinant mannose-binding lectin compositions for treating illnesses such as respiratory inflammation; thus, there is a strong need for spray-drying methods to produce recombinant human MBL compositions available for respiratory inhalation and application to external wounds.
[20]
[21 ] In order to address such problems, the present invention provides a powder formed
by adding a sugar and/or protein additives to a solution containing at least one collectin family member protein and spray-drying the solution. This powder is able to support long-term storage without losing its efficacy.
[22]
Disclosure of Invention Technical Solution
[23] The present invention provides a spray-dried powder composition containing at
least one collectin family member protein or its variant, whose particle size ranges from 0.1 to 10 0 and is capable of direct delivery to respiratory organs, other cavities and sites of microbial infection on the surface for treatment and prevention of such infections. The present invention also provides a method for producing said composition comprising the steps of:


[24] (i) producing an aqueous solution containing a sugar and at least one collectin
family member protein or its variant;
[25] (ii) spray-drying the solution of step (i) with a sufficient pressure to maintain a
spraying rate of 500 to 2,000 liters/hour(L/h) and a hot air flow rate of 400 to 1,200 L/ min wherein the hot air temperature is between 50 to 220 °C and the discharge temperature is between 42 to 150 °C; and.
[26] (iii) collecting the powder produced from step (ii).
[27] The powder produced from the above method is suitable for respiratory inhalation
and treating infections in the epidermis.
[28]
[29] To achieve the objects mentioned above, the present invention is described in detail.
[30]
[31] The present invention provides a spray-dried powder composition containing at
least one collectin family member protein or its variant, whose particle size ranges from 0.1 to 10 D and is capable of direct delivery to respiratory organs, other cavities and sites of microbial infection on the surface for treatment and prevention of such infections. The present invention also provides a method for producing the above composition comprising the steps of:
[32] (i) producing an aqueous solution including a sugar at least one collectin family
member protein or its variant;
[33] (ii) spray-drying the solution of step (i) with a sufficient pressure to maintain a
spraying rate of 500 to 2,000 liters/hour(L/h) and a hot air flow rate of 400 to 1,200 L/ min wherein the hot air temperature is between 50 to 220 °C and the discharge temperature is between 42 to 150 °C; and.
[34] (iii) collecting the powder produced from step (ii).
[35]
[36] The spray-dried composition of the present invention contains at least one member
protein from the collectin family or its variant protein as the active ingredient and has a particle size ranging from 0.1 to 10 D. It can be inhaled to the respiratory organs or directly delivered to sites of infection in the epidermis to treat or prevent microbial infections.
[37] By a collectin family member protein as used herein is meant any protein that has a
collagen domain and a carbohydrate recognition domain. These proteins have very similar physicochemical properties.
[38] By "variant" as used herein is meant a polypeptide whose sequence differs from the
original collectin family member protein sequence by virtue of at least one amino acid modification, while at the same time being a functional equivalent of the original member. These variants include homologs of the collectin family member protein

belonging to the same or different species, and also natural and artificial mutants thereof with similar functional or physiological prorperties.
[39]
[40] The collectin family includes such members as SP-A, SP-D, CL-L1, CL-P1, CL-43
and human conglutinin-like protein. Each member protein in the collectin family have a lectin domain in its C-terminus that recognizes and binds carbohydrates (Hans-Jurgen et al, Protein Science,?)-.1143, 1994). For example, CL-43 binds gpl60, a glycoprotein that constitutes the outer coat of human immunodeficiency virus (HIV), to inhibit its binding to CD4 receptors (Anderson et al, Scand. J. Immunology, 32:81, 1990). Human conglutinin-like protein is known to bind HIV-1's gpl20 to block infection (Ushijima et al, Jpn. J. Cancer Res., 83:458, 1992). There have been reports on SP-A and SP-D as well that these proteins block microbial infection by binding to the microbes (Zimmerman et al, J. Clin. Invest, 89:143, 1992; McNeely et al, J. Infect. Dis., 91, 1993; Kuan et al, J. Clin. Invest.90:97, 1992).
[41]
[42] From these findings, uses of other collectin family member proteins than MBL as
an active ingredient of the spray-dried powder composition of the present invention can be contemplated for treating and preventing microbial infections.
[43]
[44] The above collectin family member proteins can be obtained commercially or
produced with recombinant vectors by standard molecular biology protocols well-known to those skilled in the art; e. g., human CL-P1 is available as a recombinant form (catalog #2690-CL) from R&D systems (Minneapolis, MN, USA) for producing the composition of this invention.
[45]
[46] One preferred spray-dried powder formulation for the above composition may
comprise 0.001 to 60 parts recombinant MBL, 0.1 to 10 parts NaCl, 0.1 to 10 parts CaCl, and 5 to 80 parts sugar by weight. A more preferred formulation may comprise 0.001 to 0.1 recombinant MBL, 8 to 9 parts NaCl, 1 to 2 parts CaCl, 20 parts lactose or sucrose, and 0.5 parts polyvinyl alcohol (PVA) by weight. The numerical values of parts above refer to a weight ratio of the constituents forming the spray-dried composition. Since the composition of this invention is sprayed from an aqueous solution and all the solutes are non-volatile, it is evident that the ratio of solutes by weight will be maintained in the final composition. Thus, molarities and weight per volume percents are readily converted to parts by weight; for example, 10 mM CaCl (molecular weight 111) corresponds to 10x10-3 x111 = 1.11 parts by weight.
[47]
[48] The present invention preferably consists of, but limited to 0.01 to 60 parts collectin


family member protein by weight. The collectin family member protein or its variant is preferably, but not limited to a natural or recombinant mannose-binding lectin (MBL), wherein the recombinant one is more preferable. This recombinant MBL is produced preferably from, but not limited to the cell line of accession number KCTC 10472BP (Korean patent gazette No. 1020040106194).
[49] In addition, the particle size of the inventive composition is preferably around 5 D
and more preferably from 0.1 to 4 D in order to have treatment efficacy for such application routes as respiratory inhalation.
[50] The composition of the present invention may further comprise pharmaceutically
acceptable carriers. Such carrier is preferably, but not limited to, a sugar, wherein the sugar is preferably, but not limited to, sucrose or lactose. The content which such carrier takes up in the inventive composition is preferably, but not limited to, 5 to 80 % by weight.
[51] Meanwhile, the infectious microorganisms for the present invention are preferably,
viruses, bacteria and fungi: wherein the viruses are preferably, but not limited to, influenza virus, human immunodeficiency virus, herpes virus, SARS corona virus and rhinovirus; wherein the bacteria are preferably, but not limited to Staphylococcus aureus, Hemophilus influenzae and S. pyogens; and wherein the fungi are preferably, but not limited to, Candida albicans.
[52] Also the spray-dried composition of the present invention may further comprise
casein as a supporting ingredient in order to maintain a stable efficacy after formulation. The content of casein in the composition is preferably, but not limited to, from 0.1 to 20 % by weight.
[53]
[54] Meanwhile, the infectious microorganisms which are treatment and prevention
targets for the present invention are preferably, viruses, bacteria and fungi: wherein the viruses are preferably, but not limited to, viruses with outer coats including influenza virus, human immunodeficiency virus, herpes virus, SARS corona virus and rhinovirus; wherein the bacteria are preferably, but not limited to, those with gly-cosylation patterns recognizable by MBL including Staphylococcus aureus, Hemophilus influenzae and S. pyogens; and wherein the fungi are preferably, but not limited to, Candida albicans.
[55]
[56] The present invention also provides a method for producing a spray-dried powder
composition containing MBL, suitable for respiratory inhalation or direct delivery to sites of infection in the epidermis, comprising the steps of: (i) producing an aqueous solution containing a sugar and at least one collectin family member protein and its variant; (ii) spray-drying the solution of step (i) with a sufficient pressure to maintain a

spraying rate of 500 to 2,000 liters/hour(L/h) and a hot air flow rate of 400 to 1,200 17 min wherein the hot air temperature is between 50 to 220 °C and the discharge temperature is between 42 to 150 °C; and (iii) collecting the powder produced from step (ii).
[57] In step (i), the aqueous solution preferably includes, but not limited to, 0.1 to 4%
(w/v) sugar and 0.0005 to 10% (w/v) collectin family member protein or its variant, wherein the sugar is preferably, but not limited to, sucrose or lactose. Preferably, the aqueous solution of step (i) may further comprise pharmaceutically acceptable carrier or casein, wherein the casein content is preferably, but not limited to, from 0.005 to 0.2% (w/v).
[58]
[59] The present inventors have performed research on suitable formulations of collectin
family member proteins or its variants for treating bacterial respiratory infections such as pneumonia with the aim of developing aerosol inhalation routes, rather than oral or injection administration, so as to enable a direct delivery to the epithelial cells in target organs: as a result, we have discovered that powdered formulations with microscopic particles, rather than ordinary suspensions, are more efficient for administration. Freeze-drying, the conventional method for making powders, applied to produce the
composition of the present invention, however, yields particles with inadequate sizes
(less than 5 mm is appropriate) for inhalation; consequently, freeze-dried formulations are not suitable for inhalation. We have therefore contemplated spray-drying as the preferred method of choice for forming particles with the right size. Since spray-drying involves applying a strong pressure to spray a suspension and drying the suspension with hot air, it finds limited use in forming particles from heat-sensitive proteins. Only few isolated examples of spray-dried formulations have been reported for proteins, none of them being mannose-binding lectin.
[60]
[61] To address this problem, the present inventors have made attempts to produce com-
positions containing collectin family member proteins or its variants by spray-drying and analyzed whether the composition produced could maintain its activity and stablility. We have confirmed that thus obtained, spray-dried, microscopic composition has an activity comparable to that of the non-dried suspension containing the same collectin member protein or its variant, and also that this composition readily supports long-term storage. Moreover, by varying the identities and concentrations of sugars, the present inventors have established the optimal conditions for producing compositions with suitable particle sizes to support efficient inhalation. We have also established the temperature and pressure conditions for sustaining the activity of this composition. In addition, when the composition further comprises casein as a

supporting ingredient, this composition exhibits an enhanced sustenance of its activity over controls without added casein; thus, yielding a more effective treatment and preventive agent for microbial infectious diseases.
[62]
[63] The composition of the present invention is directly inhalable as aerosols. Its dose
range can vary widely according to the patient s bodyweight, age, sex, health condition, diet, means of administration, dosing intervals, clearance rate and severity of the patient s condition. Dose range is from 50 D/dose to 20 mg/dose, and preferably from 1 mg/dose to 5 mg/dose. It is preferable that the inventive composition is administered in a single or multiple daily doses.
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
Brief Description of the Drawings
[76] Figure 1 shows the particle size distribution for the spray-dried MBL composition
of the present invention.
[77] Figure 2 shows the results from the Western blot to test whether the morphology of
MBL is maintained before and after the spray-drying.
[78] Figure 3 compares a spray-dried MBL powder with a non-dried MBL solution in
terms of C4 activation.
[79] Figures 5 to 8 compare spray-dried MBL powders produced under various tem-
peratures with non-dried MBL solutions for their C4 activation.
[80] Figures 9 to 12 compare the spray-dried MBL powders produced in the presence of
different sugar additives with non-dried MBL solutions for their C4 activation.
[81] Figure 13 confirms the particle sizes of the spray-dried MBL compositions
produced in the presence of different sugar additives.
[82] Figures 14 to 17 show the scanning electron microscope images of the spray-dried
MBL compositions produced in the presence of different sugar additives.


[83] Figures 18 to 21 compare the spray-dried MBL powders produced under various
concentrations of the additive lactose, with non-dried MBL solutions for their C4
activation.
[84] Figures 22 to 26 compare the spray-dried MBL powders produced under various
concentrations of the additive sucrose, with non-dried MBL solutions for their C4
activation.
[85] Figure 27 confirms the particle sizes of the spray-dried MBL compositions
produced under different concentrations of lactose, an additive.
[86] Figure 28 confirms the particle sizes of the spray-dried MBL compositions
produced under different concentrations of sucrose, an additive.
[87] Figures 29 to 32 show the electron microscope images of the spray-dried MBL
composition produced under various concentrations of lactose, an additive.
[88] Figures 33 to 37 show the scanning electron microscope images of the spray-dried
MBL compositions produced under various concentrations of sucrose, an additive.
[89] Figures 38 to 41 compare the spray-dried MBL powder produced with a sugar
additive for their C4 activation in the presence or absence of casein with non-dried
MBL solutions.
[90] Figures 42 to 45 compare spray-dried MBL powders stored under harsh temperature
conditions with those stored at room temperature for their C4 activation.
[91] Figures 46 and 47 are plots of MBL comparing the spray-dried MBL powder
produced in the presence of different additives with non-dried MBL solutions for their
binding to microbes. [92] [93]
Mode for the Invention
[94] EXAMPLES
[95] Hereinafter, the present invention is explained in detail with examples. The
following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention. Unless otherwise mentioned, the percentage used in the examples below refers to volume-to-weight ratios.
[96]
[97]
[98] Characterization of MBL powder and establishment of the method for its analysis
[991 To formulate recombinant mannose-binding protein (MBL) into a form suitable for
inhalation, treating external wounds or into a powdered form which in turn can be formulated into tablets, the following conditions should be met: the spray-dried MBL

powder should be an aerosol with an inhalable size; MBL's structural characteristic of complex formation and the distribution of such complexes should be maintained throughout the spray-drying process; and the powder produced should activate complement in the presence of glycosylated MBL-binding proteins and serine proteases upon dissolution. In addition, a method for analyzing such properties needs to be established.
[100]
[101] As a start, the present inventors have produced an MBL powder under normal
spraying conditions for proteins. The MBL solution used for spray-drying was prepared as follows: 5 D/mL recombinant MBL (Dobeel, Korea), 150 mM NaCl, 0 mM CaCl, 1 mg/mL casein, 0.4% (w/v) sucrose. A laboratory spray-dryer (LaPlant SD-05, UK) was used. The MBL solution was fed into a nozzle (0.5 mm in diameter) at the rate of 3.5 mL/min by a peristaltic pump, with the spraying rate at 1,600 L/hr. The hot air flow through a drying duct was at 1,150 L/min, and its temperature was kept at 100 °C, while the discharge temperature was at 69 °C.
[102] A fine, white powder was obtained using the method above and the characteristics
of the MBL powder produced was analyzed by the methods described below.
[103] Example 1-1 The microscopic particle size distribution of the MBL powder
[104] It is known that drugs formulated into powders must be inhaled as aerosols in order
to achieve effective delivery to respiratory ducts and lungs, and that the required particle size for inhalation is preferably 5 mm or below. The sizes and distribution of spray-dried MBL particles were probed with a laser zeta potentiometer (ELS-8000, Otsuka Electronics, Japan).
[105]
[106] As shown in Figure 1, the MBL powder produced by the above method had particle
sizes ranging between 2.18 and 3.18 D, the average size being 2.57+0.24 0. It was thus confirmed that spray-drying can yield MBL powders that can be inhaled effectively into respiratory tracts and lungs.
[107]
[108] Example 1-2 Western blot analysis
[109] The present inventors examined whether MBL's complex formation characteristics
and their distribution can still be maintained throughout spray-drying with hot air. The MBL powder was dissolved in a suitable amount of water and the solution was subject to Western blot analysis under non-denaturing conditions to look for any differences before and after spray-drying (Figure 2).
[110]
[111] Figure 2 shows the results from the Western blot looking for morphological
changes in MBL before and after spray-drying, where M stands for molecular weight


markers, 1 is the MBL solution before spray-drying and 2 is the spray-dried MBL powder. As shown in figure 2, no structurally clear differences were detected before and after the spray-drying. It was thus confirmed that a powder formulation of MBL is possible without structural alteration.
[112] Example 1-3 C4 activation in the complement system
f 113] The present inventors examined whether the spray-dried MBL powder maintains its
activity upon dissolution of activating complement in the presence of glycosylated, MBL-binding proteins and serine proteases by the methods described below: First, in-munoassay plates (MaxiSorp Immunoplate, Nunc, Denmark) were coated with hepatitis B pre-S antigen at 500 ng/well. Either a spray-dried powder or non-dried (control) MBL was dissolved in water and the solution was added to the above plate at 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng, 6.25 ng or 3.125 ng per well and was incubated for 2 hours for binding. 100 D of MBL-free serum (Dobeel, Korea), diluted 1:100 in dilution buffer, was added to each well to provide MBL-associated serine proteases (MASPs). The plates were washed 6 times with wash buffer. 500 ng of C4 was added to the plates followed by a 2-hour incubation. Anti-C4 horseradish peroxidase (HRP) conjugate (Biogenesis, UK) was added with a dilution ratio of 1:1,500 followed by an 1-hour incubation. 150 ◘ ofo-phenylenediamine (OPD) solution was added and the col-orimetric reaction was allowed to proceed for 20 minutes to detect C4b deposits. The absorbance at 492 nm (Figure 3) was measured with an ELIS A reader after adding 50 D of 3M HCl.
[114]
[115] Figure 3 shows a plot that compares C4 activation in the spray-dried MBL
composition of the present invention (open circles) with that of control groups (filled circles). Both non-dried and spray-dried MBL induced similar levels of activation. Consequently, the present inventors were able to confirm that spray-dried MBL powders did not suffer a loss in their efficacy for respiratory infections and external wounds.
[116]
[117] As described in Example 1, the MBL powder was produced using conventional
spray-drying methods. We were able to show that the MBL powder produced had 1) an inhalable particle size, 2) its structural characteristics of complex formation and their distribution unchanged, and 3) still retained its complement-activating abilities. Thus, we have confirmed the feasibility of formulating MBL into a powder by spray-drying.
[118]
[119]
[120] The effect of air temperature during sprav-drving on the activity of MBL powders
[121] Most proteins are prone to denaturation by heat, and denatured proteins lose their


biological activities. Since spray-dryers convert liquid samples into powders using hot air flow, we have examined the differences in complement activation of MBL powders produced under different temperature conditions in the presence of glycosylated, MBL-binding proteins and serine proteases.
[122] The MBL solution used for spray-drying had the following composition: 5 D/mL re-
combinant MBL, 150 mM NaCl, 10 mM CaCl, 250 D/mL casein, 2% (w/v) sucrose. The feed/discharge temperatures for spray-drying were set at 80°C/58°C, 100°C/68°C, 115°C /78°C, 130°C/87°C and 150°C /100°C, respectively. Each powdered MBL composition produced under its own temperature condition, was dissolved in a suitable amount of water and compared with non-dried MBL solution (control) for its ability to activate C4 in the complement system (Figures 4 to 6) according to the methods of Example 1-3.
[123]
[124] Figures 4 to 6 compare the spray-dried MBL compositions above, prepared under
different temperatures, with non-dried MBL solution for their C4 activation. The feed/ discharge temperature settings for each figure are as follows: 80°C/58°C (Figure 4), 100°C/68°C (Figure 5), 115°C/78°C (Figure 6), 130°C/87°C (Figure 7) and 150°C/100°C (Figure 8). Open circles denote the spray-dried MBL composition of the present invention and filled circles represent control experiments.
[125]
[126] As shown figures 4 to 8, the MBL spray-dried at temperature settings of 80°C/58°C
and 100°C/68°C has similar activities to control MBL solutions, whereas powdered MBL compositions produced at higher temperature settings suffer from reduction in their activities as the temperature goes up. These results, therefore, have confirmed that in spray-drying, temperature settings lower than 100°C/68°C are effective for producing MBL powders with high activities.
[127]
[128]
[129] Effects of stabilizer additives on MBL: powder properties according to the added
sugar
[130] When spray-drying proteins into powder formulations using hot air, it is
commonplace to add various kinds of sugars for physical and biological stability. In the present invention, we have studied complement activation, the size distribution and shape of MBL powders according to the identity of sugar (sucrose, lactose, trehalose and pluran), a stabilizer additive. The MBL solutions used for spray-drying contained 5 ◘/mL recombinant MBL, 150 mM NaCl, 10 mM CaCl, 250 ◘/mL casein with either
0.4% sucrose, 0.5% lactose, 0.4% trehalose, or 0.4% pluran in addition. [131]

f 132] Example 3-1 C4 activation in the complement system
[1331 After spray-drying MBL solutions prepared using different sugar additives, each
MBL powder was measured for its activity using the methods of Example 1-3 and compared with non-dried MBL solution (control, Figures 9 to 12). Figures 9 to 12 compare the spray-dried MBL powders produced in the presence of different sugar additives with the MBL solution before drying for their C4 activation. Open circles denote the spray-dried MBL composition of the present invention and filled circles represent control experiments.
[134]
[135] As shown in figures 9 to 12, spray-dried MBL powders had similar activity levels
compared to controls for 0.4% sucrose (Figure 9) and 0.5% lactose (Figure 10) formulations, but suffered large losses in activity for 0.4% trehalose (Figure 11) and 0.4% pluran (Figure 12). These data establish sucrose and lactose as effective stabilizer additives for spray-drying MBL.
[136]
[ 137] Example 3-2 Particle size distribution of MBL powders
[138] The particle size distribution of spray-dried MBL powders (Figure 13 and Table 1)
was probed with a laser zeta potentiometer (ELS-8000, Otsuka Electronics, Japan). Figure 13 plots the particle sizes of the inventive, spray-dried MBL compositions in the presence of different sugar additives, wherein filled circles represent 0.4% sucrose; open circles, 0.5% lactose; filled triangles, 0.4% trehalose; and open triangles, 0.4% pluran formulations of spray-dried MBL powders.
[139]
[140] As shown in figure 13 and table 1, spray-dried MBL powders had particle sizes
ranging from 1.04 to 4.00 ◘. Addition of sucrose, lactose, or trehalose effected a more even distribution of particle sizes. Addition of pluran, however, yielded a broad size distribution ranging from 0.39 to 15.43 ◘. It was thus concluded that addition of sucrose, lactose or trehalose to the MBL solution was more effective than that of pluran for producing MBL powders expected to have good inhalation efficacies.
[141] Table 1

Particle size distribution of spray-dried MBL powders according to their sugar formulations
(Table Removed)
[142] Example 3-3 Shapes of MBL powders
[143] The shapes of MBL powders were observed with a scanning electron
microscope(Figures 14 to 17). Figures 14 to 17 show the electron microscope images of the spray-dried MBL compositions produced in the presence of different sugar additives, wherein figure 14 represents 0.4% sucrose; figure 15, 0.5% lactose; figure 16, 0.4% trehalose; and figure 17, 0.4% pluran formulations of spray-dried MBL powders
[144]
[145] As shown in figures 14 to 17, different sugar additives used for stabilizing MBL
solutions were observed to shape MBL powders into different forms. Also, the particle size distribution of MBL powders determined with a laser zeta potentiometer agreed with the distribution determined by electron microscopy.
[146]
[147] From these three sets of results, it can be concluded that sucrose and lactose are the
preferred additives for producing MBL powders suitable for inhalation and capable of a stronger C4 activation in the complement system.
[148]
[149]
[150] Characterization of MBL powders with respect to variations in the sugar content
[151] When sugar additives are used to enhance the stability of spray-dried proteins, the
outcome varies according to the identity and concentration of the sugar. The present inventors determined for each sugar, the concentration range suitable for spray-drying. The goals of this experiment were determining the effects of sugar concentration on the C4 complement activation, particle size distribution, and particle shapes of MBL powders produced when either lactose or sucrose was added to the MBL solution. The MBL solutions used for spray-drying all contained 5 D/mL recombinant MBL, 150 mM NaCl, 10 mM CaCl2 , 250 ◘/mL casein. To this common composition, sucrose was

added to a final concentration of 0.5%, 1 %, 2% or 4%, respectively before spray-drying. In the case of lactose, it was 0.5%, 1 %, 2%, 4% or 8%.
[152]
[1531 Example 4-1 C4 activation in the complement system
[154] MBL powders prepared using different sugar additives were measured for their
complement activation in the presence of MBL-binding proteins and serine proteases according to the method of Example 1-3. They were compared with non-dried MBL solutions (control) of the same composition (Figures 18 to 21, figures 22 to 26). Figures 18 and 21 compare the spray-dried MBL powders produced under various concentrations of the additive lactose, with the MBL solution before drying for their C4 activation, wherein figure 18 represents 0.5% lactose; figure 19, 1% lactose; figure 20, 2% lactose; and figure 21, 4% lactose suspensions. Open circles denote the spray-dried MBL composition of the present invention and filled circles represent control experiments.
[155]
[156] As shown in figures 18 to 21, spray-dried MBL powders had similar levels of
activity to controls for the range of lactose concentrations from 0.5% to 4%; especially, the 1% lactose formulation (Figure 19) showed a relatively high level of activity. The 0.5% formulation exhibited a small difference compared with controls. From these results, it was concluded that the lactose concentration of the MBL solution is preferably between 1 and 2% for obtaining active MBL formulations by spray-drying.
[157]
[158] Figures 22 to 26 compare the spray-dried MBL powders produced under various
concentrations of the additive sucrose, with non-dried MBL solutions for their C4 activation, wherein figure 22 represents 0.5% sucrose; figure 23, 1% sucrose; figure 24, 2% sucrose; figure 25, 4% sucrose; and figure 26, 8% sucrose suspensions. Open circles denote the spray-dried MBL composition of the present invention and filled circles represent control experiments.
[159] As shown in figures 22 to 26, spray-dried MBL powders had similar levels of
activity to controls for the range of sucrose concentrations from 0.5% to 8%; especially, the 1% (Figure 23) and 2% (Figure 24) showed relatively high levels of activity. The 8% formulation, however, exhibited a small difference compared with controls. From these results, it was concluded that the sucrose concentration of the MBL solution is preferably between 1 and 2% for obtaining active MBL formulations by spray-drying.
[160]
[161] Example 4-2 Particle size distribution of MBL powders
[162] The particle size distribution of MBL powders (Figures 27 and 28, Tables 2 and 3)

spray-dried at various sugar concentrations were probed with a laser zeta potentiometer (ELS-8000, Otsuka Electronics, Japan). Figure 27 plots the particle sizes of the inventive, spray-dried MBL compositions at various lactose concentrations, wherein filled circles represent 0.5% lactose; open circles, 1.0% lactose; filled triangles, 2.0% lactose; and open triangles, 2.0% lactose formulations of spray-dried MBL powders.
[163]
[164] Table 2
Particle sizes of spray-dried MBL powders at various lactose concentrations
(Table Removed)
[165] In the table above, DV0.1 denotes the particle diameter at 10 % accumulated
volume level, whereas DV0.9 denotes the particle diameter at 90% accumulated volume level.
[166]
[167] As shown in figure 27 and table 2, the mean particle sizes of MBL powders ranged
from 3.22 to 8.32 D. There emerged a clear trend in which an increase in the concentration of lactose, a stabilizer additive, was accompanied by an increase in the particle size; a lactose concentration higher than 2% corresponded to a particle size larger than 5 D. It has been known that in order to achieve an effective delivery of spray-dried powders to respiratory tracts and lungs, the particle size should be smaller than 5 D. The above results show that incorporating the sugar additive lactose at a concentration not higher than 2% can produce MBL powders with particle sizes effective for inhalation.
[168]
[169] Figure 28 plots the particle sizes of the spray-dried MBL compositions produced
under different concentrations of sucrose additive, wherein filled circles represent 0.5% sucrose; open circles, 1.0% sucrose; filled triangles, 2.0% sucrose; open triangles, 4.0% sucrose; and filled inverted triangles, 8.0% sucrose formulations of

spray-dried MBL powders.
[170]
[1711 Table 3
Particle sizes of spray-dried MBL powders at various sucrose
concentrations
(Table Removed)
[172] In the table above, DVO. 1 denotes the particle diameter at 10 % accumulated
volume level, whereas DV0.9 denotes the particle diameter at 90% accumulated volume level.
[173]
[174] As shown in figure 28 and table 3, the mean particle sizes of MBL powders ranged
from 3.52 to 7.07 □. There was no clear correlation between the concentration of sucrose, a stabilizer additive, and the particle size; however, a sucrose concentration higher than 4% correlated with an increase in the number of particles larger than 5 0. It has been known that in order to achieve an effective delivery of spray-dried powders to respiratory tracts and lungs, the particle size should be smaller than 5 D. The above results show that incorporating the sugar additive sucrose at a concentration not higher than 4% can produce MBL powders with particle sizes effective for inhalation.
[175]
[ 176] Example 4-3 Shapes of MBL powders
[177] The shapes of MBL powders (Figures 29 to 32 and 33 to 37) were observed with a
scanning electron microscope (JSM-5400, JEOL, Tokyo, Japan).
[178] Figures 29 to 32 show the scanning electron microscope images of the spray-dried
MBL composition produced under various concentrations of lactose, wherein figure 29 represents 0.5% lactose; figure 30,1.0% sucrose; figure 31, 2.0% lactose; and figure 32,4.0% lactose formulations of spray-dried MBL powders.

[1791
[1801 Figures 33 to 37 show the electron microscope images of the spray-dried MBL
compositions produced under various concentrations of sucrose, wherein figure 33 represents 0.5% sucrose; figure 34, 1.0% sucrose; figure 35, 2.0% lactose; figure 36, 4.0% sucrose; and figure 37, 8.0% sucrose formulations of spray-dried MBL powders.
[1811
[1821 As shown in figures 29 to 32, and 33 to 37, the shapes of MBL powders approached
a more globular shape as more sugar was added. Also, the particle size increased along with sugar concentration, as confirmed by electron microscopy.
[183]
[184] From these three sets of results, it can be concluded that preferred concentrations of
sucrose and lactose additives are in the range of 1 to 2% for producing MBL powders with effective inhalation and a strong C4 activation in the complement system.
[185]
[186] Characterization of MBL powders with respect to variations in CaCl2 . an enhancer
additive
[187] The presence of CaCl is essential for the activation of the complement system by
MBL after its binding with glycoproteins on the microbial surface. In general, blood CaCl level is known to be between 5 and 10 mM. Since this corresponds to the level required for MBL activity, there is no need for additional administration of CaCl in vascular injections. Meanwhile, when delivering MBL such targets as respiratory tracts or lungs, MBL must be dissolved in bodily fluids of the corresponding mucosa. However, CaCl2 levels in these fluids are not well understood; thus, addition of CaCl2
can be contemplated in order for inhaled MBL powders to be dissolved in such fluids and attain sufficient levels of activity.
[188]
[189] The present inventors investigated the effects of CaCl on the stability of and
production processes for spray-dried MBL powders. The MBL solutions used for spray-drying all contained 5 mg/mL recombinant MBL, 150 mM NaCl, 2% sucrose. To this common composition, CaCl was added to a final concentration of 10, 25, 50, 75, 100,150 and 200 mM respectively, before spray-drying.
[190]
[191] When the concentration of added CaCl2 was higher than 100 mM, powders suitable
for inhalation were not obtained. Inhalable aerosols were obtainable in the concentration range of 10 to 50 mM CaCl2, thus, enabling production of MBL powders capable of activating complement in the presence of glycosylated, MBL-binding proteins and serine proteases. There were no significant differences, however, that were dependent on concentration between 10 and 50 mM. These results confirm that it

is preferable to add CaCl2 at a concentration lower than 50 mM for spray-drying MBL.
[192]
[1931
[194] Characterization of MBL powders produced via co-addition of a sugar and a
protein
[195] Various protein additives are often used for stable formulation of protein drugs. The
present inventors examined the complement activation of spray-dried MBL powders (Figures 38 to 41) of which casein, a protein stabilizer additive, was or was not added (control) during the production. The composition of additives used for spray-drying was 2% sucrose or lactose with or without 250 D/mL casein.
[196]
[197] Figures 38 to 41 compare the spray-dried MBL powder produced with a sugar
additive for their C4 activation in the presence or absence of casein with non-dried MBL solutions, wherein figure 38 represents 2% sucrose with casein; figure 39, 2% sucrose without casein; figure 40, 2% lactose with casein; and figure 41, 2% lactose without casein. Open circles denote the spray-dried MBL composition of the present invention and filled circles represent non-dried control experiments.
[198]
[199] As shown 14a to 14d, an MBL powder produced with both a sugar and casein is
better at maintaining C4 activation than a powder produced without casein. Casein was especially effective when the sugar additive was lactose.
[200]
[201 ] In order to find the relationship between the characteristics of MBL powders and
casein concentration, the present inventors also spray-dried a series of MBL solutions prepared at different casein concentrations. The MBL powders produced, in turn, were analyzed and compared for their complement activation, particle size distribution, and particle shapes according to the methods of Example 1. The MBL solutions used for spray-drying all contained 5 ◘/mL recombinant MBL, 150 mM NaCl, 10 mM CaCl2 0.8% sucrose. To this common composition, casein was added to a final concentration of 0, 50, 200, 500, 1000, and 2000 0/mL respectively, before spray-drying. MBL powders produced with added casein were shown to be better at maintaining their abilities for C4 activation than those produced without casein; no concentration-dependent differences were observed in their abilities. The particle sizes of spray-dried MBL powders were in the range of 2.1 to 3.1 , which is suitable for aerosol inhalation. This size distribution was neither dependent on the presence of casein nor its concentration. These results confirm that it is preferable to add low concentrations of casein when spray-drying MBL solutions.
[202]

[2031
[204] Using bio-degradable polymers as additives for the production of MBL powders
[2051 Biodegradable polymers are widely used in the pharmaceutical industry. The
present inventors have tested for whether a bio-degradable polymer would be compatible as an additive in spray-drying of MBL. Polyvinyl alcohol (PVA), a soluble bio-degradable polymer, was added at a final concentration of 0.05, 0.1, 0.2, 0.3 or 0.4% before drying to a recombinant MBL solution containing 2% sucrose and 250 ◘/mL casein. At PVA concentrations lower than 0.4%, C4 activation by spray-dried MBL powders were almost the same as that of non-dried MBL solutions; C4 activation was lower in powders compared to non-dried solutions when PVA was added to 0.4%. These results confirm that PVA, a bio-degradable polymer, is compatible as a spray-drying additive at concentrations below 0.2%.
[2061
[2071
[2081 Stability analysis of MBL powders during long-term storage
[2091 To test whether MBL's biological activity is maintained during long-term storage,
MBL powders were tested for stability during long-term storage using harsh temperature conditions. The powders were stored either at 70°C for 2 days or 60°C for 4 days, conditions known to correspond to one year-long storage; the powders were then compared for their C4 complement activation in the presence of MBL-binding glycoproteins and serine proteases (Figures 42 to 45). The MBL solutions prepared contained 5 ◘/mL recombinant MBL, 150 mM NaCl, 10 mM CaCl2, with additional 2%
lactose or 2% sucrose and 0.05% PVA. These solutions were spray-dried and tested for their stability.
[210]
[211] Figures 42 to 45 compare spray-dried MBL powders stored under harsh temperature
conditions with those stored at room temperature for their C4 activation, wherein the figures illustrate spray-dried MBL powders stored and containing additives as follows: figure 42, 2% lactose, 2 days at 70°C; figure 43, 2% sucrose and 0.05% PVA, 2 days at 70°C; figure 44, 2% lactose, 4 days at 60°C; and figure 45, 2% sucrose and 0.05% PVA, 4 days at 60°C.
[2121
[2131 As shown in figure 42 to 45, all MBL compositions tested were confirmed as
maintaining their C4-activating activities. At room temperature, stability was maintained 100% for one year; thus, the production method of this invention enables formulation of MBL powders without any loss of activity, and such powders remain active for extended periods of time.
[214]


[215] Experimental Example 2>
[216] Analysis of binding between MBL powders and microbes
[217] Since different microbes express different glycoproteins on their surfaces, MBL
exhibits variations in binding with microbes. The present inventors compared the binding affinities of spray-dried MBL powders with those of MBL solutions (Figures 46 and 47). The MBL solutions prepared contained 5 D/mL recombinant MBL, 150 mM NaCl, 10 mM CaCl with additional 2% lactose or 2% sucrose and 0.05% PVA.
[218]
[219] Immunoassay plates (MaxiSorp Immunoplate, Nunc, Denmark) were coated with
microbes at 1x10, 1x10, 1x10 or 1x10 cells per well. Either a spray-dried powder or non-dried (control) MBL was dissolved in water at 5 D/mL. 100 D of this solution was added to each well and the plates were incubated for 2 hours to allow binding. The reaction mixture was removed and the plates were washed 6 times with wash buffer. 100 D of anti-MBL antibody (Dobeel, Korea) diluted 1:10,000 in dilution buffer, was added to each well and the mixture was allowed for binding at room temperature for an hour. After removing the reaction mixture, the plates were washed 6 times with the wash buffer. 100 D of anti-mouse antibody-HRP conjugate (Kirkegaard & Perry Laboratories (KPL), USA), diluted 1:10,000 in the dilution buffer, was then added to each well and the reaction mixture was incubated for an 1 hour. After washing 6 times with the wash buffer, 100 0 of TMB substrate solution (KPL, USA) was added and the col-orimetric reaction was allowed to proceed for 30 minutes. The reaction was stopped by adding 50 ◘ of 3N H2SO4 . Absorbance at 450 nm (Figure 46 and 47) was measured
with an ELISA reader.
[220]
[221] Figures 46 is a plot showing the binding between non-dried, solution phase MBL
and various microbes. Figure 47 is a plot showing a graph comparing the spray-dried MBL powder produced in the presence of different additives with non-dried MBL solutions for their binding to microbes. In each plot, a denotes Staphylococcus aureus ATCC29213; b, S. aureus CCARM3197; c, S. aureus CCARM3114; d, S. epidermidis ATCC12228; e, S. epidermidis CCARM35048; f, S. pyogens ATCC8668; g, Hemophilus influenzae ATCC51907; h, Enterococcus faecalis ATCC51907; i, E. faecium CCARM5028; j, Klebsiella pneumoniae ATCC 10031; and k denotes Candida alibicans. In figure 34, black bars represent spray-dried MBL compositions with 2% lactose and gray bars represent spray-dried MBL compositions with 2% sucrose and 0.05% PVA.
[222]
[223] As shown in figures 46 and 47, the non-dried MBL solution exhibited strong
binding affinities for S. aureus ATCC29213, S. aureus CCARM3197, H. influenzae

ATCC51907; weak affinities for S. aureus CCARM3114 and S. pyogens ATCC8668; almost no affinity for S. epidermidis ATCC1228, S. epidermidis CCARM35048, E. faecalis ATCC51907, E. faecium CCARM5028 and K. pneumoniaeATCC10031. Such binding behaviors were similarly repeated by the two MBL powders with additives. Small variations in the binding are observed between spray-dried and non-dried MBL, and between MBL preparations with different additives, which are considered to originate from experimental errors.
[224]
[225] For an effective delivery of MBL to respiratory organs such as the lung and
respiratory tracts, or for treatment of external wounds, it is essential that MBL be formulated into powdered compositions. Such powdered compositions of MBL should have inhalable particle sizes in the form of aerosols. Such compositions must also maintain the structural features of MBL, namely the extent and distribution of the binding complex formation by MBL. Finally, such compositions should be able activate complement in the presence of MBL-binding glycoproteins and serine proteases.
[226]
[227]
Industrial Applicability
[228] The present inventors have completed an invention that meets the above criteria as
shown in the results described in the previous paragraphs. The inventors have confirmed that the spray-dried, powder composition of MBL of the present invention has physical and biological properties sensitive to the identities and concentrations of the additives used in its production. Based on these results, they have developed for the inventive composition, a spray-drying process capable of maintaining activity and a more effective administration. Since the inventive MBL powder composition produced by the above process is able to inhibit viral, bacterial and fungal infections and has been formulated to be suitable for inhalation, the composition is useful in treating and preventing respiratory infections and external wounds from these microbes. It should be noted that the MBL powder composition of the present invention can be mixed with excipients for uses as tablet formulations.
[229]
[230]











We claim:
1. A method for producing a powder to be inhaled for treating or preventing
microbial infectious disease of the respiratory organs, comprising the steps of:
(i) preparing an aqueous solution containing 0.0005 to 10% (w/v) of a collectin family member protein, 0.1 to 4% (w/v) of a sugar, 150mM of NaCI and 10 to 50mM of CaCI2;
(ii) spray-drying the solution of step (i) at 50 to 100°C,
wherein the collectin family member protein is selected from a group consisting of mannose-binding lectin (MBL), surfactant protein A (SP-A), surfactant protein D (SP-D), collectin-liver 1 (CL-L1), collectin-placenta 1 (CL-P1) and CL-43, wherein the sugar is 0.1 to 4% (w/v) of sucrose or 0.1 to 2% (w/v) of lactose,
wherein the size of the powder is 3 to 5 µm
2. The method as claimed in claim 1, wherein the solution of step (i) further' comprises pharmaceutically acceptable carriers or diluents.
3. The method as claimed in claim 2, the pharmaceutically acceptable carrier is a
biodegradable polymer.
4. The method as claimed in claim 3, wherein the biodegradable polymer is
polyvinyl alcohol.
5. The method according to claim 1, wherein the solution of step (i) further
comprises 0.005 to 0.2% (w/v) of casein.
6. The method according to any one of claims 1 to 5, wherein the collectin family member protein is mannose-binding lectin (MBL).

Documents:

4489-DELNP-2007-Abstract-(02-05-2011).pdf

4489-DELNP-2007-Abstract-(24-08-2010).pdf

4489-delnp-2007-abstract.pdf

4489-delnp-2007-assignment.pdf

4489-DELNP-2007-Claims-(02-05-2011).pdf

4489-DELNP-2007-Claims-(24-08-2010).pdf

4489-DELNP-2007-Claims-(29-04-2011).pdf

4489-delnp-2007-claims.pdf

4489-DELNP-2007-Correspodence Others-(09-01-2012).pdf

4489-DELNP-2007-Correspondence Others-(02-05-2011).pdf

4489-DELNP-2007-Correspondence Others-(29-04-2011).pdf

4489-DELNP-2007-Correspondence-Others-(24-08-2010).pdf

4489-delnp-2007-correspondence-others-1.pdf

4489-delnp-2007-correspondence-others.pdf

4489-DELNP-2007-Description (Complete)-(02-05-2011).pdf

4489-DELNP-2007-Description (Complete)-(24-08-2010).pdf

4489-delnp-2007-description (complete).pdf

4489-DELNP-2007-Drawings-(02-05-2011).pdf

4489-DELNP-2007-Drawings-(24-08-2010).pdf

4489-delnp-2007-drawings.pdf

4489-DELNP-2007-Form-1-(02-05-2011).pdf

4489-delnp-2007-form-1.pdf

4489-DELNP-2007-Form-13-(23-08-2010).pdf

4489-delnp-2007-form-18.pdf

4489-DELNP-2007-Form-2-(02-05-2011).pdf

4489-DELNP-2007-Form-2-(24-08-2010).pdf

4489-delnp-2007-form-2.pdf

4489-DELNP-2007-Form-27-(09-01-2012).pdf

4489-DELNP-2007-Form-3-(24-08-2010).pdf

4489-delnp-2007-form-3.pdf

4489-delnp-2007-form-5.pdf

4489-DELNP-2007-GPA-(24-08-2010).pdf

4489-delnp-2007-gpa.pdf

4489-delnp-2007-pct-210.pdf

4489-delnp-2007-pct-304.pdf

4489-delnp-2007-pct-306.pdf

4489-DELNP-2007-Petition 137-(24-08-2010).pdf

4489-DELNP-2007-Petition 138-(24-08-2010).pdf


Patent Number 248281
Indian Patent Application Number 4489/DELNP/2007
PG Journal Number 27/2011
Publication Date 08-Jul-2011
Grant Date 01-Jul-2011
Date of Filing 12-Jun-2007
Name of Patentee DOBEEL CO.,LTD
Applicant Address #406 BYUCKSANTECHNOPIA 434-6 SANGDAEWON-DONG JUNGWON -GU, SEONGNAM-SI, KYONGGI-DO 462-716 REPUBLIC OF KOREA
Inventors:
# Inventor's Name Inventor's Address
1 MOON HONG MO #103-1101 BYUCKSAN APT. 295-3 HOEDUCK-DONG KWANGJU-SI, KYONGGI-DO 464-120 REPUBLIC OF KOREA
2 YUM JUNG SUN # 115-502, CHUNGGU APT.HANSOL MAEL JUNGJA-DONG BUNDANG-GU, SEONGNAM-SI, KYONGGI-DO 463-914 REPUBLIC OF KOREA
3 LEE JOO YOUN #T-1202 PUNGRIM I-ONE, SEOHYUN-DONG. BUNDANG-GU, SEONGNAM-SI, KYONGGI-DO 463-862, REPUBLIC OF KOREA
4 MOON JE YOUNG # 102-1003 SAMIK APT., 152-3, SANGDAEWON-DONG, JUNGWON-GU, SEONGNAM-SI, KYONGGI-DO 462-120, REPUBLIC OF KOREA
5 KIM HYUNJUNG #10-204 SIYEONG APT. JAMSIL-4DONG SONGPA-GU, SEOUL 138-794 REPUBLIC OF KOREA
6 AHN BYUNG CHEOL #508 -207 KUNYOUNG VILA KKACHI MAEL 13, KUMI-DOING, BUNDANG-GU SEONGNAM SI KYONGGI-DO 463 -808 REPUBLIC OF KOREA
PCT International Classification Number A61K 9/14
PCT International Application Number PCT/KR2005/004682
PCT International Filing date 2005-12-30
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10-2004-0117100 2004-12-30 Republic of Korea