AU2015215955B2 - Technology for preparation of macromolecular microspheres - Google Patents

Abstract

Micro spheres are produced by contacting an aqueous solution of a protein or other macromolecule with an organic solvent and a counterion, and chilling the solution. The micro spheres are useful for preparing pharmaceuticals of defined dimensions.

Description

-1- 2015215955 21 Aug 2015

Technology for Preparation of Macromolecular Microspheres RELATED APPLICATIONS

Benefit of priority Is claimed to U.S. Provisional Application No. 60/762,002, filed 24 January 2006, entitled “Technology for Preparation of Macromolecular Microspheres." Where permitted, this application is incorporated by reference In its entirety.

This application also is related to U.S. Application Serial No.11/657,812 (Attorney Docket No. 21865-004001/6504, filed 24 January 2007). This application also Is related to U.S. Publication Nos. US20050004020 A1 and US20050112751 A1. Where permitted, each of these applications is incorporated by reference in its entirety.

BACKGROUND

The administration of proteins to animals, including humans, in nutritional supplements or as therapeutics has been known for some time. Proteins tor therapeutic or nutritional administration generally are available either as (1) concentrates or powders that are administered directly or are reconstituted in a liquid of choice prior to use; or (2) liquid formulations.

The preparation and delivery of therapeutic proteins of interest In powder or particle form Is an area of concentrated research and development activity in the pharmaceutical industry. For therapeutic efficacy, it is desirable to have a uniform formulation. For example, for pulmonary administration, the protein ideally Is prepared in the form of discrete microspheres, which are solid or semi-soiid particles having a diameter of between 0.5 and 5.0 microns. It also is desirable for the particles to have a protein content that is as high as possible and that maintains its activity for concentrated delivery and therapeutic efficacy.

Previous methods of producing protein microparticles or nanoparticles have involved complex steps, such as blending with organic polymers and/or forming a lattice array with polymers; spray drying, spray freeze-drying or supercritical fluid antisolvent techniques that use specialized and complex equipment; or lyophilization followed by pulverization or milling that often results in non-uniform particles that must further be sorted. Often previous - 2 - 2015215955 21 Oct 2015 methods of producing solid protein formulations involve processing steps, such as heating, that denature the protein and compromise Its activity, in addition, some methods do not pfcwide,fiigft fecdvefy'Trofn solution into the solid formulation.

Accordingly, there is a need for a method for producing protein and other :S macromoieGuiar microparticles that does not require complex or specialized equipment and that produces uniform-sized microparticles for delivery. There further is a need for a method of producing microparticles that contain high concentrations of the protein or macromDleculerelative to other components, that are stable and maintain their activity for long periods ef time when stored at ambient temperature and that do not confairiva 10 significant: amount of denatured protein. There also is a need for a method of producing microparticles df proteins and other macromolecuies wherein substantially all of the protein armapromolecule in the starting materia! is recovered in the microparticle formulation, with minimal loss. There also Is a need for microparticles of proteins or other macromolecules containing these properties for-administration, for example, as a. 15 therapeutic or nutritional supplement.

Any discussion of the prior art throughout the specification should in ho way be considered as an admission that such prior art is widely known orforms part of common ge neraI knowledge in the field.

It is an object of the present Invention to overcome or ameliorate at least one of 20 the disadvantages of the prior art., or to provide a useful alternative.

SUMMARY

According to a first aspect of the invention, there is provided a composition comprising microparticles, wherein the microparticles comprise: a protein comprising 8EQ ID NO: 17, histidine, trehalose, and magnesium sulfate. 25 Unless the context clearly requires otherwise; throughout the description and the claims, the words “comprise”, “comprising”, and. the like are to be construed in an inclusive sense as opposed to an;exclusive or exhaustive sense;1 that is to say, in the sense of ‘Including, but not limited to”.

Provided herein in preferred embodiments are: methods for producing protein 30 and other macrornolecuiar mioroparticies that do not require complex or specialized equipment and that produces; uniform-sized microparticles for delivery; methods for producing microparticles: that contain high concentrations: of protein or macromoiecute relative to other components,: that are stable and maintain their activity for long periods of time when stored at ambient temperature and that do hot contain a significant 35 amount of denatured protein. Alse provided are methods; for producing microparticles of -3- 2015215955 21 Oct 2015 proteins and other mscromolecules where substantially all of the protein of macromoieouie ip the starting materia! is recovered in the microparticle formulation, with minimal loss. Also provided are microparticles of proteins and other rnacramolecules containing these properties for administration, for example, as a therapeutic or 5 nutritional supplement,

The methods of making a protein-based composition, the protein-based compositions themseives, combinations and articles of manufacture provided below are characterised by a variety of component ingredients, steps of preparation, and biophysical, physical, biochemical and chemical parameters. As would be apparent to 10 one of skill in the art, the compositions and methods provided herein include any and all permutations and combinations of the ingredients, steps and/or parameters described below.

Provided herein in preferred embodiments are methods of making a protein-based composition. The method provided herein can be used: to make-compositions 15 from other macromoiecuies besides proteins, including DNA, RNA, PNAt lipids, Oligosaccharides and combinations thereof.

The methods provided herein can induge the steps of: a) adding: a counterion to a solution containing the protein in an aqueous solvent; 20 b) adding: an organic solvent to the solution; and e) gradually cooling the solution to a temperature beiow about 25 °C, whereby a composition containing microparticles comprising the protein is formed, wherein steps a), b) and 0): are performed simultaneously, sequentially, intermittently, or in any order.

In one embodiment, the steps are performed sequentialiy a), b) and then c). in 25 another embodiment, the method of making a protein-based composition includes performing steps a) and b) simultaneously or sequentialiy In any order, followed by step e),

The resulting microparticles can be obtained by precipitation, by phase separation or by colloid formation, in some aspects, the methods provided herein 30 further comprise separating the mieroparticlestfrom the; solution to remove components other than the microparticles. Thisseparation step can be performed following the above-mentioned step c). The separation can be effected by, for example, sedimentation, filtration and/or freeze-drying.

The methods provided herein include the addition of an organic solvent;to an 35 aqueous solvent containing the protein. In certain embodiments, the organic solvent is miscible or partially miscible with the aqueous solvent, in .. - 4" 2015215955 21 Aug 2015 further embodiments of the methods provided herein, the organic solvent is selected from among aliphatic alcohols, aromatic alcohols, chloroform, dimethyl chloride, polyhydric sugar alcohols, aromatic hydrocarbons, aldehydes, ketones, esters, ethers, dioxanes, alkanes, alkenes, conjugated 5 dienes, dlchloromethane, acetonitrile, ethyl acetate, polyols, polyimides, polyesters, polyaldehydes and mixtures thereof. For example, where the organic solvent is an aliphatic alcohol, the organic solvent can be isopropanol. The amount of organic solvent added can vary in the methods provided herein. For example, the amount of organic solvent added can be from about 10 0.1 % or 0.1 % to about 50% or 50% v/v. In other embodiments, the amount of organic solvent added is from about 1% or 1% to about 30% or 30% v/v, from about 5% or 5% to about 30% or 30% v/v, from about 10% or 10% to about 30% or 30% v/v or from about 15% or 15% to about 20% or 20% v/v.

The counterion used in the methods provided herein can be an anionic 15 compound, a cationic compound and/or a zwitterionic compound. For example, when the counterion is an anionic compound, the counterion can be glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesium sulfate, potassium sulfate or calcium sulfate. The concentration of organic solvent added to the solution can vary in the methods provided herein. For example, 20 the concentration of counterion added to the solution can be from about 0.1 mM or 0.1 mM to about 100 mM or 100 mM. In other embodiments, the concentration of counterion added to the solution is from about 0.2 mM or 0.2 mM to about 50 mM or 50 mM, from about 0.3 mM or 0.3 mM to about 30 mM or 30 mM, from about 0.5 mM or 0.5 mM to about 20 mM or 20 mM or from 25 about 1 mM or 1 mM to about 10 mM or 10 mM. In a particular embodiment, the concentration of counterion added to the solution is about 5 mM or 5 mM. in one aspect of the methods provided herein, the pH of the solution that contains the protein is at or below the pi of the protein, in some aspects, the pH of the solution is from about 4.0 or 4.0 to about 9.0 or 9.0. In other 30 aspects, the pH of the solution is from about 4.5 or 4.5 to about 8.0 or 8.0, from about 4.5 or 4.5 to about 6.5 or 6.5, or from about 4.5 or 4.5 to about 5.5 or 5.5. -5- 2015215955 21 Aug 2015

The protein that is used in the methods provided herein to make a protein-based composition can be selected from among slalidases, slalidase fusion proteins, proteases, protease inhibitors, cytokines, insulin, human growth hormone, calcitonin, recombinant human DNase, interferons and 5 parathyroid hormone. In one embodiment, where the protein is a protease inhibitor, the protein is human protease Inhibitors (Pi8). in another embodiment, the protein Is a slalidase fusion protein. In some aspects where the protein is a sialidase fusion protein, the sialidase fusion protein contains a catalytic domain of a sialidase and an anchoring domain, wherein the catalytic to domain of the sialidase is the only portion of the sialidase in the sialidase fusion protein. The sialidase can be, for example, an Actinomyces viscosus sialidase, a Clostridium periringens sialidase, an Arthrobacter ureafaciens sialidase, a Micromonospora viridifaciens sialidase, a human Neu2 sialidase or a human Neu4 sialidase. 15 In one aspect, where the sialidase is an Actinomyces viscosus sialidase, the amino acid sequence of the catalytic domain contains the sequence of amino acid residues beginning at any of the amino adds from amino add 270 to amino add 290 and ending at any of the amino adds from amino add 665 to amino acid 901 of the sequence of amino acids set forth in 20 SEQ ID NO:1. For example, in one embodiment, the sequence of toe sialidase catalytic domain contains the sequence of amino acid residues set forth in SEQ ID NO:2. In another embodiment, the sequence of the catalytic domain comprises the sequence of amino acid residues beginning at amino add 274 and ending at amino acid 681 of the sequence of amino acids set 25 forth in SEQ ID NO:1. in a different embodiment, toe sequence of toe catalytic domain comprises the sequence of amino acid residues beginning at amino acid 274 and ending at amino add 666 of the sequence of amino acids set forth in SEQ ID NO:1. In another embodiment, the sequence of toe catalytic domain comprises the sequence of amino acids beginning at amino 30 acid 290 and ending at amino acid 681 of toe sequence of amino acids set forth in SEQ ID NO:1. -6- 2015215955 21 Aug 2015 ln one aspect, where the protein that is used in the methods provided herein to make a protein-based composition is a sialidase fusion protein that contains an anchoring domain, the anchoring domain is a glyoosaminoglycan (GAG)-binding domain, in a further aspect, the GAG-binding domain is 5 selected from among the GAG-binding domain of human platelet factor 4, the GAG-binding domain of human interleukin 8, the GAG-binding domain of human antithrombin ill, the GAG-binding domain of human apoprotein E, the GAG-binding domain of human angio-associated migratory protein and the GAG-binding domain of human amphiregulin. In particular embodiments, the 10 amino acid sequence of the GAG-binding domain contains the sequence of amino acid residues set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

In some aspects where the protein that is used in the methods provided herein to make a protein-based composition is a sialidase fusion 15 protein, the amino acid sequence of the sialidase fusion protein contains the sequence of amino acid residues set forth in SEQ ID NO:9. the sequence of amino acid residues set forth in SEQ ID NO:10, the sequence of amino add residues set forth in SEQ JD NO:11,the sequence of amino acid residues set forth in SEQ ID NO;12, the sequence of amino acid residues set forth in SEQ 20 ID NO: 13, the sequence of amino acid residues set forth in SEQ ID NO:14, or the sequence of amino acid residues set forth in SEQ ID NO:17.

In one aspect, the amount of protein in the microparticles produced by the methods provided herein, relative to the total amount of protein in the solution of step a) is about 80% or 80% to greater than about 99% or 99%, In 25 another aspect, the resulting microparticle composition produced by the methods provided herein can further comprise acid-resistant coating agents, protease-resistant coating agents, enteric coating agents, bulking agents, excipients, inactive ingredients, stability enhancers, taste and/or odor modifiers or masking agents, vitamins, therapeutic agents, anti-oxidants, 30 Immuno-modulators, trans-membrane transport modifiers, anti-caking agents, . . chitosans orfiowabiiity enhancers. -7- 2015215955 21 Aug 2015

The solution and/or the resulting composition of the methods provided heroin can, In one aspect, further comprise an active agent. In some embodiments, the active agent is selected from among antidlabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium 5 antagonists, anesthetics, antimicrobials, antimalarials, antiparasltics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, aipha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diagnostics, diuretics, electrolytes, enzymes, 10 hypnotics, hormones, hypoglycemics, hyperglycemias, muscle contractants, muscle reiaxants, neoplasties, glycoproteins, nucieoproteins, lipoproteins, ophthalmics, psychic energizers, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, urinary tract drugs, vaccines, vaginal drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin 15 converting enzymes, polynucleotides, polypeptides and polysaccharides. In another embodiment, the active agent is a nutritional supplement.

The methods provided herein involve gradually cooling the solution to a temperature below about 25 °C. In one embodiment, the temperature is between about 4 °C to about -45 °C. In another embodiment, the temperature 20 is between about 2 °C to about-20 "C. In a further embodiment, the temperature is between about 2 °Cto about -15 aC, In another embodiment, the temperature is between about 0 aC or 0 °C to about -2 aC or -2 °C to from about -15 °C or -15 °C to about -20 aC or -20 °C. Hie gradual cooling can be performed at a variety of rates. For example, cooling can be effected at a rate 25 of from about 0.01 ac/min or 0.01 eG/min to about 20 °C/min or 20 aC/min. In other embodiments, the gradual cooling is at a rate of from about or at 0.05 *C/min or about or at 0.1 aC/min to about or at 10 °C/min or about or at 15 °C/min. about or at 0.2 °C/min to about or at 5 °C/min, or about or at 0.5 °C/min to about or at 2 °C/min. in a particular embodiment, foe gradual 30 cooling is performed at a rate of about or at 1 °C/min. in one aspect, the size of the microparticles produced by the methods provided herein is from about 0.001 pm or 0.001 pm to about 50 pm or 50 pm. -8- 2015215955 21 Aug 2015 ln other embodiments, the size of the microparticles is from about 0.3 pm or 0.3 pm to about 30 pm or 30 pm, from about 0.6 pm or 0.5 pm to about to pm or 10 pm, from about 0.5 pm or 0.5 pm to about 5.0 pm or 5.0 pm, from about 1.0 pm or 1.0 pm to about 5.0 pm or 5.0 pm or from about 1.0 pm to about 5 2.0,3.0, 4.0 or 5.0 pm.

In some aspects of the methods provided herein, the resulting protein-based composition has a shelf life of from about one week to about 1 month, from about 1 month to about six months, from about six months to about one year, from about 1 year to about 2 years, or from about 2 years to about 5 10 years at a temperature of about 55 eC, 50 "C, 45 °C, 44 eC. 42 aG, 40 °C, 39 °C, 38 eC, 37 "C or below. 54. In another aspect, the moisture content of the microparticles is adjusted whereby at least about 90% of the activity of the protein Is retained after storage for about six months to about 1 year at a temperature of about 25 “C. in another aspect, the moisture content of the 15 microparticles is adjusted whereby at (east about 90% of the microparticles are not aggregated after storage for about six months to about 1 year at a temperature of about 25 “C.

In a certain aspect of the methods provided herein, the protein is a fusion protein containing a sialidase catalytic domain and an anchoring 20 domain, wherein the sialidase catalytic domain is the only portion of the sialidase in the fusion protein, the organic solvent is added in an amount of about 5% or 5% to about 20% or 20% v/v, the counterion is added in an amount of about 1 mM or 1 mM'to about 5 mM or 5 mM, and the pH of the solution is adjusted to about 4.5 or 4.5 to about 5.5 or 5.5. In one embodiment 25 of this aspect, the sialidase catalytic domain is from Actinomyces viscosus and the anchoring domain is the GAG-binding domain from human amphiregulin. Further still, the pH is about 5.0 and/or the counterion is selected from among glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesium sulfate, potassium sulfate or calcium sulfate. In another 30 embodiment of this aspect, the organic solvent is isopropanoi. In one embodiment of this aspect, the resulting composition contains the microparticles containing the protein as the only active ingredient (/. e. consists -9- 2015215955 21 Aug 2015 essentially of). In another embodiment, the method includes separating the microparticles from the solution to remove components other than the microparticles, such as by sedimentation, filtration and/or freeze-drying, in some embodiments of tilts aspect, the moisture content of the microparticles 5 is from about 6% to about 12%, or from about 7% to about 10.5%.

Provided herein are compositions containing microparticles of a siatidase or a slalldase fusion protein. Where the protein is a sialidase fusion protein, the sialidase fusion protein can comprise a catalytic domain of a stalidase and an anchoring domain. In some aspects, the sialidase in the 10 composition is an Actinomyces viscosus sialidase, a Clostridium peifringens siatidase, an Arihrobacter ureafaciens sialidase, a Micromonospora viridifadens sialidase, a human NeuZ sialidase, or a human Neu4 sialidase.

In one aspect, where the sialidase of the composition is an Actinomyces viscosus sialidase, the amino add sequence of the catalytic 15 domain comprises the sequence of amino acids beginning at any of the amino acid residues from amino acid 270 to amino acid 290 and ending at any of the amino acid residues from amino acid 665 to amino acid 901 of the sequence of amino acids set forth in SEQ ID NO:1. For example, the sequence of the catalytic domain can comprise the sequence of amino acids beginning at 20 amino acid 274 and ending at amino acid 681 of the sequence of amino acids set forth in SEQ ID NO:1, the sequence of amino acids beginning at amino add 290 and ending at amino acid 666 of the sequence of amino acids set forth in SEQ ID NO:1 or the sequence of amino acids beginning at amino acid 290 and ending at amino acid 681 of the sequence of amino adds set forth in 25 SEQ ID NO:1. In another embodiment, the sequence of the sialidase catalytic domain comprises the sequence of amino acids set forth in SEQ ID NO:2.

In one aspect, where the composition comprises microparticles of a sialidase fusion protein, the anchoring domain of the sialidase fusion protein is 30 a glycosaminoglycan (GAG)-binding domain, tn a further aspect, the GAG-binding domain is selected from among the GAG-bindtng domain of human platelet factor 4, the GAG-binding domain of human interleukin 8, the GAG- -10- 2015215955 21 Aug 2015 binding domain of human antithrombin III, the GAG-blndlng domain of human apoprotein E, the GAG-binding domain of human angio-associated migratory protein and the GAG-binding domain of human amphiregulln. In particular embodiments, the amino acid sequence of the GAG-binding domain contains 5 the sequence of amino acid residues set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID ΝΟ:β.

The sialidase fusion proteins of the compositions provided herein can contain, for example, the sequence of amino add residues set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12f SEQ ID NO:13, SEQ 10 ID NO:14, or SEQ ID NO:17.

The amount of protein in the microparticles of the compositions provided herein can vary. For example, the amount of protein in the microparticles can be from about 60% to greater than about 99% w/w. In one embodiment the amount of protein in the microparticles is from about 65% to 15 about 90% w/w, In another embodiment, the amount of protein In the microparticles Is from about 70% to about 85%, 86%, 87%, 88%, 89% or 90% w/w. The amount of protein in the microparticles of the compositions provided herein also can be from about 90% to about 99% w/w.

The microparticles in the compositions provided herein can further 20 contain acid-resistant coating agents, protease-resistant coating agents, enteric coating agents, bulking agents, excipients, inactive ingredients, stability enhancers, taste and/or odor modifiers or masking agents, vitamins, therapeutic agents, anti-oxidants, immuno-modulators, trans-membrane transport modifiers, anti-caking agents, chitosans orfiowability enhancers. 25 In one aspect, the compositions provided herein can further contain an active agent. The active agent can be a nutritional supplement, antidiabetics, anticonvulsants, analgesics, antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, 30 alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diagnostics, diuretics, electrolytes, enzymes, -11- 2015215955 21 Aug 2015 hypnotics, hormones, hypoglyeemics, hyperglycemics, muscle contractants, muscle relaxants, neoplasties, glycoproteins, nucleoproteins, lipoproteins, ophthalmlcs, psychic energizers, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, urinary tract drugs, vaccines, vaginai 5 drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin converting enzymes, polynucleotides, polypeptides and polysaccharides.

The compositions provided herein can have a shelf-life of varying length. In one aspect, the shelf life is from about one week to about 1 month, from about 1 month to about six months, from about six months to about one 10 year, from about 1 year to about 2 years, or from about 2 years to about 5 years at a temperature of about 5S eC, 50 °C, 45 °C, 44 "C, 42 °C, 40 °C, 39 °C, 38 eC, 37 eC or below. In a certain aspect, the moisture content of the microparticles is adjusted whereby at least about 90% of the activity of the protein is retained after storage for about six months to about 1 year at a 15 temperature of about 25 °C.

The microparticles in foe compositions provided herein can further contain a counterion, such as, for example, an anion, a cation, or a zwitterion. In certain embodiments, foe counterion is selected from among glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesium sulfate, potassium 20 sulfate or calcium sulfate, The amount of counterion in a microparticle can be varied. For example, the amount of counterion in the microparticles can be from about 0.5% or 0.5% to about 5% or 5% w/w, from about 0.5% or 0.5% to about 2% or 2% w/w, or from about 1% or 1 % to about 2% or 2% w/w.

In some embodiments, the moisture content of the microparticles in foe . 25 compositions provided herein is from about 6% or 6% to about 12% or 12%, or from about 7% or 7% to about 10.5% or 10.5%.

The compositions provided herein can be formulated for a variety of modes of administration. For example, the compositions can be orally e.g. by ingestion, intravenously, intranasally, parenterally, subcutaneously.or 30 intramuscularly administered. The compositions also can be formulated for pulmonary or ophthalmic administration. In a certain aspect, foe composition provided herein is for inhalation. -12- 2015215955 21 Aug 2015

The compositions provided herein can be formulated as tablets, caplets, gels, vials, pre-filled syringes, inhalers, electrostatic devices and other devices for delivery. The delivery dosage of the compositions can be from between about 0.5 mg protein per dose to about 100 mg protein per 5 dose, or about 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg protein per dose. The frequency of administration of a dose, for example, for the treatment or prophylaxis of Influenza, can be from three or more times a day, to two times a day, to once a day, to two times a week, to once a week, to once every two 10 weeks or less frequent than once every two weeks. For prophylaxis, the administration generally can be of the order of about once every twoweeks or Jess frequent, such as once every three weeks oronde every four weeks or longer.

The size of the microparticles in the compositions provided herein can 15 vary. For example, the size of the microparticles can be from about 0.001 pm or 0.001 pm to about 50 pm or 50 pm. In certain embodiments, the size of the microparticles is from about 0.3 pm or 0.3 pm to about 30 pm or 30 pm, from about 0.5 pm or 0,5 pm to about 10 pm or 10 pm, from about 0.5 pm or 0.5 pm to about 5.0 pm or 5.0 pm, from about 1.0 pm or 1.0 pm to about 5.0 pm 20 or 5.0 pm or from about 1.0 pm to about 2.0,3.0,4.0 or 5.0 pm.

Also provided herein are articles of manufacture that contain a composition containing microparticles of a sialidase or a sialidase fusion protein, a packaging material for the composition and a label that indicates that the composition is for a therapeutic indication. In one embodiment, the 25 therapeutic indication Is influenza. The article of manufacture also can contain an inhafer for pulmonary administration of tire composition, in certain embodiments, the inhaler is a dry powder inhaler, a metered dose inhaler or an electrostatic delivery device. DETAILED DESCRIPTION 30 A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as Is commonly understood by one of skill in the art -13- 2015215955 21 Aug 2015 to which the inventions) belong. All patents, patent applications, published applications and publications, Genbank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event 5 that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a U RL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the 10 availability and public dissemination of such information.

As used herein, the term "macromolecule" Is used to mean a molecule composed of two or more monomeric subunits, or derivatives thereof, which are linked by a bond, or any macromolecule that can form tertiary structure. A macromoiecuie can be, for example, a polynucleotide, a nucleic acid 15 molecules Including DNA, RNA and peptide nucleic acid (PNA) a polypeptide, a protein, a carbohydrate, or a lipid, or derivatives or combinations thereof, for example, a nucleic acid molecule containing a peptide nucleic add portion or a glycoprotein, respectively. The methods, compositions, combinations, kits' and articles of manufacture provided herein, although described with 20 reference to proteins, can be adapted for use with other macromolecules as defined and provided herein.

The term “substantially" or "substantial” as used herein generally means at least about 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher relative to a reference 25 such as, for example, a nucleic acid or protein sequence or the original composition of an entity. Thus, a composition containing microparticles separated from “substantially" all other contaminants and/or ingredients including counterions, salts and solvents from the cocktail solution means that at least about 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 30 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amounts of contaminants and/or reagents have been removed from the cocktail solution in which the microparticles are formed. The term “substantially identical” or "substantially -14- 2015215955 21 Aug 2015 homologous" or similar varies with the context as understood by those skilled in the relevant art and generally means at least about 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 85%, 00%, 95%, 96%, 97%, 98%, 99% or higher identity. 5 The term "consists essentially of or “consisting essentially of as used herein refers to an entity from which substantially all other components/ingredients that are not associated with the entity or its properties have been removed or separated from the entity. Thus, a composition “consisting essentially of microparticles means that all other ingredients such 10 as contaminants and solvents have substantially been removed from the sotution/suspenslon containing the microparticles.

The term “microparticle" as used herein is interchangeable with “microsphere" and refers to particles In the size range (average length, width or diameter) of about or at 0.001 micron (pm) to about or at 500 microns that 15 contain a macromolecule and deliver an agent of interest, such as a drug or nutritional supplement, to a subject. The agent can be the macromolBcule, for example, a protein, nudelc acid, lipid or polysaccharide, or the macromolecule forming the microparticle can be a carrier for the active agent, such as a drug or a nutritional supplement. The microparticles also can 20 contain synthetic macromolecules induding polymers, such as polyethylene glycol (PEG), polylactic acid (PIA), polylactic-co-glycollc acid (PLGA), and natural polymers such as albumin, gelatin, chitosan and dextran. The "microparticles* as described herein can contain and can be made from a particular natural or synthetic macromolecule alone, or from more than one 25 type of the same natural or synthetic macromolecule (e.g., more than one type of protein), or from combinations of more than one different type of natural or synthetic macromolecule (e.g„ a protein and a nucleic acid or a protein and a synthetic polymer).

The term “mfcropartide" as used herein also generally refers to a 30 partide that is not a solid form of the entire solution from which it is produced, although frozen and/or dried particles of a solution containing macromolecules also are contemplated herein. Rather, the mfcropartide as used herein -15- 2015215955 21 Aug 2015 gen erally is an assembly of a fhacfion of the components of a solution, including salts, counterions, solvents and other Ingredients, that is formed by a process including, but notlfmited to, precipitation, sedimentation, phase separation and colloid formation. 5 The term “precipitation" as used herein refers to a process whereby a solute or solutes of interest in a solution, such as the components of a microparticle, no longer stay in solution and form a phase that is distinct from the solvent or solvents that were used to form the solution. Precipitation of a microparticle and controlling the size of the precipitated microparticle can be 10 accomplished by a variety of means including, but not limited to, adjusting temperature, ionic strength, pH, dielectric constant, counterion concentration, organic solvent concentration, the addition of polyelectratytes or polymers, surfactants, detergents, or a combination thereof.

The term "phase separation" as used herein refers to the 15 transformation of a single homogeneous phase, such as a solution, into two or more phases, such as a suspension of a solid particle in a solvent or solution.

The term "sedimentation1' as used herein refers to the motion of particles, such as microparticles, which are in a suspension in a liquid or which are formed in a solution in response to an external force such as 20 gravity, centrifhga I force o r electric force.

The term “solution" is used interchangeably with “cocktail solution” herein and refers to a homogeneous mixture of two or more ingredients in a single phase, solid, liquid, or gas, where the distinct ingredients only are recognizable at the molecular level. The solution can be a liquid in which one 25 or more solutes, such as salts, are dissolved in a solvent, such as water or alcohol, or dissolved in a mixture of miscible solvents, such as a mixture of water and ethyl alcohol. The solution also can be a frozen form of a liquid solution.

The term "miscible” as used herein refers to the ability of one or more 30 components, such as liquids, solids and gases, to mix together to form a single, homogeneous phase. Thus, two liquids are miscible if they can be mixed to form a single, homogenous liquid whose distinct components are -16- 2015215955 21 Aug 2015 recognized only at the molecular level. When components are "partially miscible,” it means that they can be mixed to form a single homogenous phase in a certain concentration'range, but not at other concentration ranges. As used herein, when a solvent is "partially miscible’’ with another solvent, it 5 means that it is miscible at a concentration of about or at 50%, 45%, 40%, 35%, 30%, 25%. 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or below volume/volume (v/v), when mixed with the other solvent.

As used herein, "immiscible” means that when two or more components, such as liquids, solids or gases are mixed, they form more than 10 one phase. For example, when an organic solvent Is Immiscible with an aqueous solvent (e.g., hexane and water), the organic solvent is visible as a distinct layer that does not mix with the layer of aqueous solvent

As used herein, the term "polypeptide,” means at least two amino acids, or amino acid derivatives, including mass modified amino acids and IS amino acid analogs, that are linked by a peptide bond, which can be a modified peptide bond. The terms "polypeptide," "peptide" and "protein" are used essentially synonymously herein, although the skilled artisan will recognize that peptides generally contain fewer than about fifty to about one hundred amino acid residues, and that proteins often are obtained from a 20 natural source and can contain, for example, post-translational modifications! A polypeptide or protein can be translated from a polynucleotide, which can include at least a portion of a coding sequence, or a portion of a nucleotide sequence that (s not naturally translated due, for example, to it being located in a reading frame other than a coding frame, or it being an 25 intron sequence, a 3' or 5’ untranslated sequence, a regulatory sequence such as a promoter, or the like. A polypeptide also can be chemically synthesized and can be modified by chemical or enzymatic methods following translation or chemical synthesis. A polypeptide can be post-translationally modified by phosphorylation (phosphopnoteins), glycosyiation (glycoproteins, 30 proteoglycans), and the like, which can be performed in a cell or In a reaction in vitro. 17- 2015215955 21 Aug 2015

As used herein, the term "fusion protein" refers to a protein that is a conjugate of domains obtained from more than one protein or polypeptide. A domain can be a polypeptide tag, such as a Hisetag. The conjugates can be prepared by linking the domains by chemical conjugation, recombinant DNA 5 technology, or combinations of recombinant expression and chemical conjugation. A variety of chemical linkers are known to those of skill in the art and include, but are not limited to, amino acid and peptide linkages, typically containing between one and about 60 amino acids, more generally between 10 about 10 and 30 amino adds, heferobifunctionai cleavable cross-linkers, including but are not limited to, N-succtnimidyl (4-iodoacetyl)-aminobenzoate, suifosuccinimidyl (4-iodoacetyl)-air)inobenzoate, 4-succinimidyl-oxycarbonyl-a- (2-pyridyldithio)toluene, suifosuccinimidyi-6- [a-methyl-a-{pyridyldithlol)-toluamidoj hexanoate, N-succinimidyl-3-(-2-pyridyIdithio) - propionate, 15 succinimidyl 6[3(-(-2-pyridy!dlthio)-propionamldo] hexanoate, suifosuccinimidyl 6[3(-(-2-pyridyIdithto^prapionamido] hexanoate, 3-(2-pyridyldithioj-propiony] hydrazide, Eilman's reagent, dichlorotriazinic add, and S-(2-thiopyridyl)-L-cysteine.

The term “sialidase fusion protein" as used herein refers to a fusion 20 protein in which one or more domains is a sialidase or a portion thereof that retains at least about 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or more of its catalytic activity. A sialidase fusion protein as used herein also can refer to a fusion protein that contains a protein or polypeptide that is substantially homologous to a 25 sialidase and possesses the enzymatic activity of a sialidase.

The term "catalytic domain" of a protein as used herein refers'to a protein or polypeptide in which the only portion of the sequence that is substantially homologous to a sialidase Is a sequence of amino acid residues that includes the domain responsible for the catalytic activity of the protein 30 (e.g,, residues 274-666 of SEQ ID NO: 1 are identified as the catalytic domain of Actinomyces v/scosus sialidase) or catalytically active fragments thereof. The catalytic domain or catalytically active fragment thereof retains at least -18- 2015215955 21 Aug 2015 about 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the catalytic activity of the protein.

As used herein, the term "flowability characteristic" refers to a property that renders the ability to “flow,” where “flow" is a property that can permit a 5 substance to be poured and to assume the shape of a container that it is poured Into, without hindrance due to, for example, aggregation. Fluids generally have the property of "flow,” which generally renders them deformable, /.e., they can change their shape. The term “fluid” as used herein encompasses colloids containing liquids. Including emulsions, aerosols and 10 gases. Liquids, aerosols and gases with suspensions of solid particles, such as microparticles, also are considered “fluid" as defined herein.

As used herein, an emulsion is defined as a colloid of two immiscible liquids, a first liquid and a second liquid, where the first liquid is dispersed in the second liquid. 15 As used herein, surfactants (or "surface-active agents") are chemical or naturally occurring entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the Interfacial tension between two or more phases in solution. The surfactant molecules generally are amphiphilic and contain hydrophilic head groups and hydrophobic tails. The 20 surfactant molecules can act as stabilizers and/or improve flowability characteristics of the microparticles provided herein.

As used herein, a combination refers to any association between two or among more items for a purpose. For example, a combination of microparticles and an inhaler can be used for pulmonary delivery of a 25 therapeutic agent.

As used herein, a composition refers to any mixture. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

As used herein, a kit refers to a combination in which components are 30 packaged optionally with instructions for use and/or reagents and apparatus for use with the combination. -19- 2015215955 21 Aug 2015

As used herein, the term “enzyme” means a protein that catalyzes a chemical reaction or biological process. Enzymes generally facilitate and/or speed up such reactions and processes. In addition, enzymes generally are specific for a particular reaction or process, converting a specific set of 5 reactants Into specific products.

As used herein, the term “colloid" refers to a dispersion of solid particles, such as microparticles, In a liquid, such as the solution in which the microparticles are formed. The term “colloidal stability” refers to a colloid in which the particles are not substantially aggregated. For example, a stable 10 colloid is one in which about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the solid particles, such as microparticles, have formed aggregates.

The term "agglomerates* refers to the association of one or more particles, such as microspheres, loosely held together by van derWaals 15 forces or surface tension or electrostatic or combinations thereof. In some instances, associations held by electrostatic forces can be defined as "Flocculates." For the purposes herein, “Agglomeratesalso encompass “Flocculates”. Agglomerates can generally readily be broken apart by shear forces within the air or liquid. The term “dispense” or “dispersivrty” refers to the 20 ability of the particles to “flow." i.e., the extent to which the movement is not impeded by the presence of, for example, aggregates.

The term "aggregates” refers to the association of one or more particles, such as microspheres, amorhous precipitates, crystal- or glass-like particles or combinations thereof. Aggregates generally are not easily broken 25 apart which inhibits their ability to disperse or form homogeneous suspensions or to form aerosols with desirable properties.

The term “non-denatured" as used herein is in reference to proteins and means a conformation of a protein, /.e., Its secondary structure, tertiary structure, quaternary structure or combinations thereof, which essentially is 30 unaltered from the protein in its naturally occurring state. The terms “non-denatured” and “native” are used interchangeably herein and mean a protein that retains all or at least about 50%, 60%, 70%, 80%, 85%, 90% 91%, 92%; -20- 2015215955 21 Aug 2015 93%, 94%, 95%, 95%, 97%, 98% or 99% of Its length and/or natural conformation. The terms “non-denatured" or "native” as used interchangeably herein include the natural state of a protein in a cell, such as it’s length and conformation including secondary, tertiary and quaternary structures. As 5 defined herein, the "'non-denatured" or “native" proteins including those in the compositions provided herein generally retain all or at least about 50%, 60%, 70%. 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the normal activity or function of the proteins in their natural state, e.g., as a nutrient to provide amino acid building blocks, an antioxidant, an enzyme, an 10 antibody, a regulator of gene expression, a scaffold, eta

As used herein, the terms “activity” dr “function" are interchangeable with "biological activity" and refer to the in vivo activities of a compound, such as a protein, vitamin, mineral or drug, or physiological responses that result upon in vivo administration of a compound, composition or other mixture. 15 Activity, thus, encompasses therapeutic effects and pharmaceutical activity of compounds, compositions and mixtures. Biological activities also can be observed in in vitro systems designed to test or use such activities.

As used herein, "functional activity” also is interchangeable with “activity," "biological activity” or "function" and refers to a polypeptide or 20 portion thereof that displays one or more activities associated with the native or non-denatured protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity (ability to bind to or compete with a polypeptide tor binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, and the ability to specifically bind to 25 a receptor or ligand for the polypeptide.

The term “denatured" as used herein refers to a protein that is altered from its native or non-denatured conformation, /.e., its secondary, tertiary or quaternary structure or combinations thereof. The altered conformation generally occurs by processing steps that include pasteurization, radiation, 30 heat, chemicals, enzyme action, exposure to acids or alkalis, and ion- exchange and any combinations thereof. Denaturation of a protein generally results in diminishing all or some, generally more than 50% and at least about -21- 2015215955 21 Aug 2015 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the original properties including activity and function of the protein in it® native or non-denatured state.

As used herein, the term "nutritional supplement” means a substance 5 or composition that provides nutrients, including vitamins, minerals, fatty acids, amino acids, carbohydrates, enzymes, proteins, biochemicals and their metabolites, herbs and plants, to a host, such as an animal, including a human being. Nutrients that are supplied to the host through nutritional supplements can include nutrients essential for survival, good health, curing 10 disease or preventing disease that are missing or deficient in a hosts diet, and nutrients that are believed to augment good health, prevent disease or cure disease but are not considered essential for survival or good health.

As used herein, "hydrophobic? refers to a substance that is not charged or charge-polarized, or Is not sufficiently charged or charge-polarized to bond 15 with water or other polar solvents. Hydrophobic ligands can associate with each other or with other non-polar molecules or solvents in the presence of water or a polar solvent, through hydrophobic interactions. A hydrophobic iigand generally also is more soluble in non-polar solvents than in polar solvents. Examples of non-polar solvents include alkanes such as hexane, 20 alkyl ethers such as diethyl ether, aromatic hydrocarbons such as benzene and alkyl halides such as methylene chloride and carbon tetrachloride, mono-, di- and triglycerides, fatty acids, such as oleic, linoleic, palmitic, stearic, conjugated forms thereof and their esters.

As used herein, the term "therapeutic agent" means an agent which, 25 upon administration to a host, including humans, effectively ameliorates or eliminates symptoms or manifestations of an inherited or acquired disease or that cures said disease.

As used herein, "shelf life" or “stability" refers to the time after preparation 30 of the microparticle composition that the composition retains at least about or 70%, 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the initial protein activity that is present in the composition and other -22- 2015215955 21 Aug 2015 general physical characteristics of microspheres such as size, shape, and aerodynamic particle size distribution. Thus, for example, a composition that is stable for or has a shelf life of 30 days at room temperature, defined herein as a range of between about 18 °Cto about 25°C, 26*C, 27°C or 28°C, would 5 have at least about 70%, 80%. 85%, 90% 91%, 92%, 93%, 94%. 95%, 96%. 97%, 98% or 99% of the initial amount of the activity of protein present in the composition at 30 days following storage at 18 "C to about 25 “C, 26 °C, 27 °C or 28 °C. The shelf life of the microparticle compositions provided herein generally is at least about 10 days at 55 DC, at least about 2-3 weeks at 42 °C, 10 and at least about eight months or greater at 25 °C, however, microparticle compositions of any length of shelf life at any temperature that are produced by the methods provided herein are contemplated herein.

As used herein, "a biologically active agent, "an active agent," "a biological agent," or "an agent,” is any substance which when introduced into 15 the body causes a desired biological response, such as altering body function at the cellular, tissue or organ level and/or altering cosmetic appearance, such as body weight and shape. Such substance can be any synthetic or natural element or compound, protein, ceil, or tissue including a pharmaceutical, drug, therapeutic, nutritional supplement, herb, hormone, ortho like, or any 20 combinations tiiereof. Hie terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, pnodmgs, active metabolites, isomers, fragments, analogs, and the like. When the terms "biologically active agent,” "biological agent” and "agent" 25 are used, or when a particular active agent is specifically identified, it is intended to include the active agent perse as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, active metabolites, isomers, fragments and analogs.

As used herein, a "subject" is defined as an animal, including a 30 mammal, typically a human.

As used herein, “therapeutically effective amount" refers to an amount of the active agent for a desired therapeutic, prophylactic, or other biological -23- 2015215955 21 Aug 2015 effect or response when a composition is administered to a subject in a single dosage form. The particular amount of active agent in a dosage will vary widely according to conditions such as the nature of the active agent, the nature of the condition being treated, the age and size of the subject, 5 As used herein, “pharmaceutically acceptable derivatives" of a compound include salts, esters, enol ethers, eno) esters, acids, bases, solvates, hydrates or prodrugs thereof. .Such derivatives can be readily prepared by those of skill in this art using known methods for such derivatlzation. The compounds produced can be administered to animals or 10 humans without substantial toxic effects and either are pharmaceutically active or are prodrugs. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkyiamines, ethylenedlamine, N-methylglucamine, 15 procaine, N-benzylphenethylamine. 1-para-chlorobenzyl-2-pynolidin-1’- ylmethyibenzimidazole, diethylamine and other alkylamines, piperazine and trls(hydroxymethyi)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not 20 limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic adds, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, 25 valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cydoalkyl and heterocyclyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric adds, phosphinic acids, sulfonic acids, sulfinic acids and boronic adds. 30 As used herein, "treatment" means any manner in which one or more of the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any -24- 2015215955 21 Aug 2015 pharmaceutlcal use of the compositions herein, such as use for treating influenza.

As used herein, “organic solvent” refers to a solvent that is an organic compound, which is any member of a large class of chemical compounds 5 whose molecules contain carbon and hydrogen. Such solvents can include, for example, compounds from the following classes: aliphatic or aromatic alcohols, polyols, aldehydes, alkanes, alkenes, alkynes, amides, amines, aromatics, azc compounds, carboxylic acids, esters, dioxanes, ethers, haloalkanes, imines, imides, ketones, nitriles, phenols and thiols. 10 As used herein, an “aqueous solvent” refers to water, or a mixture of solvents that contains at least about 50% or 50%, at least about 60% or 60%, at leaet about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amounts of water. The term “aqueous solvent” as used herein also refers to solutions containing water as a solvent, such as is buffers, salt solutions, solutions containing counterions, and other solutes that are soluble in water.

As used herein, the term “pi” or “isoelectric point' refers to the pH at which there is no net charge on a protein or polypeptide.

As used herein, the term "counterion" refers to a charged or charge-20 polarizable molecule that can Initiate formation of a microparticle from a macromolecuie, such as a protein, nucleic acid, lipid or oligosaccharide. For example, in the case of the DAS181 fusion protein (SEQ ID NO:17), sodium sulfate is a counterion because it can initiate the formation of microparticles in the methods provided herein, whereas glycine, sodium chloride or sodium 2S acetate generally are not suitable as counterions for DAS181. Whether a charged molecule is a counterion can be determined empirically based on parameters Including, but not limited to, the type of protein, the pH, the ionic strength, the type of organic solvent used, and the presence of salts and additional ingredients such as active agents. As provided and described 30 herein, counterions can be anionic or having a net negative charge or charge-polarizab'le gnoup(s), cationic or having a net positive charge or charge- -25- 2015215955 21 Aug 2015 polarizable group(s), or zwitterionic and possessing both negative and positive charged or charge-polarizable groups.

As used herein, the term "cooling" refers to a lowering of temperature to a desired temperature for obtaining microparticles or, once the 5 microparticles are obtained, further lowering the temperature to a desired temperature for obtaining dry preparations of the microparticles by volatilizing solvents (e.g„ for freeze-drying). The term "gradual cooling" or "gradually cooling" or "gradually cooled" as used herein means that the lowering of temperature to a desired temperature from ambient temperature (about or at 10 18 °C to about or at 30 “CJ for microparticle formation occurs at a rate or tor an amount of time that is suitable tor generating microparticles in a solution before the solution becomes frozen. Thus gradual cooling Is different from, for example, snap freezing, spray drying or spray freeze-drying, whereby the entire solution is converted to a solid form without the generation of distinct 15 microparticles.

The rate of gradual cooling is empirically determined based on the type of macromolecule, solvents, counterions and other Ingredients as well as the method of cooling (e.g., a heat exchanger, refrigerator or freezer or freeze-dryer) and can vary, for example, tor an amount of time tor microparticle 20 formation of between about or at 1 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h or 10 h to about or at 1.5 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h, 10 h or 15 h.

As used herein, the term "cooling" refers to a lowering of temperature 25 to a desired temperature for obtaining microparticles or, once the microparticles of desired dimensions are obtained, further lowering the temperature to a desired temperature for obtaining dry preparations of the microparticles by volatilizing solvents (e.g„ for freeze-drying). The term “gradual cooling” or "gradually cooling" or “gradually cooled" as used herein 30 means that the lowering of temperature to a desired temperature from ambient temperature at which the solution cocktail was formed (about or at -15 °C to about or at 50 °C, generally about or at 18 °C to about or at 30 °C) -26- 2015215955 21 Aug 2015 for microparticle formation occurs at a rate or for an amount of time that is suitable for generating microparticles of desired dimensions in a solution before the solution becomes frozen. Thus gradual cooling is different from, for example, snap freezing, spray drying or spray freeze-drying, whereby the 5 entire solution is converted to a solid form without the generation of distinct microparticles.

The rate of gradual cooling is empirically determined based on the type of macromolecule, solvents, counterions and other ingredients as well as the method of cooling (e.g., a heat exchanger, refrigerator or freezer or freeze-10 dryer) and ca n vary, for example, for an amount of time for microparticle formation of between about or at 0.1 sec, 0.2 sec, 0.6 sec, 1 sec, 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 1 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h or 10 h to about or at 1.5 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h, 10 h 15 or15h.

Microparticles of desired size also can be formed, for example, by rapidly chilling the cocktail (e.g. using a heat exchanger) and allowing the suspension of microparticles to be maintained for a certain period of time without significant temperature changes, then snap freezing the cocktail. 20 The temperature at which microparticles are formed also is empirically determined based on the type of macromolecule, solvents, counterions and other ingredients as well as the method and uniformity of cooling and can vary from about or at 15 °C, 10 “C, 8 °C, 5 °C, 3 °C, 2 °C, 1 °C, -2 °C, -5 °C, -7.5 eC, -10 °C, -15 “C, -20 eC, -25 eC, -30 °C, -35 °C, -40 °C or -45 °C. 25 As used herein, the term “spray drying" refers to a process wherein a solution containing a macromolecule, such as a protein, is transformed into a dry particulate form by atomizing into a hot drying medium, generally for a period of about a few milliseconds to 1-2 seconds to a few tens of seconds. The term "spray freeze-drying" as used herein refers to a process wherein a 30 solution containing a macromolecule, such as a protein, is atomized into a cryogenic medium, such as liquid nitrogen, to obtain frozen droplets of solution that can then be dried by lyophilization. The term “snap freezing” or -27- 2015215955 21 Aug 2015 “rapid freezing” or “quick freezing" as used interchangeably herein refers to freezing a solvent or solution, including solutions containing macromolecules, such as proteins, by immersing the container with good heat transfer properties (e.g. thin-wal! glass or metal test tube) holding the solvent or 5 solution in liquid nitrogen or pouring the solution directly into liquid nitrogen. “Snap freezing” and "rapid freezing" generally occur within a period of about a few milliseconds to 1 -2 seconds to a few tens of seconds.

The term “iyophilize" or "lyophilizatiorV1 as used herein Is synonymous with “freeze drying” and refers to a process wherein a solution, including an 10 emulsion, colloid or suspension, is frozen and the solvents are volatilized directly into the vapor state, leaving behind the solid components. B. Methods for Preparing Macromolecuiar Microparticle

Compositions

Provided herein are methods of making microspheres having a high 15 content of a macromolecule, such as a protein. The microspheres provided herein are prepared by controlled precipitation in the presence of a counterion and an organic solvent. The microspheres are suitable for preparing pharmaceutical compositions which can be delivered to a patient by delivery routes including pulmonary, parenteral and oral administration routes. The 20 method also can be performed in a batch or continuous mode, for Increased efficiency and production.

The microspheres obtained by the methods provided herein are useful as prophylactic, therapeutic or diagnostic agents for treating or diagnosing disease states in a subject in vivo or in vitro. The sizes of the microspheres 25 obtained by the methods provided herein can be controlled by adjusting parameters including type and concentration of organic solvent, protein or macromolecule concentration, ionic strength, counterion type and concentration, rate and time of cooling, to provide microspheres in a wide range of sizes, from 0.001 micron to 50 microns or greater, that can deliver 30 therapeutic agents via a desired route including pulmonary (e.g., 1 micron to 5 micron particles for delivery to foe throat, trachea and bronchi for treatment of influenza and other respiratory infections), subcutaneous, intramuscular, -28- 2015215955 21 Aug 2015

Intravenous and other routes (e.g„ using particles of tens of microns In size), render active components of inhalant medicines for human subjects.

The steps of the method provided herein include: combining a solution containing the macromolecule with a counterion and an organic solvent, and 5 gradually cooling the resulting solution to a temperature whereby microparticles are formed. In one embodiment, the steps can be described as follows: 1) To a solution containing a macromolecule, such as a protein, nucleic acid, oligosaccharide or lipid, adding a counterion and an organic 10 solvent at concentrations that do not cause precipitation of the macromotecule at ambient temperature; 2) Precipitation: cooling the macromolecule/solvent cocktail to initiate formation of microspheres; and 3) Dehydration: freezing of the suspension and removal of organic 15 solvent and water by sublimation (freeze-drying, e.g., at a temperature of about -20 °C to about -80 °C, or about -30 °C to about -80 °C, or about -40 °C to about -80 °C, or about -45 °C to about -80 °C, or about -45 °C to about -75 °C),

The above steps of the method can be performed sequentially, 20 intermittently or simultaneously in any order. In one embodiment, the counterion and the organic solvent are added simultaneously or sequentially in any order to the solution containing the macromolecule, followed by chilling. In other embodiments, the solution containing the macromolecule can be prechilled to a temperature suitable for microsphere formation, prior to adding the 25 counterion and organic solvent. For example, a prechilled aqueous solution of a macromolecule, such as a protein, can be combined with ammonium sulfate and acetonitriie to form microspheres.

The resulting suspension of microparticles can be converted Into a dry powder by further cooling to a temperature below freezing point and 30 subsequent removal of volatiles (water, organic solvent and where possible the counterion) by, for example, sublimation using a standard freeze dryer. -29- 2015215955 21 Aug 2015 ln one embodiment, the microspheres formed by contacting the macromotecule with a counterion and organic solvent and exposed to low temperature, are separated from the suspension by methods including sedimentation or filtration techniques. After separation from the original 5 precipitation mix, the microspheres can be washed and/ar combined with other materials that improve and/or modify characteristics of macromolecules and microspheres.

In another embodiment, the microspheres prepared by the methods provided herein do not have a direct therapeutic effect, but serve as micro-10 carriers for other therapeutic agent(s) or active agentfs) i nciuding nutritional supplements. Therapeutic agents can be added at the time of precipitation or can be added to the suspension of formed microspheres prior to lyophlllzation. Alternatively, therapeutic agents can be blended into dry powder consisting of microspheres. 15 Without being bound by any theory, in one aspect, the methods provided herein can permit the formation of microspheres by: (1) neutralization of charges on the surface of macro molecule by the counterion and (2) decreased solubility of the macromolacule caused by the combined effects of added organic solvent and gradual cooling. 20 By choosing a suitable pH in the range of about or at pH 2.0 to about or at pH 10.5 or greater, depending on the macromolecule, counterion, and organic solvent, in the presence of a suitable amount of the counterion, a substantial number of the charged groups, in some embodiment all charged groups, on the surface of the macromalecule can become neutralized. A 25 decrease in the polarity of the solution by adding a suitable organic solvent can then initiate the formation of microspheres by precipitation, phase separation, colloid formation, or other such method.

Alternatively, without being bound by any theory, in some embodiments, the observed phenomenon of the precipitation of microspheres 30 also can be explained by the kosmotropfc (structure forming) effect of counterions and organic solvents due to interactions with the water molecules of the aqueous solution containing the macromolecule at low temperatures. -30- 2015215955 21 Aug 2015

Regardless of the underlying mechanism, in the methods provided herein, the addition of relatively small amounts of organic solvent and counterion to an aqueous or other hydrophilic solution containing a macromolecule and cooling of the solution results in the production of compositions containing 5 microspheres of the macromolecule(s).

In one embodiment, gradual coding of the cocktail solution can be performed by passing the cocktail solution through a heat exchanger. The temperature of the heat exchanger and the flow rate of the cocktail through the heat exchanger can be adjusted so that the cocktail Is either pre-chilled 10 prior to formation of the microspheres, or is chilled to a temperature whereby microspheres are formed.

In another embodiment, the microspheres farmed by the methods provided herein are concentrated or separated from the suspension by methods such as sedimentation or filtration techniques. Upon formation of the 15 microspheres, their growth (size) can be controlled by adjusting the ionic strength, polarity, pH, or other parameters of the suspension. The separation □f microspheres from the liquid phase of the cocktail solution can be performed by centrifugation, filtration (hollow fiber, tangential flow, etc.), or other techniques. The resulting microspheres or concentrated suspensions 20 thereof can be lyophilized or air dried.

In some embodiments, the microspheres separated from the original precipitation mix or the dried microspheres can be reconstituted prior to administration as a therapeutic agent or a carrier, or can be suspended in solutions that contain agents that modify characteristics of the microspheres. 25 The modifying agents can include but are not limited to bulking agents, excipients, inactive ingredients, stability enhancers, taste and/or odor modifiers or masking agents, vitamins, therapeutic agents, antl-oxidants, immuno-modulators, trans-membrane transport modifiers, anti-caking agents, enteric coating agents, agents that confer acid resistance, such as against the 30 acids of the digestive system, agents that confer protease resistance, chitosans, polymers, and flowability enhancers. -31- 2015215955 21 Aug 2015

The formation and characteristics of the microspheres produced by the methods provided herein can empirically be determined by varying parameters, including: nature and concentration of the macromolecule, pH of the cocktail solution, nature and concentration of the counterion, nature and 5 concentration of the organic solvent, ionic strength and the cooling rate by which gradual cooling is effected. The steps of the methods provided herein render the method amenable to high-throughput screening, such as in a microplate format, for determining suitable combinations of macromolecule, organic solvent, counterion, pH, ionic strength and cooling ramp for the 10 generation of microspheres.

Macromolecules

Macromolecules that can be used to form microspheres according to the methods provided herein include a variety of therapeutic agents, diagnostic agents, nutritional agents and other active agents. Therapeutic 15 agents indude antibiotics, vaccines, hematopoietics, anti-infective agents, antiulcer agents, antiallergic agents, antipyretics, analgesics, antiinflammatory agents, antidementia agents, antiviral agents, antitumoral agents, antidepressants, psychotropic agents, cardiotonics, antlarrythmic agents, vasodilators, antihypertensive agents, antidiabetic ^gents, 20 anticoagulants, and cholesterol lowering agents. Other examples of suitable macromolecules include proteins, peptides, nucleic acids, carbohydrates, protein conjugates, viruses, virus particles, and mixtures thereof.

The macromolecules can be characterized by their ability to interact with the counterion and organic solvent, such as citrate (counterion) and 25 isopropanoi (solvent), to form Intact, discrete microspheres containing a high content of macromolecule. The content of the macromolecule in the microspheres can vary from about or at 30%, 40%, 45%, 50%, 55%, 60%, 65%. 70%. 75%, 80%, 85%, 90%, 91%, 92%. 93%, 94%, 95%. 96%, 97%, 98%, 99% or greater weight/weight (w/w) of the microspheres. in some 30 embodiments, the macromolecule content of mlcrasphere is substantially the same as the amount of macromolecule initially in solution, prior to forming the microspheres. -32- 2015215955 21 Aug 2015

The macromolecules used to prepare microspheres by the methods provided herein can include peptides, including polypeptides and proteins, carbohydrates, including polysaccharides and nucleic acids (DNA, RNAor PMA). In some embodiments, the macromolecules are proteins, including 5 therapeutic proteins such as DAS181 (the sialidase fusion protein having the sequence of amino add residues set forth in SEQ ID NO:17), aiphat-antitrypsin, PI8, eglin c, Ecotin, aprotlnin, recombinant human DNase, insulin, interferons, recombinant human DNAse (rhDNAse, useful, for example, in the treatment of cystic fibrosis as an inhalation therapeutic (Genentech); see also 10 Shak et al., Proc. Natl. Acad. Sci. USA, 87:9188-9192 (1990)), human serum albumin, human growth hormone, parathyroid hormone and calcitonin. In some embodiments, the protein Is DAS181, the counterion is sodium sulfate or sodium citrate, and the organic solvent is isopropanoi.

The methods provided herein can avoid the use of conditions, such as 15 heat, that can denature the protein and reduce its activity. The microspheres provided according to the methods provided herein therefore can be used to prepare vaccines or other therapeutic medications that require proteins or peptides to be present In their native conformation.

The concentration of the macromolecule in solution, used during 20 precipitation of the microspheres, can be between about or at 0.1 mg/m! to about or at 0.2. 05, 0.8,1,0,2.0, 5.0,10.0,12.0, 15.0, 20.0,25.0,30.0, 35.0, 40.0, 45.0, 50.0,60.0, 70.0, 80.0, 90.0,100, or 200 mg/ml. In some embodiments, the concentration Is between about or at 1 mg/ml and about or at 20 mg/ml. Depending on the characteristics of the macromolecule (pi, 25 hydrophobicity, solubility, stability, etc.) and other process parameters, the concentration of macromolecute can empirically be determined to achieve formation of microspheres of a desired size. In general, macromolecules with lower solubility in the solvent (generally, aqueous solvent) prior to adding counterion and organic solvent can be used at tower concentrations (0.1 - 5 30 mg/ml) to form microspheres according to the methods herein, while macromoiecules with higher solubility can be used at 1 - 20 mg/ml or higher. If the formation of amorphous aggregates or aggregated microspheres is -33- 2015215955 21 Aug 2015 observed, the concentration of the macromolecule generally should be decreased to reduce or prevent such aggregation.

Nature and concentration of counterion The counterion can be any compound capable of neutralizing one or 5 more oppositely charged groups on the macromolecule at the pH at which the method is performed. Depending on the characteristics of the macromolecule (pK, pi, nature and quantity of charged groups, distribution of charge groups on the surface, solubility and structural stability under different pH conditions), the pH can empirically be determined for microsphere formation. In general, If 10 precipitation is performed at a pH below the pK of the macromolecule, anionic counterions can be used. In general, if precipitation is performed at a pH above the pK of the macromolecule, cationic counterions can be used. The counterion can empirically be selected based on its suitability to initiate microsphere formation, in some embodiments, the counterion can have a 15 molecular weight of 60 Da or greater, or about 75 Da or greater.

The counterions can be anionic, cationic or zwitterionic. Anionic counterions can be inorganic (phosphate, sulphate, thiocyanate, thiosulfate, hypochlorate, nitrate, bromine, iodine, etc.) or organic compounds that carry charge-polarizable groups Including enol, hydroxy, -SH, carboxylic, 20 carboxymethyl, sulfopropyl, sulfonic, and phosphoric. Organic compounds carrying other anionic groups or having negative charge due to other molecular characteristics also can be used. Compounds that can be used as anionic counterions also include, but are not limited to, the following: oxaloaceiate, maiate, maleate, oxalate, piruvate, citrate, succinate, fumarate, 25 ketoglutarate, butanetricarboxylic acid, hydromuconic acid, cydobutanedicarboxytic acid, dimethyl maleate, deoxyribonucleic acid, polyglutamic acid, folic acid, lactic acid, ascorbic add, carminlc add, sorbic acid, malonic add, EDTA, MOPS, TES, MES, PIPES, pyrfcfine, tricine, glycine, glycylglycine, betaine, sulfuric add, thiosuifuric acid, phosphoric acid, 30 adenosine triphosphate, nitric acid, itaconic acid, pivalic acidi dimethylmalonic acid, and perchloric acid. In some embodiments, itaconic, pivalic, -34- 2015215955 21 Aug 2015 dimethylmalonfc, and succinic adds are used as counterions in the methods provided herein.

Cationic counterions can be inorganic (ammonium, phosphonium, sulfonium, cesium, rubidium, etc.) or organic compounds that cany groups 5 known as amine, amide, imlne, imide, guanidine, imidazole, dioxane, aniline. Organic compounds carrying other cationic groups or that have positive charge polarizability due to other molecular characteristics also can be used. Compounds that can be used as cationic counterions also indude, but are not limited to, the following: Tris, Bis-Tris, Bis-Tris propane, diaminopropane, 10 piperazine, piperadine, pentyiamine, diaminobutane, propylamine, trimethyiamine, triethyiamine, spermine, spermidine, putresdne, cadaverine, ethanolamine, diethanolamine, triethanolamine, imidazole, tetramethyiammonium, trimethylammonium, ammonium, cesium, rubidium, Imidazole, poiyethileneimfne, DEAE, TEAE, QAE IS Zwitteiionic counterions possessing any charged groups in any combination can also be used. Compounds that can be used as zwitterionic counterions include, but are not limited to, the following: HEPES, B1CINE, glycine, glycyiglycine, 6-aminohexanoic add, piperidlc add, natural and non-natural amino acids (e.g., histidine, glutamine, arginine, lysine). 20 The counterions can be used as acids (e.g. sulfuric acid) or bases (e.g. imidazole) or their salts (e.g. sodium sulfate or imidazole-HCI). Counterions that can be used In the methods provided herein include those listed by the National Formulary, United States Pharmacopeia, Japanese Pharmacopeia, or European Pharmacopeia, the clinical safety of which has been 25 demonstrated (citric add, malic add, amino acids, sulfate, etc.). In some embodiments, counterions used In the methods provided herein Include ones for which safety has been established or as falling Into the GRAS (generally regarded as safe) category. The counterions (or their salts) can be solid at room temperature (about 25 °G), or at toe intended temperature of use and 30 storage). Combinations of two or more counterions also can be used. Volatile and liquid counterions also can be used in the methods provided herein. -35- 2015215955 21 Aug 2015

The concentration of counterion generally is maintained between about 0.1 mM and about 0.2,0.5, 0.8, 1.0,2.0, 3.0, 5.0,7.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0,60.0,70.0,80.0,90.0 and 100,0 mM. in some embodiments, the concentration cf the counterion is between about or at 0.5 mM and about or at 5 20 mM. Depending on the characteristics of macromolecule (pi, hydrophobiclty, solubility, stability, etc.) and other process parameters, the concentration of the counterion can empirically be determined using, for example, a high-throughput format as provided herein. In general, the formation of oversized microspheres, amorphous aggregates or aggregated 10 mtcrospheres indicates that the concentration of counterion should be decreased, while failure to form microspheres (broken glass-like crystals or flakes) or formation of microspheres below the desired size indicates that the -concentration of counterion should be increased.

Nature and concentration of organic solvent 15 An organic solvent added to the cocktail in the methods provided herein generally can be water miscible and selected from among alcohols (methanol, ethanol, 1-propanol, isopropanol, butanol, tert-bulyl alcohol), chloroform, dimethyl chloride, polyhydric sugar alcohols (glycerin, erythriol, arabitpl, xylitol, sorbitol, mannitol), aromatic hydrocarbons, aldehydes, 20 ketones, esters, ethers (di-ethyi ether), alkanes (hexane, cyclohexane,' petroleum ether), alkenes, conjugated dienes, toluene, dichloromethane, acetonitrile, ethyl acetate, polyols, polyimids, polyesters, polyaldehydes, and mixtures thereof. In some embodiments, the organic solvent can be volatile. In other embodiments, when incorporation of the organic solvent into the 25 microspheres is desired, non-volatile organic solvents can be used that provide, for example, novel characteristics to the microspheres (e.g., sustained release or added mechanical strength). The concentration of the organic solvent generally can be maintained between about or at 0.1 %, to about or at 0.5%, 1 %, 2%, 5%, 10%, 15%. 20%, 25%, 30%, 40% or 50%, 30 votume/volume (v/v). In some embodiments, the concentration of the organic solvent Is between about or at 1 % to about or at 30%, v/v. Organic -36- 2015215955 21 Aug 2015 compounds that are partially miscible or completely immiscible with water also can be used.

Organic solvents that can be used in the methods provided herein Include alcohols and others listed as Class 3 and 2 solvents in International 5 Conference on Harmonisation (ICH) Harmonised Tripartite Guideline {Impurities: Guideline for Residual Solvents), safe handling of which has been established in pharmaceutical and food industries.

Depending on the characteristics of the macromolecule (hydrophobidty, solubility, stability, etc.) and other process parameters, the 10 choice and concentration of the organic solvent can be optimized, for example, using high-throughput screening on microtiter plates or similar chips or other device. In general, uncontrolled precipitation before the initiation of cooling, the formation of oversized microspheres, amorphous aggregates, aggregated microspheres or sticky aggregates indicates that the 15 concentration of organic solvent should be decreased, while failure to form microspheres (broken glass-like crystals or flakes) or formation of microspheres below the desired size indicates that the concentration of the organic solvent should be increased.

pH 20 in addition to initiating microsphere formation, the counterion also can serve as a buffer. Alternately, in some embodiments, a buffering compound can be used to obtain the desired pH. In some embodiments, the buffering compound is 60 Da or larger. Depending on the characteristics of the macromolecule (pi, hydrophobiGity, solubility and stability at a specific pH, 25 etc.) and other process parameters, the optimal pH can empirically be adjusted to achieve formation of microspheres of desired dimensions and preserve the activity of the macromolecule. In general, failure to form microspheres (broken gtass-like crystals or flakes) indicates that the protein may be too soluble under the conditions used. Formation of amorphous 30 aggregates can Indicate that precipitation is not well controlled and the protein may not be stable or soluble at the pH used. -37- 2015215955 21 Aug 2015

When file macromolecule is a protein, it has been observed that certain protein/counterion combinations can cause immediate and uncontrolled precipitation at certain pH values. The high-throughout screening methods provided herein can be used to empirically determine the appropriate 5 combination of protein, pH and counterion to form microspheres of desired dimensions. This is easily remedied by changing the pH of the cocktail, by using a different counterion or by decreasing concentration of the protein in cocktail. In general, for forming protein-based microspheres, a pH value that is below the pi of the protein provides optimal micro sphere formation 10

Ionic strength

The ionic strength of the cocktail solution can be modulated by the concentration of the counterion or by other salts such as chlorides or acetates. In some embodiments, no additional salt is required to produce 15 microspheres. In certain embodiments, the ionic strength can be adjusted to preserve the structural integrity and activity of the macromolecule. Examples of other applications where the presence of specific salts can be beneficial include formulations of parenteral and olher drugs, or foods where specific tonicity or buffering capacity may be required upon reconstitution of 20 microspheres.

Cooling ramp

The cocktail containing a macromolecule, a counterion and an organic solvent initially Is prepared, prior to cooling, at a temperature at which the macromolecule is soluble, generally about -15 °C to about 30 °C. In some 25 embodiments, the initial temperature, prior to cooling is at ambient temperature (16-25 °C). The microspheres are formed by a process such as precipitation, phase separation or colloid formation upon gradual cooling to a temperature below the temperature at which the macromolecule is dissolved and in solution. The rate at which cooling is performed can control the 30 formation and other characteristics such as size of the microspheres. In general, when the macromolecule is protein, Hash-freezing in liquid nitrogen does not generate microspheres -38- 2015215955 21 Aug 2015

The rate at which cooling and freezing of the cocktail (cooling ramp) is performed can determine the final size of the microspheres. In general, a faster cooling ramp yields smaller microspheres whereas a slower cooling ramp yields larger mictospheres. Without being bound by any theory, the S cooling rate can determine the rate of: (1) nucteation that produces initial smaller microspheres and (2) a fusion process in which the initial microspheres coalesce (aggregate) and anneal Into larger microspheres. Fusion of the smaller particles into larger ones is a time dependent process that can be determined, for example, by the duration for which liquid 10 suspension of microspheres exists prior to freezing. Due to the reversible nature of the bonds between certain macromolecules, such as some proteins, in the microsphere compositions provided herein, smaller microspheres annealing into larger particles can generate microspheres with smooth surfaces. Depending on the size of microparticles desired, the cooling rate 15 can be from about 0.01 "C/min or 0.01 "C/min to about 20 “C/min or 20 “C/min; from about or at 0.05 °C/mln or about or at 0.1 “C/min to about or at 10 °C/min or about or at 15 “C/min, from about or at 0.2 "C/min to about or at 5 “C/min, from about or at 0.5 “C/min to about or at 2 “C/min, or about or at 1 “C/min. In some embodiments, the cooling ramp can be between 0,1 “C per 20 minute and about 40 °C per minute. In other embodiments, a cooling ramp can be between about 0.5 °C per minute and 15 °C per minute.

Depending on the specific needs, in some embodiments it can be desirable to adapt the production process to the specific equipment, in some embodiments, a lyophilizer with temperature-controlled shelves can be used 25 for the cooling. If the microspheres produced are larger than desired, other parameters of the process including concentration of macromolecule, organic solvent, ccunterion, Ionic strength and/or pH can be modified to achieve the desired reduction in size of the microspheres.

For a faster cooling ramp (smaller particle size), the cocktail solution 30 can be passed through a heat exchanger, such as that used in a continuous mode, if the size of microspheres needs to be increased, increased concentrations of one of the cocktail ingredients (macromolecule, organic -39- 2015215955 21 Aug 2015 soivent, counterion) can provide the desired increase in the size of microspheres. in general, the cooling should be performed uniformly and at a steady rate to prevent the formation of aggregates and crystals. Depending on the 5 concentration of the organic solvent, the precipitation of the macromolecule into microspheres can occur in several ways. At higher concentrations of organic solvent (about 5% - 40%, dependent on the actual components used) the microspheres generally can form when the cocktail solution is still in liquid form. At lower concentrations of organic solvent (2 - 25%, dependent on the 10 actual components used) ice crystals can form first, following which the expelled macromolecules and organic solvent reach can reach a critical local concentration and precipitate. A further decrease of temperature in the nearbottom layer of the lyopbilizer tray can lead to complete solidification of foe liquid suspension and further expulsion of fiie organic solvent Into the top IS layer. An excess of organic solvent in the top layer can cause uncontrolled precipitation of the macromolecule and aggregation of microspheres. This effect usually can be alleviated by selecting appropriate ratios of the components—macromolecule, counterion, organic solvent, salts, etc. in the cocktail. In addition, maintaining a thin layer of cocktail in the lyophilizatlon 20 tray or mixing of the cocktail while befog chided can prevent formation of aggregates and crystals and yield uniform microspheres. For example if a relatively low concentration of Isopropanol (e.g. 2-6%) is used, and a thin layer of cocktail (10-20 mm) Is filled into the tray, and the tray Is placed on a pre-chilled shelf (-30 -75°C) uniform microspheres can be obtained. 25 The methods provided herein can lead to substantially all or all the protein or other macromolecule being incorporated from the solution into the microspheres

High-throughout screening of microparticle formation conditions and optimization of particle formation 30

Depending on the characteristics of the macromolecule, tha composition of the cocktail solution used to prepare the microspheres according to the methods provided herein can be optimized. The optimization -40- 2015215955 21 Aug 2015 can rapidly be performed in a medium or high throughput format using, for example microtiter p!ate(s) or chips where tens to hundreds to thousands to tens of thousands of cocktails can be screened simultaneously. In some embodiments, a number of pH values in conjunction with cationic, anionic or 5 zwitterionic counterions and organic solvents at various concentrations can be screened. For example, the screening can be performed using several identical microtiter plates, to each of which the macromolecule of interest is added at various concentrations. Each set of test conditions can be screened in duplicate. In some embodiments, microplates with flat-bottom wells can be 10 used with foe skirt of foe microtHer plate broken off to permit good heat transfer between the lyophilizer sheif and foe bottoms of foe wells. The micropiafes can be placed on the shelves of foe lyophilizer and cooled to form mlcrospheres and to subsequently solidity the suspensions. Upon freezing of foe contents of foe wells, a vacuum can applied. At the end of lyophilfzatlon, 15 one of foe duplicate plates can be reconstituted with water or a buffer of choice to observe if certain conditions rendered the macromolecule Insoluble or reduced its activity. Conditions that resulted in material that can readily be resolubilized or provide mlcrospheres with desirable characteristics can be subjected to further analysis by spectroscopic, chromatographic, enzymatic or 20 other assays to confirm that native structure and activity are preserved. LyophllEzed material in a duplicate plate can be used for microscopy to determine whether microspheres are formed. Conditions that produced mlcrospheres can further be modified and fine-tuned to produce microspheres of desirable size and characteristics. 25 Kits for performing high-throughput screens can be provided and can contain alf foe ingredients used In the methods provided herein Including one or more of a macromolecule, such as a protein, buffers, pre-dispensed cocktail of known composition (organic solvent, counterion) and/or salts. Kits can contain 3,4,5,10,15,20,30,40,50,100 or more (typically 96 or more) 30 buffers with predetermined pH, counterion, ionic strength and organic solvent in each microtiter plate. The microtiter plate supplied with the kit can be -41- 2015215955 21 Aug 2015 modified so that bottoms of wells am in direct contact with the shelf of lyophilizer. C. Macromolecular Microparticle Compositions

The macromolecules contained in the microparticle compositions 5 obtained by the methods provided herein are substantially structurally and chemically unchanged by the methods. For example, when the macromolecule is Green Fluorescent Protein or Red Fluorescent Protein, their fluorescence and native conformation and activity of the proteins am retained in the microparticles. The dry microspheres, obtained by volatilizing 10 substantially all of the solvents and/or moisture except for the solvent and other components associated with the microspheres, can be stored and their activity can substantially be recovered upon reconstitution. The relatively low moisture content of the microparticles provided herein, for example, between about or at 0.1% to about or at 0.2%, 0.3%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 15 5.0%, 5.5%, 6.0%, 6.5%. 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 14%, 15%, 16%, 17%, 18% 19%, or 20%, can provide improved stability. The microsphems obtained by the methods provided herein also are homogeneous in size and shape, and can be obtained raproducibly with the desired characteristics. Other techniques 20 traditionally used for preparation of dry formulations (salt precipitation, alcohol or acetone precipitation, lyophilization, e.g.) can result in complete or partial denaturation of the macromolecules, such as proteins. In addition, the microspheres prepared by the methods provided herein avoid foe need for complex or specialized spray drying, spray freeze-drying, supercritical fluid 25 anti-solvent based processes or milling processes (See, for example, Laube BL. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination. Respir Care 2005; 50(9):1161-1176; Taylor G, Gumbleton M. Aerosols for Macromolecule Delivery: Design Challenges and Solutions. American Journal of Drug Delivery 2004; 2(3):143-155; Smyth HDC, Hickey 30 AJ. Carriers In Drug Powder Delivery. Implications for Inhalation System Design. American Journal of Dreg Delivery 2005; 3(2):117-132: Cryan SA. Carrier-based strategies for targeting protein and peptide drugs to the lungs. -42- 2015215955 21 Aug 2015 AAPS J 2005; 7(1):E20-E41; UCalsi C, Maniaci MJ, Christensen Tf Phillips E. Ward GH, Wftham C. A powder formulation of measles vaccine for aerosol delivery. Vaccine 2001; 19(17-19):2629-2636: Maa YF, Prestrelski SJ. Biopharmaceutical powders: particle formation and formulation 5 considerations. Curr Pharm Biotechnol 2000; 1(3):283-302; Maa YF, Nguyen PA, Hsu SW. Spray-drying of air-liquid interface sensitive recombinant human growth hormone. J Pharm Sci 1998; 87(2):152-159; Vanbever R, Mintzes JD, Wang J et al. Formulation and physical characterization of large porous particles for inhalation. Pharm Res 1999; 16(11):1735-1742: Bot Al, Tarara 10 TE, Smith DJ, Sot SR, Woods CM, Weers JG. Novel lipid-based hollow-porous microparticles as a platform for immunoglobulin delivery to the respiratory tract. Pharm Res 2000; 17(3):275-283: Maa YF, Nguyen PA, Sweeney T, Shire SJ, Hsu CC. Protein inhalation powders: spray drying vs spray freeze drying. Pharm Res 1999; 16(2):249-254: Sellers SP, Clark GS, 15 Sievers RE, Carpenter JF. Dry powders of stable protein formulations from aqueous solutions prepared using supercritical CO(2)-asslsted aerosoiization. J Pharm Sci 2001; 90(6):785-797; Garcla-Contreras L, Morcol T, Bell SJ, Hickey AJ. Evaluation of novel particles as pulmonary delivery systems for insulin in rats. AAPS PharmSci 2003; 5(2):E9; Pfutzner A, Flacke F, Pohi R et 20 al. Pilot study with technosphere/PTH(1-34)-a new approach for effective pulmonary delivery of parathyroid hormone (1-34). Horm Metab Res 2003; 35(5):319-323; Alcock R, Blair JA. O’Mahony DJ, Raoof A, Quirk AV. Modifying the release of leuprolide from spray dried OED microparticles. J Control Release 2002; 82(2-3) :429-440; Grenha A, Seijo B, Remunan-Lopez 25 C. Microencapsulated chitosan nanoparticles for lung protein delivery. Eur J Pharm Sci 2005; 25(4-5):427-437; Edwards DA, Hanes J, Caponetti G et al. Large porous particles for pulmonary drug delivery. Science 1997; 276(5320):1868-1871; McKenna BJ, BJrkedal H, Bartl MH, Deming TJ, Stucky GD. Micrometer-sized spherical assemblies of polypeptides and smalt 30 molecules by add-base chemistry. Angew Chem Int Ed Engl 2004; 43(42):5652-5655; Oh M, Mirkin CA. Chemically tailorable colloidal particles from infinite coordination polymers. Nature 2005; 438(7068):651-654; U.S. -43- 2015215955 21 Aug 2015

Pat. No. 5,981,719; U.S. Pat. No. 5,849,884 and U.S. Pat No. 6,090,925; U.S. Patent application No, 20050234114; U.S. Pat. No. 6,051,256).

The microparticles obtained by the methods provided herein can be of any shape and can have sizes (mean width or diameters) in the range of from 5 about or at 0.001 micron to about or at 0.002,0.005,0.01,0.02,0.03,0.05, 0.1,0.2, 0.3,0.5,1.0,2.0, 2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5, 7.0,7.5, 8.0. 8.5, 9.0, 9.5,10.0,15.0, 20.0,25.0, 30.0,35.0,40.0,45.0, or 50.0 or greater microns. For pulmonary administration to the alveoii, the size can be from about 0.1 micron or less to about 0.5 micron. For pulmonary 10 administration to the throat, trachea and bronchi, the size can be from about or at 0.5 microns to about or at 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5,0,5.5, 6.0, or 6.5 microns, or in soma embodiments from about or at 1.0 micron to about or at 2.0 microns. In some embodiments, die microparticles are substantially spherical in shape. 15 The macromolecules thatcanbeusedto form microparticles according to the methods provided heroin can include therapeutic and diagnostic agents, processed foods, dietary supplements and polymers. In some embodiments, cross-linking agents, salts, or other compounds can be included in foe formulation cocktail to modiiy solubility of the microspheres 20 and/or enhance their mechanical strength. In some embodiments, microspheres that are insoluble in most aqueous or organic solvents can be used to manufacture particles such as chromatographic resins and dispersible abrasives, in other embodiments, microspheres with partial solubility in solvents such as pharmaceutical vehicles for delivery can be useful in die 25 manufacture of sustained release active agent or therapeutic formulations.

In some embodiments, the microparticles provided herein can be used in combination with an inhaler device to deliver a therapeutic dose of macromolecular microspheres to the respiratory airways and lungs of a subject. For example, when the macromofecuie is the DAS 181 protein 30 (sequence set forth in SEQ ID NO: 17), microspheres of about 0.5 micron to about 8 microns, or about 1 micron to about 5 micron can be obtained by the methods provided herein, using sodium sulfate as the counterion and -44» 2015215955 21 Aug 2015 isopropanol as the organic solvent For DAS181 microspheres, which are administered to prevent or treat viral infections that initiate In the respiratory tract, such as influenza, it can be desirable to deposit the microspheres in the throat, trachea or bronchi. The DAS181 fusion protein formulated as 5 microspheres can act by degrading toe receptor sialic acids in the throatftrachea/bronchl, thus preventing viral binding and infection at these sites. For optimal delivery of the DAS181 microspheres to sites where respiratory viral infection can be initiated, ;.e.t in toa throat, trachea or bronchi, toe microspheres must not be (a) so big that they are trapped at toe front end 10 in the mouth (/.e., microspheres are too big, about 8 microns or greater); or (b) so small that they are absorbed deep in the lungs and absorbed systemically into the.biood stream through the alveoli where they are not active and/or can be toxic {/.e., 0.5 micron or smaller). For delivery of the OAS181 microspheres to the throat, trachea and bronchi, a size range of 15 about 1 micron to about 5.5 - 6 microns generally can be suitable.

The inhaler can be used to treat any medical condition in which the protein or other macromotecule can be administered by inhalation therapy. Typical inhaier devices can include dry powder Inhalers, metered dose inhalers, and electrostatic delivery devices. Typical applications of the delivery 20 apparatus include the deep lung delivery of insulin and other therapeutic proteins. in some embodiments, toe microspheres obtained by the methods provided herein also can be delivered by oral ingestion, intranasaliy, intravenously, intramuscularly, subcutaneously, and by other delivery 25 methods suitable tor the delivery of therapeutic molecules. The microsphere formulations for pulmonary delivery generally can be in a size range of about 0.5 micron to about 5»6 microns, while those designed for other types of delivery, such as subcutaneous delivery, parenteral delivery or intramuscular delivery can be in a range of from about or at 10 micron to about or at 30,4Q 30 or 50 microns.

In some embodiments, the mtcrospheres provided herein have no direct therapeutic effect but can serve as micro-earners for other therapeutic -45- 2015215955 21 Aug 2015 agent(s). Examples of macromolecules useful for preparation of such microspheres include but are not limited to polysaccharides, glycans, proteins, peptides, polymers or combinations thereof. Therapeutic agents or other active agents can be added at the time of microsphere formation or added to 5 the suspension of formed microspheres. Alternatively, therapeutic agents can be blended with the dry microsphere compositions by mixing, tumbling or other techniques practiced in pharmaceutical and food industries.

In some embodiments, cross-linking agents, lipophilic substances, salts such as those with poor solubility in aqueous solvents, or combinations 10 thereof or other compounds can be Included In the formulation cocktail solution to modify the solubility of the microspheres and/or enhance their mechanical strength. Slow dissolution of the microspheres can be useful in sustained release of therapeutics delivered by oral ingestion, inhalation, intranasally, intravenously, intramuscularly, subcutaneously, and by other 15 delivery methods suitable for the delivery of therapeutic molecules. In some embodiments, the microspheres can be delivered by oral ingestion in a form of a pill or capsule with an enteric coating, endocytosed from the duodenum, and the macromolecule released into the blood stream or other site of action.

In some embodiments, the microspheres can be rendered insoluble by 20 partial denaturation of the macromolecule, which upon delivery becomes renatured and biosvailable.

In other embodiments, the microspheres are substantially spherical in shape, and can have mean diameters within the range of from about 0.1 microns to 30.0 microns. In yet other embodiments, the mean diameter of the 25 microspheres can be within the range of from about 0.5 microns to 5.0 microns, or from about 1.0 microns to 2.0 microns. in yet another aspect, provided herein are devices and methods for delivering the microspheres to a subject, such as an animal or human patient In need of medical treatment Suitable delivery routes can include parenteral, 30 such as i.m., i.v. and s.c.. and non-parenteral, such as oral, buccal, intrathecal, nasal, pulmonary, transderma!, transmucosal, and the like delivery routes. Delivery devices can include syringes, both needleless and needle -46- 2015215955 21 Aug 2015 oontaining, and inhalers.

The delivery devices can contain a single dose of the microspheres for treating a condition that is treatable by rapid or sustained release of the macromolecuie In vivo. The number of microspheres present in the single 5 dose is dependent on the type and activity of the macromolecule. The single dose can be selected to achieve sustained release over a period of time that has been optimized for treating the particular medical condition. For example, when the macromolecule is the DAS181 fusion protein (SEQ ID NO:17), the delivery dosage of microsphere compositions containing DAS181 can be from 10 between about 0.5 mg protein per dose to about 100 mg protein per dose, or about 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg. 40 mg, 45 mg, 50 mg, 55 mg or 60 mg protein per dose.

The macromolecuie component of the microsphere can be any molecule capable of forming microspheres according to the methods provided 15 herein. In some embodiments 1he macromolecuie is a protein, including enzymes and recombinant proteins, peptides, carbohydrates, polysaccharides, carbohydrate- or polysaccharide-protein conjugates, nucleic acids, virus, virus particles, conjugates of small molecules (such as a hapten) and proteins, or mixtures thereof. An organic or inorganic natural or synthetic 20 pharmaceutical compound or drug can be incorporated into the microspheres by attaching the drug to a macromolecuie, such as a protein, and then forming the microspheres from the macromoiecule-drug complex or conjugate. It will be understood by those skilled in the art that a compound incapable of having a tertiary and quaternary structure can ba formed Into a microsphere by 25 Incorporation or coupling of the compound into a carrier molecule that has a tertiary and quaternary structure. It will further be understood by those of skill in the art that the macromolecuie can be a portion of a molecule such as, for example, a peptide, a single-stranded segment of a double-stranded nucleic acid molecule, or a virus particle, or other macromolecuie having a tertiary 30 and quaternary structure.

The term "macromolecuie" also can include a plurality of macromolecules and includes combinations of different macromolecules such -47- 2015215955 21 Aug 2015 as a combination of a pharmaceutical compound and an affinity molecule for targeting the pharmaceutical compound to a tissue, organ or tumor requiring treatment An affinity molecule can be, for example, a ligand or a receptor. Examples of ligands can include viruses, bacteria, polysaccharides, or toxins 5 that can act as antigens to generate an immune response when administered to an animal and cause the production of antibodies.

In some embodiments, the macromolecule is a therapeutic protein including, but not limited to, a sialidase, a sialidase fusion protein, a fusion protein containing a sialidase catalytic domain fused to a GAG-binding 10 domain, a protease, a protease inhibitor, insulin, interferons, human growth hormone, calcitonin, rhDNase or parathyroid hormone, and the protein content of the microspheres can be from about or at 50% to about or at 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater. For pulmonary administration, the microspheres can have an average size in the 15 range of from about or at 0.5 microns to about or at 5.0 microns, and in some embodiments, between about or at 1 micron and about or at 2 microns.

Other proteins that can be used to form microspheres by the methods provided herein can include, but are not limited to, therapeutic proteins including DAS181 (DAS181; SEQ ID NO:17), a1-antitrypsin, Ecotin, eglin c, 20 serpin, Pulmozyme (rhDNase), betaxolol™., diclofenac™,, doxorubicin, acetyl cysteine, rifampin™., leuprolide acetate, luteinizing honnone releasing hormone (LHRH), (D-Tryp6)-LHRH. nafarelin acetate, insulin, sodium insulin, zinc insulin, protamine, lysozyme, alpha-iactalbumin, basic fibroblast growth factor (bFGF), beta-Iactoglobulin, Trypsin, calcitonin, parathyroid hormone, 25 carbonic anhydrase, ovalbumin, bovine serum albumin (BSA), human serum albumin (HSA), phosphorylase b, alkaline phosphatase, beta-galactosidase, IgG, fibrinogen, poly-L-lysine, IgM, DNA, desmopressin acetate, growth honnone releasing factor (GHRF), somatostatin, antide, Factor VIII, G-CSF/GM-CSF, human growlh hormone (hGH), beta interferon, antithrombin 30 ill, alpha interferon, alpha interferon 2b.

An inhaler device can be used to deliver a therapeutic protein such as those listed above or other macromolecule-based microspheres to the -48- 2015215955 21 Aug 2015 respiratory airways and lungs of a subject. The protein microspheres can be prepared, for example by contacting an aqueous solution of the protein with a carboxylic acid such as citrate, or sulfate or other counterion and an organic solvent such as isopropanol, and cooling the solution to form the 5 micraspheres. The protein can be a therapeutic protein, such as a sialidase, a protease inhibitor, insulin, human growth hormone, calcitonin, rhDNase or parathyroid hormone, and the protein content of the microspheres can be about or at 70% to about or at 90% or more, 95% or more, or at least about 99% or more. For pulmonary administration, the microspheres, for example 10 DAS181 microspheres, can be sized to have a mean diameter in the range of from about 0.5 microns to 5.0 microns, or between about 1 micron to about 2 microns.

Incubation conditions for forming the microspheres can be optimized to incorporate at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 15 95%, 96%, 97%, 98%, or 99% or greater of the total amount of macromolecule present in the solution prior to formation of the microspheres, by adjusting parameters including pH, temperature, concentration of macromolecule, or duration of reaction or incubation. in some embodiments, a molecule or compound that does not produce 20 microspheres of desirable characteristics, can be incorporated into microspheres having desirable characteristics, e.g., of size, delivery profile, mechanical strength, by Incorporation or coupling of the compound with a carrier molecule that can form microspheres with desirable characteristics. In some embodiments, tire carrier macromolecule is a protein, and the molecule 25 or compound is bound inside and/or on the surface of the microsphere, in some embodiments, the moiecute or compound also can serve as the counterion and initiate and/or facilitate the formation of microspheres.

When preparing microspheres containing a protein, a protein stabilizer such as glycerol, fatty acids, sugars such as sucrose, ions such as zinc, 30 sodium chloride, or any other protein stabilizers known to those skilled in the art can be added prior to cooling the cocktail during microsphere formation to minimize protein decatenation. -49- 2015215955 21 Aug 2015 ln some embodiments the mierospheres can further be coated on the surface with suitable molecules and/or coating agents, such as those that lend resistance to adds, such as digestive acids, or proteases. In other embodiments, the microspheres can be non-covalently coated with 5 compounds such as fatty acids or lipids. The coating can be applied to the mierospheres by immersion in the solubilized coating substance, then spraying the mierospheres with the substance, or by using other methods known to those of skill in the art. In some embodiments, the fatty acids or lipids are added directly to the microsphere-form'mg cocktail solution. 1 o Formation of the mierospheres by decreasing temperature can be performed by a multitude of conventional methods in batch or continuous modes. Microsphere formation can further be triggered by other methods inducting, but not limited to, modulating atmospheric pressure, g-force or surface expansion. Including seeding. Microsphere formation can occur 15 immediately upon exposure to these conditions or can require an extended period of time as provided herein.

Proteins

Exemplary proteins that can be used to form microparticles by the methods provided herein are described below. 20 Slalldases

Sialidases, also referred to as neuraminidases and N-acylneuraminosyiglycohydrolases, are a family exogiycosidases that catalyze the removal of terminal sialic acid residues from sialo-glycaconjugates. Sialic acids are a family of a keto acids with 9-carbon backbones that are usually 25 found at the outermost positions of foe oligosaccharide Ghains attached to glycoproteins and glycolipids. These molecules are involved in a variety of biological functions and processes, such as foe regulation of innate immunity, cell adhesion, and the interaction between inflammatory cells and target cells, possibly mediated through foe binding of various iectins (Varkf etal. (1992) 30 Curr Opin Ceil Biol 4:257-266). Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. Further still, siaiic acids on eukaryotic ceils can be used as receptors or coreceptors for -50- 2015215955 21 Aug 2015 pathogenic microorganisms, including, but not limited to, influenza virus, parainfluenza virus, some coronavirus and rotavirus, Haemophilus influenzae. Streptococcus pnei/moniae, Mycoplasma pneumoniae, Moraxella catarrhalis, Helicobacter pylori and Pseudomonas aeruginosa. The most prominent 5 member of the sialic acid family is N-acetylneuraminic acid (NeuSAc), which is the biosynthetic precursor for most of the other types. Two major linkages between Neu5Ac and the penultimate galactose residues of carbohydrate side chains are found in nature, Neu5Ac a(2,3)-Ga! and NeuSAc a(2,6)-Gai. Both NeuSAc a(2,3)-Gal and NeuSAc a(2,6)-Gal molecules can be recognized to by influenza viruses and used as the receptor through which the virus binds and initiates infection. Human influenza viruses, however, seem to prefer NeuSAc a(2,6)-Gaf, while avian and equine influenza viruses predominantly recognize Neu5Ac a(2,3)-Gal (ito et al. (2000) Microbiol Immunol 44:423-730). The human respiratory epithelium expresses both forms of sialic acids, 15 but a(2,6}-Iinked sialic acid is more abundant than a(2,3)- linked sialic acid. The low abundance of 0(2,3)- linked sialic add is most likely the basis for the species barrier for avian viruses, and indicates that reducing the level of a receptor sialic acid expressed on the airway epithelium would likely reduce the infectivity of an influenza virus. Thus, sialidases, which remove terminal 20 sialic add residues from sialo-glycoconjugates, present themselves as potential influenza virus therapeutic agents that function to reduce the levels of receptor sialic acids. Sialidases also can act as therapeutic agents tor any other pathogen that utilizes sialic adds in the Infection process Including, but not limited to, M. pneumoniae, M. catarrhalis, H. pylori, H. influenzae, S. 25 pneumoniae, P. aeruginosa, parainfluenza viruses and some coronaviruses and rotaviruses.

Sialidases tend to be highly substrate specific. They can target particular types of complex molecules, such as glycoproteins or glycolipids; specific sugar linkages (e.g. 2-^3,2-6, or 2-8); or can be sensitive to the 30 nature of the linkage sugar itself (e.g. D-galactose, N-acetyl-D- galactosamine). Substrate molecules include, but are not limited to, oligosaccharides, polysaccharides, glycoproteins, gangliosides, and synthetic -51- 2015215955 21 Aug 2015 molecules. For example, a sialidase can cleave bonds having a(2,3)-Gal, a(2,6H3al, or a(2,8)-Gal linkages between a sialic acid residue and the remainder of a substrate molecule. A sialidase also can cleave any or all of the linkages between the sialic acid residue and the remainder of the S substrate molecule. Many sialidase proteins have been purified from microbes and higher eukaryotes and of these, several have been shown to catalyze the removal of terminal sialic acid residues than can serve as receptors for pathogenic microorganisms. For example, among the large bacterial sialidases are those that that can degrade the influenza receptor sialic adds 10 NeuSAo ct(2,6)-Gal and NeuSAc a(2,3)-Gal, Including sialidases from

Clostridium perfringens, Actinomyces viscosus, Arthrobacter ureafaciens, and Mscmmonospora vlridifadens. Other sialidases that can serve as therapeutic agents include the human sialidases, such as those encoded by the genes NEU2 and NEU4. 15 Sfalidase-GAG fusion proteins

Sialidase-GAG fusion proteins are proteins that are made up of a sialidase protein, or catalytically active portion thereof, fused to a glycosaminoglycan (GAG)-binding sequence. As such, these proteins effectively contain an anchoring domain (the GAG-binding sequence) and a 20 therapeutic domain (the sialidase protein, or catalytically active portion thereof). The sialidase-GAG fusion proteins are designed to bind to the epithelium and remove the sunrounding sialic acids, and can therefore be used as a therapeutic agent against pathogens that utilize sialic acids in the infection process. The ability of the fusion protein to bind to the epithelium 25 increases its retention when the fusion protein is administered, for example, as an inhalant to treat influenza infection. The GAG-binding sequence acts as an epithelium-anchoring domain that tethers the sialidase to the respiratory epithelium and increases its retention and patency.

Heparan sulfate, closely related to heparin, is a type of 30 glycosaminoglycan (GAG) that is ubiquitously present on cell membranes, including the surface of respiratory epithelium. Many proteins specifically bind to heparlnfheparan sulfate, and the GAG-binding sequences in these proteins -52- 2015215955 21 Aug 2015 have been identified. For example, the GAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NQ:3), human interteukin 8 (IL8) (SEQ ID NO:4), human antithrombin III (AT III) (SEQ ID NO:5), human apoprotein E (ApoE) (SEQ ID NO:6), human angio-associated migratory cell protein 5 (AAMP) (SEQ ID NO:7), or human amphiregulin (SEQ ID NO:8) have been shown to exhibit high affinity tor heparin (Lee et al. (1991) PNAS 88:2768-2772; Gogeretal.. (2002) Blochem. 41:1640-1646; Witt et al. (1994) Curr Bio 4:394-400; Welsgraber et al. (1986) J Bio Chem 261:2068-2076). The GAG-binding sequences of these proteins are distinct from their receptor-binding 10 sequences, so they do not induce the biological activities associated with the full-length proteins or the receptor-binding domains. These sequences, or other sequences that can bind heparin/heparan sulfate, can be used as epithelium-anchoring-domains in sialidase-GAG fusion proteins.

In the context of a sialidase-GAG fusion protein, the sialidase can 15 include the entire sialidase protein, or a catalytically active portion thereof. For example, sialidase-GAG fusion protein can contain the 901 amino acid sialidase protein from A. viscosus set forth in SEQ ID NO:1. In another example, the sialidase-GAG fusion protein can contain the 394 amino acid catalytically active portion of a sialidase protein from A. viscosus set forth in 20 SEQ ID NO:2. The GAG-binding sequence can be linked to the sialidase by recombinant methods. In some examples, the fusion protein can Indude an amino acid linker, such as four glycine residues. Furthermore, linkage can be via the N- or C-terminus of the GAG-binding sequence, or tee N-or C-temnlnus of tee sialidase. Exemplary examples of slatldase-GAG fusion proteins include 25 those polypeptides set forth in SEQ ID NOS: 9-13, and 17. In a further example, tee sialidase and GAG-binding sequence components can be linked using chemical or peptide linkers, by any method known in the art,

Proteinase inhibitors

Proteinase inhibitor 8 (PI8), also known as Serpln B8, is a serine 30 protease inhibitor (serpin). Serpins are a large superfamily of structurally related proteins teat are expressed in viruses, insects, plants and higher organisms, but not in bacteria or yeast. Serpins regulate the activity of -53- 2015215955 21 Aug 2015 proteases involved in many biological process, including .coagulation, fibrinolysis, inflammation, call migration, and tumorigenesis. They contain a surface-exposed reactive site loop (RSL), which acts as a “bait" for proteases by mimicking a protease substrate sequence. On binding of the target 5 protease to the serpin, the RSL is cleaved, after which the protease is covalently linked to the serpin. The protease in the newly formed serpin-protease complex is inactive (Huntington et a). (2000) Nature 407:923-926). P18 is a member of a suhfamily of serpins of which chicken ovalbumin is the archtype. Like other serpins that belong to this family, PI8 lacks a typical 10 cleavable N-terminal signal sequence, resulting in a 374 amino acid protein (SEQ ID NO':14) that resides mainly Iniracellutarly. Other members of this human ovalbumin-like subfamily include plasminogen activator inhibltortype 2 (PAI-2), monocyte neutrophil elastase inhibitor (MNEI), squamous cell carcinoma antigen (SCCA)-1, leupin (SCCA-2) maspln (PI5), protease 15 Inhibitor 6 (PI6), protease inhibitor (PI9) and bomapin (PH0). Within this family the serpins P16, PI8, and PI9 show the highest structural homology (up to 68% amino acid identity) (Sprscher et al. (1995) J Biol Chem 270:29854-29861). Pi-8 has been shown to inhibit trypsin, thrombin, factor Xa, subtllisin A. furin, and also chymotrypsin in vitro, it Is released by platelets and appears 20 to be involved in the regulation of furin activity and, therefore, platelet aggregation (LeBlond et al. (2006) Thromb Haemost 95:243-252).

In addition to their role in the regulation of endogenous biological processes, such as coagulation, serine protease inhibitors also can function to inhibit the biological activities of exogenous microorganisms. For example, a 25 number of serine protease inhibitors have been shown to reduce influen2a virus activation in cultured cells, chicken embryos and in the lungs of infected mice. The serpins bind to hemagglutinin (HA) molecules on the surface of the influenza virus and inhibit its activity; thus reducing the irifectlvity of the virus. For example trypsin inhibitors, such as: aprotlnln (Zhirnov et al. (2002) J Virol 30 76:8682-8689), leupeptin (Zhirnov et al. (2002) J Virol 76:8682-8689; Tashiro et al. (1987) J Gen Virol 68:2039-2043), soybean protease inhibitor (Barbey-Morel et al. (1987) J Infect Dis 155:667-672), e-aminocaprolc acid (Zhirnov et -54- 2015215955 21 Aug 2015 al.. 1982. Arch Virol 73:263-272) aid n-p-tosyI-L-iysine chloromethylketone <TLCK) (Barbey-Morel et al. (1987) J Infect Dis 156:667-672) have all been shown to inhibit influenza virus infection, and are candidate therapeutic agents for use in the treatment of influenza virus infection. Thus, as a related 5 trypsin inhibitor, PI8 also can be used as a therapeutic agent in the treatment of influenza virus infection. *

Surface Active Agents

The compositions provided herein can contain one or more surface active agents that are added in an amount sufficient to form a stable 10 . emulsion. The appropriate amount of surfece active agent is a function of the non-denatured protein, optionally additional active agents for delivery, and other components present in the emulsion, since some agents can have seif-emuislfying properties and other agents and components affect surface tension. 15 The surface active agents for use herein are substances which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous phase and the oil phase, to form a stable oil In water or water in oil emulsion. The surfactant molecules are amphiphilic and contain hydrophilic head groups and hydrophobic tails. The 20 surfactant molecules form various macro-molecular structure in an emulsion, such as micelles, inverse micelles, lipid bilayers (liposomes) and cubrisomes. The exact macromolecular structure which is formed depends on the relative sizes of the hydrophilic and hydrophobic regions of the surface active molecule, in certain embodiments, the surface active agent is selected from 25 sodium iauryl sulfate; sorbitan iaurate, sorbitan palmitate, sorbitan stearate (available under the tradename Span® 20-40-60 etc.); polysorbates such as polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate (available under the tradename TWEENS® 20-40-60 etc.); benzalkonium chloride, mixed 30 chain phospholipids, cationic lipids, oligolipids. phospholipids, carnitines, sphingosines, sphingomyelins, ceramides, giycoiipids, lipoproteins, apoproteins, amphiphilic proteins, amphiphilic peptides, amphiphilic synthetic -55- 2015215955 21 Aug 2015 polymers, and combinations thereof. Other exemplary surface active agents for use herein include, but are not limited to i) Natural lipids, i.e. Cholesterol, Sphingosine and Derivatives, Gangliosides, Sphingosine derivatives (Soy Bean), Phytosphingosine and 5 derivatives (Yeast), Choline (Phosphatidylcholine), Ethanolamine (Phosphatidylethanolamine), Glycerol (Phosphatidyl-DL-glyceroi), Inositol (Phosphatidylinositol), Serine (Phosphatidylserine (Sodium Sait)), Cardiolipin, Phosphatidic Acid, Egg Derived, Lyso (Mono Acyl) Derivatives (Lysophosphatides), Hydrogenated Phospholipids, Lipid Tissue Extracts, 10 H) Synthetic lipids, i.e. Asymmetric Fatty Acid, Symmetric Fatty

Acid - Saturated Series, Symmetric Fatty Add - Unsaturated Series, Acyl Coenzyme A (Acetoyl Coenzyme A, Butanoyl Coenzyme A, Crotanoyl Coenzyme A, Hexanoyl Coenzyme A, Octanoyi Coenzyme A, Decanoyl Coenzyme A, Lauroyl Coenzyme A, Myristoyf Coenzyme A, Palmitoyi 15 Coenzyme A, Stearoyl Coenzyme A, Oleoyl Coenzyme A, Arachidoyl

Coenzyme A, Arachldonoyl Coenzyme A, Behenoyl Coenzyme A, Tricosanoyl Coenzyme A, Lignoceroyl Coenzyme A, Nervonoyl Coenzyme A, Hexaoosanoyl Coenzyme A, lit) Sphingoliplds, i.e. D-erythro (018) Derivatives (Sphingosine, 20 such as: D-erythro Sphingosine (synthetic), Sphingosine -1 -Phosphate, N,N Dimethylsphingosine, N ,N,N-Trimethylsphingosine, Sphingosylphosphorylcholine, Sphingomyelin and Glycosylated Sphingosine), Ceramide Derivatives (Ceramides, D-erythro Ceramide-1-Phosphate, Gtycosolated Ceramides), Sphinganine (Dihydrosphtngosine) (Sphinganine-1-25 Phosphate, Sphinganine (C20), D-erythro Sphinganine, N-Acyl-Sphinganine C2, N-Acyl-Sphinganine C8, N-acyl-Sphinganlne C16, N-Acyl-Sphinganine C18, N-Acyl-Sphinganine C24, N-Acyi-Sphinganine C24:1), Glycosylated (C18) Sphingosine and Phospholipid Derivatives (Glycosylated -Sphingosine) (Sphingosine, β D-Gtucosyl, Sphingosine, β D-Galactosyl,

30 Sphingosine, β D-Lactosyl), Glycosylated — Ceramide (D-Glucosyl-fi1-T

Ceramide (C8), D-Galactosyl-B1-1' Ceramide (C8), D-Lactosyl~B1-1' Ceramide (C8), D-Glucosyl-ΒΙ-Γ Ceramide (C12), D-Galactosyl-B1-V -56- 2015215955 21 Aug 2015

Ceramide (C12), D-LactosyI»B1-1' Ceramlde ¢012)), Glycosylated -Phosphatidylethanolamlne (1,2-Dioteoyl-sn-Glycero-3-Phosphoethanolamine-N-Lactose), D-erythro (C17) Derivatives (D-erythro Sphlngosine, D-erythro Sphingosine-1-phosphate), D-erythro (C20) Derivatives (D-erythro 5 Sphingosine), L-threo (C18) Derivatives (L-threo Sphingosine, Saflngol (L-threo Dihydrosphingosine)), Sphingosine Derivatives (Egg, Brain &amp; Milk) (D-erythro-SphingosIne, Sphingomyelin, Ceramides, Cerebrosides, Brain Sulfiatides), Gangliosides (Gangliosides Structures, Gangliosides - Ovine Brain, Gangliosides - Porcine Brain), Sphingosine Derivatives (Soy Bean) 10 (Glucosylceramide), Phytosphingosine Derivatives (Yeast) (Phytosphingosine. D-ribo-Phytosphingosine-1-Phosphate, N-Acy! Phytosphingosine C2, N-Acyl Phytosphingosine C8, N-Acyl Phytosphingosine C18, iv) Acyl coenzyme A, i.e. Acetoyl Coenzyme A (Ammonium Salt), Butanoyl Coenzyme A (Ammonium Salt), Crotanoyl Coenzyme A (Ammonium 15 Salt), Hexanoyl Coenzyme A (Ammonium Salt), Octanoyl Coenzyme A (Ammonium Sait), Decanoyl Coenzyme A (Ammonium Salt), Lauroyl Coenzyme A (Ammonium Salt), Myristoyl Coenzyme A (Ammonium Salt), Palmitoyl Coenzyme A (Ammonium Salt), Stearoyi Coenzyme A (Ammonium Salt), Oleoyi Coenzyme A (Ammonium Salt), Arachidoyt Coenzyme A 20 (Ammonium Sait), Arachidonoyl Coenzyme A (Ammonium Salt), Behenoyi Coenzyme A (Ammonium Salt), Tricosanoy) Coenzyme A (Ammonium Salt), Lignoceroyl Coenzyme A (Ammonium Salt), Nervonoyl Coenzyme A (Ammonium Salt), Hexacosanoyl Coenzyme A (Ammonium Salt), Docosahexaenoyl Coenzyme A (Ammonium Salt), 25 v) Oxidized lipids, i.e. 1 -Palmitoyl-2-Azefaoyl-sn-Glycero-3-

Phosphochotine, 1-0-Hexadeoyl-2“AzelaoyI-sn-Glycero-3-Phosphocholine, 1-Palmitoyl-2-G!utaroyl-sn-GIycero-3-Phosphocholine (PGPC), 1-Palmitoyl-2-(9’-oxo-Nonanoyl)-sn-Giycero-3-Phosphocholine, 1 -Palmitoyl-2-(5-oxo-Valeroyl)-sn-G1ycero-3-Phosphocholine, 30 vt) Ether lipids, i.e.: Diether Lipids (Dialkyl Phosphatidylcholine,

Diphytanyl Ether Lipids), Alkyl Phosphochollne (Dodadylphosphocholine), O-Alkyi diacylphosphatidylcholinium (1,2-Diacy!-sn-Glycero-3- -57- 2015215955 21 Aug 2015

EthylphosphoGholine), Synthetic PAF &amp; Derivatives (1-A1kyl-2-Acyl-Glycero-3-Phosphocholine &amp; Derivatives), vii) Fluorescent lipids, l.e.: Glycerol Based (Phosphatidylcholine (NBD). Phosphaiidic Acid (NBD), Phosphatidyiethanolamine (NBD), 5 Phosphatidylglycerol (NBD), Phosphatidylserine (NBD)), Sphingosine Based (Ceramlde (NBD), Sphingomyelin (NBD), Phytosphingosine (NBD),

Galactosyl Cerebroside (NBD)), Headgroup Labeled Upids (Glycerol Based) (Phosphatidyiethanolamine (NBD), Phosphalidylethanolamine (Lissamine Rhodamine B), Dioleoyl Phosphatidyiethanolamine (Dansyl, Pyrene, 10 Fluorescein), Phosphatidylserine (NBD), Phosphatidylserine (Dansyl)), 25-NBD-Cholesterol, viii) Other lipids including, but not limited to Lecithin, U|tralec-P (ADM), Soy powder, ix) Surfactants including, but not limited to polyethylene glycol 400; 15 sodium lauryl sulfate; sorbifan laurate, sorbitan palmitate, sorbitan stearate (available under the tradename Span® 20-40-60 etc.); polysorbates such as . polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate (available under the tradename TWEENS® 20-40-60 etc.); benzalkonium chloride. 20 In certain embodiments, the phospholipids for use are phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, phosphatidyllnositols, phosphatidic adds, mixed chain phospholipids, lysophosphollpids, hydrogenated phospholipids, partially hydrogenated phospholipids, and mixtures thereof. 25 In certain embodiments, the surface active agent is selected from polysorbate-80, lecithin and phosphatidylcholine. The surface active agents are present in an amount suffident to form a stable emulsion.

The amount of surface active agent can be empirically determined and is a function of the agent selected, and the desired form of the resulting 30 composition. The amount included can be from less than 0.1 % by weight up to 35% or more, in certain embodiments, the surface active agent Is present at a concentration of about 1 %, 2%, 3%. 4%, 5%, 6%, 7%, 8%, 9%, 10%, -58- 2015215955 21 Aug 2015 15%, 20%, 25% by weight up to about 30 % by weight of the total weight of the composition. In certain embodiments, the surface active agent is present at a concentration of about 1 weight % up to about 20 weight % of the total weight of the composition. In certain embodiments, the surface active agent is 5 present at a concentration of about 1 weight % up to about 15 weight % of the total weight of the composition, in other embodiments, the surface active agent is present at a concentration of about 1 weight % up to about 10 weight % of the total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 1 weight % up to about 8 10 weight % of the total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 1 weight % up to about 6 weight % of toe total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 1 weight % up to about 4 weight % of the total weight of the composition. In 15 other embodiments, the surface active agent is present at a concentration of about 20 weight % of the total weight of the composition, in other embodiments, the surface active agent is present at a concentration of about 15 weight % of the total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 13 weight % of the 20 total weight of the composition. In other embodiments, toe surface active agent is present at a concentration of about 11 weight % of the total weight of the composition. .In other embodiments, the surface active agent is present at a concentration of about 8 weight % of the total weight of toe composition. In other embodiments, the surface active agent is present at a concentration of 25 about 6 weight % of the total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 4 weight % of the total weight of the composition. In other embodiments, the surface active agent is present at a concentration of about 2 weight % of the total weight of the composition: In other embodiments, the surface active 30 agent is present at a concentration of about 1 weight % of toe total weight of the composition. -59- 2015215955 21 Aug 2015

The stable emulsions provided herein can contain one or more delivery vehicles selected from among micelles, liposomes and cubosomes and mixtures thereof, or macromolecular assemblies of non-denatured proteins such as tubes, helices, spheres and the like, that can encapsulate additional 5 nutrients or active agents. The delivery vehicles encapsulating the active agent are then absorbed in the epithelium where the non-denatured proteins and/or additional nutrients/active agents are delivered.

Optional additional Agents

The compositions provided herein can optionally, in addition to non-10 denatured proteins, contain one or more pharmaceutical or nutraceutical or other such agent for ingestion by a subject. Generally the agents are those that have a function in a host, e.g., immune regulation, regulation of biochemical processes, or enzymatic activity. Any agent that can be formulated as described herein can ba administered in the compositions 15 provided herein. Where the agent is a therapeutic the compositions contain a therapeutically effective amount of an agent to be delivered. The particular amount of active agent in a dosage will vary widely according to the nature of the active agent, the nature of the condition being treated, the age and size of the subject, and other parameters. 20 . Generally, the amount of additional active agent or nutrient besides the non-denatured proteins in the composition will vary from less than about 0.01% by weight to about 20% by weight of the composition, or more and typically are formulated for single dosage administration. A single dosage can vary from about 0.01 to 10 mg of an agent per kilogram of body weight of 25 the host, with dosages from about 0.1 pg to 1 mg/kg being commonly employed. These concentrations, however, are general guidelines only and particular amounts and dosages may be selected based on the active agent being administered, the condition being treated, and the treatment regimen being employed means an amount of a drug or an active agent that is 30 sufficient to provide the desired local or systemic effect and performance at a reasonable benefit/risk ratio to a subject attending any medical treatment. PCT/US2007/001914 WO 2007/114881 2015215955 21 Aug 2015 -60-

Agents can be selected from inorganic and organic drugs including, but not limited to drugs that act on the peripheral nerves, adrenergic receptors, cholinergic receptors, nervous system, skeletal muscles, cardiovascular system, smooth muscles, blood circulatory system, synaptic sites, neuro-5 effector junctional sites, endocrine system, hormone systems, immunological system, reproductive system, skeletal system, autocoid systems, alimentary and excretory systems, histamine systems, and the like. The active agents that can be delivered using the compositions provided herein include, but are not limited to, anticonvulsants, analgesics, antiparkinsons, anti-10 inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalariais, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, aipha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diagnostics, diuretics, electrolytes, enzymes, 15 hypnotics, hormones, hypogiycemics, hyperglycemics, muscle contractants, muscle relaxants, neoplasties, glycoproteins, nucleoproteins, lipoproteins, ophthalmics, psychic energizers, sedatives, steroids, sympathomimetics, parasympathomimetics, tranquilizers, urinary tract drugs, vaccines, vaginal drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs, angiotensin 20 converting enzymes, polynucleotides, polypeptides, polysaccharides, and nutritional supplements including herbal supplements.

The level of agent to be delivered is from about 0.01 % up to about 50%, from about 0.1% up to about 40 %, from about 0.1% up to about 30 %, from about 0.1% up to about 20 %, from about 0.1% up to about 10 %, from about 0.1% 25 up to about 9 %, from about 0.1 % up to about 8 %, from about 0.1 % up to about 7 %, from about 0.1 % up to about 6 %, from about 0.1% up to about 5 %, from about 0.1 % up to about 4 %, from about 0.1% up to about 3 %, from about 0.1 % up to about 2 %, from about 0.1% up to about 1 % by weight of the composition. The agent to be delivered can be water soluble, slightly 30 water soluble, or oil soluble. In certain embodiments, the agent to be delivered is selected from anticonvulsants, analgesics, antiparkinsons, antiinflammatories, calcium antagonists, anesthetics, antimicrobials, antimalariais, -61 - 2015215955 21 Aug 2015 antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diagnostics, diuretics, electrolytes, enzymes, 5 hypnotics, hormones, hypoglycemtcs, hyperglycemias, muscle contractants, muscle relaxants, neoplasties, glycoproteins, nudeoproteins, lipoproteins, non denatured whey protein, ophthalmias, psychic energizers, sedatives, steroids, sympathomlmetics, parasympathomimetics, tranquilizers, urinary tract drugs, vaccines, vaginal drugs, vitamins, minerals, nonsteroidal antl-infiammatory 10 drugs, angiotensin converting enzymes, polynucleotides, polypeptides, polysaccharides, and nutritional supplements including herbal supplements.

In certain embodiments, the active agent is selected as follows: α-Adrenergic agonists such as Adrafinil, Adrenoione, Amldephrine, Apraclonidlne, Budralazine, Clonidine, Cyciopentamine, Detomidine, 15 Dimetofrine, Dipivefrin, Ephedrine, Epinephrine, Fenoxazotine, Guanabenz, Guanfacine, Hydroxyamphetamine, lbopamine, Indanazoiine, Isometheptene, Mephentermine, Metaramlnoi, Methoxamine Hydrochloride, Methylhexaneamine, Metizolene, Midodrine, Naphazoline, Norepinephrine, Norfenefrine, Octodrine, Octopamine, Oxymetazoline, Phenylephrine 20 Hydrochloride, Phenylpropanolamine Hydrochloride,

Phenylpropylmethylamlne, Pholedrine, Propylhexedrine, Pseudoephedrine, Rllmenidine, Synephrine, Tetrahydrozoiine, Tlamenidine, Tramazoline, Tuaminoheptane, Tymazoline, Tyramine and Xylometazoiine; p-Adrenergic agonists such'as Albuterol, Bambuterol, Bitolterol, 25 Carbuterol, Clenbuteiol, Clorprenatine, Denopamine, Dioxethedrine, Oopexamine, Ephedrine, Epinephrine, Etafedrine, Ethylnorepinephrine, Fenoterol, Formoterol, Hexoprenaline, lbopamine, Isoetharine, Isoproterenal, Mabuterol, Metaproterenol, Methoxyphenamlne, Oxyfedrlne, Pirbuteroi, Prenalteroi, Procateral, Protokylol, Reproterol, Rimiterol, Ritodrine, Soterenol, 30 Terbuterol and Xamoterol; -62- 2015215955 21 Aug 2015 α-Adrenergic blockers such as Amosulalol, Arotinolol, Dapiprazole, Doxazosin, Ergoloid Mesylates. Fenspiride, Indoramln, Labetaiol, Nicergoline, Prazosin, Terazosin, Tolazoline, Trimazosin and Yohimbine; β-Adreneiigic blockers such as Acebutolo], Alprenolol, Amosulalol, 5 Arotinolol, Atenolol, Befunolol, Betaxolol, Bevantoiol, Bisoprolol, Bopindolo!, Bucumolol, Befetolol, Bufuralol, Bunltrolol, Bupranolol, Butidrine Hydrochloride, Butofilolol, Carazofol. Carteolol, Carvedilol, Celiprolol, Cetamolol, Cioranolol, Dilevalol, Epanolol, Esmolol, Indenolol, Labetaiol, Levobunolol, Mepindotol, Metipranatol, MetoproFof, Moprolol, Nadoxolol, 10 NIfenalol, Nipradifol, Oxprenolol, Penbutolol, Pindolol, Practolol, Pronethalol, Propranolol, Sotatal, Sulfinalol, Talinolol, Teriatolol, Timolol, Toliprolo! and Xibenolol;

Alcohol deterrents such as Calcium Cyanamide Citrated, Disulfiram, Nadlde and Nitrefazole; 15 Aldose reductase inhibitors such as Epalrestat, Ponalrestat, Sorbinil and Totrestat;

Anabolics such as Androisoxazoie, Androstenediol, Bolandiol, Bolasterone, Clastebol, Ethylestrenol; Formyldienolone, 4-Hydroxy-l9-nortestosterone, Methandriol, Methenoione, Methyltrienolone, Nandralone, 20 Nandrolone Decanoate, Nandrolone p-Hexyloxyphenylpropionate,

Nandrolone Phenpropionate, Norbolethone, Oxymesterone, Pizotyline, Quinbolone, Stenbotone and Trenbolone;

Analgesics (dental) such as Chlorobutanol, Clove and Eugenol;

Analgesics (narcotic) such as Aifentanil, Allylprodine, Alphaprodine, 25 Anileridine, Benzylmorphine, Bezitramide, Buprenorphine, Butorphanol, Clonitazene, Codeine, Codeine Methyl Bromide, Codeine Phosphate,

Codeine Sulfate, Desomorphine, Dextromoramlde, Dezocine, DFampromide, Dihydrocodeine, Dlhydrocodeinone Enol Acetate, Dihydromorphine, Dimenoxadol, Dimepheptanol, Dlmethylthiambutene, Dioxaphetyl Butyrate, 30 Dipipanone, Epiazocine, Ethoheptazine, Ethylmethlythlambutene,

Ethylmorphlne, Etonitazene, Fentanyl, Hydrocodone, Hydrocodone Bitartrate, Hydromorphone, Hydroxypethidine, isomethadone, Ketobemidone, 2015215955 21 Aug 2015 -63-

Levorphanol, Lofentanll, Meperidine, Meptazinol, Metazocine, Methadone Hydrochloride, Metopon, Morphine, Morphine Derivatives, Myrophine, Nalbuphine, Narceine, Nicomorphine, Norlevorphanol, Normethadone, Normorphine, Norpipanone, Opium, Oxycodone, Oxymorphone, 5 Papaveretum, Pentazocine, Phenadoxone, Phenazocine, Pheoperidine, Piminodine, Piritramide, Proheptazine, Promedol, Properidine, Proplram, Propoxyphene, Sufentanil and Tilidine;

Analgesics (non-narcotic) such as Acetaminophen, Acetaminosafol, Acetanilide, Acetylsalicyisaficyiic Add, Aldofenac, Alminoprofen, Aloxiprin, 10 Aluminum Bis(acetylsalicylate),Aminochforthenoxazin,2-Amina-4-picaline, Aminopropylon, Aminopyrine, Ammonium Salicylate, Antlpyrlne, Antlpyrine Salicylate, Antrafenine, Apazone, Aspirin, Benorylate, Benoxaprofen, Benzpiperylon, Benzydamine, p-Bromoacetaniiide, 5-Bromosalicylic Acid Acetate, Buceti.n, Bufexamac, Bumadizon, Butacetin, Calcium IS Acetylsalicyiate, Carbamazepine, Carbetidine, Carbiphene, Carsalam, Chloralantipyrine, Chlorthenoxazin(e), Choline Salicylate, Cinchophen, Ciramadoi, Clometacin, Crapropamide, Crotethamide, Dexoxadrol, Difenamizole, Diflunisal, Dihydroxyaluminum Acetylsalicyiate, Dipyrocetyl, Dipyrone, Emorfazone, Enfenamic Acid, Epirizole, Etersalate, Ethenzamide, 20 Ethoxazene, Etodolac, Felbinac, Fenoprofen, Floctafenine, Flufenamic Acid, Fluoresone, Flupirdne, Fluproquazone, Flurbiprofen, Fosfosal, Gentlsic Acid, Glafenine, Ibufenac, Imidazole Salicylate, Indomethacin, Indoprofen, lsofezolac, tsoladol, Isonixln, Ketoprofen, Ketorolac, p-Lactophenetide, Lefetamine, Loxoprofen, Lysine Acetylsalicyiate, Magnesium Acetylsalicyiate, 25 Methotrimeprazine, Metofoline, Miroprofen, Morazone, Morpholine Salicylate, Naproxen, Nefopam, Nifenazone, 5' Nitro-2* prapoxyacetanilide, Parsalmide, Perisoxal, Phenacetin, Phenazopyridine Hydrochloride, Phenocoll, Phenopyrazone, Phenyl Acetylsalicyiate, Phenyl Salicylate, Phenyramidol, Pipebuzone, Piperytone, Prodilidlne, Propacetamol, Propyphenazone, 30 Proxazole, Quinine Salicylate, Ramifenazone, Rimazolium Metilsulfote, Salacetamide, Sallcin, Salicylamide, Salicylamide Ο-Acetic Acid, Salicylsuifuric Acid, Salsatte, Salverine, Simetride, Sodium Salicylate, -64» 2015215955 21 Aug 2015

Sulfamipyrine, Suprofen, Talnfflumate, Tenoxicam, Terofenamate, Tetradrine, Tinoridine, Tolfenamic Acid, Tolpronine, Tramadol, Vimino], Xenbucin and Zomeplrac;

Androgens such as Androsterone, Boldenone, 5 Dehydroeplandrosterane, Ruoxymesterone, Mestanolone, Mesterolone, Methandrostenolone, 17-MethyItestosterone, 17cc»Me1hyltestosterone 3-Cyclopentyi Enol Ether, Norethandrolone, Normethandrone, Oxandrolone, Oxymesterone, Oxymetholone, Prasterone, StanioEone, Stanozoiol, Testosterone, Testosterone 17-Chlorai Hemtacetal, Testosterone 17β-10 Cyplonate, Testosterone Enanthate, Testosterone Nlcotinate, Testosterone Pheynylacetate, Testosterone Propionate and Tiomesterone;

Anesthetics such as Acetamidoeugenol, Alfadolone Acetate, Alfaxalone, Amucaine, Amolanone, Amylocaine Hydrochloride, Benoxinate, Benzocaine, Betoxycaine, Bfphenamine, Bupivacaine, Butacaine, Butaben, 15 Butanilicaine, Burethamine, Buthaiitai Sodium, Butoxycaine, CarBcaine, 2-Chloroprocaine Hydrochloride, Cocaethylene, Cocaine, Cyclomethycaine, Dibucaine Hydrochloride, Dimethisoquin, Dimethacaine, Diperadon Hydrochloride, Dyclonine, Ecgonldine, Ecgonine, Ethyl Aminobenzoate, Ethyl Chloride, Ettdocaine, Etoxadrol, β-Eucaine, Euprocin, Fenalcomine, 20 Fomocalne, Hexobarbltal, Hexylcaine Hydrochloride, Hydroxydione Sodium, Hydroxyprocaine, Hydroxytetracaine, isobutyl p-Aminobenzoate, Kentamine. Leucinocaine Mesylate, Levoxadroi, Lidocaine, Mepivacaine, Meprylcaine Hydrochloride, Metabutoxycaine Hydrochloride, Methohexital Sodium, Methyl Chloride, Midazolam, Myrtecaine, Naepalne, Octacaine, Orthocaine, 25 Oxethazalne, Parethoxycaine, Phenacaine Hydrochloride, Phencyclidine, Phenol, Piperocaine, Piridocaine, Polidocanol, Pramoxine, Prilocaine, Procaine, Propanidid, Propanocaine, Proparacalne, Propfpocaine, Propofol, Propoxycaine Hydrochloride, Pseudococaine, Pyrrocaine, Quinine Urea Hydochloride, Risocaine, Sallcyl Alcohol, Tetracaine Hydrochloride, 30 Thialbarbital, Thimylal, Thtobutabarhital, Thiopental Sodium, Tolycaine, Trimecafne and Zoiamine; 2015215955 21 Aug 2015 -65-

Anorexics such as Amlnorex, Amphecloral, Amphetamine, Benzaphetamine, Chlorphentermina, Clobenzorex, Cloforex, Clortermlne, Cydexednne, Destroamphetamine Sulfate, Diethylpropion, Diphemethoxidlne, N-Ethyiamphetamine, Fenbuirazate, Fenfluramine, Fenproporex, 5 Furfurytmethylamphetamlne, Levophacetoperate, Mazindol, Mefenorex, Metamfeproamone, Methamphetamine, Norpseudoephedrine, Phendimetrazine, Phendimetrazine Tartrate, Phenmetrazine, Phenpentermine, Phenylpropanolamine Hydrochloride and Plcllorex;

Anthelmintics (Cestodes) such as Arecoline, Aspidin, Aspidlnol, 10 D(chlorophen(e}, Embelin, Kosin, Napthalene, Niclosamide, Pellertierine, Pellertierine Tannate and Quinacrtne;

Anthelmintics (Nematodes) such as Alantolactone, Amoscanate, Ascaridole, Bephenium, Bitoscanate, Carbon Tetrachloride, Carvacrol, Cyclobendazole, Diethylcarbamazine, Diphenane, Dithiazanine Iodide, 15 Dymanthine, Gentian Violet, 4-Hexylresorcinol, Kainic Acid, Mebendazole, 2-Napthol, Oxantel, Papain, Piperazine, Piperazine Adipate, Piperazine Citrate, Piperazine Edetate Calcium, Piperazine Tartrate, Pyrantel, Pyrvinium Pamoate, α-Santonin, Stilbazium Iodide, Tetrachloroethylene, Tetramisole, thiabendazole. Thymol, Thymyl N-lsoamylcarbamate, Triclofenol Piperazine 20 and Urea Stibamine;

Anthelmintics (Onchocerca) such as Ivermectin and Suramin Sodium;

Anthelmintics (Schistosoma) such as Amoscanate, Amphotalide, Antimony Potassium Tartrate, Antimony Sodium Gluconate, Antimony Sodium Tartrate, Antimony Sodium TTiioglycollate, Antimony Thioglycollamide, 25 Becanthone, Hycanthone, Lucanthone Hydrochloride, Nirldazole,

Oxamniquine, Praziquantel, Stibocaptate, Stibophen and Urea Stibamine;

Anthelmintic (Trematodes) such as Anthiolimine and Tetrachloroethylene;

Antiacne drugs such as Adapelene, Atgestone Acetophenide, Azelaic 30 Acid, Benzoyl Peroxide, Cyoctol, Cyproterone, Motretlnide. Resorcinol, Retinoic Acid, Tetroquinone and Tretinonine; -66- 2015215955 21 Aug 2015

Antlallergics such as Amlexanox, Astemizote, Azelastlne, Cromolyn, Fenpiprane, Histamine, Ibudilast, Nedocromil, Oxatomide, Pentigetide, Poison Ivy Extract, Poison Oak Extract, Poison Sumac Extract, Repirinast, Tranrlast, Traxanox and Urushiol; 5 Antiamebics such as Arsthinol, Blalamicol, Carbarsone, Cephaellne,

Chlorbetamide, Chioroquine, Chlorphenoxamide, Chlortetracycline, Dehydroemetine, Dibromopropamldine, Difoxanide, Dephetarsone, Emetine, Fumagillin, Glaucarubin, Glycobiarsol, 8-Hydroxy-7-iodo-5-quino[lnesuIfonic Add, lodochlorhydroxyquin, todoquinol, Paromomycin, Phanquinone, 10 Phearsone Sulfoxylate, Polybenzarsol, Propamidine', Quinfamide, Secnidazole, Sulfarside, Teclozan, Tetracycline, Thiocarbamizine, Thiocarbarsone and Tinidazole;

Antiandrogens such as Bifluranol, Cyoctol, Cyproterone, Delmadinone Acetate, Flutimide, Nilutamlde and Oxendoione; 15 Antianginals such as Acebutolol, Alprenolol, Amiodarone, Amlodipine.

Arotinolol, Atenolol, Bepridil, Bevantolol, Bucumoioi, Bufetolol, Bufuralol, Bunitrolo!, Bupranolcl,.Carozolol, Carteolot, Carvediiol, Celipraiol, Cinepazet Maleate, Diltiazem, Epanolol, Felodipine, Gallopamil, Imolamine, Indenolol, Isosorbide Dinitrate, Isradipine, Umaprost, Mepindolol, Metoprotol, 20 Molsidomine, Nadolol, Nicardipine, Nifedipine, Nifenalol, Nilvadlpine,

Nipradilol, Nisoldipine, Nitroglycerin, Oxprenolol, Oxyfedrine, Ozagrel, Penbutolol, PentaerythritolTetranitrate, Pindolol, Pronethalol, Propranolol, Sotaiol, Teroditine, Timolol, Toliprotol and Verapamil;

Antiarrhythmics such as Acebutol, Acecaine, Adenosine, AJmaline, 25 Alprenolol, Amiodarone, Amoproxan, Aprindine, Arotinolol, Atenolol,

Bevantolol, Bretyiium Tosyiate, Bubumolol, Bufetolol, Bunaftine, BunitroioJ, Bupranolol, Butidrine Hydrochloride, Butobendine, Capobenic Acid, Canazoiol, Carteolot, Crfenline, Cloranolol, Disopyramide, Encalnide, Esmolol,

Flecainide, Gallopamil, Hydroqufnidine, Indecainide, Indenolol, Ipratropium 30 Bromide, Lidocaine, Lorajmine, Lorcainide, Meobentine, Metipranolol,

Mexiletlne, Moricizlne, Nadoxolol, Nifenalol, Oxprenolol, Penbutolol, Pindolol, Pirmenol, Practoloi, Prajmaline, Procainamide Hydrochloride, Pronethalol, 2015215955 21 Aug 2015 -67-

Propafenone, Propranolol, Pyrinoline, Quinldine Sulfate, Quinidlne, Sotalol, Talinolol, Timolol, Tocainlde, Verapamil, Viquidil and Xibenolol;

Antiarteriosderotics such as Pyridlnol Carbamate;

Anifarthritic/Antirheumatics such as Allocuprelde Sodium, Auranofin, 5 Aurothioglucose, Aurothloglycanide, Azathioprine, Calcium 3-Aurothto-2-propanoM -sulfonate, Ceiecoxib, Chloroquine, Clobuzarit, Cuproxoline, Diacereln, Glucosamine, Gold Sodium Thiomalate, Gold Sodium Thiosulfate, Hydroxychloroquine, Kebuzone, Lobenzarit, Melittin, Methotrexate, Myoral and Penicillamine; 10 Antibacterial {antibiotic) drugs including: Aminoglycosides such as

Amikacin, Apramyctn, Arbekacin, Bambermyclns, Butirosin, Dibekacin, Dihdrostreptomycin, Forb‘micin{s), Gentamicin, Ispamlcin, Kanamycin, Micronomicin, Neomycin, Neomycin Undecylenate, Netilmicin, Paromomycin, Ribostamycln, Sisomicin, Spactinomycin, Streptomycin, Streptonlcozid and 15 Tobramycin;

Amphenicols such as Azidamfenicol, Chloramphenicol, Chloramphenicol Palmitate, Chloramphenicol Pantothenate, Florfenicol and Thiamphenicol;

Ansamycins such as Rifamide, Rifampin, Rifamycin and Rifeximin; 20 β-Lactams, including: Carbapenems such as Imipenem;

Cephalosporins such as Cefactor, .Cefadroxil, Cefamandoie, Cefetrizine, Cefazedone, Cefazolln, Cefixfme, Gefmenoxime, Cefodlzima, Cefonicid, Cefoperazone, Cefbranide, Cefotaxime, Cefotiam, Cefpimizole, Cefpirimide, Cefpodoxime Proxetil, Cefroxadlne, Cefsulodin, Ceftazidime, 25 Cefteram, Ceftezole, Ceftibuten, Ceftizoxlme, Ceftriaxone, Cefuroxime,

Cefuzonam, Csphacetrile Sodium, Cephalexin, Cephaloglycin, Cephaloridine, Cephalosporin, Cephalothin, Cephaplrin Sodium, Cephradine and Pivcefalexin;

Cephamycins such as Cefbuperazone, Cefmetazoie, Cefminox, 30 Cefetan and Cefoxitin;

Monobactams such as Aztreonam. Carumonam and Tlgemonam;

Oxacephems such as Romoxef and Moxolactam; -68- 2015215955 21 Aug 2015

Penicillins such as Amldinacillin, Amdinocillin Pivoxil, Amoxicillin, AmpicIIIan, Apalcillin, Aspoxlcillln, Azldoctllan, Azlocillan, Bacampiclllin, Benzylpenicillinic Acid, Benzylpenicillin Sodium, Carbenicilliri, Carfecillin Sodium, Carindacillfn, Clometocili in, Cloxacill in, Cyclaclllin, Didoxacillin, 5 Diphenlcillin Sodium, Epicillin, Fenbenicillin, Floxicillin, Hetarillin,

Lenampicillin, Metamplciilin, Methidilin Sodium, Mezlocillin, Nafctllin Sodium, Oxacillin, Penameciliin, Penethamate Hydriodide, Penicillin G Benethamlne, Penicillin G Benzathine, Penlciliin G Benzhydrylamine, Peniciilin G Calcium, Penicillin G Hydrabamfne, Penicillin G Potassium, Peniciilin G Procaine, 10 Penicfllen N, Penicillin O, Penicillin. V, Penicillin V Benzathine, Peniciilin V Hydrabamine, Penimepicycline, Phenethicillin Potassium, Piperacillin, Pivaplcillin, Propicillin, Quinacillin, Sulbenicillin, Talampiciilin, Temociltln and TicarcilHn;

Uncosamides such as Clindamycin and Lincomycin; 15 Macroirdes such as Azithroimycin, Carbomycin, Clarithromycin,

Erythromycin, Erythromycin Acistrate, Erythromycin Estolate, Erythromycin Glucoheptonate, Erythromycin Lactobionate, Erythromycin Propionate, Erythromycin Stearate, Josamycin, Leucomycins, Midecamycins, Miokamycin. Oleandomycin, Primycin. Rokitamycin, Rosaramicin, Roxithromycin, 20 Spiramycin and Troleandomycin;

Polypeptides such as Amphomycin, Bacitracin, Capreomycin, Colistin, Enduracidln, Envlomycin, Fusafungine, Gramicldin(s), Gramicidin S, Mikamycin, Polymyxin, Polymyxin B-Meihanesulfcnic Acid, Pristinamycin, Ristocetin, Teicoplanin, Thiostrepton, Tuberactinomycin, Tyrocidine, 25 Tyrothrtcln, Vancomycin, Viomycin, Vlomycin Pantothenate, Virginiamycin and Zinc Bacitracin;

Tetracyclines such as Apicycline, ChlortetracycJfne, Ciomocycline, Demeclocycline. Doxycydfne, GuamecycIJne, Lymecydine, Medocydfne. Methacycline, Minocycline, Oxytetracycline, Penimepicycline, Pipacycline, 30 Rolitetracycline, Sancycline, Senociclrn and Tetracydine; and other antibiotics such as Cycloserine, Mupirocin and Tuberin; 2015215955 21 Aug 2015 -69-

Antibacteriai drugs (synthetic), including: 2,4-DiaminopyrimId[nes such as Brodimoprim, Tetroxoprlm and Trimethoprim;

Nitrofurans such as Furaltadone, Furazolium Chloride, Nifuradene, Nifuratel, Nifurfoline, Nrfurpirinol, Nifurprazine, Nifurtoinol and Nitrofurantoin; 5 Quinolones and Analogs such as Amifloxacin, Cinoxacin, Ciprofloxacin,

Difloxacin, Enoxacin, Reroxacin, Fiumequine, Lomefloxacin, Miloxacln, Nalidixic Acid, Norfloxacin, Ofloxacin, Oxolinic Acid, Pefloxacin, Pipemidic Acid, Piromidic Acid, Rosoxacin, Temafloxacin and Tosuftoxacin;

Sulfonamides such as Acetyl Sulfamethoxypyrazine, Acetyl 10 Sulfisoxazole, Azosutfamide, Benzylsulfamide, Chloramine-B, Chloramine-T, Dichloramine T, Formosulfathiazole, Nz Formyisulfisomidine, N2 -p-D-Glucosylsulfanilamlde, Mafenide, 4'-(Methylsulfamoyl)sulfani!anilider p-Nitrosulfathiazole, Noprylsulfamide, Phthalylsulfacetamide, Phthalylsulfathiazole, Salazosulfadimidine, Succinylsulfathiazole, 15 Sulfabenzamide, Sulfacetamide, Sulfdchlorpyridazine, Sulfachrysoidine, Sulfacytine, Sulfadiazine, Suifadicramide, Sulfadimethoxine, Sulfadoxine, Sulfaethidole, Sulfaguanidine, Sulfaguanol, Suifalene, Sulfaloxic Acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfamethizole, Sulfamethomidine, Sulfamethoxazole, Sulfamethoxypyridazine, Sulfametrole, 20 Sulfamidochrysoidine, Sulfamoxote, Sulfanilamide,

Sulfanilamidornethanesulfonic Acid Triethanolamine Salt, 4-SuifanilamidosalicyJic Acid, N-Sulfanilylsulfanilamide, Sulfanilylurea, N-Sulfanilyl-3,4-xylamide, Sulfanltran, Sulfaperine, Sulfaphenazole, Sulfaproxyline, Sulfapyrazine, Suifapyridlne, Sulfasomizole, Sulfasymazine, 25 Sulfathiazole, Suifathiourea, Sulfatolamide, Sulfisomidine and Sulfisoxazole;

Sulfones such as Acedapsone, Acediasulfone, Acetosulfone Sodium, Dapsone, Diathymosulfone, Glucosulfone Sodium, Solasulfone, Succisulfone, Sulfanilic Acid, p-SuIfanilylbenzylamine, ρ,ρ-Sulfonyldlaniline- N.N'digalactoside, Sulfoxone Sodium and Thiazolsulfbne; and 30 others such as Clofoctol, Hexedine, Methenamine, Methenamine

Anhydromethylene-cltrate. Methenamine Hippurate, Methenamine Mandelate, Methenamine Suffosalicylate, Nitroxoline and Xibomoi; -70- 2015215955 21 Aug 2015

Anticholinergics such as Adiphenine Hydrochloride, Alverine, Ambutonomium Bromide, Aminopentamide, Amixetrine, Amprotropine Phosphate, Anisotropine Methylbromide, Apoatropine, Atrapine, Atropine N-Oxide, Benactyzine, Benapryzine, Benzetimide, Benzilonlum Bromide, 5 Benztropine Mesylate, Bevonium Methyt Sulfate, Bjperiden, Butropium Bromide, N-Butylscopoiammonium Bromide, Buzepide, Camylofine, Caramiphen Hydrochloride, Chlorbenzoxamine, Chlorphenoxamine, Cimetropium Bromide, Clldinlum Bromide, Cyclodrine, Cyctonium Iodide, Cycrimine Hydrochloride, Deptropine, Dexetlmide, Dibutoline Sulfate, 10 Dicyclomine Hydrochloride, Diethazine, Dlfemerine, Dihexyverine, Diphemanil Methylsulfate, N-(1,2-DIphenylethyl) nicotinamide, Dipiproverlne, Diponium Bromide, Emepronium Bromide, Endobenzyline Bromide, Ethopropazlne, Ethybenztroplne, Eihylbenzbydramine, Etomidoiine, Eucatropine, Fenpiverinium Bromide, Fentonium Bromide, Flutropium Bromide, 15 Glycopyrrofate, Heteronium Bromide, Hexocycilum Methyl Sulfate, Homatropine, Hyoscyamine, Ipratropium Bromide, Isopropamide,

Levomepate, Mecloxamine, Mepenzolate Bromide, Metcaraphen, Methanthellne Bromide, Methixene, Methscopolamine Bromide, Octamylamine, Oxybutynin Chloride, Oxyphencyciimine, Oxyphenonium 20 Bromide, Pentaplperide, Penthienate Bromide, Phencarbamide,

Phengtutarimide, Pipenzolate Bromide, Piperidolate, Piperilate, Poldine Methysulfate, Pridinol, Prifinium Bromide, Procyclidlne, Propantheline Bromide, Propenzolate, Propyromazlne, Scopolamine, Scopolamine N-Oxlde, Stilonium Iodide, Stramonium, Sultroponfum, Thihexinol, Thiphenamil, 25 Tfemonium Iodide, Timepidium Bromide, Tfquizlum Bromide, Tridihexethyl Iodide, Trihexyphenidyl Hydrochloride, Tropacine, Tropenzile. Tropicamide, Trospium Chloride, Valethamate Bromide and Xenytropium Bromide: Anticonvulsants such as Acetylpheneturide, Albutoin, Aloxidone, Aminoglutethlmide, 4-Amino-3-hydroxybutyric Acid, Atrolactamide, Bectamide, 30 Buramate, Calcium Bromide, Carbamazepine, Cinromide, Clomethiazole, Clonazepam, Decimemide, Diethadione, Dimethadione, Doxenitoin,

Eterobarb, Ethadione, Ethosuxlmlde, Ethotoin, Fluoresone. Garbapentin, 5- -71- 2015215955 21 Aug 2015

Hydroxytryptophan, Lamotrigine, Lomactil, Magnesium Bromide, Magnesium Sulfate, Mephenytoin, Mephobarbital, Metharbital, Methetoln, Methsuximfde, S-Methyl-5-(3-phenanthryl)hydantoin, 3-MethyI-5-phenyihydantoin, Narcabarbital, Nimetazepam, Nitrazepam, Paramethadione, Phenacemlde, 5 Phenetharbital, Pheneturide, Phenobarbital, Phenobarbital Sodium,

Phensuximlde, Phenylmethyibarbiturlc Acid, Phenytoin, Phethenylate Sodium, Potassium Bromide, Pregabatln, Primidone, Progabide, Sodium Bromide, Sodium Valproate, Solanum, Strontium Bromide, Suclofenide, Sulthfame, Tetrantoin, Tiagabine, Trimethadione, Valproic Acid, Valpromide, Vlgabatrin 10 and Zonisamide;

Antidepressants, including: Bicyciics such as Binedaline, Caroxazone, Citalopram, Dimethazan, indatpine, Fencamine, Fiuvoxamine Maleate, tndeloxazine Hydrochloride, Nefopam, Nomifensine, Oxitriptan, Oxypertine, Paroxetine, Sertraline, Thiazesim, Trazodone, Venlafaxine and Zometapine; 15 Hydraades/Hydrazines such as Benmoxine, Iproclozide, Iproniazid,

Isocarboxazid, Nialamide, Octamoxtn and Phenelzine;

Pyrroiidones such as Cotinine, Rolicyprine and Rolipram;

Tetracyclics such as Maprotiline, Metralindole, Mianserin and Oxaprotillne; 20 Tricyclics such as Adinazolam, Amitriptyline, Amitriptyirnoxide,

Amoxapine, Butriptyline, Clomipramine, Demexiptiline, Desipramine, Dibenzepin, Dimetracrine, Dothiepin, Doxepin. Fluactzine, Imlpramine, imipramine N-Oxide, Iprindole, Lofepramine, Melitracen, Metapramlne, Nortriptyline, Noxiptilin, Opipramol, Pizotyline, Proplzepine, Protriptyline, 25 Quinupramine, Tlaneptine and Trimipramine; and others such asAdraffnii, Benactyzlne, Bupropion, Butacetin, Deanol, Deanol Acegiumate, Deanol Acetamidobenzoate, Dioxadrol, Etopertdone, Febarbamate, Femoxetine, Fenpentadiol, Fluoxetine, Fiuvoxamine, Hematoporphyrin, Hypercinin, Levaphacetoperane, Mediibxamine, Minaprine, 30 Moclobemide, Oxaflozane, Piberaline, Prolintane, Pyrisuccideanol, Rubidium Chloride, Sulpiride, Sultopride; Tenlioxazine, Thozaiinone, Tofehacin, Toloxatone, Tranylcypromine, L-Tryptophan, Viloxazine and Zimeldine; -72- 2015215955 21 Aug 2015

Antldiabetics, including; Biguanides such as Buformin, Metformin and Phenlbimin;

Hormones such as Glucagon, Insulin, Insulin Injection, Insulin Zinc Suspension, Isophane Insulin Suspension, Protamine Zinc Insulin Suspension 5 and Zinc Insulin Crystals;

Sulfonylurea derivatives such as Acetohexamide, 1-Butyl-3-metanilylurea, Carbutamide, ChJorpropamide, Glibomuride, Glidazide, Glipizide, Gliquidone, Glisoxepid, Glyburide, Glybuthiazol(e), Glybuzole, Glyhexamide, Glymldine, Giypinamide, Phenbutamide, Tolazamide, 10 Tolbutamide and TolcycIamEde; and others such as Acarbose, Calcium Mesoxalate and Mrglitol;

Antidiantieal drugs such as Acetyltannic Acid, Albumin Tannate, Alkofanone, Aluminum Salicylates-Basic, Catechin, Difenoxin, Diphenoxylate, Lfdamldine, Loperamide, Mebiquine, Trillium and.Uzarln; . 15 Antidiuretics such as Desmopressin, Felypressin, Lypressin, O rn i pressin, -Oxycinchophen, Pituitary-Posterior, Terlipressin and Vasopressin;

Antfestrogens such as Delmadinone Acetate, Ethamoxytrlphetoi, Tamoxifen and Toremrfene; ' 20 Antifungal drugs (antibiotics), including: Polyenes such as

Amphotericin-B, Candlctdin, Dermostatin, Filipin, Fungichromin, Hachimycin, Hamycin, Lucensomycin, Mepartricln, Natamycin, Nystatin, Pecilocin and Perimycin; and others such as Azaserine, Griseofulvin, OHgomycins,

Neomycin Undecylenate, Pyrroinitrin, Siccanin, Tubercidin and VIridin; 25 Antifungal drugs (synthetic), Including; AJIylamines such as Naftifine and Terbinafine;

Imidazoles such as Bifonazole, Butoconazole, Chlordantoln, Chtormidazole, Cloconazole, Clotrimazole, Econazoie, Eniiconazole, Fentlconazole, isoconazole, Ketoconazole, Miconazole, Omoconazole, 30 Oxiconazole, Nitrate, Sulconazole and Tioconazole;

Triazoles such as Fluconazole, Itraconazole and Terconazole; and 2015215955 21 Aug 2015 -73- others such as Acrisorcin, Amorolflne, Biphenamine, Bromosallcylchloranilrde, Buclosamide, Calcium Propionate, Chlophenesfn, Cfcloplrox, Cloxyquin, Coparafflnate, Dramthazoie, Dihydrochloride, Exaiamide, Flucytosine, Halethazole, Hexetidine, Loflucarban, Nlfunatel, 5 Potassium iodide, Propionic Acid, Pyrithione, Salicyianilide, Sodium Propionate, Sulbentlne, Tenonttrozole, Tolciclate, Tollndate, Tolnaftate, Tricetln, Ujothion, Undecylenic Add and Zinc Propionate;

Antiglaucoma drugs such as Acetazoiamide, Befunolof, Betaxolol, Bupranolol, Carteolol, Dapiprazoke, Dlchlorphenamide, Dipivefrin, 10 Epinephrine, Levobunolol, Methazolamlde, Metipranolol, Pilocarpine, Pindolol and Timolol;

Antigonadotrapins such as Danazol, Gestrinone and Paroxypropione;

Antigout drugs such as Altopurinol, Carprofen, Colchicine, Probenecid and Sulfinpyrazone; 15 Antihistamines, including: Aikylamine derivatives such as Acrivastine,

Bamipine, Brompheniramine, Chlorpheniramine, Dlmethindene, Metron S, Pheniramine, Pyrrobutamine, Thenaldine, Toipropamine and Triproiidine;

Aminoalkyl ethers such as Bietanautine, Bromodiphenhydramine, Carbtnoxamine, Clemastine, Diphenlypyraline, Doxylamine, Embrammine, 20 Medrylamine, Mephenphydramine, p-Methyldiphenhydramine, Orphenadrine, Phenyftoloxamine, Piprinhydrinate and Setasine;

Ethylenediamine derivatives such as Alloclamide, p-Bromtripelennamine, Chioropyramine, Chlorothen, Histapyrrodine, Methafurylene, Methapheniiene, Methapyrilene, Phenbenzamine, Pyrllamine, 2S Talastine, Thenyldiamine, Thonzylamine Hydrochloride, Tripelennamlne and Zolamine;

Piperazines such as Cetirizine, Chiorcydizine, Cinnarizine, Clocinizine and Hydroxyzine;

Tricyclics, including: Phenothlazines such as Ahistan, Etymemazine, 30 Fenethazine, N-Hydroxyethylpromethazlne Chloride, Isopromethazine,

Mequitazine, Promethazine, Pyrathiazine and Thlazlnamium Methyl Sulfate; and -74- 2015215955 21 Aug 2015 others suph as Azatadine, Clobenzepam, Cyproheptadine, Deptropine, Isothipendyl, Loratadine and Prothipendyl; and other antihistamines such as Antazoline, Astemizole, Azelastine, Cetoxime, Clemizole, Ctobenztropine, Diphenazollne, Diphenhydramine, 5 Fluticasone Propionate, Mebhydroline, Phenindamine, Terfenadlne and Tritoqualine;

Antihyperlipoproteinemics, including: Aryioxyalkanoic acid derivatives such as Beclorbrate, Bazafibrate, Binifibrate, Ciprofibrate, Ciinoflbrate, Clofibrate, Clofibric Acid, Etonfrbrate, Fenoflbrate, Gemfibrozil, Nicofibrate, 10 Pirifibrate, Ronifibrate, SImfibrate and Theofibrate;

Bile acid sequesterants such as Cholestyramine Resin, Colestipol and Polidexide; HMG CoA reductase inhibitors such as Fluvastatin, Lovastatin, Pravastatin Sodium and Simvastatin; 15 Nicotinic acid derivatives Aluminum Nicotinate, Acipimox, N [central,

Nicoclonate, Ntcomol and OxinlacicAcfd;

Thyroid hormones and analogs such as Etiroxate, Thyropropic Acid and Thyroxine; and others such as Aclfran, Azacosterol, Benfluorex, β-Benzalbutyramide, 20 Carnitine, Chondroitin Sulfate, Ciomestone, Detaxtran, Dextran Sulfate Sodium, 5l8,11,14,1T’-Elcosapentaenofc Acid, Eritadenine, Furazboi,

Meglutol, Melinamfde, Mytatrlenediol, Ornithine, γ-Oryzanol, Pantethine, Penataerythritol Tetraacetate, o-Phenylbutyramlde, Pirozadil, Probucol, a-Sitosterol, Sultosilic Acid, Piperazine Salt, Tladenol, Triparanol and Xenbucin; 25 Antihypertensive drugs, including: Arylethanolamine derivatives such as Amosulaiol, Bufuralol, Dilevalol, Labetalol.'Pronethalol, Sotatoi and Sulfinalol;

Aryloxypropanolamin© derivatives such as Acebutoloi, Alprenolol, Arotinolol, Atenofol, Betaxolol, Bevantolol, Bisoproloi, Bopindolol, Bunitrolol, 30 Bupranoiol, Butofilolol, Carazolol, Cartezofol, Carvedilol, Ceilprolol, Cetamolol, EpanoIoJ, Indenoiol, Mepindolol, Mettpranolol, Metoprolol, Moprolol, Nadolol, -75- 2015215955 21 Aug 2015

Nipradilof, Oxprenolol, Penbutolol, Pindolol, Propranolol, Taltnolol, Tetraolol, Timolol and Toliprolol:

Benzothiadlazins derivatives such as Aithiazide, Bendroflumethiazide, Benzthiazide, Benzylhydrochlorothiazide, Buthiazide, Chlorothiazide, 5 Chlorthalidone, Cyclopenthlazide, Cyolothiazide, Diazoxide, Epithiazide, Ethiazide, Fenquizone, Hydrochlorothiazide, Hydroflumethiazide, Methyclothiazide, Meticrane, Metolazone, Parafiutizide, Poiythiazide, Tetrachlormethiazfde and Trichlormethiazide; N-Carboxyalkyl (peptide/lactam) derivatives such as Alacepril, 10 Captoprii, Cilazapril, Delapril, Enalapril, Enalaprilat, Fosinopril, Lisinopril, Moveitiprfl, Perindopril, Quinapril and Ramiprll;

Dihydropyridine derivatives such as Amlodlpine, Felodipine, isradlpine, Nicardipine, Nifedipine, Nllvadlpine, Nisoldipine and Nitnendipime;

Guanidine derivatives such as Bethanidine, Debrisoquin, Guanabenz, 15 Guanacline, Guanadrei, Guanazodine, Guanethidine, Guanfacine, Guanochlor, Guanoxabenz and Guanoxan;

Hydrazines and phthalazines such as Budrafazine, Cadralazine, Dihydralazine, Endralazine, Hydracarbazine, Hydralazine, Pheniprazine, Pildralazine and Todralazine; 20 Imidazole derivatives such as Clonidine, Lofexidine, Phentolamine,

Phentolamine Mesylate, Tiamenidine and Tolonidine;

Quaternary ammonium compounds Azamethonium Bromide, Chlorisondamlne Chloride, Hexamethonium, Pentacynium Bis(methyl sulfate), Pentamethonium Bromide, Pentolinlum Tartate, Phenactopinium Chloride and 25 Trimethldiunum Methosulfate;

Quinazoiine derivatives such as Alfuzosin, Bunazosin, Doxazosin, Prasosin, Terazosin and Trimazosfn;

Reserpine derivatives such as Bietaserpine, Deserpidine, Rescinnamine, Reserpine and Syrosingopine; 30 Sulfonamide derivatives such as Ambuside, Clopamide, Furosemide,

Indapamide., Quinethazone, Tripamide andXipamlde; and -76- 2015215955 21 Aug 2015 others such as AJmaline, γ-Aminobutyric Acid, Bufeniode, Candesattan, Chlorthalidone, Cicletaine, Ciclosidomine, Cryptenamine Tannates. Eprosartan, Fenoldopam, Flosequinan, Indoramin, Irbesartan, Ketanserm, Losartan, Metbutamate, Mecamylamine, Methyldopa, Methyl 4-PyridyI Ketone 5 Thfosemicarbarzone, Metoiazone, Minoxidil, Muzolimine, Pargyllne,

Pempldine, Pinacidil, Ptperoxan, Primaperone, Protoveratrines, Raubasine, Rescimetoi, Rllmenfdene, Saralasin, Sodium Nitroprusside, TIcrynafen, Trimethaphan Camsylate, Tyrosinase, Urapidil and Valsartan;

Antihyperthyrofds such as 2-Amino-4-methylthiazole, 2-Aminothiazoie, 10 Carbimazole, 3,5-Dibromo-L-tyrosine, 3,5-Diiodotyrosine, Hinderin, Iodine, lothlouracil, Methimazole, Methylthlouracii, Propylthiouracil, Sodium Parchlorate, Thibenzazoline, Thiobarbltal and 2-Thiouracil;

Antihypotensive drugs such as Amezinium Methyl Sulfate, Angiotensin Amide, Dimetofrine, Dopamine, Etifelmin, Ettlefrin, Gepefrine, Metaraminoi, 15 Mfdodrine, Norepinephrine, Pholedrinead and Synephrine;

Antihypothyroid drugs such as Levothyroxlne Sodium, Liothyronine, Thyroid, Thyroidin, Thyroxine, Tiratricol and TSH;

Anti-Inflammatory (non-steroidal) drugs, including: Aminoarylcarboxytic acid derivatives such as Enfsnamic Acid, Etofenamate, Flufenamic Acid, 20 Isonixin, Mectofenamic Acid, Mefanamlc Acid, NHJumic Acid, Talnlffumate,

Terofenamate and Tolfenamic Acid;

Arylacetic acid derivatives such as Acemetacin, Alclofenac, Amfenac, Bufexamac, Cinmetacin, Clopirac, Diclofenac Sodium, Etodolac, Felbinac, Fenclofenac, Fenclorac, Fenclozic Acid, Fentiazac, Glucametacin, Ibufenac, 25 indomethacin, Iscfezolac, Isoxepac, Lonazolac, Metiazinic Acid,

Oxametacine, Proglumatacin, Sulindac, Tiaramide, Tolmetrn and Zomepirac;

Arylbutyrfc acid derivatives such as Bumadizon, Butibufen, Fenbufen and Xenbucin;

Aryicarboxylto acids such as Clidanac, Ketorolac and Tinoridine; 30 Arylpropionic acid derivatives such as Aiminoprofen, Benoxaprofen,

Bucloxic Acid, Carprofen, Fenoprofen, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Indoprofen, Ketoprofen, Loxoprofen, Miroprofen, Naproxen, 2015215955 21 Aug 2015 -77-

Oxaprozin, Piketoprofen, Pirprofen, Pranoprofen, Protizinic Acid, Suprofen and Tiaprofenic Acid;

Pyrazoles such as Dtfenamizole and Epirizole;

Pyrazolones such as Apazone, Benzplperylon, Feprazone, 5 Mofebutazone, Morazone, Oxyphenbutazone, Phenybutazone, Pipebuzone, Propyphenazone, Ramifenazone, Suxibuzone and Thiazolinobutazone;

Salicylic acid derivatives such as Acetaminosalol, Aspirin, Benorylate, Bromosaligenin, Calcium Acetylsalicylate, Diflunisal, Etersalate, Fendosali Gentisic Acid, Glycol Salicylate, Imidazole Salicylate, Lysine Acetylsalicylate, 10 Mesalamine, Morpholine Salicylate, 1-Narhthyl Salicylate, Olsalazine, Parsalmlde, Phenyl Acetylsalicylate, Phenyl Salicylate, Salacetamide, Sallcylamine Ο-Acetic Acid, Salicylsulfuric Acid, Salsalate and Sulfasalazine;

Thlaztnecarboxamides euch as Droxicam, Isoxicam, Piroxicam and Tenoxicam; and 15 others such as s-Acetamidocaprolc Acid, S-Adenosylmethlonine, 3-

AmIno-4-hydroxybutyricAcid, Amixetrlne, Bendazac, Benzydamine, Bucolome, Difenpiramide, Dltazol, Emorfazone, Guaiazulene, Nabumetone, Nlmesulide, Orgoteln, Oxaceprol, Paranyline, Perisoxal, Pifoxime, Proquazone, Proxazole and Tenidap; 20 Antimalarial drugs such as Acedapsone, Amodiaquln, Arteethsr,

Artemether, Artemisinin, Artesunate, Bebeerine, Berberine, Chlrata, Chiorguanide, Chloroqulne, Chlorproguanil, Cinchona, Cinchonidine, Cinchonine, Cycloguanil, Gentiapicrin, Halofantrine, Hydroxychloroquine, Mefloquine Hydrochloride, 3-MethyIarsacetin, Pamaquine, Plasmocld, 25 Primaquine, Pyrimethamine, Qulnacrine, Quinine, Quinine Bfsulfate, Quinine carbonate, Quinine Dihydrobromlde, Quinine Dihydrochloride, Quinine Ethylcarbonate, Quinine Formate, Quinine Gluconate, Quinine Hydriodlde, Quinine Hydrochloride, Quinine Salicylate, Quinine Sulfate, Quinine Tannate, Quinine Urea Hydrochloride, Quinocide, Quinoline and Sodium Arsenate 30 Diabasfc;

Antimigraine drugs such as Alpiropride, Dihydroergotamine,'Eletriptan, Ergocomine, Ergocorninine, Ergocryptine, Ergot, Ergotamine, Fiumedroxone -78- 2015215955 21 Aug 2015 acetate, Fonazine, Llsuride, Methysergid(e), Naratriptan, Oxetorone, Pfeotyline, Rizatriptan and Sumatriptan;

Antinauseant drugs such as Acetylleucine Monoethanolamine, Alizapride, Benzqufnamide, Bietanautine, Bromopride, Buclizine, 5 Chlorprortiazine, Clebopride, Cyclizine, Dimenhydrinate, Dipheniodol, Domperidone, Granisetron, Meclizine, Methailtal, Metoclopramlde, Metoplmazine, Nabilone, Ondansteron, Oxypendyl, Pipamazine, Piprinhydrinate, Prochlorperazine, Scopolamine, Tetrahydrocannabfnols, Thiethylperazine, Thioproperzaine and Trimethobenzamide; 10 Antineoplastic drugs, including: Alkylating agents, such as AJkyi sulfonates such as Busuifan, Improsulfan and Piposulfan;

Aziridines such as Benzodepa, Carboquone, Meturedepa and Uredepa;

Ethylenimines and methylmelamines such as Altretamine, 15 Triethylenemelamine, Triethylenephosphoramide,

Triethylenethiophosphoramide and Trimethylotomelamine;

Nitrogen mustards such as Chlorambucil, Chlornaphazine, Chdophosphamide, Estramustine, Ifosfamide, Mechlorethamine, . Mechlorethamine Oxide Hydrochloride, Melphalan, Novembichin, 20 Phenesterine, Prednimustine, Trofosfamtde and Uracii Mustard;

Nitrosoureas such as Carmustine, Chiorozotocin, Fotemustine,

Lomustine, Nimustine and Ranimustine; and ethos such as Camptotheoin, Daoarbazine, Mannomustine, Mitobronitol, Mitolactol and Pipobroman; 25 Antibiotics such as AciacJnomycins, Actinomycin Fi, Anthramycin,

Azaserine, Bleomycins, Cactinomycin, Carubicin, Carzrnophilin, Chromomycins, Dactinomycin, Daunorubfcin, 6-Dlazo-S-oxo-L-norleucine, Doxorubicin, Epirubicin, Mitomycins, Mycophenolic Acid, Nogalamycln, Olivomycins, Peplomycin, Plicamycin, Porfiromycin, Puromycin, Streptonigrfn, 30 Streptozocin, Tubercidln, Ubenimex, Zinostatin and Zorubicin;

Antimetabolites, including: Folic add analogs such as Denopterin, Methotrexate, Pteropterin and Trimetrexate; -79- 2015215955 21 Aug 2015

Puifne analogs such as Fludarablne, 6-Mercaptopurine, Thiamiprine and Thioguanaine; and

Pyrimidine analogs such as Ancitabine, Azadtldlne, 6-Azauridine, Camnofur, Cytarabine, Doxifluridine, Enocitablne, Floxuridine Fluroouracil and S Tegafen

Enzymes such as L-Asparaginase; and others such as Aceglafone, Amsacrhne, Bestrabucll, Bisantrene, Bryostatin 1, Carboplatin, Cisplatln, Defofamide, Demecolclne, Diaziquone, Elfomithlne, Elllptinium Acetate, Etoglucld, Etoposlde, Gallium Nitrate, 10 Hydroxyurea, Interferon-a, lnterferon-β, Interferon-γ, lnterieukine-2, Lentinan, Letrozole, Lonrdamine, Mitoguazone, Mitoxantrone, Mopidamol, Nitracrine, Pentostatin, Phenamet, Plrarubicin, Podophyllinicc Acid, 2-Ethythydrazide, Polynitrocubanes, Procarbazine, PSK7, Razoxane, Sizofiran, Spirogermanium, Taxol, Tenlposide, Tenuazonic Acid, Triaziquone, 2.2'.2"-15 Trich I orotriethy lamine, Urethan, vinblastine, Vincristine, Vindesine and

Vinorelbine;

Antineoplastic (hormonal) drugs, including: Androgens such as Calusterone, Dromostanolone Propionate, Epitiostanol, Mepitiostane and Testolactone; 20 Antiadrenals such as Aminoglutethimfde, Mitotane and Trllostane;

Antiandrogens such as Flutamlde and Nilutamide; and

Antiestrogens such as Tamoxifen and Toremlfene;

Antineoplastic adjuncts including foiic add replenishes such as Frolinic

Acid; 25 Antiparkinsonian drugs such as Amantadine, Benserazide,

Bietanautine, Biperiden, Bromocriptine, Budipine, Cabergoline, Carbidopa, Deprenyl (a/k/a L-deprenyl, L-deprenil, L-deprenaline and selegiline), Dexetimide, Diethazine, Diphenhydramine, Droxidopa, Ethopropazine,

Ethyl benzhydramine, Levodopa, Naxagolide, Pergolide, Piroheptine, 30 Pramipexole, Pridinol, Prodipine, Quinpirole, Remacemide, Ropinirole. Terguride, Tigloidine and Trihexyphenidyl Hydrochtoride; -80- 2015215955 21 Aug 2015

Antipheachromocytoma drugs such as Metyrosine, Phenoxybenzamine and Phentolamine;

Antipneumocystis drugs such as Effornithine, Pentamidine and Sulfamethoxazole; 5 Antiprostafic hypertrophy drugs such as Gestonorone Caproate,

Mepartricin, Oxendolone and Proscar7;

Antiprotozoal drugs (Leshmanla) such as Antimony Sodium Gluconate, Ethylstibamine, Hydroxystilbamidine, N-Methylglucamine, Pentamidine, Stilbamidine and Urea Stibamine; 10 Antiprotozoal drugs (Trichomonas) such as Acatarsone, Aminrtrozole,

Anisomydn, Azanidazole, Forminitrazole, Furazolidone, Hachimycln, Lauroguadine, Mepartricin, Metronidazole, Niiuratel, Nifuroxime, Nimorazole, Secnidazote, Silver Picrate, Tenonitrozoie and Tinfdazole;

Antiprotozoal drugs (Trypanosma) such as Benznidazole, Eflomithine, 15 Melarsoprol, Nifurtimox, Oxophenarsine, Hydrochloride, Pentamidine, Propamidine, Puromycin, Quinapyramine, Stilbamidine, Suramin Sodium, Trypan Red andTryparasmide;

Antipuritios such as Camphor. Cyproheptadine, Dichlorisone, Glycine, Halometasone, 3-Hydroxycamphor, Menthol, Mesulphen, Methdilazine, 20 Phenol, Polidocanol, Risocaine, Spirit of Camphor, Thenaldine, Tolpropamine and Trimeprazine;

Antipsoriatic drugs such as Acitretin, Ammonium Salicylate, Anthralin, 6-Azauridine, Bergapten(e), Chrysarobln, Etretinate and Pyrogallol;

Antipsychotic drugs, Including: Butyrophenones such as Benperidol, 25 Bromperidol, Droperidol, Fluanisone, Haloperidol, Melperone, Moperone, Pipamperone, Sniperone, Timiperone and Trifluperidol; .

Phenothlazines such as Acetophenazlne, Butaperazine, Carphenazlne, Chlorproethazine, Chiorpromazine, Ciospirazlne, Cyamemaztne, Dlxyrazine, Fluphenazine, Imidopazine, Mepazlne, Mesoridazine, Methoxypromazine, 30 Metofenazate, Oxaflumazine, Perazine, Pericyazine, Perimethazine,

Perphenazine, Piperacetazfne, Pipotiazine, Prochlorperazine, Promazine, -81- 2015215955 21 Aug 2015

Sulforidazine, Thiopropazate, Thioridazine, Trifluoperazine and T rif lupromazine;

Thioxanthenes such as Chlorprolhixene, Clopenthixol, Flupentixol and

Thiothixene; 5 other tricyclics such as Benzquln'amide, Carpipramine, Clocapramine,

Ciomacran, Clothiapine, Clozapine, Opipramol, Prothipendyl, Tetrabenazine, and Zotepine; and others such as Allzapride, Amlsulpride, Buramate, Fiuspirilene, Molindone, Penfluridol, Pimozide, Sptrilene and Sulpiride; 10 Antipyretics such as Acetaminophen, Acetaminosalol, Acetanilide,

Aconine, Aconite, Aconitine. Atdofenac, Aluminum Bis(acetylsalicylate), Aminochiorthenoxazin, Aminopyrine, Aspirin, Benorylate, Benzydamine, Berberine, p-Bromoacetanilide, Bufexamac, Bumadizon, Calcium Acetysallcylate, Chlorthenoxazin(e), Choline Salicyiatp, Ciidanac, 15 Dihydroxyaluminum Acetylsalicylate, Dipyrocetyl, Dipyrone, Epirizole,

Etersalate, imidazole Salicylate, Indomethacin, tsofezolac, p-Lactophenetide, Lysine Acetylsalicylate, Magnesium Acetylsalicylate, MeclofenamlcAcid, Morazone, Morpholine Salicylate, Naproxen, Nifenazone, 51-Nitro-2'-propoxyacetanilide, Phenacetin, Phenicarbazide, Phenocoil, Phenopyrazone, 20 Phenyl Acetylsalicylate, Phenyl Salicylate, Pipebuzone, Propacetamoi,

Propyphenazone, Ramifenazone, Salacetamide, Saiicylamide O-Acetic Add, Sodium Salicylate, Sulfamipyrine, Tetrandrineand Tinoridine;

Antirickettslal drugs such as p-Aminobenzoic Acid, Chloramphenicol, Chloramphenicol Palmitate, Chloramphenicol Pantothenate and Tetracycline; 25 Antiseborrheic drugs such as Chlonoxine, 3-O-Lauroylpyridoxol

Diacetate, Plroctone, Pyrithione, Resorcinol, Selenium Sulfides and Tloxolone; . Antiseptics, including: Guanidines such as Alexidine, Ambazone, Chlorhexidine and Picloxydine; · 30 Halogens and halogen compounds such as Bismuth Iodide Oxide,

Bismuth iodosubgailate, Bismuth Tribromophenate, Bornyl Chloride, Calcium iodste, Chlorinated Lime, Cloflucarban, Flurosalan, Iodic Acid, Iodine, Iodine -82- 2015215955 21 Aug 2015

Monochloride, Iodine Trichloride, Iodoform, Methenamine Tetraiodine, Oxychlorosene, Povidone-Iodine, Sodium Hypochlorite, Sodium lodate, Symcfosene, Thymol Iodide, Triclocarban, Tridosan and Troclosene Potassium; 5 Mercurial compounds such as Hydragaphen, Meralein Sodium,

Merbramin, Mercuric Chloride, Mercuric Chloride, Ammonlated, Mercuric Sodium p-Phenoisuifonate, Mercuric Succinimlde, Mercuric Sulfide, Red, Mercurophen, Mercurous Acetate, Mercurous Chloride, Mercurous Iodide, Nitromersol, Potassium Tetratodomercurate(II), Potassium Triiodomercurate 10 (II) Solution, Thlmerfonate Sodium and Thlmerosal;

Nitrofurans such as Furazolidone, 2-(Methoxymethyl)-&amp;~nitrofuran, Nidroxyzone, Nifuroxime, Nifurzfde and Nitrofurazone;

Phenols such as Acetomeroctol, Bithfono!, Cadmium Salicylate, Carvacrol, Chloroxylenol, Clorophene, Cresote, Cresol(s), p-Cresol, Fenticlor, 15 Hexachlorophene, 1-Napthy! Salicylate, 2-Napthyl Salicylate, 2,4,6-Tribromo-m-cresol, and S'^'.S'-Trichlorosalicylanilide;

Quinolines such as Aminoquinuride, Benzoxiqulne, Broxyquinoline, Chloroxine, Chlorquinaldof, Cioxyquin, Ethylhydrocupreine, Euprocin, Halquinol, Hydrastlne, 8-HydroxquinoIine, 8-Hydroxqufnoline Sulfate and 20 lodochlorhydroxyquin; and others such as Aluminum Acetate Solution, Aluminum Subaoetate Solution, Aluminum Sulfate, 3-Amino-4-hydroxybutyric Acid, Boric Acid, Chiorhexidine, Chloroazodin. m-Cresyl Acetate, Cupric Sulfate, Dibromopropamidlne, Ichthammol, Negatol7, Noxytiolin, Omidazole, β-25 Propiolactone, α-Terpineol;

Antispasmodlc drugs such as Alibendol, Ambucetamide, Aminopromazlne, Apoatropine, Bevonium Methyl Sulfate, Bletamiverlne, Butaverine, Butropium Bromide, N-Butylscopolammonium Bromide, Caroverine, Cimetropium Bromide, Cinnamedrine, Clebopride, Coniine 30 Hydrobromide, Coniine Hydrochloride, Cyclonium Iodide, Dlfemerine, Diisopromine, Dioxaphetyl Butyrate, Diponium Bromide, Drofenine, Emepronium Bromide, Ethaverine, Feclemine, Fenalamide, Fenoverine, 2015215955 21 Aug 2015 -S3 -

Fenpiprane, Fenpiverinium Bromide, Fentonlum Bromide, Flavoxate, Flopropione, Gluconic Acid, Guaiactamlne, Hydramitrazine, Hymecromone, Leiopyirole, Mebeverine, Moxaverine, Nafiverine, Ociamylamine, Octaverine, Pentapiperide, Phenamacide Hydrochloride, Phlorogtuclnol, Pinaverium 5 Bromide, Piperiiate, Plpoxolan Hydrochloride, Pramiverln, Prifinium Bromide, Properidine, Propivane, Propyromazine, Prozapine, Racefemine, Rociverine, Spasmolytol, Stilonlum Iodide, Suliroponlum, Tiemonium Iodide, Tiquizium Bromide, Tiropramide, Treplbutone, Tricromyl, Trifolium, Trimebutine, N,N-ΙΤπΓπβϋΊγΡβ,^ϊρηθηγΙ-ρΓορνΙθηπΙηβ, Tropenzite, Trospium Chloride and 10 Xenytroplum Bromide;

Antithrombotic drugs such as AnagreUde, Argatroban, Cilostazol, Chiysoptin, Daltroban, Defibrotide, Enoxaparin, Fraxiparine7, Indobufen, Lamoparan, Qzagrel, Picotamtde, Pl’afibride, Reviparin, Tedelparin, Ticlopidine, Triflusal and Warfarin; 15 Antitussive drugs such as Aitocamide, Amicibone, Benproperine,

Benzonatate, Bibenzonlum Bromide, 8romoform, Butamirate, Butethamate, Caramiphen Ethanedrsulfbnate, Carbetapentane, Chlophedianol, Clobutinol, Cloperastine, Codeine, Codeine Methyl Bromide, Codeine N-Oxide, Codeine Phosphate, Codeine Sulfate, Cyclexanone, Dextromethorphan, Dibunate 20 Sodium, Oihydrocodefne, Dihydrocodeinone Enol Acetate, Dimemoifan,

Dimethoxanate, a.a-DIphenyl^-piperidinepropanol, Dropropizine, Drotebanol, Epraz/none, Ethyl Dibunate, Ethylmorphine, Fominoben, Guiaiapate, Hydrocodone, Isoaminile, Levopropoxyphene, Morclofbne, Narceine, Normethadone, Noscapine, Oxeladin, Qxolamine, Phoicodine, Picoperine, 25 Pipazethate, Piperidione, Prenoxdiazine Hydrochloride, Racemethorphan, Taziprinone Hydrochloride, Tipepidine and Zipeprol;

Antiulcerative drugs such as Aceglutamide Aluminum Complex, s-Acetamidocaproic Acid Zinc Salt, Aceioxolone, Arbaprostil, Benexate Hydrochloride, Bismuth Subcitrate Sol (Dried), Carbenoxolone, Cetraxate, 30 Cimetldine, Enprostil, Esaprazole, Famotidine, Ftaxilide, Gefamate,

Guaiazulene, Irsogladine, Misoprostol, Nizatidine, Omeprazole, Ornoprosffl, y~ Oryzanol, Pifamine, Pirenzepine, Plaunotol, Ranitidine, Rioprostil, -84- 2015215955 21 Aug 2015

Rosaprostol, Rotraxate, Roxatidine Acetate, Sofalcone, Spizofurone, Sucralfate, Teprenone, Trlmoprostil, Thrithioane, Troxipide and Zoiimidine;

Anfiuroiithic drugs such as Acetohydroxamic Acid, Allopurlnol, Potassium Citrate and Succinimlde; 5 Antivenin drugs such as Lyovac7 Antivenin;

Antiviral drugs, including: Purines and pyrimldlnones such as Acyclovir, Cytarabine, Dideoxyadenoslne, Dideoxycytidine, Dldeoxyinosine, Edoxudine, Floxuridfne, Ganciclovir, Idoxurldine, Inosine Pranobex, MADU, Penciclovir, Trifluridine, Vidrarbine and Zidovudiine; and 10 others such as Acetytleucine Monoeihanolamine, Amantadine,

Amfdinomycin, Cosalane, Cuminaldehyde Thiosemicarbzone, Foscarnet Sodium, fmiquimod, Interferon-a, lnterferon-β, interferon-/, Kethoxal, Lysozyme, Methisazone, Moroxydine, Podophyltotoxin, Ribavirin, Rimantadine, Stallimycin, Statolon, Tromantadtne and Xenazoic Acid; 15 Anxiolytic drugs, including: Aryipiperazines such as Buspirone,

Gepirone, Ipsapirone and Tondospirone;

Benzodiazepine derivatives such as Alprazolam, Bromazepam, Camazepam, Chiordiazepoxide, Clobazam, Clorazepate, Chotiazepam, Cloxazofam, Diazepam, Ethyl Loflazepate, Etizolam, Fiutdazepam, 20 FJutazoiam, Flutoprazepam, Halazepam, Ketazolam, Lorazepam, Loxapine, Medazepam, Metadazepam, Mexazolam, Nordazepam, Oxazepam, Oxazolam, Pinazepam, Prazepam and Toiisopam;

Carbamates such as Cyciarbamate, Emylcamate, Hydroxyphenamate, Meprobamate, Phenprabamate and Tybamate; and 25 others such as Alpidem, Benzoctamine, Captodiamlne,

Chlormezanone, Etifoxine, Flesinoxan, FJuoresone, Glutamic Acid, Hydroxyzine, Lesopitrbn, Medoralurea, Mephenoxafone, Mirtazepine, Oxanamide, Phenaglycodol, Suridone and Zatosetron;

Benzodiazepine antagonists such as Flumazenll; 30 Bronchodilators, including: Ephedrine derivatives such as Albuterol,

BambuteroJ, Bitolterol, Carbuterol, CienbuteroJ, Cloiprenallne, Dioxethedrine, Ephedrine, Epinrphrine, EprozinoI.Etafedrine, Ethylnorepinephrine, Fenoterol, 2015215955 21 Aug 2015 -85-

Hexoprenaline, Jsoetharine, Isoproterenol, Mabuterol, Metaproterenol, N-Mettiytephedrine, Pirbuterol, Pracalerol, Protokylol, Reproterol, Rimiterol, Salmetenol, Soterenol, Terbutalineand Tulobuterol;

Quaternary ammonium compounds such as Bevonium Methyl Sulfate, 5 Clutropium Bromide, Ipratropium Bromide and Oxitropium Bromide;

Xanthine derivatives such as Acefylllne, Acelytline Piperazine, Ambuphylline, Aminophylline, Bamlfyiiine, choline Theophyllinate, Doxolylline, Dyphylline, Enproiylline, Etamiphyllin. Etofylline, Guaithylline, Proxyphylline, Theobromine, 1-Theobromineacetic Acid and Theophylline; and 10 others such as Fenspiride, Medibazine, Montekulast,

Methoxyphenanime, Tretoquinol and Zafirkulast;

Calcium channel blockers, Including: Arylalkylamines such as Bepridil, Ditlazem, Fendiline, Gallopanil, Prenylamine, Terodiline and Verapamil;

Dihydropyridine derivatives such as Felodipine, Isradipine, Nicardipine. 15 Nifedipine, Nilvadipfne, Nimodipine, Nisoldipine and Nitrendipine;

Piperazine derivatives such as Cinnarizine, Flunarisine and Lidoflazine; and others such as Bencyclane, Etatenone and Perhexiline;

Calcium regulators such as Caicrfedioi, Calcitonin, Calcitriol, Clodronlc 20 Acid, Dihydratachysterol, Elcatonin, Etidronic Acid, Ipriflavone, Pamidronic Acid, Parathyroid Hormone and Teriparatide Acetate;

Cardiotonics such as Acefylline, Acetyldigititoxins, 2-Amlno-4-picoline, Amrinone, Benfurodll Hemlsuccinate, Buclasdesine, Cerberoside, Camphotamide, Convallatoxin, Cymarin, Denopamine, Deslanoside, DItalin, 25 Digitalis, Digitoxin, Digoxin, Dobutamine, Dopamine, Dopexamine,

Enoximone, Erythrophlelne, Fenalcomlne, Gitalin, Gitoxin, Glycocyamine, Heptaminol, Hydrastinine, Ibopamine, Lanotodises, Metamivam, Milrinone, . Neriifalin, Oieandrin, Ouabain, Oxyfedrine, Prenalterol, Proscillaridin,

Resibufogenin, Scillaren, Scillarenin, Strophanthin, Sulmazole, Theobromine 30 and Xamoterol;

Chefating agents such as Deferozmine, Ditfocarb Sodium, Edetate Calcium Disodlum, Edetate Disodium, Edeate Sodium, Edetate Trisodium, -86- 2015215955 21 Aug 2015

Penicillamine, Pentetafe Calcium Trisodlum, Pentectic Acid, Succimer and Trientine;

Cholecystokinin antagonists such as Proglumide;

Choleiithoiytic agents such as Chenodlol, Methyl tert-Butyl Ether, 5 Monooctanoin and Ursodlol;

Choleretics such as Alibendof, Anethole Trithion, Azlntamide, Cholic Acid, Cicrotoic Acid, Clanobutin, Cyciobutyrol, Cyclovalone, Cynarin(e). Dehydrocholic Acid, Deoxycholic Acid, Dtmecrotic Acid, a-Ethylbenzyl Alcohol, Exiproben, Feguprol, Fenclbutirol, Fenipentol, Florantyrone, 10 Hymecromone, Menbutone, 3-(o-Methoxyphenyl)-2-phenylacrylic Acid, Metochalcone, Maquizone, Osalmld, Ox Bile Extract, 4.4-Oxydi-2-butanoI, Piprozolin, Prozaplrte. 4-Salicyloylmorpholine, Sincalide, Taurocholic Acid, Timonaclc, Tocamphyl, Trepibutone and Vanitiolide;

Cholinergic agents such as Acedidine, Acetylcholine Bromide, 15 Acetyicholide Chloride, Adatonium Napadisiiate, Benzpyrinium Bromide,

Bethanechol chforide, Carbachol, Carpronium chloride, Demecarium Bromide, Dexpanthenol, Diisopropyl Paraoxon, Echothiophate iodide, Edrophomium chloride, Eseridine, Furtrethonium, isoflurophate, Methacholine chloride, Muscarine, Neostigmine, Oxapropanium Iodide, Physostigmfne and 20 Pyridostigmine Bromide;

Cholinesterase inhibitors such as Ambenonium Chloride, Distigmine Bromide and Gaianthamine;

Choiinesterase reactivators such as Obidoximine Chloride and Pralidoxime Chloride; 25 Central nervous system stimulants and agents such as Amineptine,

Amphetimine, Amphetamlnil, Bemegride, Benzphetamine, Brucine, Caffeine, Chlorphentermine, Clofenciclan, Clortermlne, Coca, Demanyl Phosphate, Dexoxadrol, Dextroamphetamine Sulfate, Diethlpropron, N-Ethylamphetamine, Ethamivan, Etifelmin, Etryptamine, Fencamfamine, 30 Fenethylline, Fenosolone, Flurothyl, Gaianthamine, Hexacydonate Sodium, Homocamfin, Mazindol, Megexamide, Methamphetamlne, Methylphenidate, Nikethamide, Pemoline, Pentylenetetrazols, Phenldimetrazine, 2015215955 21 Aug 2015 - 87-

Phenmetrazine, Phentermine, Picrotoxin, Pipradrol, Prolintane and Pyrovalerone;

Decongestants such as Amidephrine, Cafaminol, Cyclopentamine, Ephedrine, Epinephrine, Fenoxazoline, Indanazollne, Metizolfne, 5 Naphazoline, Nordefrin Hydrochloride, Octodrine, oxymetazollne, Phenylephrine Hydrochloride, Phenylpropanolamine Hydrochloride, Phenylpropylmethylamlne, Propylhexedrine, Pseudoephedrine,

Tetrahydrozoline, Tymazoline and Xylometazoline;

Dental agents, including: Bisphosphonates (anti-periodontai disease io and bone resorption) such as Alendronate, Clodronate, Etidronate,

Pamidronate and Tiludronate; Carries Prophylactics such as Arginine and Sodium Fluoride;

Desensitizing Agents such as Potassium Nitrate and Citrate Oxalate;

Depigmentors such as Hydroquinine, Hydroquinone and 15 Monobenzone;

Diuretics, including: Organomercurials such as Chlormerodrin, Meralluride, Mercamphamide, Mercaptomerin Sodium, Mercumallylic Add, Mercumatilin Sodium, Mercurous Chloride and Mersalyl;

Pterldines such as Furterene and Triamterene; 20 Purines such as Acefylline, 7-MorpholinomethyKheophylllne,

Pamabrom, Protheobromine and Theobromine;

Steroids such as Canrenone, Oleandrin and Spironolactone;

Sulfonamide derivatives such as Acetazolmide, Ambuside, Azosemide, Bumetanide, Butazolamlde, Chloraminophenamide, Clofenamide, Clopamide, 25 Clorexoiene, Dlphenylmethane-^-disulfbnamide, Disuifamide,

Ethbxzolamide, Furosemide, Indapamide, Mefruside, Methazolamide, Pfretanide, Quinethazone, Torasemide, Tripamide and Xlpamide;

Uracils such as Aminometradine and Amlsometradine; others such as Amanozine, Amiioride, Arbutin, Chlorazanil, Ethacrynic 30 Acid, Etozotln, Hydracarbazine, Isosarblde, Mannitol, Metochalcone, Muzoiimine, Perhexlline. Ticrynafen and Urea; -88- 2015215955 21 Aug 2015

Dopamine receptor agonists such as Bromocriptine, Dopexamine, Fanoldopam, Ibopamine, Lisurtde, Naxagoiide and Pergolide;

Ectoparasiticfdes such as Amitraz, Benzyl Benzoate, Carbaryl. Crotamiton, DDT, Dixanthogen, IsobomylThiocyanoacetate-Technical, Lime 5 Sulfurated Solution, Lindane, Malathion, Mercuric Oleate, Mesulphen and Sulphur—Pharmaceutical;

Enzymes, including: Digestive enzymes such as α-Amylase (Swine Pancreas), Lipase, Pancreltpase, Pepsin and Rennln;

Mucolytic enzymes such as Lysozyme; 10 Penicillin inactivating enzymes such as Penicillinase; and

Proteolytic anz/mes such as Coilagenase, Chymopapain, Chymotrypsins, Papain and Trypsin;

Enzyme inducers (hepatic) such as Flumecinol;

Estrogens, Including: Nonsteroidal estrogens such as Benzestrol, 15 Broparoestroi, Chlorotrianfsene, Dienestroi, Diethyistilbestrol, Diethylstilbestroi Diproprionate, Dimestroi, Fosfestrol, Hexestrol, Methallenestrii and Methestnoi; and

Steroidal estrogens such as Colpormon, Conjugated Estrogenic Hormones, Equiienin, Equiiin, Estradiol, Estradiol Benzoate, Estradiol 17β-20 Cypionate, Estrtol, Estrone, Ethinyl Estradiol, Mestranoi, Moxestroi, Mytatrienedfol, Qulnestradiol and Quinestrol;

Gastric secretion inhibitors such as Enterogastrone and Octreotide;

Glucocorticoids such as 21-Acetoxyprefnenolone, Aalclometasone, Algestone, Amicinontde, Beclomethasone, Betamethasone, Budesonide, 25 Chloroprednisone, Ciobetasol, Blovetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone, Gortivazoi, Deflazacort, Desonide, Desoximetasone, Dexamelhasone, Diflorasone, Diflucortolone, Difluprednate, Enoxofone, Fluazacort, Flucloropide, Flumehtasone, Flunisoiide, Ffuocinoione Acetonide, Fluocinonide, RuocorHn Butyl, Fluocortoione, Fluoromethoione, 30 Fluperoione Acetate, Fluprednidene Acetate, Fluprednisolone,

Fiurandrenolide, Formocortal, Halcinonide, Halometasone, Halopredone Acetate, Hydrocortamate, Hydrocortisone, Hydrocortisone Acetate, 2015215955 21 Aug 2015 -89- ydrocortisone Phosphate, Hydrocortisone 21-Sodium Succinate, Hydrocortisone Tebutate, Mazipredone, Medrysone, Meprednrsone, Methyolprednisolone, Mometasone Furoate, Paramethasone, Prednicarbate, Prednisolone, Prednisolone 21-Diethylaminoacetate, Prednisone Sodium 5 Phosphate, Prednisolone Sodium Succinate, Prednisolone Sodium 21 -m-Sulfobenzoate, Prednisolone 21-Stearoylglycolate, Prednisolone Tebutate, Prednisolone 21-Trimethylacetate, Prednisone, Prednival, Prednylidene, Prednylidene 21-Diethylaminoacetate, Tixocortal, Triamcinolone, Triamcinolone Acetonlde, Triamcinolone Benetonide and Triamcinolone 10 Hexacetonlde;

Gonad-Stimulating principles such as Buserelin, Clomlphene, Cyclofenil, Epimestrol, FSH, HCG and LH-RH;

Gonadotropic hormones such as LH and PMSG;

Growth hormone inhibitors such as Octreotide and Somatostatin; 15 Growth hormone releasing factors such as Semorelin;

Growth stimulants such as Somatotropin;

Hemolytic agents such as Phenylhydrazine and Phenylhydrazine Hydrochloride;

Heparin antagonists such as Hexadimethrine Bromide and Protamines; 20 Hepatoprotectants such as S-Adenosytmethionlne, Betaine, Catechin,

Citolone, Malotilate,· Orazamide, Phosphorylcholine, Protoporphyrin IX, Silymarin-Group, Thiotic Acid and Tiopronin;

Immunomodulators such as Amiprilose, Buciliamine, Ditiocarb Sodium, Inosine Pranobex, lnfcerferon-y. lnterieukin-2, Lentinan, Munoctasin, Platonin, 25 Procodazole, Tetramisole, Thymomodulin, Thymopentin and Ubenlmex;

Immunosuppressants such as Azathioprine, Cyclosporins and Mizoribine;

Ion exchange resins such as Carbacrylic Resins, Cholestyramine Resin, Colestipol, Polidexide, Resodec and Sodium Polystyrene Sulfonate; 30 Lactation stimulating hormone such as Prolactin; LH-RH agonists such as Buserelin, Goserelin, Leuprolide, Nafarelin, and Trfptorelin; -90- 2015215955 21 Aug 2015

Lipotropic agents such as N-Acetylmethlonine, Choline Chloride, Choline Dehydrocholate, Choline Dihydrogen Citrate, Inositol, Lecithin and Methionine;

Lupus erythematosus suppressants such as Bismuth Sodium 5 Trlglycollamate, Bismuth Subsalicylate. Chloroquine and Hydroxychloroquine;

Mineralcorticoids such as Aldosterone, Deoxycorticosterone, Deoxycorticosterone Acetate and Fludrocortisone:

Miotic drugs such as Carbachoi, Physostigmine, Pilocarpine and Pilocarpus; 10 Monoamine oxidase inhibitors such as Deprenyl, Iproclozlde,

Iproniazid, Isocarboxazid, Modobemide, Octomoxin, Pargyiine, Phenelzine, Phenoxypropazine, Pivaiylbenzhydrazine, Prodipine, Toloxatone and Tranylcypromine;

Mucolytic agents such as Acetylcysteine, Bromhexine, Carbocysteine, 15 Domiodol, Letosteine, Lysozyme, Mecysteine Hydrochloride; Mesna,

Sobreroi, Stepronin, Tiopronin and Tyioxapol;

Muscle relaxants (skeletal) such as Afloqualone, Alcuronium, Atracurium Besylate, Baclofen, Benzoctamine, Benzoquinonlum Chloride, C-Calebassine, Carisoprodol, Chlormezanone. Chlorphenesin Carbamate, 20 Chlorproethazlne, Chlozoxazone, Curare, Cydarbamate, Cyclobenzaprine, Dantrolene, Decamethonlum Bromide, Diazepam, Eperlsone, Fazadlnium Bromide, Ffumetramide, Gallamine Triethiodide, Hexacarbacholine Bromide, Hexafluorenium Bromide, IdrocHamlde, Lauexium Methyl Sulfate, Leptodactyline, Memantine, Mephenesin, Mephenoxalone, Metaxalone, 25 Methocarbamol, Metocurine Iodide, Nimetazepam, Orphenadrine,

Pancuronium Bromide, Phenprobamafe, Phenyramidol, Pipecurium Bromide, Promoxolane, Quinine Sulfate, Styramate, Succlnylchollne Bromide, Succinylcholine Chloride, Sucdnylchoiine Iodine, Suxethonium Bromide, Tetrazepam, Thiocolchicoside, Tizanldine, Tolperisone, Tubocurarine 30 Chloride, Vecuronium Bromide and Ζοχοί amine; -91- 2015215955 21 Aug 2015

Narcotic antagonists such as Amiphenazole, Cydazocine, Levallorphan, Nadide, Nalmfene, Nalorphine, Nalorphine Dinicotinate, Naloxone and Naltrexone;

Neuroprotectiva agents such as Dizocilpine; 5 Nootropic agents such as Acaglutamide, Acetylcarnltlne, Aniracetam,

Bifematiane, Exifone, Fipexide, Idebenone, Indefoxazune Hydrochloride, Nizofenone, Oxiracetam, Piracetam, Propentofylline, Pyritinol and Tacrine;

Ophthalmic agents such as 15-ketoprostaglandins;

Ovarian hormone such as Relaxin; 10 Oxytocic drugs such as Carboprost, Cargutocin, Deaminooxytodn,

Ergonovine, Gemeprost, Methyiergonovine, Oxytocin, Pituitary (Posterior), Prostaglandin £2, Prostaglandin F2h and Sparteine;

Pepsin inhibitors such as Sodium Amylosuifate;

Peristaltic stimulants such as Cisapride; 15 Progestagens such as Allylestrenol, Anagestone, Chlormadinone

Acetate, Delmadlnona Acetate, Demegestona, Desogestrel, Dimethisterone, Dydrogesterone, Ethisterone, Ethynodiol, Flurogestone Acetate, Gestodene, Gestonorone Caproate, Haloprogesterone, 17-Hydroxy-16-methylene— progesterone, 17a-Hydroxy progesterone, 17a-Hydroxygesterone Caproate, 20 Lynestrenol, Medrogestone, Medroxyprogesterone, Megestrol Acetate, Mefengestrol, Norethindrone, Norethynodrel, Norgesterone, Norgestimate, Norgestrel, Norgestrienone, Norvinisterone, Pentagestrone, Progesterone, Promegesfone, Quingestrone and Trengestone; ·

Prolactin Inhibitors such as Metergoline; 25 Prostaglandins and prostaglandin analogs such as Arbaprostil,

Carboprost, Enprostil, Bemeprost, Limaprost, Misoprostol, Ornoprostll, Prostacyclin, Prostaglandin Ej, Prostaglandin E2, Prosiagland in F2B, Rioprostii, Rosaprostol, Sulprostone and Trimoprostil;

Protease inhibitors such as Aprotinin, Camostat, Gabexate and 30 Nafamostat;

Respiratory stimulants'such as Almitrine, Bemegrida, Carbon Dioxide, Cropropamide, Crotethamide, Dimefline, Dlmorpholamine, Doxapram, -92- 2015215955 21 Aug 2015

Ethamivan, Fominoben, Lobeline, Mapixanox, Metamivam, Nikethamide, Picrotoxfn, Pimecfone, Pyridotylline, Sodium Succinate and Tacrine;

Sclerosing agents such as Ethanolamrne, Ethylamine, 2-HexyIdecanoic Add, Poiidocanol, Quinine Bisulfate, Quinine Urea Hydrochloride, Sodium 5 Ricinoleate, Sodium Tetradecyl Sulfate and Tribenoside;

Sedatives and hypnotics, induding: Acyclic ureides such as Acecarbromal, Apronalide, Bomisovalum, Capuride, Carbromal and Ectylurea;

Alcohols such as Chlorhexadol, Ethchlorvynol, Meparfynol, 4-Methyl-5-thiazoleethanol, tert-Pentyl Alcohol and 2,2,2-Trichloroethanol; 10 Amides such as Butoctamide, Diethylbramoacetamlde, Ibrotamide,

Isovaleryl Diethylamide, Niaprazine, Tricetamide, Trimetozlne, Zolpidem and Zopiclone;

Barbituric add derivatives such as Allobarbltai, Amobarbital, Aprobarbita), Barbital, Brallabarbita), Butabarbital Sodium, Butalbital, 15 Butallylonal, Butethal, Carbubarb, Cyclobarbital, Cyclopentobarbital, Enallylpropymal, 5-Ethyl-5-(1-piperidyl) barbituric Acid, 5-Furfuryl-5-isopropylbarbituric Acid, Heptabarbital, Hexethal Sodium, Hexobarbital, Mephobarbital, Methitural, Narcobarbital, Nealbarbital, Pentobarbital Sodium, Phenaliymal, Phenobarbitai, Phenobarbital Sodium, Phenylmethylbarbiturlc 20 Acid, Probarbital, Propallylonal, Proxibarbal, Reposal, Secobarbital Sodium, Talbutai, Tetrabarbital, Vinbarbital Sodium and Vinylbltal;

Benzodiazepine derivatives such as Brotizolam, Doxefazepam, Estazolam, Flunitrazepam, Flurazepam, Hatoxazolam, Loprazolam, Lormetazepam, Nitrazepam, Quazepam, Temazepam and Triazolam; 25 Bromides such as Ammonium Bromide, Calcium Bromide, Calcium

Bromolactobionate, Lithium Bromide, Magnesium Bromide, Potassium Bromide and Sodium Bromide;

Carbamates such as Amyl Carbamate-Tertiary, Ethinamate, Hexaprpymate, Meparfynot Carbamate, Novonal and Tricholorourethan; 30 Chloral derivatives such as Carbocloral, Chloral Betaine, Chloral

Formamide, Chloral Hydrate, Chloraiantipyrine, Dichloralphenazone, Pentaerythritol Chloral and Triclofos; -93- 2015215955 21 Aug 2015

Plperidinediones such as Glutehimide, Methyprylon, Piperidlone, Pyrithyldione, Taglutimide and Thalidomide;

Quinazolone derivatives such as Etaqualone, Mecloqualone and Methaqualone; and 5 others such as Acetal, Acetophenone, Aldoi, Ammonium Valerate,

Amphenidone, d-Bomyl a-Bromoisovalerate, d-Bomyl Isovaferate, Bromoform, Calcium 2-Ethylbutanoate, Carfinata, a-Chlorolose, Clomethiazole, Cypripedium, Doxylamine, Etadroxizlne, Etomldate, Fenadiazole, Homofenazine, Hydrobromic Acid, Mecloxamine, Menthyl 10 Valerate, Opium, Paraldehyde, Perlapine, Propiomazine, Rilmazafone, Sodium Oxybate, Sulfonethylmethane and Sulfonmathane;

Thrombolytic agents such as APSAC, Plasmin, Pro-Urokinase, Streptokinase, Tissue Plasminogen Activator and Urokinase;

Thyrotropic hormones such as TRH and TSH; 15 Uricosurics such as Benzbromarone, Ethebenecid, Orotic Acid,

Oxycinchophen, Probenecid, Sulfinpyrazone, Tlcrynafen and Zoxazolamine;

Vasodilators (cerebral) sUGh as Bencydane, Cinnarizine, Citicoline, Cyctandelate, Ciclonicgte, Diisopropylamine Dichloractetate, Eburnamorine, Fenoxedil, Flunarizine, Ibudilast, Ifenprodil, Nafronyl, Nicametate, Nicergoline, 20 Nimodlpine, Papaverine, Pentifyiline, Tinofedrine, Vincamine, Vinpocetine and Vlquidil;

Vasodilators (coronary) such as Amotriphene, Bendazoi, Benfurodii Hemisuccinate, Benzlodarone, Chioacizine, Chromonar, Clobenfurol, Clonitrate, Dilazep, Dipyridamole, Droprenifamine, Efloxate, Erythritol, 25 Erythrityl Tetranitrate, Etafenone, Fendlline, Floredil, Ganglefene, Hexestrol Bis(p-diethyiaminoethyl ether), Hexobendine, Itramin Tosylate, Khellin, Lidoflazine, Mannitol Hexanitrate, Medibazine, Nicorandil, Nitroglycerin, Pentaerythritol Tetranitrate, Pentrinitrol, Perhexlline, Pimefyiline, Prenylamine, Propatyi Nitrate, Pyridoiylline, Trapidil, Tricromyl, Trimetazldlne, Trolnitrate 30 Phosphate and Visnadine;

Vasodilators (peripheral) such as Aluminum Nicotinate, Bamethan, Bencydane, Betahistlne, Bradykinin, Brovlncamine, Bufonlode, Buflomedil, -94- 2015215955 21 Aug 2015

Butalamine. Cetiedil, Ciclonfcate, Cinepazide, Cinnarfeine, Cyclandeiato, Diisopropylamine Dichloracetate, Eledoisin, Fenojddil, Flunaiisine, Heronicate, Ifenprodil, Inositol Niadnate, Isoxsuprine, Kaliidin, Kallikrein, Moxisylyte, Nafronyl, Nicametate, Nicergollne, Nicofuranose, Nicotinyl 5 Alcohol, Nylfdrin, PenfriyJJine, Pentoxifylline, Pirlbedil, Protaglandin Bu Suloctidil and Xanthinal Niadnate;

Vasoprotectants such as Benzarone, Bioflavonoids, Chromocarb, Clobeoslde, Diosmin, Dobesilate Calcium, Escin, Rolescutol, Leucocyanidin, Metescufylline, Quercetin, Rutin and Troxerutin; 10 Vitamins, vitamin sources, and vitamin extracts such as Vitamins A, B, C, D, E, and K and derivatives thereof, Calciferols, Glycyrrhiza and Mecobalamin;

Vulnerary agents such as Acetylcysteine, AHantoin, Asiaticoside, Cadexomer iodine, Chitin, Dextranomer and Oxaceprol; 15 Anticoagulants such as heparin;

Miscellaneous such as Erythropoietin (Hematinic), Filgrastim, Finasteride (Benign Prostate Hypertrophy) and Interferon β l-α (Multiple Sclerosis). in certain embodiments, the agent to be delivered is one or more 20 proteins, hormones, vitamins or minerals. In certain embodiments, the agent to be delivered Is selected from insulin, IGF-1, testosterone, vinpocetin, hexarelin, GHRP-6 or calcium. In certain embodiments, the compositions contain two or more agents.

The above list of active agents is based upon those categories and 25 species of drugs set forth on pages THER-1 to THER-28 of The Merck

Index, 12th Edition, Merck &amp; Co. Rahway, N.J. (1996). This reference is incorporated by reference herein In its entirety. D. Uses of the compositions

Therapeutic and diagnostic applications of the microspheres Include 30 drug delivery, vaccination, gene therapy, and in vivo tissue or tumor imaging.' Routes of administration include oral or parenteral administration; mucosal administration; ophthalmic administration; intravenous, subcutaneous, intra -95- 2015215955 21 Aug 2015 articular, or intramuscular Injection: inhalation administration; and topical administration.

The diseases and disorders can include, but are not limited to neural disorders, respiratory disorders, immune system disorders, muscular 5 disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, cancerous diseases and inflammation.

The microparticles provided herein can be used to treat infectious diseases, such as arbovtrai infections, botulism, brucellosis, candidiasis, 10 campylobacteriosis, chicken pox, chlamydia, cholera, coronovirus infections, staphylococcus infections, coxsackie virus infections, CreutzFeldt-Jakob disease, cryptosporidiosis, cydospora infection, cytomegalovirus infections, Epstein-Barr virus infection, dengue fever, diphtheria, ear infections, encephalitis, influenza virus infections, parainfluenza virus infections 15 giardiasis, gonorrhea, Haemophilus influenzae infections, hantavirus infections, viral hepatitis, herpes simplex virus infections, HiV/AIDS, helicobacter infection, human papillomavirus (HPV) infections, infectious mononucleosis, legionellosis, leprosy, leptospirosis, listeriosis, lyme disease, lymphocytic choriomeningitis, malaria, measles, marburg hemorrhagic fever, 20 meningitis, monkeypox, mumps, mycobacteria infection, mycoplasma infection, norwalk virus infection, pertussis, pinworm infection, pneumococcal disease, Streptococcus pneumoniae infection, Mycoplasma pneumoniae infection, Moraxelia catarrhatis Infection, Pseudomonas aeruginosa infection, rotavirus Infection, psittacosis, rabies, respiratory syncytial vims infection 25 (RSV), ringworm, rocky mountain spotted fever, rubella, salmonellosis, SARS, scabies, sexually transmitted diseases, shigellosis, shingles, sporotrichosis, streptococcal infections, syphilis, tetanus, trichinosis, tuberculosis, tularemia, typhoid fever, viral meningitis, bacterial meningitis, west nile virus infection, yellow fever, yersiniosis zoonoses, and any other Infectious respiratory, 30 pulmonary, dermatological, gastrointestinal and urinary tract diseases.

Other diseases and conditions include arthritis, asthma, allergic conditions, Alzheimer’s disease, cancers, cardiovascular disease, multiple -96- 2015215955 21 Aug 2015 sclerosis (MS), Parkinson's disease, cystic fibrosis (CF), diabetes, non-viral hepatitis, hemophilia, bleeding disorders, blood disorders, genetic disorders, hormonal disorders, kidney disease, liver disease, neurological disorders, metabolic diseases, skin conditions, thyroid disease, osteoporosis, obesity, 5 stroke, anemia, inflammatory diseases and autoimmune diseases. E. Combinations, Kits, Articles of manufacture

Combinations and kits containing the combinations provided herein including microparticles or ingredients for forming the microparticles such as a protein or other macromotecule, counterions, solvents, buffers, or salts and 10 optionally including instructions tor administration are provided. The combinations include, tor example, the compositions as provided herein and reagents or solutions tor diluting the compositions to a desired concentration for administration to a host subject, including human beings. The combinations also can include the compositions as provided herein and 15 additional nutritional and/or therapeutic agents, including drugs, as provided herein.

Additionally provided herein are kits containing the above-described combinations and optionally instructions tor administration by oral, subcutaneous, transdermal, intravenous, intramuscular, ophthalmic or other 20 routes, depending on the protein and optional additional agent(s) to be delivered.

The compositions provided herein can be packaged as articles of manufacture containing packaging material, a composition provided herein, and a label that indicates that the composition, e.g., a DAS181 formulation, is 25 formulated tor oral, pulmonary or other delivery.

The articles of manufacture provided herein can contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Patent Nos. 9,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials 30 include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, botiles, and any packaging material suitable tor a selected formulation and intended mode of administration and treatment. 2015215955 21 Aug 2015 -97-

The following examples are Included for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1

Preparation of microspheres of the sialidase fusion protein, DAS181 5 A. Purification of DAS181 OAS181 is a fusion protein containing the heparin (glysosamino glycan, or GAG) binding domain from human amphiregulln fused via its N-termlnus to the C-terminus of a catalytic domain of Actinomyces vlscosus (sequence of amino acids set forth in SEG ID N0:17), The DAS181 protein was purified as 10 described in Malakhov ef a/., Antimicrob. Agents Chemother., 1470-1479, 2006, which Is incorporated in its entirety by reference herein. Briefly, the DNA fragment coding for DAS 131 was cloned into the plasmid vector pTrc99a (Pharmacia; SEQ ID NO:16) under the control of a IPTG (isopropyl-S-D-thiogalactopyranosidej-inducible promoter. The resulting construct was 15 expressed in the BL21 strain of Escherichia coll (£. coU).

The E. coil cells containing the expressed construct were lysed by sonication in 50 mM phosphate buffer, pH 8.0; 0.3 M NaCI and 10% glycerol. The clarified lysate was passed through an SP-Sepharose column. Proteins were eluted from the column with lysis buffer that contained 0.8 M NaCI. The 20 fraction eluted from SP-Sepharose was adjusted to 1.9 M ammonium sulfate ((NH^SCU), clarified by centrifugation, and loaded onto a butyi-Sepharose column. The column was washed with two volumes of 1.3 M (NH^SO^ and the DAS181 fusion protein was eluted with 0.65 M (Ni-UjzSO*

For the final step, size exclusion chromatography was performed on 25 Sephacryi S-200 equilibrated with phosphate-buffered saline (PBS). The protein purity was determined to be greater than 98% as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase high-pressure liquid chromatography, and enzyme-linked immunosorbent assay with antibodies generated against E. colicell proteins. The purified DAS181, 30 molecular weight 44,800 Da, was dialyzed against 2 mM sodium acetate buffer, pH 5.0. B. Activity of DAS 181 -98- 2015215955 21 Aug 2015

The sialidasa activity of DAS181 was measured using the fluorogenic substrate 4-methylumbel!iferyl-/V-acetyi-a-D-neuraminic acid (4-MU-NANA; Sigma). One unit of siaildase Is defined as the amount of enzyme that releases 10 nmol of MU from 4-MU-NANA in 10 minutes at 37 °C (50 mM 5 CHgCOOH-NaOH buffer. pH 5.5) in a reaction that contains 20 nmol of 4-MU-NANA In a 0.2 ml volume (Potier eta!., Anal. Biochem., 94:287-296.1979). The specific activity of DAS181 was determined to be 1,300 U/mg protein (0.77 pg DAS181 protein per unit of activity). C. Preparation of microspheres using purified DAS181 10 DAS181 (10 mg/ml), purified and prepared as described under Section A above, was used to form 200 pi cocktails as shown below. The cocktails contained either glycine or citrate as counterions, and isopropanol as organic solvent, as follows: 1) DAS181 + 5 mM glycine, pH 5.0; 15 2) DAS181 + 5 mM glycine, pH 5.0 + 10% Isopropanol; 3) DAS181 + 5 mM sodium citrate, pH 5.0; 4) DAS181 + 5 mM sodium citrate, pH 5.0 + 10% isopropanol;

Plastic micracentrifuge tubes containing the cocktails with ingredients as described in 1) — 4) above were gradually cooled from: 20 (a) ambient temperature (about 25 °C) to 4 °C by placing the cocktails in a refrigerator, followed by: (b) cooling to —20 *C by placing the resulting cocktail from (a) in a freezer, followed by: (c) freezing to -80 'C by placing the resulting cocktail from b) in a 25 freezer.

Under optimal conditions, microspheres would be expected to form between about 4 °G to about -20 °C (generally in the range of about -2 °C to about -15 °C). Freezing to -80 °c is carried out to remove ingredients from the cocktail other than the microspheras (e.gr., solvent, efc.) by freeze-drying. 30 Cocktail 4) was prepared in triplicate, two aliquots in plastic tubes and one in a glass tube. One aliquot (in a plastic tube) was cooled as described above, while the two other aliquots (one in a plastic tube and one in a glass tube) 2015215955 21 Aug 2015 -99- were subjected to snap cooling/ffeezing by dipping the tubes into liquid nitrogen.

Upon freezing, all tubes were placed into the lyophilizer and the volatiles (water and isopropanol) were removed by sublimation, leaving the •5 dry pellets.

Results: The dry pellets recovered from the cocktails treated as described above, were tested tor the presence of microspheres. Of the above samples, microspheres with good dispersivity characteristics, about 2 microns (pm) in size, were observed only with cocktail 4) containing citrate counterion 10 and isopropanoi and subjected to gradual cooling. The counterion glycine did not prove to be optimal for the DAS181 protein (cocktail 2)), showing a mixture of glass-like crystals and agglomerates with only a few mfcrospheres. When no organic solvent was present, a giass-like mass of lyophilized DAS181 protein was obtained and no microspheres were observed (cocktails 15 1) and 3)). Snap-freezing of cocktail 4) in a glass tube produced glass-like crystals and no microspheres, while snap-freezing of cocktail 4) in a plastic tube (cooling rate is slightly slower due to slower diffusion of heat through plastic than through glass) produced agglomerated microspheres.

This example demonstrates that microspheres with narrow size 20 distribution and good dispersivity (minimal agglomeration) can be produced by a combination of appropriate protein, counterion, organic solvent and gradual cooling, using the methods provided herein. EXAMPLE2 25 Size of DAS181 microspheres as a function of organic solvent concentration DAS181 was purified and used to prepare microspheres as described above in Example 1 (see cocktail 4)), using a combination of DAS181 protein 30 (10 mg/ml), citrate counterion (sodium citrate, 5 mM) and isopropanol organic solvent (10%, 20% or 30%). The resulting cocktail solutions were cooled from ambient temperature (about 25 eC) to 4 *C, followed by cooling to -20 *C, followed b'y freezing to -80 Ό, as described in Example 1. Upon freezing to - -100- 2015215955 21 Aug 2015 80 °C, the tubes are placed in a lyophJIlzer and the volatiles (water and isopropanol) were removed by sublimation, leaving the dry powder containing microspheres.

Results; Microsphere formation was observed with all three 5 concentrations: 10%, 20%, or 30%, of the organic solvent Isopropanol. The dimensions of the micraspheres however varied, depending on the concentration of the organic solvent. The sizes of the microspheres as determined by comparing the particles to a grid on a hemocytometer were estimated to be 2 microns using 10% isopropanol, 4 microns using 20% 10 .isopropanol, and 5-6 microns ueing 30% isopropanol. These results demonstrate that the size of the microparticles can be engineered as desired using an appropriate concentration of organic solvent. EXAMPLE 3

Size.of DAS1S1 microspheres as a function of-protein concentration 15 DAS181 was purified and used to prepare microspheres as described above in Example 1 (see cocktail 4)), using a combination of DAS181 protein (5 mg/ml or 10 mg/ml), citrate counterion (sodium citrate, 5 mM) and isopropanol (5% or 20%). The resulting cocktail solutions were cooled from 20 ambient temperature (about 25 °C) to 4 °C, followed by cooling to -20 "C, followed by freezing to -80 “C, as described in Example 1. Upon freezing to -80 °C, the tubes were placed in a lyophiiizer and the volatiles (water and isopropanol) were removed by sublimation, leaving the dry powder containing microspheres. 25 Results: Microsphere formation was observed with both concentrations of protein (5 mg/ml and 10 mg/ml), and both concentrations of organic solvent (5% or 20%). The dimensions erf the microspheres however varied. Cocktails containing 5 mg/ml or 10 mg/ml protein and 5% isopropanol produced microspheres estimated to be about 1.5 micron in size. The cocktail 30 containing 5 mg/ml protein and 20% isopropartol produced microspheres of an estimated size of about 3 microns, while the cocktail containing 10 mg/ml protein and 20% isopropanol produced microspheres of an estimated size of 2015215955 21 Aug 2015 -101- about 4 microns. These results demonstrate that the size of the microparticles can be engineered as desired using an appropriate concentration of protein, or an appropriate combination of concentration of organic solvent and concentration of protein. 5 EXAMPLE 4

Size of DAS 181 microspheres as a function of counterion concentration DAS181 was purified and used to prepare microspheres as described above in Example 1 (see cocktail 4)), using a combination of DAS 181 protein 10 (10 mg/ml), citrate counterion (sodium citrate; 2 mM, 3 mM or 8 mM) and isopropanol (20%). The cocktail solutions were mixed in glass vials and cooled from +20 *C to —40 °C at a freeze ramp of 1 °C per minute in a Mlllrock Lab Series lyophilizer. Volatiles (water and Isopropanol) were removed by sublimation at 100 mTorr with primary drying at -30 *C for 12 hours and 15 secondary drying at 30 'C for 3 hours, leaving the dry powder containing microspheres.

Results: Microsphere formation was observed at all three tested concentrations of citrate counterion. The size of the microspheres increased from 1 micron at 2 mM citrate, to 3 microns at 3 mM citrate, to 5 microns at 6 20 mM citrate. Addition of 1 mM sodium acetate or 1 mM sodium chloride to die cocktail containing 2 mM citrate did not affect formation'of the microspheres triggered by the citrate counterion. These results demonstrate that the size of • the microparticles can be engineered as desired using an appropriate concentration of counterion. 25 EXAMPLE 5 DAS181 microspheres formed in the presence of surfactants

The addition of surfactants to macromolecular (e.g., protein) microspheres often can improve characteristics of the microspheres that render them suitable for administration to a subject, such as flowabillty, 30 dispersivity and disposition for a particular route of administration, such as intranasal or oral inhalation. To test whether surfactants can be incorporated into the methods of manufacturing microspheres as provided herein, the -102- 2015215955 21 Aug 2015 production of DAS181 microsphems was undertaken as described In Example 1 above, except that in addition, a surfactant was added to the solution.

To a cocktail solution containing 5 mg/ml DAS181,5 mM sodium citrate, and 20% isopropanoi, was added a surfactant (3,5% w/w lecithin, 5 0.7% w/w Span-85® (sorbitan trioleate), or 3.5% w/w oleic acid). The microspheres were formed by cooling the solutions to 4 "C, followed by cooling to —20 °C, followed by freezing to —80 °C for lyophilization as described above in Example 1. Upon freezing, the tubes were placed into a lyophiiizer and the volatiles (water and isopropanoi) were removed by sublimation, 10 leaving the dry powder containing microspheres.

Results: The microspheres resulting from treatment of each of the above cocktails as described above were spread on glass slides using cover slips rubbed in a circular motion. Efficient microsphere formation was observed in all cases. When the samples containing surfactant were 15 compared to the sample containing all the remaining ingredients but no added surfactant, it was noted that the microspheres formed in the presence of surfactant had improved dispersivity (lesser agglomeration or aggregation). EXAMPLE 6

Preparation of microspheres of bovine serum albumin (BSA) by 20 selection of suitable types and concentrations of organic solvents and counterions

As described herein, the methods provided herein can empirically be optimized in high-throughput format to obtain microspheres having desired 25 characteristics including size, flowabillty and dispersivity. The purpose of this experiment was to demonstrate that by varying types and concentrations of organic solvents and counterions, as well as pH of the cocktail, size and quality of microspheres of a protein of interest, in this case bovine serum albumin (BSA), can be adjusted. 30

Cocktail solutions containing 5 mg/ml of BSA and various organic solvents and counterions at indicated pH and concentrations (see Table 1) were placed in a microtiter plate, (final volume perwell of 0.1 ml). Cocktails 2015215955 21 Aug 2015 - 103- were cooled from +20 °C to -40 'C at a freeze ramp of 1 ,"C per minute in a Mill rock Lab Series lyophilizer. Volatiles were removed by sublimation at 100 mTorr, with a primary drying at -30 ’C for 12 hours and secondary drying at 30 *C for 3 hours. S' Results: The results are shown In Table 1 below. For the BSA protein, combinations (of counterion and organic solvent, respectively) that produced the most uniform microspheres with minimal crystallization or aggregation

Include: (1) citrate + isopropanol 10 (2) citrate + acetone (3) itaconic acid + 1-propanol (4) glycine + dioxane (5) glycine + 1-propano! (6) rubidium + 1-propano! 15 (7) perchlorate + 1-propano!

Table 1: High-throughput screening of BSA microspheres Ibrmed under different conditions

Counterion pH Organic Solvent Product description 5 mM pivalic acid 4.0 5% Cyclohexanol 0.5- 1 micron microspheres with occasional crystals 5 τηΜ pivalic acid 4.0 5% 1- propanol 0.5- 1 micron microspheres with some aggregates 5 mM pivalic acid 4.0 5% butyl alcohol Aggregated microspheres 5 mM pivalic acid 4.0 5% p-Dioxane Aggregated microspheres 5 mM rubidium chloride 9.0 5% Cyclohexanol 0.5-1 micron microspheres. Aggregates and occasional crystals 5 mM rubidium chloride 9.0 5% 1- propanol 0.5-1 micron microspheres 5 mM rubidium chloride 9.0 5% butyl alcohol Few microspheres (0.5- 1 micron). Mostly aggregates and crystals 5 mM rubidium chloride 9.0 5% p- Dioxane 1- 2 microns microspheres with some aggregates 5 mM sodium bromide 4.0 5% Cyclohexanol 1-2 microns microspheres with some aggregates 2015215955 21 Aug 2015 -104- Counterion pH Organic Solvent Product description 5 mM sodium bromide 4.0 5% 1~ propanol Few microspheres (0.5- 2 micron). Mostly aggregates and crystals 5 mM sodium bromide 4.0 5% butyl alcohol Few microspheres (0.5-1 mlcion). Mostly aggregates and crystals 5 mM sodium bromide 4.0 5% p- Dioxane 1- 2 microns microspheres with some aggregates 5 mM sodium perchlorate 4.0 5% Cyclohexanol 0-5- 2 microns microspheres with some crystals and aggregates 5 mM sodium perchlorate 4.0 5% 1- propanol 0.5- 1 micron microspheres 5 uiMsodium perchlorate 4.0 5% butyl alcohol Few 1-2 microns microspheres. Mostly crystals and aggregates 5 mM sodium perchlorate 4.0 5% p- Dioxane Aggregated microspheres 5 mM calcium phosphate 4.0 5% Cyclohexanol Few 1- 2 microns microspheres, mostly aggregates 5 mM calcium phosphate 4.0 5% 1- propanol' 1- 2 microns microspheres with some aggregates 5 mM calcium phosphate 4.0 5% butyl alcohol Few 1- 2 micron microspheres. Mostly crystals and aggregates 5 mM calcium phosphate 4.0 5% p- Di.oxane Aggregated microspheres 5 mM triethylamine 9.0 5% Cyclohexanol 0.5- 1 micron microspheres with some crystals and aggregates 5 mM triethylamine 9.0 5% 1- propanol 1- 2 micron microspheres with some aggregates 5 mM triethylamine 9.0 5% butyl alcohol Fetw 1- 2 micron microspheres. Mostly crystals and aggregates 5 mM triethylamine 9.0 5% p- Dioxane Aggregated microspheres 5 mM glycine 9.0 5% Cyclohexanol 0.5- 1 micron microspheres with some crystals and agjpregates 5 mM glycine 9.0 5% 1-propanol 0.5- 2 micron microspheres with occasional aggregates 5 mM glyoine 9.0 5% butyl alcohol Few 1- 2 micron microspheres. Mostly crystals and aggregates 5 mM glycine 9.0 5% p- Dioxane 1- 2 micron microspheres 5 mM sodium citrate 4.0 15¾ isopropanol 1-2 micron microspheres 5 mM sodium citrate 4.0 15% acetone 0.5-1 micron microspheres 5 mM itaconic acid 4.0 15% 1-nropanol 1-2 micron microspheres 2015215955 21 Aug 2015 -105-

These results demonstrate that, for each protein, multiple formulations can readity be screened for the best microsphere formation (desired dimensions, uniformity, dispersivity, minimal aggregation and crystal formation, etc.) in high-throughput format. The combinations of reagents and 5 conditions (counterion, organic solvent, pH, concentrations) selected from the initial screen can then further be fine-tuned as desired. EXAMPLE 7

Preparation of microspheres using a variety of proteins 10

The methods provided herein can be used to prepare microspheres using a variety of proteins. In addition to DAS181 and BSA exemplified above, the methods were used to prepare microspheres from trypsin, hemoglobin, DNase (, lysozyme, ovalbumin, RNAse A, hexahistidine-tagged 15 human proteinase inhibitor 8 (PI8, having tire sequence of amino acids set forth in SEQ ID NO: 15), red fluorescent protein (RFP) and green fluorescent protein (GFP). DNase I, trypsin and hemoglobin were purchased from Worthington. Lysozyme, ovalbumin, and RNAse A were purchased from Sigma. 20 Purification of 6xHis tagged PI 8, GFP and RFP; 6xHis tagged PI8, GFP and RFP were expressed and purified essentially as described for DAS131 in Example 1 above, with the following modifications:

Purification of 6xHis tagged GFP and 6xHis tagged RFP: Constructs encoding Red Fluorescent protein and Green Fluorescent protein with N-25 terminal Hise tags were expressed in S. coll as 6xHis-tagged proteins.

Expression of Red Fluorescent protein was allowed to proceed overnight in LB medium with 1 mM IPTG. Green Fluorescent protein was induced for 3 hour in TB medium with 1 mM IPTG. Cell lysates from 4 liters of induced cultures were clarified by centrifugation and the proteins were purified by 30 metal chelate affinity chromatography on Fast-Flow Chelating resin (GE Healthcare) charged with Nickel and packed into C-10 columns (GE Healthcare). 2015215955 21 Aug 2015 -106-

The proteins were further purified by Gel Filtration Chromatography on a 0.5 cm x 70 cm Sephacryl 200 column equilibrated with phosphate buffered saline. The proteins were dialyzed against 2 mM sodium acetate buffer, pH 5.0, and concentrated on a Centriprep (Amfcon). 5 Purification of 6xHis tagged Pi8: A construct encoding PI8 with an N- terminal HIse lag was expressed in E colias 6xHis-tagged PI8. Purification was performed as described for 6xHis RFP and 6xHis GFP above, with tile exception.that all buffers used in the various chromatographic purification steps contained 1 mM TCEP (Tris(2-carboxyetbyl)phosphine hydrochloride). io Preparation of microspheres: Cocktail solutions containing 5 mg/ml of protein and various counterions, organic solvents and pH as listed below were prepared in a microtiter piate as described above in Example 6. 15

Table 2: Combinations Used to Produce Microspheres of Different Proteins Protein Counterion pH Organic Solvent Microsphere Size (microns) Trypsin 5mM arginine 8.0 5% isopropanol 0.5-1 Lysozyme 5 mM citrate 8.0 5% isopropanol 4-5 PIN 168 (PIS) 5 mM citrate 5.0 7% isopropanol 2-5 DNase I 5 mM citrate 4.0 5% isopropanol 0.4-1 RNase A 5 mM citrate 4.0 5% Isopropanol 1 O Hemoglobin 5 mM glycine 5.0 10% isopropanol o t o Ovalbumin 5 mM pivallc acid 4.0 10% isopropanol 0,5-1 Red fluorescent protein 5 mM pivalic acid 7.0 10% 1-propanol 1-4 (occasional aggregates) Green fluorescent protein 5 mM pivalic acid 7.0 10% 1-propanol 0.5-1.5 2015215955 21 Aug 2015 -107-

The microtiter plate was cooled from +20 "C to -40 "C at a freeze ramp of 1 °C per minute in a Millrock Lab Series lyophilizer. Volatiles (water and isopropanol) were removed by sublimation at 100 mTorr with primary drying at -30 "C for 12 hours and secondary drying at 30 *C for 3 hours, leaving the dry 5 powder containing microspheres.

The dry powders were spread on glass slides and microphotography was performed through either 32x or 100x objective. All the combinations listed in Table 2 above produced microspheres of good quality (uniform size distribution, dlspersivity, with few aggregates and/or crystals). The 10 microspheres varied in size from about 0.4 -1 micron (RNAse A, DNAse i) to about 2-5 microns (6xHis PIS, lysozyme), depending on the protein. This example demonstrates that the methods provided herein can be used to produce microspheres from a wide variety of proteins. EXAMPLE 8 IS Aerodynamic particle size distribution of DAS181 microspheres for inhalation: a comparison of the methods provided herein with spraydrying

As described herein, the methods provided herein can be used to 20 produce microspheres in any desired size range, including a range of about 0.5 micron to about 6-8 microns for delivery w*a inhalation. A. Preparation of microspheres

To test the aerodynamic particle size distribution of DAS181 dry powder (microspheres) formulated for delivery by inhalation, DAS181 25 microspheres were prepared using two methods as follows: (a) A DAS181 aqueous solution containing 14 mg/ml DAS181,5 mM sodium citrate, pH 5.0 was spray dried into an air stream at 55 °C, to produce microspheres. (b) Alternately, DAS181 microspheres were produced according to the 30 methods provided herein. To a DAS181 aqueous solution containing 14 mg/ml DAS181,5 mM sodium citrate, pH 5.0, was added 5% isopropanol as organic solvent. The resulting solution was cooled from +20 °C to -40 ‘C at a freeze ramp of 1 °C per minute in a Millrock Lab Series lyophilizer. Volatiles 2015215955 21 Aug 2015 -108- (water and Isopropanol) were removed by sublimation at 100 mTorr with primary drying at -30 °C for 12 hours and secondary drying at 30 °C for 3 hours, leaving the dry powder containing microspheres. B. Aerodynamic particle size distribution of microspheres 5 The microspheres prepared as described in Example 8A were tested by Andersen Cascade Impaction. The deposition of pharmaceuticals in the respiratory tract can be predicted by the aerodynamic behavior of particles (microspheres) on the stages/collection plates of the cascade Impactor.

The cascade impaction experiment was performed using DAS181 10 microspheres prepared by one of the two alternate methods described in section A above, f.e., either by spray-drying or by the methods provided herein. The microspheres (10 mg) were loaded into gelatin capsules. The gelatin capsules were placed into a CycloHaler (PharmaChemie) dry powder inhaler and subjected to cascade impaction. An 8-stage, ηοπ-viable Andersen 15 Cascade impactor (Thermo Electron, Boston) modified for use at 90 liters per minute of airflow and equipped with a USP throat, induction cone and no preseparator, was used. The collection plates of the Impactor representing various areas/stages of deposition post-inhalation (trachea, primary and secondary bronchi, terminal bronchi, alveoli, etc.) were coated with silicon 20 spray to prevent bouncing of the microspheres. Hie microspheres from the stages and collection plates were recovered into a phosphate buffered saline containing 0-1% Tween, and the amount of deposited DAS181 recovered from each stage and collection plate was quantified by measuring absorbance at 280 nm. 25 Results: The geometric size of microspheres produced by the two methods was assessed by light microscopy and found to be essentially identical (range of 1.5 — 3.0 microns) for both methods. As shown in Table 3 below, however, the aerodynamic particle size distribution of the two preparations differs significantly between the two methods. For the 30 microspheres produced according to a method as provided herein (/.e., method (b) as set forth In section A above), less than 25% remained trapped in the mouth (throat/cone of the impactor assembly), while greater than 70% 2015215955 21 Aug 2015 -109- of the microspheres were delivered to the trachea and lungs (with greater than 40% in the terminal bronchi and alveoli), in comparison, less than 50% of the DAS181 microspheres formed by spray-drying (method (a) as set forth in section A above) was delivered to the trachea and lungs (less than 20% in 5 the terminal bronchi and alveoli). The results demonstrate that methods provided herein can produce microspheres for delivery into deep lungs, and that the microspheres produced by methods provided herein have superior disagglomeration and tlowability properties (provide a higher delivered-dose) compared to microspheres produced by a spray-drying method. 10

Table 3: Results of Cascade Impaction Analyses of DAS 181 Microspheres

Percent Deposition of DAS181 Component , of the Andersen Cascade Impactor Corresponding Size Cut-Off (microns) Expected Deposition in Respiratory Airways Microspheres Produced by. Method (a) (i.e., Spray Drying) Microspheres -Produced by Method (b) Throat + Cone >10 oral cavity 42.9 16.6 -2 (S +P) 8.0-10 oral cavity 3.7 4.9 -1 (S + P) 6:5-8.0 oropharynx 5.9 5.5 -0 5.2-6.5 pharynx 5.8 4.0 1 3.5-5.2 trachsa/bronchi 12.5 9.3 2 2.6-3,5 secondary bronchi 11.6 12.6 3 1.7-2.6 terminal bronchi 11.0 24.0 4 1.0-1.7 alveoli 4.5 19.2 5 0.43-1.0 Alveoli 1.4 3.5 EXAMPLE 9

Large scale manufacture of Microspheres

This example demonstrates that the methods provided herein can be scaled for the manufacture of large quantities of DAS181. The Batch Process' 2015215955 21 Aug 2015 -llO- described herein is suitable for the manufacture of high quality dry powder microspheres In an amount ranging from, for example, milligrams to about a kilogram and is limited by the capacity of the mixing tank and/or lyophilfzer shelf space. An alternative "continuous” process described herein can be 5 used to manufacture amounts ranging from, for example, hundreds of grams to hundred or more kilograms (100 grams to 100 kg and above). Additional advantage of continuous process Is a better control over the chilling of the cocktail.

The large scale manufacture by a batch process or by a continuous 10 process can follow, for example, one or more of the steps described below in any combination of steps or specific alternative methods: • Precipitation of protein into microsoheres. This step can be performed in a batch mode by placing the cocktail solution containing the desired concentration of protein, organic solvent and counterion in 15 lyophilization tray(s) and placing the tray(s) onto lyophilizer shelves.

Alternatively, trays can be chilled and frozen on a chilled platform or other type of equipment (e.g., a freezer) and stored for a period of time frozen and lyophitized later. Alternatively, the microspheres can be formed by precipitation in a vessel with stirring, wherein the vessel is placed onto a cold surface or a cooling coil is immersed into liquid or while the cocktail is being recirculated through a heat exchanger using a peristaltic pump. Alternatively, the microspheres can be formed by precipitation in a continuous mode, by passing the cocktail solution through a heat exchangers) once using a.peristaltic pump. :5 · Removal, of_bulk liquid· The suspension of the microspheres can be concentrated using standard centrifugation, continuous flow centrifugation {e.g., CARR ViaFuge Pilot), or filtration (e.g., on glass fiber, sintered glass, polymer filters, hollow fiber cartridges (e.g., those manufactured by GE Healthcare) or tangential flow filtration cassettes t> (TFF cassettes, such as those manufactured by Mlllipore or Sariorius)).

The removal of bulk liquid (50% or greater) can result in a faster drying cycle and higher efficiency and throughput. 2015215955 21 Aug 2015 -111 - • Drying the microspheres. The recovered microspheres formed by any mode, can be dried by conventional lyophilization. Alternatively, the microspheres can be dried under ambient temperature and atmospheric pressure, eliminating the use of lyophilizer. 5

Results: DAS1S1 protein was successfully processed into dry powder (microspheres) by a continuous mode as described herein. Cocktail containing 10 mg/ml DAS181,20% isopropanol, 2 mM sodium sulfate was passed through 35 SERIES heat exchanger (Exergy, Garden City, NY) 10 coupled with a NESLAB circulating cryostat using a peristaltic pump so that during the passage the cocktail was cooied from about 25°C to about -12°C. The resulting suspension of microspheres exiting the heat exchanger was pumped into a prechilied lyophilization tray (-40 °C), frozen and iyophilized or, alternatively, pumped directly into liquid nitrogen and then Iyophilized. The 15 resulting microspheres, which were analyzed by microscopy and cascade impaction, showed uniform microspheres with minimal aggregation and good dispersivity and were similar in dimensions and aerodynamic particle size distribution to the microspheres produced by batch mode. When the formulated DAS181 cocktail solution was not chilled (not passed through heat 20 exchanger, thus no precipitation of microspheres was induced) and poured directly into liquid nitrogen, no microspheres were observed and, instead, glass-like crystals were observed after lyophilization. EXAMPLE 10

Batch mode process and formulation of DAS131 microspheres for 25 delivery to upper and central respiratory airways

Tills example describes formulation and a process for manufacture of DAS181 microspheres. The contents of the DAS181 cocktail solution and their relative amounts are shown in Table 4 below. 30 Table 4: Batch Manufacturing Formula for DAS181 Microspheres.

Ingredient Amount for one batch*1* Final Function Stock solution Amount concentration “ concentration added In formulated cocktail 2015215955 21 Aug 2015 -112- DAS181 protein 19.55 β(L 3.306 L, API solution 12 80- Active ingredient Sodium acetate™ 1.12 mM 0.688 mM pH buffer Acetic acid™ 0.63 mM 0.0387mM pH buffer Sodium Sulfate 500 raM 0.0215 L 2 mM Microparticle formation agent (counterion) Isopropanol 100% v/v 0.269 L 5% v/v Microparticle formation agent Calcium chloride 500 mM 0.0028 L 0.268 mM Stability enhancing agent Water for irrigation neat 1.79 L NA Diluent ^ Batch size: final volume of formulated cocktail 5.38 L. Theoretical yield 74 g of bulkDAS181 Dry Powder. ® Components of the DAS181 protein (API) stock solution. 5 A. Production of Bulk Drug Substance

The terms Drug Substance, Active Pharmaceutical Ingredient, and API are used Interchangeably In this example and referto the DAS181 protein. Production of DAS181 protein In bulk was conducted as follows. First, bulk amounts of DAS181 were expressed in E cofi (BL21 strain) essentially as 10 described In Example 1. The E colt cells expressing the DAS181 protein were washed by diafiltration in a fermentation harvest wash step using Toyopearl buffer 1, UFP-500-E55 hollow fiber cartridge (GE Healthcare) and a Watson-Marlow peristaltic pump.

The recombinant DAS181 protein was then purified in bulk from the 15 cells. The detailed specifications of the components and buffers used in the bulk purification of DAS181 are provided in Tables 5 and 6 below. The harvested and washed cells were lysed in a homogenization step by passing the cells twice through using Niro-Soave Panda cell disruptor. The homogenate thus obtained was clarified by microfittrailon using the Toyopearl 20 buffer 1, Hydrosart 0.2 micron TFF cassette and a Watson Marlow pump.

The clarified homogenate was then concentrated by allowing the lysate to recirculate without fresh buffer feed. Next,DAS181 protein was captured from the clarified homogenate an a Toyopearl SP-550C resin which was washed in a series of buffers (see Table 5) before the DAS181 protein was 25 eluted from the resin. The sodium chloride concentration of the eluate was 2015215955 21 Aug 2015 - 1X3 - adjusted to 1.0 M in a final buffer of 50 mM phosphate at pH 8.0. The DAS 181 -containing eluate was then passed through a Toyopearl Hexyl-650C resin for further purification using a Toyopearl Buffer 4. The resin eluate containing DAS181 protein was then buffer-exchanged into 5 mM sodium 5 acetate in a diafiltration step (see step 8 in Table 5). The concentrated protein was next passed through a Sartorius Q SingleSep Filter in order to remove DNA in a flow-through mode. Isopropanol was added to the Q SingleSep filtrate to a final concentration of 20% v/v. The DAS181 protein in the buffer was passed through an Amberchrome CG300M resin equilibrated 10 with an Amberchrom buffer (see step 11 in Table 5). The purified bulk DAS181 protein was then buffer-exchanged into formulation buffer and concentrated by diafiltration (see step 12 of Table 5).

Table 5: Purification of bnlkDASlSl drug substance 1 Purpose 1 Perm notation Harvest Wash Cartridge GE UFP-SOO-E55 Specifications Activity I Buffer Name Infer PSI Diaffttroitoa 1 Toyopearl Buffer 1 25-35 2 Purpose Homogenization Activity Step I Buffer Name Equilibration EmiiKb rattan I Harvest Buffer HotnoKcaimlion 1st Pass j Sample Load HomOKcnizatlon 2nd Pass 1 Sample Loed 3 Faroose I Horaojienate Clarification fDicfiJtmliori) I TBF Cartridge 1 KvdroSartlOK 0,6 nr1 | Specifications Actlviiv 1 Buffer Nome J Inlet PSi Recirculation | Sample Load Dlofiltnitton 1 Toyopearl Buffer 1 1 <50 A Purpose Permeate Concentration Specifications TFFGnrtridec HvdroSart lOKO.tJmi1 Activity BnfTcr Name Inlet PSI Redtcufndon 'Semple Load NS Concentration Sample Load <50 s Purpose OASl 81 captureperformed in bind and dole mode Resin Tovopcart SP-550C Activity St CD Buffer Name Loading Samolc Load Cfar* Homonsnate Wash SPWaehl TPvopearl Buffer 1 SP Wash 2 Toyopearl Buffer 2 SP 'Wash 3 Tovoocarl Buffer 3 2015215955 21 Aug 2015 -114- S?TVwh4 Toyopeari Buffer 2 SPWtishS Toyopeari Buffer 1 Elution Elution Toyopeari Buffer4

6 Pit mo so Adlust NaCI Concentration Method Add NaCI to I.OM Flout Buffer 50mM ohospliMe. 1.0 MNeCL pH 8.0 7 Purpose DAStSI purification in flow-throtieh mode Resin Tovooearl Hcxyl-SSOC Activity SlCD Buffer Name Loading Sample Land Cond. Hexyl Load 6 Furooje Concentration &amp; Diafiltration TFFCttrtndce HvdroSart 1 OK 0.5 m* Specifications Aeilvltv Buffer Nome ReeTrc. L/aiTn* Rcarcufation Toyonatrl Buffers 15-16 Concentration Hexyl Product Pool 15-15 Diafiltration ToyotMirl Buffers 15-16 Recirculation Tuvourarl Buffer 6 NS 9 Purpose Remove DNA in flow-through mode Ruin Sartoriua 0 SlngleSeu Filter Activity Step BufTerNimo Loadinc Sample Load 10 Purpose Buffer Adiustment Method Add Isoorooanol to 20¾ Final BufRi· S mM Accta te.-20°A Tsotwooanol. pH 5.0 Π Purpose DAS1B1 Dolishlnaln flow-throuEh made Resin Atnfastciromc CG300M Activity Step Buffer Name Loading Sample Load Ambirchrom Load J2 Parnosc Concentration &amp; DFttEltmtlon TFF Cnitridee HvdroSart 1 OK 0.6 m* Specifications Activity Buffer Name Rccfrc. LAniii* Recirculation Formulation Buffer 15-16 Concentration Ambcrehrom Product Foot 15-16 Dinnitratlon Formulalloti Buffer 15-16 •'Volume* ίη Hteis, except 4xdeflotes.tnu1tiptG5 ofitieiretCTtale volume CV * Colun*t Volumes NR e Nor Recorded NS*· Not Specified

Table (Ss Buffers used thiriairl AeDASlSl nitrification oroccss BufTcrNorne Buffer Composition Toyopeari Buffer 1 50 mM BOtassfumnUosohate. 03 M NeCl. pH 8.0 Tovooearl Suffer 2 1.1 mM potassium phosphite. 2.9 mM sodium phosphate. 154 mM NaCI. pH 7A Toyanearl Buffer 3 1 el aiM potassium phosphate, 23 mM sodium phosphate, 154 mM NcCh 1½ Triton Ji-100rC. 1¾ SOS. 0.5¾ sodium deoxvchalate. pH 7.4 Tovooearl Buffer 4 50 mM potassium phosphate. 1.0 M NaCI. dH 8.0 Toyoncarl Buffer 5 50 mM uDtas&amp;tum nhosphaie.. 0,5 M NaCI, dK 8.0 -115- *0 ii 1 β s mM sodium acetate. oH 3.0 ΤογοηβατΙ Buflcr7 3 irM sodium acetate. 60¾ Isoprooauol, pH 3.0 Formulation Buffer 1.75 tnM sodium acetate, pH 3.0 3% Isooronvl Alcohol 3%5sojiiODanal Amberotirom Buffin' 5 mM sodium acetate. 20% Isocnrnranut, pH 5.0 adiusted with acetic acid LON NaOH 3% IsoDrooanol 1.0 N NaOH. 3» isootwanol 1 .ON NaOH LONNaOH O-SNNaOH 0.3 N NaOH 0.1 N NaOH 0.1 N NaOH 70% IsomroDV) Alcohol 70%rsoorooanol 20%£tOH 20% ethyl alcohol 2015215955 21 Aug 2015 B. Batch Manufacturing Process

The Ingredients set forth in Table 4 above were combined to form 5 DAS181 microspheres In a large scale batch process as described below. Step I: Thawing of bulk Drug Substance

Frozen 0.2 pm-filtered bulk Drug Substance in plastic bottles was thawed overnight at ambient temperature (25±3 °C).

Step il: Weighing of the excipients and preparation of solutions 10 35.51 g of Sodium Sulfate anhydrous powder was weighed and Q.S. to 500 mL with Water For Irrigation, then stirred to obtain a clear solution. 18.38 g of Calcium Chloride dihydrate powder was weighed and Q.S. to 250 mL with Water For Irrigation, then stirred to obtain a dear solution.

Step HI: Preparation of die DAS181 cocktail solution 15 To 3.3 L of concentrated Drug Substance (19.55 g/L), 1,79 L of Water

For irrigation was added slowly with stirring, followed by 0.0215 L of Sodium Suffate solution, 0.0028 L of Calcium Chloride solution and 0.269 L of isopropanol. The solution was stirred to ensure complete mixing of components. 20 Step IV: Filtration of formulated cocktail solution through 0.2 um filter

The formulated cocktail solution of Step III was filtered through a 0.2 pm filter into sterile media bags to control particulates and btoburden.

Step V: Filling Into tvophilization travs

The formutated filtered solution was dispensed into autoclaved 25 Lyoguard lyophilization trays. To ensure even cooling of the solution and formation of high quality microspheres, 6 trays were each filled with 0.9 L or less of cocktail solution. 2015215955 21 Aug 2015 -116»

Step VI: Freezing and ivophilization

The trays were placed onto lyophilizer (Hull 120FSX200) shelves pre* chilled to -45 ±5 °C and the solution was allowed to chill and freeze. Formation of microspheres occurred while the solution was being frozen. The 5 freezing fs allowed to proceed for 1-2 h to ensure complete solidification. The product temperature was verified by reading the thermocouples attached to two of die six trays.

The lyophilization cycle steps are as follows: a) Set vacuum to 160 microns and allow to evacuate to 100 - 200 10 microns; b) Ramp shelf temperature to +10 °C over 3 h; c) Hold shelf temperature at +10 °C for 36 h (primary drying);

d) Thermocouple traces examined to verify that primary drying phase is completed and the product temperature has stabilized at +10°C ± 5eC 15 for 15-30 h. e) Ramp shelf temperature to +30°C over 1 h and hold for 3-5 h (secondary drying).

Step VII: Transfer of bulk DAS181 microsnheres Into container and mixing A section on the bottom film of each Lyoguard lyophilization tray was 20 cleaned using sanitizing wipes and a 3 x 3 cm opening was made with a scalpel. The dry microspheres were transferred into a plastic bottle. The bottle was capped and tumbled forty times, changing directions with each inversion. The tumbling was to ensure uniformity of bottle content Samples tor analytical testing were taken and the bottle was recapped and sealed into 25 plastic bags for storage.

In the DAS181 microsphere bulk manufacturing process as described above, sulfate was demonstrated to be a safe substance for use as a counterion, and reproducibly produced microspheres with a narrow size distribution. Further, the organic solvent isopropanol was a good solvent of 30 choice because (1) It is a class 3 solvent, (2) it can produce microspheres in a wide range (2 - 30%, v/v) of concentrations, and (3) it has a relatively high freezing point so its vapors can efficiently be trapped during lyophilization. 2015215955 21 Aug 2015 -117-

The protein concentration in the final formulation could be varied (10 -14 mg/ml), as could the concentration of counterion (1-5 mM) and isopropanol (2 - 30% v/v), without substantial impact on the physical properties of the microspheres or the activity of the DAS181 protein in the 5 microspheres. At higher concentrations of isopropanol (15 - 30%), the microspheres formed while the cocktail was still fully liquid. At tower concentrations (2 ~ 15%), ice crystals began to form first, followed by precipitation to form microspheres. C. Yield of DAS181 in the microspheres 10 The theoretical yield of DAS181 in the dry microspheres is calculated according to the following formula:

Theoretical yield = DAS181 protein, g * protein fraction in Dry Powder (microspheres)

The protein fraction value (0.866) was established empirically by 15 analysis of several manufactured batches of DAS181 microspheres. The theoretical yield for the amounts as set forth in Table 2 is 64.56 g + 0.866 = 74.55 g. The actual yield of DAS181 Dry Powder was found to be 64 g.

Results: The suitability of the microspheres prepared as described in section B above for administration by oral inhalation was tested by Andersen 20 - Cascade Impaction. The results are summarized in Table 7 below. The deposition of pharmaceuticals in the respiratory tract can be predicted by deposition of particles (microspheres) on the stages/coilection plates of the cascade impactor. For a pharmaceutical, e.g., DAS181 microspheres, that is administered to prevent or treat viral infections that Initiate in the respiratory 25 tract, such as Influenza, it is desirable to deposit the pharmaceutical in the threat, trachea and bronchi (upper and central respiratory airways). The DAS181 fusion protein delivered to upper and central respiratory airways cleaves off the receptor sialic acids from mucous membranes, thus preventing viral binding and infection at these sites. For optimal delivery of the DAS181 30 microspheres to sites where respiratory viral infection can be initiated, i.e., in the throat trachea or bronchi, the microspheres must not be (a) so big that they are trapped at the front end in toe mouth {i.e., microspheres are too big, 2015215955 21 Aug 2015 -118- about 8 microns or greater); or (b) so small that they are deposited in deep lungs and absorbed systemlcally into the blood stream (l.e„ 0.5 microns or smaller). For delivery of the DAS181 microspheres to the throat, trachea and • bronchi, a size range of about 1 micron to about 5.5—6 microns generally is 5 suitable. DAS181 microspheres manufactured as described above were characterized by Andersen cascade impaction and found to be suitable for delivery to upper and central respiratory airways with sufficiently low percentage (<5%) deposited in the alveoli. 10

Table 7: Aerodynamic Particle Size Distribution of DAS181 dry powder at 60 liters per minnte.__

Component of Andersen Cascade Impactor Corresponding size aut-orf, microns Expected deposition in respiratory airways DAS1S1 protein deposited (la mg) Percent of total DAS181 protein recovered Inhaler (Cyclohaler) 1.57±0.I1 20.13% Throal/Cone >10 * Oral cavity 0.93 * 0.19 11.92% —l (Stage+Plate) S.6-10 Oral cavity 0.50 ±0.10 6.41% -0 (Stage+Plate ) 6.5-8.6 oropharynx 0.40 ±0.03 5.13% I (Stage+Plate) 4.4-6.5 pharynx 0.53 ±0.03 744% 2 (Stage+Plate) 3.3—4.4 trachea/bronchi 6.83*0.07 10,64% 3 (Stage + Plate) 2.0-33 Secondary bronchi 1.80 ±0.09 23.08% 4 (Stage + Plate) 1.1-2.0 Terminal bronchi 0.82*0.08 10.51% 5 (Stage+Plate) 0.54-1.1 alveoli 0.23 ± 0.03 2.95% 6 (Stage + Plate) 0.25 - 0.54- alveoli 0.14± 0.03 1.79% EAC1 (Emitted) 6J24±0.10 80.00% 10’±~1.0 mg of DASl81 Dry Powder (8.5 mg± 10%DAS181 protein) was filled into HPMC capsule 15 SAC1 (Emitted) flection is the sum of all material recovered from USP Throat, Induction

Cone and stages -I to 6. DAS181 microspheres were further characterized by laser diffraction, which demonstrated, consistent with the cascade impaction results, that the majority 20 of the microspheres produced by the method described in this Example are .within a size range of between 1 micron and 5 microns in size.

Scanning Electron Microscopy (FEI Quanta 200 Scanning Electron ' Microscope, Everhart Thornley (ET) detector) of.the DAS181 microspheres . prepared according to the method described in this Example revealed that the 25 microspheres are present as agglomerates of hundreds and thousands of 2015215955 21 Aug 2015 -119- individual particles approximately 0.5 -3 micron in size. The agglomerates however are easily dissipated by air turbulence produced during the actuation through dry powder inhaler (as demonstrated by Andersen Cascade Impaction or laser diffraction). Light microscopy of microspheres dispersed in 5 a liquid surfactant (e.g. Triton X-100 or Tween 20) or non-polar solvent (e.g., alcohol, acetone, or acetonitrile) that does not dissolve the microspheres, confirmed that aggregates are easily dissipated Into individual uniform microspheres. EXAMPLE 11 10 Preparation of DAS181 microspheres using sulfates other than the sodium salt

Studies have shown that in certain instances, e.g., in some asthmatics, the presence pf sodium in formulations for pulmonary administration could cany a risk of inducing airway hyperresponsiveness (Agrawal et ah, Lung, 15 183:375-387 (2005)). This example therefore tested alternate salts, such as salts of other metals such as potassium, magnesium and calcium. DAS181 microspheres were manufactured as described above in Example 1. Cocktail solutions containihg 12 mg/mL DAS181 and 5% (v/v) isopropanol contained as counterions the indicated sulfates at 2 mM 20 concentration, pH 4.5 — 5.0. The microspheres were formed by cooling the solutions from +25 "C to -45 “C. Upon freezing, the volatiles (water and isopropanol) were removed by sublimation, leaving the dry powder containing microspheres.

The aerodynamic particle size distribution of the dry powder was 25 assessed by Andersen Cascade Impaction, and the amount of DAS181 per stage was determined by UV measurement at 226 nm (A226). The results are shown below in Table 8. The results demonstrate that sulfate salts otherfhan the sodium salt can be used as counterion to obtain DAS181 microsphems of a size range such that the majority are delivered to the throat, trachea and 30 bronchi, in an amount that is comparable to the amount delivered when sodium sulfate is used as the counterion. 2015215955 21 Aug 2015 -120-

Aerodynamic Particle Size Distribution ofDASISl microspheres formulated with or without sodium

Table 8:

Percent BASIS! ocr stupe Corresponding sine cut-off, microns Expected, deposition in respiratory airways. Sodium Sulfate Potassium Sulfate Magnesium Sulfate Calcium Sulfate Inhaler 19.86% 28.58% 21.41% 16.71% Capsule 2,07% 2.30% 1.83% - 0.00% XhroaH-Cone >10 Oral cavity 11.67% 9.00% 12.91% ' 16.79% ~1(S+P) 8.6-10 Oral cavity 10.00% 3.43% 7.86% 14.87% -OfS+Pl 6.5-8.6 oropharynx 5.30% 3.08% 4.71% 7-77% lfS+Pl 4.4-6.5 pharynx 6.97% 5.86% 6.58% 7.54% 2fS+P) 3.3-4.4 trachealbronebi 755% 8.24% 6.90% 6.43% 3fS+P) 2.0-3.3 Secondary bronchi 1957% 20.21% 17.01% 12.65% 4fS+Pl 1.I-2.0 Terminat bronchi 12.39% 14.00% 13.00% 10.39% 5CS+P1 0.54- LI alveoli 2.80% 2,99% 4.31% 4.69% 6f3+P) 0-25-0.54 alveoli | 1.82% 231% 3.44% 2.16% 5 The dry powders also were incubated at +37 °C or +53°C for a duration as indicated in Table 9 and tested for sialldase activity using the 4-MU-NANA assay'as described in Example 1- and incorporated by reference herein. The relative activity compared to ηοη-Iyophilized DAS181 microspheres stored at -80 °C is shown in Table 9. The results show that the stability of the 10 microspheres prepared using the various metal sulfates as counterions were ' comparable to that of sodium sulfate, with retention of almpst all or all the activity for over 2-months at-37 °C and retention of almost ail (sodium and :· potassium sulfates) or-over 85% (magnesium and zinc sulfaites) of the activity . for over 10 days at 53 eCThis experiment demonstrates that various non-1S sodium containing counterions can produce microspheres with desirable characteristics.

Table 9: Sialldase activity of DA5181.microsph.ere formulations: accelerated stability studies.

Percent Activity Remaining Temperature 37°C S3°C incubation Days 42 Days 69 Days 11 Days 39 Days 2raM Sodium SulfktcH- 0.268mM CaClg 2mM Potassium Sulfate+ 0.268mM CaCIy 2mM Magnesium Sulfate+ 0.268mM C&amp;dz 13.34mM Calcium/ 2mM Sulfate 107.14% 105.62% 97.37% 104.00% 123.81% 107.29% 116.67% 93.20% 110.66% 23.66% 101.54% 52.76% 85.93% 60.00% 87.12% 40.48% 2015215955 21 Aug 2015 -121- EXAMPLE 12

Stability of DAS181 microspheres 5 The stability of the DAS181 protein in the microspheres was assessed by measuring sialidase activity over time using the 4-MU-NANA activity assay as described above in Example 1 and as incorporated by reference herein. The production of dry DAS181 microspheres was undertaken in a cocktail solution containing 10 mg/mL DAS181,2 mM sodium sulfate, 5% v/v 10 isopropanol. To some solutions, 0.01 % w/v sugar (sorbitol, mannitol, trehalose or sucrose) was added. The microspheres were formed by cooling the solutions from +25 °C to -45 eC. Upon freezing, the volatiles (water and isopfopanol) were removed by sublimation, leaving the dry powders containing microspheres. 15 A. Stability of DAS181 microspheres without sugars

The DAS181 dry powder microspheres formulated without sugars were stored at room temperature (25 DC) In a container next to Drierite desiccant (Hammond Drierite, Xenia, OH). The dry powder retained its original potency (as measured by sialidase activity using 4-MU-NANA according to Exampiel 20 and as incoiporated by reference herein; results shown in Table 10) and aerodynamic particle size distribution (as measured by Andersen Cascade impaction; Table 11) for at least 8 months.

Table 10: Specific activity of DAS 183 dry powder.

Test TimeO 3 months 8 months Sialidase Activity with reference to time 0 100% 102.0% 99.9%

Table 11: Aerodynamic particle size distribution of PAS181 dry powder ACI Component Corresponding size cut-off, .microns Expected deposition in respiratory airways TimeO 3 Months 8 Months Throat +· Cone >10 Oral cavity 19.57 ± 2.43 26.00 ± 0.30 18.57 ± 4.14 Stage -1 8.6-10 Oral cavity 17.87 ± 0.51 12.87 db 1.56 15,13 ± 2.41 Stage -0 6.5-8.6 oropharynx 10.27 ± 7.07 * 9.80 =fc 2015215955 21 Aug 2015 - 122 - ACI Comnoneut Corresponding size cut-off, microns Expected deposition in respiratory airways TimeO 3 Months 8 Months 0.93 0.32 1.80 Stage 1 4.4-6.5 pharynx 8.57* 0.49 8.80 £ 0.26 7.73 ± 0.57 Stage 2 33-4.4 trachea/bropchi 10.67 ± 033 10.70 ± 0.35 9.30 ± 0.82 Stage 3 2.0-3.3 Secondary bronchi 21.10 ± 0.75 21.80 ± 0.52 21.90 ± 0.87 Stage 4 1.1-2.0 Terminal bronchi 10.10 ± 0.75 10.63 * 0.80 14.50 * 3.22 Stage 5 0.54-1.1 alveoli 1.47 ± 0.23 1.73 ± 0.06 2.37 ± 0.06 Stage 6 0.25-0.54 alveoli 0.33 ± 0.06 0.40 ± 0.10 0.73* 0.06 1 Table 11: Aerodynamic particle distribution was assessed by Andersen. Cascade Impaction and expressed as % of total DAS 181 protein recovered. Capsules were filled with 10 mg ofDASISl dry powder and actuated using Cyclohaler dry powder 5 inhaler as delivery device. Air flow rate was 50 Liters per minute. Assays were performed in triplicate, mean and standard deviation are shown. B. Stability of DAS181 microspheres formulated with sugars

The sialidase activity of DAS181 in the dry powder microsphere formulations containing sugars and in foe unlyophilized microsphere 10 formulations stored at «80 eC, were measured using fluorescent substrate 4- MU-ΝΑΝΑ as described in Example 1 and as incorporated by reference herein. The dry powder formulations containing no sugar or various sugars as indicated below in Table 12 were stored at +42 ”C for 4 weeks (forced degradation). The results are shown in Table 12. Relative to unlyophilized is formulations stored at -80 ec, the formulation containing no sugar retained almost 80% of its activity. The addition of various sugars increase the stability so that about 88-98% of the activity is retained, depending on the sugar.

Table 12

Sugar Percent Slalidase Activity Remaining after 4 weeks at 42 °C No Sugar 79.82 Sorbitol 91.23 -123-

Mannitol 89.47 Trehalose 97.37 Sucrose 88.60 2015215955 21 Aug 2015

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims (4)

1, A composition comprising microparticles, wherein the microparticles comprise: a protein comprising SEQ ID NO: 17, histidine, trehalose, and magnesium sulfate,

2, The composition of claim 1, wherein the microparticles are 85% to 90¾ w/w a protein comprising SEQ ID N.Q:17.

3, The composition of claim 1 or claim 2,which is a dry powder,

4> The com position pf any one of claims 1 to 3, wherein; the size of the microparticles: is between 1 micron and 1:0 microns.