JP2023553703A - Lipocalin mutein dry powder formulation for the treatment of asthma - Google Patents

Abstract

The present invention provides for the treatment of asthma in a human subject by administering to the subject a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, by oral inhalation. Regarding treatment. The present invention also relates to dry powder formulations comprising anti-IL-4 receptor alpha (IL-4Rα) lipocalin muteins, or variants or fragments thereof.

Description

The present invention provides for the treatment of asthma in a human subject by administering to the subject a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, by oral inhalation. Regarding treatment. The present invention also relates to dry powder formulations comprising anti-IL-4 receptor alpha (IL-4Rα) lipocalin muteins, or variants or fragments thereof.

Asthma is a chronic, complex and heterogeneous respiratory disease characterized by various pathogenic features including pneumonitis, mucus hypersecretion, variable airway obstruction and airway remodeling. It is defined by a history of respiratory symptoms such as wheezing, shortness of breath, and cough, and varies over time and in severity. Both symptoms and airway obstruction can be caused by a variety of factors, such as exercise, exposure to inhaled irritants, allergens or respiratory infections. Patients are at risk for worsening (exacerbation) of their asthma. These asthma exacerbations can be life-threatening and can significantly impact a patient's quality of life. Treatment of most asthma patients consists of a treatment regime of control agents and bronchodilator therapy. Inhaled corticosteroids (ICS) are considered the "gold standard" for controlling asthma symptoms, and long-acting beta agonists (LABAs) are the most effective bronchodilators currently available. Oral corticosteroids remain the standard treatment for severe asthma, but are associated with significant side effects. On the other hand, anti-IgE monoclonal antibodies omalizumab, benralizumab, mepolizumab, and reslizumab, anti-IL-5 antibody, and IL-4Rα and IL-13 monoclonal antibody blocker dupilumab (USA) are limited to severely ill patients. Provide a number of choices. Furthermore, patients often remain uncontrolled even with ICS/LABA and a limited number of alternative therapies, highlighting important unmet needs (Ray, A., et al., 2016, Current concepts of severe asthma. J. Clin. Invest. 126, 2394-2403).

Signal transducers and activators of interleukin-4, interleukin-13, interleukin-4 receptor alpha, and transcription factor-6 are important in the development of airway inflammation, mucus production, and airway hyperresponsiveness in asthma. It is a component.

Lipocalins are proteinaceous binding molecules that have naturally evolved to bind ligands. Lipocalin is present in many organisms including vertebrates, insects, plants, and bacteria. Members of the lipocalin protein family (Pervaiz, S., & Brew, K. (1987) FASEB J. 1, 209-214) are typically small secreted proteins with a single polypeptide chain. They are characterized by a range of different molecular recognition properties: their variety binds to mainly hydrophobic molecules (e.g. retinoids, fatty acids, cholesterol, prostaglandins, bilivergins, pheromones, tastants, and odorants) capacity, their binding to specific cell surface receptors, and their formation of macromolecular complexes. Although classified primarily as transport proteins in the past, it is now clear that lipocalins serve a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in regulating immune responses and mediating cellular homeostasis (see, eg, Flower, D.R. (1996) Biochem. J. 318, 1-14 and Flower, D.R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).

Lipocalins share an unusually low level of overall sequence conservation, often less than 20% sequence identity. In contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded antiparallel β-sheet closed upon itself to form a serially hydrogen-bonded β-barrel. This β-barrel forms a central cavity. One end of the barrel is sterically blocked by an N-terminal peptide segment across its bottom, as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to solvent and contains a target binding site formed by four flexible peptide loops. This diversity of loops within the rigid lipocalin scaffold gives rise to a variety of different binding modes, each capable of accommodating targets of different size, shape, and chemical properties (e.g., Flower, DR. (1996), supra, Flower, D.R. et al. (2000), supra, or reviewed in Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).

Human tear lipocalin (TLPC or Tlc), also called lipocalin-1, tear prealbumin, or von Ebner gland protein, was originally identified as the major protein (approximately one-third of the total protein content) of human tears. Although described, it has also been identified in several other secretory tissues, including the prostate, adrenal glands, thymus, mammary glands, testes, nasal and tracheal mucosa, and corticotropin-secreting cells of the pituitary gland. Homologous proteins have been found in rhesus monkeys, chimpanzees, rats, mice, pigs, hamsters, cows, dogs, and horses. Tear lipocalin is an unusual lipocalin member in that it exhibits unusually broad ligand specificity when compared to other lipocalins and in its high promiscuity toward relatively insoluble lipids (Redl, B. 2000) Biochim. Biophys. Acta 1482; 241-248). This characteristic of tear lipocalin is thought to be due to the protein's ability to inhibit bacterial and fungal growth in the cornea. A remarkable number of lipophilic compounds of different chemical classes such as fatty acids, fatty alcohols, phospholipids, glycolipids and cholesterol are endogenous ligands of this protein. Interestingly, in contrast to other lipocalins, the strength of the ligand (target) binding to tear lipocalins correlates with the length of the hydrocarbon tail for both alkylamides and fatty acids. Therefore, tear lipocalin binds most strongly to the least soluble lipids (Glasgow, B.J. et al. (1995) Curr. Eye Res. 14, 363-372, Gasymov, O.K. et al. 1999) Biochim. Biophys. Acta 1433, 307-320). The 1.8-A crystal structure of tear lipocalin revealed an unusually large cavity within its β-barrel (Breustedt, D.A. et al. (2005) J. Biol. Chem. 280, 1, 484 -493).

International Patent Application No. 2005/19256 discloses muteins of tear lipocalin having at least one binding site for a different or the same target ligand and provides methods for the production of such muteins of human tear lipocalin. According to this PCT application, certain amino acids extend within the primary sequence of tear lipocalin, specifically amino acids 7-14, 24-36, 41-49, 53-66, 69- of mature human tear lipocalin. The loop regions containing 77, 79-84, 87-98, and 103-110 are subjected to mutagenesis to generate muteins with binding affinity. The resulting muteins have binding affinities for the selected ligands (K _D ) in the nanomolar range, in most cases >100 nM. International Patent Application No. 2008/015239 discloses muteins of tear lipocalin that bind to certain non-natural ligands, including IL-4 receptor alpha. Binding affinity is in the nanomolar range. International Patent Application No. 2011/154420 describes high affinity muteins of human tear lipocalin that bind to human IL-4 receptor alpha in the nanomolar range, and methods for producing such high affinity muteins. ing. International Patent Application No. 2013/087660 describes the use of human tear lipocalin muteins to treat diseases in which the IL-4/IL-13 pathway contributes to disease development, including asthma.

International Patent Application No. 2020/200960 describes the use of muteins of human tear lipocalin to treat asthma by inhalation, e.g. by nebulization, wherein the delivered dose of lipocalin mutein is from about 0.1 mg to about 160 mg. be.

Advantages of pulmonary delivery of active agents such as lipocalin muteins include convenience of patient self-administration, potential reduction of drug side effects, ease of delivery by inhalation, elimination of needles, etc. Many clinical studies using inhaled proteins, peptides, DNA and small molecules have demonstrated that efficacy can be achieved both intrapulmonally and systemically. However, many molecules, especially biological molecules, that require high payloads for delivery present problems for the development of inhalable formulations. The formulation must provide stability to the biological payload, e.g., lipocalin mutein, and allow for scalable manufacturability while also maintaining desirable physical properties to facilitate delivery to the patient's lungs. Must have.

The present invention provides a pre-formulation study, a phase I crossover study in humans to evaluate the pharmacokinetics (PK effects) of anti-IL-4 receptor alpha (IL-4Rα) human tear lipocalin AZD1402 by inhalation in healthy subjects. Based on clinical trials and a two-part Phase IIa study to evaluate the efficacy and safety of AZD1402 administered to human patients with asthma as a dry powder. The amino acid sequence of AZD1402 is shown in Table 70 as SEQ ID NO:1. AZD1402 antagonizes IL-4 receptor alpha (IL-4Rα) and is designed for inhalation.

Based on these studies, the present invention provides a method of treating asthma in a human subject, which method comprises administering a therapeutically effective amount of anti-IL-4 receptor alpha (IL-4 receptor alpha) comprising the amino acid sequence set forth in SEQ ID NO: 1. 4Rα) administering to said subject by inhalation a dry powder formulation comprising a lipocalin mutein, or variant or fragment thereof, wherein said lipocalin mutein, or variant or fragment thereof, at a nominal delivered dose of about 0.1 mg to about 30 mg; Administered to the above subjects by oral inhalation.

The present invention also provides a dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject. and the method comprises administering a dry powder formulation by inhalation, wherein a nominal delivered dose of about 0.1 mg to about 30 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. The anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

Furthermore, the present invention provides a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of asthma in a human subject. wherein the treatment comprises administering the dry powder formulation by inhalation, the lipocalin mutein, or variant or fragment thereof, at a nominal delivered dose of about 0.1 mg to about 30 mg, orally. The anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, which is administered to the subject by inhalation, comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

In any of the above aspects of the invention, a nominal delivered dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of said lipocalin mutein, or variant or fragment thereof, is delivered to said subject by oral inhalation. may be administered.

In any of the above aspects of the invention, the lipocalin mutein at a nominal delivered dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg, or The variant or fragment may be administered to the subject by oral inhalation.

In some embodiments, a nominal delivered dose of about 0.5 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 1 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 2 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 3 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 4 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 5 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 6 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 10 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation. In some embodiments, a nominal delivered dose of about 30 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject by oral inhalation.

In another aspect, the invention also provides a method for treating asthma in a human subject, the method comprising: a therapeutically effective amount of an anti-IL-4 receptor alpha ( IL-4Rα) administering to said subject by inhalation a dry powder formulation comprising a lipocalin mutein, or variant or fragment thereof, in a nominal metered dose of about 0.2 mg to about 40 mg of said lipocalin mutein, or variant or fragment thereof; A method is provided in which the fragment is administered to the subject by oral inhalation.

The present invention also provides a dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject. and the method comprises administering a dry powder formulation by inhalation, wherein a nominal metered dose of about 0.2 mg to about 40 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. The anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

Furthermore, the present invention provides a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of asthma in a human subject. wherein the treatment comprises administering the dry powder formulation by inhalation, wherein the lipocalin mutein, or a variant or fragment thereof, in a nominal metered dose of about 0.2 mg to about 40 mg is administered orally. The anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, which is administered to the subject by inhalation, comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

In any of these embodiments, a nominal metered dose of the lipocalin mutein, or variant thereof, of about 0.55 mg, about 0.6 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg. Alternatively, the fragment may be administered to the subject.

In any of these embodiments, about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11 A nominally metered dose of .1 mg, about 12 mg, or about 36 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject.

In some embodiments, a nominal metered dose of about 0.55 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 0.6 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 1.1 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 2.2 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 3.3 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 4.4 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 5.5 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 6.6 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominal metered dose of about 11.1 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominally metered dose of about 12 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject. In some embodiments, a nominally metered dose of about 36 mg of the lipocalin mutein, or variant or fragment thereof, may be administered to the subject.

In any of the aspects or embodiments of the invention described above, the lipocalin mutein, or variant or fragment thereof, may be administered to the subject at least once a day. In some embodiments, the lipocalin mutein, or variant or fragment thereof, is administered to the subject once daily. In some embodiments, the lipocalin mutein, or variant or fragment thereof, is administered to the subject twice a day.

In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 2.5 mg, about 5 mg, about 10 mg, about 15 mg. , about 20 mg, or about 25 mg.

In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 2.5 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 5 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 10 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 15 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 20 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject once daily is about 25 mg.

In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg.

In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg.

In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.5 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 1 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 2 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 3 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 4 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 5 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 6 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 10 mg. In some embodiments, the nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 30 mg.

In some embodiments, a total nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg, e.g., the total nominal delivered dose per day is For example, about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg is administered to the subject in one, two, or more doses per day.

In some embodiments, a total nominal delivered dose of about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 20 mg, or about 60 mg of the lipocalin mutein, or variant or fragment thereof, is , delivered to the subject per day such that the total nominal delivered dose per day is about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 20 mg, or about 60 mg, It is administered to the subject in one, two, or more doses per day, for example, once per day.

In some embodiments, a total nominal delivered dose of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg of the lipocalin mutein, or variant or fragment thereof, per day is delivered to the subject. administered, e.g., once, twice per day, such that the total nominal delivered dose per day is about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg. or more doses are administered to the subject.

In some embodiments, a total nominal delivered dose of about 1 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 2 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 2.5 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 4 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 5 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 6 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 8 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 10 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 12 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 15 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 20 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 25 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. In some embodiments, a total nominal delivered dose of about 60 mg per day of the lipocalin mutein, or variant or fragment thereof, is administered to the subject.

In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.55 mg, about 0.6 mg, about 1 .1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg.

In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.55 mg, about 0.6 mg, about 1 .1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 36 mg.

In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 0.55 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 0.6 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 1.1 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 2.2 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 3.3 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 4.4 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 5.5 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 6.6 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 11.1 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 12 mg. In some embodiments, the nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 36 mg.

In some embodiments, the lipocalin mutein at a total nominal metered dose of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg per day. , or a variant or fragment thereof, is administered to the subject, e.g., at a total nominal daily metered dose of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, One, two, or more doses per day of about 24 mg, or about 72 mg are administered to the subject.

In some embodiments, about 1.1 mg, about 1.2 mg, about 2.2 mg, about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22.2 mg, A total nominal metered dose of about 24 mg, or about 72 mg of the lipocalin mutein, or variant or fragment thereof, per day, e.g., a total nominal metered dose of about 1.1 mg, about 1.2 mg, about 2.2 mg per day. , about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22.2 mg, about 24 mg, or about 72 mg per day to the subject.

In some embodiments, a total nominal metered dose of about 1.1 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 1.2 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 2.2 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 4.4 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 6.6 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 8.8 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 11 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 13.2 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 22.2 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 24 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal metered dose of about 72 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day.

In any of the described aspects and embodiments of the invention, the formulation may include a plurality of microparticles, the microparticles comprising the lipocalin mutein described above, or a variant or fragment thereof.

In some embodiments of any of the described aspects of the invention, the dry powder formulation comprises trehalose, leucine and/or trileucine, either alone or in any combination thereof. For example, in some preferred embodiments, the dry powder formulation may include (i) trehalose, (ii) trehalose and leucine, (iii) trehalose, leucine and trileucine, or (iv) trehalose and trileucine.

In some embodiments of any of the described aspects of the invention, the dry powder formulation comprises a phosphate buffer as a buffering agent, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate. and isotonic agents such as sodium chloride.

In another aspect, the invention provides a dry powder formulation comprising an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof. including.

In some embodiments, the dry powder formulation includes trehalose, leucine, and/or trileucine, either alone or in any combination thereof. For example, in some preferred embodiments, the dry powder formulation may include (i) trehalose, (ii) trehalose and leucine, (iii) trehalose, leucine and trileucine, or (iv) trehalose and trileucine. In the most preferred embodiment, the dry powder formulation includes trehalose, leucine, and trileucine.

In some embodiments, the dry powder formulation further comprises a buffering agent such as, for example, a phosphate buffer, e.g., potassium dihydrogen phosphate and/or anhydrous disodium phosphate, and a tonicity agent such as sodium chloride. include.

In some embodiments, the dry powder formulation further comprises a buffer, such as a histidine buffer, and a tonicity agent, such as sodium chloride.

In some embodiments, the dry powder formulation provides a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg when the formulation is administered to the subject by oral inhalation. do.

In some embodiments, the dry powder formulation provides a nominal delivered dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of the above when the formulation is administered to the subject by oral inhalation. Lipocalin muteins, or variants or fragments thereof, are provided.

In some embodiments, the dry powder formulation contains about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, when the formulation is administered to the subject by oral inhalation. A nominal delivered dose of about 10 mg or about 30 mg of the lipocalin mutein, or variant or fragment thereof, is provided.

In some embodiments, the dry powder formulation delivers a nominal delivery of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg when the formulation is administered to the subject by oral inhalation. A dose of the lipocalin mutein, or variant or fragment thereof, is provided.

In some embodiments, the nominal delivered dose is about 0.5 mg. In some embodiments, the nominal delivered dose is about 1 mg. In some embodiments, the nominal delivered dose is about 2 mg. In some embodiments, the nominal delivered dose is about 2.5 mg. In some embodiments, the nominal delivered dose is about 3 mg. In some embodiments, the nominal delivered dose is about 4 mg. In some embodiments, the nominal delivered dose is about 5 mg. In some embodiments, the nominal delivered dose is about 6 mg. In some embodiments, the nominal delivered dose is about 10 mg. In some embodiments, the nominal delivered dose is about 15 mg. In some embodiments, the nominal delivered dose is about 20 mg. In some embodiments, the nominal delivered dose is about 25 mg. In some embodiments, the nominal delivered dose is about 30 mg.

In some embodiments, a dry powder formulation comprises a nominal metered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.2 mg to about 40 mg for administration to the subject via oral inhalation.

In some embodiments, the dry powder formulation is about 0.55 mg, about 0.6 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg for administration to the subject by oral inhalation. , or a nominally measured dose of about 36 mg of the lipocalin mutein, or a variant or fragment thereof.

In some embodiments, the dry powder formulation is about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about A nominal metered dose of the lipocalin mutein, or variant or fragment thereof, of 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg or about 36 mg is provided.

In some embodiments, the nominal metered dose is about 0.55 mg. In some embodiments, the nominal metered dose is about 0.6 mg. In some embodiments, the nominal metered dose is about 1.1 mg. In some embodiments, the nominal metered dose is about 2.2 mg. In some embodiments, the nominal metered dose is about 3.3 mg. In some embodiments, the nominal metered dose is about 4.4 mg. In some embodiments, the nominal metered dose is about 5.5 mg. In some embodiments, the nominal metered dose is about 6.6 mg. In some embodiments, the nominal metered dose is about 11.1 mg. In some embodiments, the nominal metered dose is about 12 mg. In some embodiments, the nominal metered dose is about 36 mg.

In any aspect or embodiment of the invention, the dry powder formulation may include a plurality of microparticles, the microparticles comprising the lipocalin mutein described above, or a variant or fragment thereof.

The present invention also provides a method for treating asthma in a human subject, the method comprising administering to the subject by oral inhalation a dry powder formulation according to any of the embodiments above.

The present invention also provides a dry powder formulation according to any of the above embodiments for use in a method for treating asthma in a human subject, the method comprising administering the dry powder to the subject by oral inhalation. including administering the formulation.

Furthermore, the present invention provides the use of a dry powder formulation according to any of the above embodiments for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of asthma in a human subject, the treatment comprising: comprising administering a dry powder formulation to the subject by oral inhalation.

Embodiments and experiments illustrating the principles of the invention are described herein with reference to the accompanying drawings.
16 shows a study flowchart for the Phase I clinical trial described in Example 16. Test Product A: AZD1402 nebulizer solution at a nominal dose of 18 mg administered via an InnoSpire Go nebulizer. Test Product B: AZD1402 inhalation powder at a nominal delivery dose of 10 mg administered via a Plastiape Monodose inhaler. Test Product C: AZD1402 inhalation powder at a nominal delivery dose of 30 mg administered via a Plastiape Monodose inhaler. Geometric mean (*/SD) serum concentration (ng/mL) of AZD1402 vs. time after inhalation via the InnoSpire Go nebulizer (18 mg nominal delivered dose), Plastiape Monodose inhaler (10 mg or 30 mg nominal delivered dose). Kinetic Analysis Set - Linear Scale). A: Nebulizer solution administered via InnoSpire Go nebulizer (18 mg nominal delivered dose), B: Inhalation powder administered via Plastiape Monodose inhaler (10 mg nominal delivered dose), C: Via Plastiape Monodose inhaler Inhalation powder administered (30 mg nominal delivered dose). SD: standard deviation. N: Number geometric mean of subject/SD: exp (average [log{PK concentration}]-std[log{PK concentration}]). Geometric mean *SD: exp(average [log{PK concentration}] + std[log{PK concentration)}]). Vertical lines represent the geometric mean */SD. Geometric mean (*/SD) serum concentration (ng/mL) of AZD1402 vs. time after inhalation via the InnoSpire Go nebulizer (18 mg nominal delivered dose), Plastiape Monodose inhaler (10 mg or 30 mg nominal delivered dose). Kinetic analysis set - semi-logarithmic scale). A: Nebulizer solution administered via InnoSpire Go nebulizer (18 mg nominal delivered dose), B: Inhalation powder administered via Plastiape Monodose inhaler (10 mg nominal delivered dose), C: Via Plastiape Monodose inhaler Inhalation powder administered (30 mg nominal delivered dose). SD: standard deviation. N: Number of targets. Geometric mean/SD: exp(average [log{PK concentration}]-std[log{PK concentration}]). Geometric mean *SD: exp(average [log{PK concentration}]+std[log{PK concentration)}). Vertical lines represent the geometric mean */SD. AZD1402 steady-state serum concentrations are shown after 12 days of once-daily and once-every-two-day administration of doses of 5 mg and 1 mg, respectively.

Aspects and embodiments of the invention will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

The present invention relates to a method of treating asthma in a human subject. Asthma may be, for example, moderate or severe asthma. In moderate asthma, the patient can speak in phrases, the respiratory rate is increased, the accessory muscles are not used, the pulse rate is 100-120 bpm, and the oxygen saturation (in air) is 90-95%. Yes, and peak expiratory flow (PEF) is >50% predicted or best (see Pocket Guide for Asthma Management and Prevention, Global Initiative for Asthma, Updated 2020). In severe asthma, the patient speaks in words, the respiratory rate is >30/min, accessory muscles are used, the pulse rate is >120 bpm, and the oxygen saturation (in air) is <90%. In some embodiments, the asthma is allergic asthma.

A method of treating asthma comprises administering a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

"Therapeutically effective amount" means a dose that produces the effect for which it is administered. A "therapeutically effective amount" of a lipocalin mutein described herein depends on factors such as age, weight, general health, gender, diet, time of administration, drug interactions, and severity of the condition. Variations may be necessary and this will be ascertainable by one skilled in the art by routine experimentation. A therapeutically effective amount, as used in this application, is also one in which any toxic or detrimental effects of the lipocalin mutein are outweighed by the therapeutically beneficial effects.

Interleukin-4 receptor alpha chain (IL-4Rα) is a type I transmembrane protein that can bind interleukin-4 and interleukin-13 to regulate IgE antibody production in B cells. In T cells, the encoded protein can also bind to interleukin-4 and promote Th2 cell differentiation.

Lipocalin muteins Lipocalin muteins specific for IL-4 receptor alpha (IL-4Rα), particularly human IL-4Rα, are disclosed in International Patent Publication No. 2008/015239, International Patent Publication No. 2011/154420, and International Patent Publication No. 2013/087660. has been disclosed. The human interleukin-4 receptor alpha chain may have the amino acid sequence of SWISS PROT Data Bank Accession No. P24394, set forth as SEQ ID NO: 4, or a fragment thereof. Illustrative examples of fragments of human interleukin-4 receptor alpha chain include amino acids 26-232 of IL-4 receptor alpha.

The IL-4Rα-specific lipocalin mutein having the amino acid sequence shown in SEQ ID NO: 1 is a mutein of human tear lipocalin.

As used herein, a "mutein" preferably refers to a protein that is 1 compared to a native (wild type) nucleic acid or protein "reference" scaffold, which is mature human tear lipocalin, set forth as SEQ ID NO:3. Refers to the exchange, deletion, or insertion of one or more nucleotides or amino acids. The "reference scaffold" also includes a mutein described herein, or a fragment or variant thereof.

The amino acid sequence of human tear lipocalin is provided by SWISS-PROT Databank Accession No. P31025, shown in SEQ ID NO:2. Mature human tear lipocalin lacks the N-terminal signal peptide contained in the sequence of SWISS-PROT Accession No. P31025, ie, the N-terminal signal peptide (amino acids 1-18) contained in the sequence of SWISS-PROT Accession No. P31025. The amino acid sequence of mature human tear lipocalin is shown in SEQ ID NO:3.

Lipocalin muteins for use in the invention include SEQ ID NO: 1, or a variant or fragment thereof. The lipocalin mutein set forth as SEQ ID NO: 1 is a variant of the mature human tear lipocalin set forth as SEQ ID NO: 3, which lacks the first four amino acids and, inter alia, is a variant of the mature human tear lipocalin set forth as SEQ ID NO: 3. contains the following amino acid substitutions at positions corresponding to the sequence positions of the amino acid sequence. Arg26→Ser, Glu27→Arg, Phe28→Cys, Glu30→Arg, Met31→Ala, Asn32→Val, Leu33→Tyr, Glu34→Asn, Met55→Ala, Leu56→Gln, Ile57→Arg, Ser58→Lys, Cys 61→ Trp, Glu63→Lys, Asp80→Ser, Lys83→Arg, Glu104→Leu, Leu105→Cys, His106→Pro, and Lys108→Gln.

In some embodiments, the lipocalin mutein used in the invention comprises SEQ ID NO: 1, or a variant or fragment thereof. The lipocalin mutein set forth as SEQ ID NO: 1 is a variant of the mature human tear lipocalin set forth as SEQ ID NO: 3, which lacks the first four amino acids and, inter alia, is a variant of the mature human tear lipocalin set forth as SEQ ID NO: 3. contains the following amino acid substitutions at positions corresponding to the sequence positions of the amino acid sequence. Arg26→Ser, Glu27→Arg, Phe28→Cys, Glu30→Arg, Met31→Ala, Asn32→Val, Leu33→Tyr, Glu34→Asn, Val53→Phe, Met55→Ala, Leu56→Gln, Ile57→Arg, Ser 58→ Lys, Cys61→Trp, Glu63→Lys, Val64→Tyr, Ala66→Leu, Asp80→Ser, Lys83→Arg, Tyr100→His, Cys101→Ser, Glu104→Leu, Leu105→Cys, His106→Pro, Lys1 08→Gln, Arg111→Pro, Lys114→Trp, and Cys153→Ser.

Variants As used herein, the term "variant" relates to derivatives of proteins or polypeptides that contain mutations, for example, by substitution, deletion, insertion, and/or chemical modification of amino acid or nucleotide sequences. In some embodiments, such mutations and/or chemical modifications do not reduce the function of the protein or peptide. Such substitutions may be conservative, ie, an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are substitutions between members of the following groups: 1) alanine, serine, and threonine, 2) aspartic acid and glutamic acid, 3) asparagine and glutamine, 4) arginine and lysine, 5) isoleucine, leucine, methionine, and valine, and 6) phenylalanine, tyrosine, and tryptophan. Such variants include proteins or polypeptides in which one or more amino acids are present in their respective D-stereoisomers or in amino acids other than the 20 naturally occurring amino acids (e.g., ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline, etc.). Such variants include, for example, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N-terminus and/or the C-terminus, e.g. deletion of four amino acids from the N-terminus, and/or or deletion of two amino acids from the C-terminus. Generally, a variant will be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% different from the native sequence protein or polypeptide. %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity. A variant preferably retains the biological activity of the protein or polypeptide from which it is derived, eg, binding to the same target.

Accordingly, a variant of a lipocalin mutein according to the invention comprising an amino acid as set forth in SEQ ID NO:1 may be at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity and retains the ability to bind to IL-4 receptor alpha, particularly human IL-4Rα, or a fragment thereof. Preferably, the lipocalin mutein variant is capable of inhibiting binding of IL-4 to IL-4Rα.

In some embodiments, a variant of a lipocalin mutein comprising an amino acid set forth in SEQ ID NO: 1 according to the invention has at least about 50%, at least about 60%, the amino acid sequence of mature human tialipocalin, set forth as SEQ ID NO: 3. , at least about 65%, at least about 70%, at least about 72%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 79%, at least about 80%, at least about 85% have at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity, and the IL-4 receptor Retains the ability to bind alpha, particularly human IL-4Rα, or a fragment thereof. Preferably, the lipocalin mutein variant is capable of inhibiting binding of IL-4 to IL-4Rα.

As used herein, the terms "sequence identity" or "identity" refer to the property of sequences that measures their similarity or relationship. As used in this disclosure, the term "sequence identity" or "identity" refers to the difference between the sequences of a protein or polypeptide of this disclosure and the sequence in question after (homologous) alignment of these two sequences. In terms of the number of longer residues, it is meant the proportion of identical residues in pairs. Sequence identity is determined by dividing the number of identical amino acid residues by the total number of residues and multiplying by 100.

Those skilled in the art will be familiar with available computer programs for determining sequence identity using standard parameters, such as BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990), FASTA (using the method of Pearson and Lipman (1988)), Altschul et al. (1990) described the TBLASTN program, GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA) and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981). you will understand. Percentages of sequence identity are determined herein using, for example, BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is calculated from the entire protein or polypeptide sequence (matrix: BLOSUM62, gap cost: 11.1, cut Based on the arrangement with an off value of 10 ⁻³ ). It is calculated as the percentage of the number of "positives" (homologous amino acids) shown as a result of the BLASTP program output divided by the total number of amino acids selected by the program for alignment. Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps, which are spaces in the alignment that are the result of additions or deletions of amino acids. do. Generally, default parameters are used, with a gap creation penalty equal to 12 and a gap expansion penalty equal to 4.

Specifically, in order to determine whether the amino acid residues of the amino acid sequence of lipocalin (mutein) are different from the lipocalin mutein having the amino acid sequence shown in SEQ ID NO: 1, one skilled in the art can use methods known in the art. Means and methods, such as algorithms, can be performed manually or using computer programs such as BLAST 2.0 (which stands for Basic Local Alignment Search Tool) or ClustalW, or any other suitable program suitable for generating sequence alignments. Can be used with any of the following: Thus, SEQ ID NO: 1 can serve as a "reference sequence," whereas the amino acid sequence of a lipocalin that is different from the lipocalin mutein having the amino acid sequence set forth herein as SEQ ID NO: 1 may serve as a "query sequence." ”.

Fragment The term "fragment" as used herein in connection with the lipocalin mutein of the present disclosure is a protein or peptide derived from a lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, which comprises an N-terminal and/or or C-terminally truncated, ie, lacking at least one of the N-terminal and/or C-terminal amino acids. Such fragments may contain up to 1, up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to It may be missing 25 or up to 30 (including all numbers in between). As an illustrative example, such a fragment may lack one, two, three, or four N-terminal and/or one or two C-terminal amino acids. It is to be understood that the fragment is preferably a functional fragment of the full-length lipocalin (mutein), which means preferably comprising the binding pocket of the full-length lipocalin (mutein) from which it is derived. As an illustrative example, such functional fragments include positions 5-158, 1-156, 5-156, 5-153, 26-153, 5, which correspond to the linear polypeptide sequence of mature human tear lipocalin. ~150, 9-148, 12-140, 20-135, or 26-133 may contain at least an amino acid. Such fragments include at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous amino acids of the sequence set forth as SEQ ID NO:1. and is usually detectable in an immunoassay for a lipocalin mutein having amino acid sequence number 1. The fragment has at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% of the amino acid sequence set forth in SEQ ID NO:1. %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Preferably, the fragment retains the ability to bind to IL-4 receptor alpha, particularly human IL-4Rα, or a fragment thereof. Preferably, the lipocalin mutein fragment is capable of inhibiting IL-4 from binding to IL-4Rα.

A "fragment" for the corresponding target IL-4Rα of the present disclosure (described in UniProt P24394 and shown as SEQ ID NO: 4 without the 25 residue signal peptide) is an N-terminally and/or C-terminally truncated IL-4Rα -4Rα or protein domain. The fragments of IL-4Rα described herein retain the ability of full-length IL-4Rα to be recognized and/or bound by the lipocalin muteins of the present disclosure. As an illustrative example, the fragment may be the extracellular domain of IL-4Rα, such as the extracellular domain comprising amino acid residues 26-232 of UniProt P24394 set forth as SEQ ID NO:5.

Administration and Delivered Doses In methods of treating asthma, a nominal delivered dose of a lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg is administered to a subject by oral inhalation of a dry powder formulation. In some embodiments, a nominal delivery dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of a lipocalin mutein, or variant or fragment thereof, is administered to a subject by oral inhalation of a dry powder formulation. Ru. In some embodiments, a nominal delivered dose of a lipocalin mutein, or variant or fragment thereof, of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg is , administered to a subject by oral inhalation of a dry powder formulation. In some embodiments, the nominal delivered dose is about 0.5 mg. In some embodiments, the nominal delivered dose is about 1 mg. In some embodiments, the nominal delivered dose is about 2 mg. In some embodiments, the nominal delivered dose is about 3 mg. In some embodiments, the nominal delivered dose is about 4 mg. In some embodiments, the nominal delivered dose is about 5 mg. In some embodiments, the nominal delivered dose is about 6 mg. In some embodiments, the nominal delivered dose is about 10 mg. In some embodiments, the nominal delivered dose is about 30 mg. The nominal delivered dose is the target amount of lipocalin mutein, or variant or fragment thereof, that a subject receives when inhaling the dry powder formulation. For example, it can be the amount of lipocalin mutein, or variant or fragment thereof, delivered from the mouthpiece of a device used to administer a dry powder formulation. Calculation of the nominal delivered dose is based on the efficiency of the dry powder inhalation device.

A nominal metered dose is the target amount of lipocalin mutein, or variant or fragment thereof, present in a dry powder formulation for addition to an inhalation device. For example, the nominal metered dose can be a target amount of lipocalin mutein, or a variant or fragment thereof, filled into a capsule as a dry powder formulation, where the target amount is the average fill weight times the average active pharmaceutical ingredient (API) content. . In the methods of treating asthma described herein, a nominally metered dose of a lipocalin mutein, or variant or fragment thereof, of about 0.2 mg to about 40 mg is administered to a subject by oral inhalation of a dry powder formulation. In some embodiments, a nominal measured dose of about 0.65 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg of lipocalin mutein, or a variant or fragment thereof, is a dry powder. Administered to the subject by oral inhalation of the formulation. In some embodiments, about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11. A nominally metered dose of 1 mg, about 12 mg, or about 36 mg of a lipocalin mutein, or variant or fragment thereof, is administered to a subject by oral inhalation of a dry powder formulation.

Frequency of Administration Lipocalin muteins may be administered to a human subject at least once a day, preferably twice per day, ie, twice daily. A nominal delivered dose of a lipocalin mutein, or variant or fragment thereof, of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg is administered to a subject twice per day, i.e., twice per day. When administered twice daily, each dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg is administered to a subject twice per day; twice daily). Thus, the total nominal delivered dose of lipocalin mutein, or variant or fragment thereof, administered to a subject per day is about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg. A lipocalin mutein, or variant or fragment thereof, at a nominal delivery dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg is administered to the subject twice a day. about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg when administered twice per day, i.e., twice daily. Each dose is administered to the subject twice per day, ie, twice daily. Accordingly, the total nominal delivered dose of lipocalin mutein, or variant or fragment thereof, administered to a subject per day is about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 20 mg, or It is about 60 mg. Similarly, a nominal metered dose of a lipocalin mutein, or variant or fragment thereof, of about 0.65 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg is administered to a subject twice daily. each dose of about 0.65 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg when administered twice per day, i.e., twice daily. , is administered to the subject twice per day, ie, twice daily. Accordingly, the total nominal metered dose of lipocalin mutein, or variant or fragment thereof, administered to a subject per day is about 1.1 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about It is 72 mg. about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about When a nominal metered dose of 36 mg of lipocalin mutein, or variant or fragment thereof, is administered to a subject twice per day, i.e., twice daily, about 0.55 mg, about 0 Each dose of .6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 36 mg It is administered twice per day, ie, twice daily. Accordingly, the total nominal metered dose of lipocalin mutein, or variant or fragment thereof, administered to a subject per day is about 1.1 mg, about 1.2 mg, about 2.2 mg, about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22.2 mg, about 24 mg, or about 72 mg.

In some embodiments, a total nominal delivered dose of about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg of a lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. In some embodiments, a total nominal delivered dose of a lipocalin mutein, or variant or variant thereof, of about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 20 mg, or about 60 mg per day. A fragment is administered to a subject. In some embodiments, a total nominal metered dose of a lipocalin mutein, or variant or fragment thereof, of about 1.1 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg per day. is administered to the subject. In some embodiments, about 1.1 mg, about 1.2 mg, about 2.2 mg, about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22 mg per day. A total nominal metered dose of .2 mg, about 24 mg, or about 72 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. The total nominal delivered dose or total nominal metered dose can be achieved by any number of doses per day, eg, 1, 2, 3, 4, or 5 doses per day.

In some embodiments, a total nominal delivered dose of a lipocalin mutein, or a variant or fragment thereof, of about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg is delivered once daily. This can be accomplished by administering to a subject a nominal delivered dose of 6 mg, about 20 mg, or about 60 mg of a lipocalin mutein, or a variant or fragment thereof. Similarly, a total nominal metered dose of a lipocalin mutein, or variant or fragment thereof, of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg, a nominally metered dose of a lipocalin mutein, or a variant or fragment thereof, of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg once daily; This can be achieved by administering to a subject.

In some embodiments, a total nominal delivered dose of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg of a lipocalin mutein, or variant or fragment thereof, is administered to a subject once daily. This can be accomplished by administering to a subject a nominal delivered dose of a lipocalin mutein, or a variant or fragment thereof, of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg.

Dry Powder Inhalation Lipocalin muteins according to the invention are administered to human subjects by inhalation of a dry powder formulation. Suitably, the dry powder formulation is formulated for pulmonary delivery, including inhalation via a dry powder inhaler (DPI). Inhalation of dry powder formulations is usually by oral inhalation. Dry powder formulations may, for example, be present in capsules.

Means and devices for inhalation administration of dry powder formulations of lipocalin muteins are known to those skilled in the art. Such means and devices include single dose dry powder inhalers, such as capsule-based single dose dry powder inhalers. Other suitable means and devices directed to the pulmonary inhalation administration of dry powder formulations of lipocalin muteins are also known in the art, such as multi-dose dry powder inhalers.

Preferred capsule-based dry powder inhalers are rotary capsule inhalers, such as the Plastiape Monodose inhaler, such as the RPC Plastiape S. p. A RS01 Monodose Model 7. This inhalation device delivers drug formulations to the lungs in dry powder form and is intended for self-administration by patients at home, out of the home, and in facilities such as hospitals and clinics.

Dry Powder Formulation The lipocalin mutein used in the present invention is typically administered in the form of a dry powder formulation, which can contain at least one ingredient in addition to the lipocalin mutein. Accordingly, pharmaceutical compositions for use in accordance with the present invention will contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. But that's fine. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredients.

For example, in addition to the active agent, lipocalin mutein, the dry powder formulation can include stabilizers, aerosol performance enhancers, buffering agents, and tonicity agents, as described in more detail below.

As used herein, a "dry powder formulation" preferably has less than about 20% moisture, more preferably less than 10% moisture, less than about 5-6% moisture, or less than about 3% moisture. Refers to a formulation comprising a plurality of solid microparticles in a powder composition containing water. As described herein, dry powder formulations may preferably be utilized for delivery via inhalation to the patient.

Microparticles As used herein, "microparticles" refer to solid particles having a size mass mean diameter (MMD) of less than 20 μm. Mass average diameter is a measure of the average particle size of microparticles, as measured using any suitable method including, for example, centrifugal sedimentation, electron microscopy, light scattering, laser diffraction.

The dry powder formulations described herein suitably contain a plurality of microparticles. As used herein, "plurality" refers to two or more items, preferably 5 or more, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, etc. refers to

Stabilizers In preferred embodiments, the dry powder formulations described herein further include stabilizers to assist in stabilizing the formulation, particularly stabilizing the active agent. Stabilizers stabilize the active agent (preferably polypeptide) in the dry powder formulation, preferably by displacing water on the surface of the active agent during drying or by inhibiting the degradation process; Refers to an excipient that forms an amorphous solid containing an active agent. Examples of stabilizers include amorphous sugars, polymeric sugars, buffers, salts, or synthetic polymers (eg, poly-L-glycolic acid), as well as mixtures of such components. In a preferred embodiment, the stabilizer is an amorphous sugar. Most preferably, the stabilizer is an amorphous sugar with a high glass transition temperature, such as trehalose. The chemical structure of trehalose is shown below.

Preferably, trehalose is present from about 0.5% to about 99.5% (weight percentage, w/w %) of the dry powder formulation. In some embodiments, trehalose is present at about 5% to about 98% (w/w%) of the dry powder formulation. In a preferred embodiment, trehalose is present at about 5% to about 95% (w/w%) of the dry powder formulation.

Aerosol performance enhancer The dry powder formulation of the present invention preferably comprises an aerosol performance enhancer such as trileucine and/or leucine.

"Trileucine," as utilized herein, refers to three leucine molecules linked together in a peptide as leucine-leucine-leucine (Leu-Leu-Leu), C ₁₈ H ₃₅ N ₃ O ₄ Refers to compounds that The chemical structure of trileucine is shown below.

As used herein, "leucine" refers to the amino acid leucine ( _{C6H13NO2 )} , whether present as _a single amino acid or as the amino acid component of a peptide, and in a racemic mixture. or its D or L forms, as well as modified forms of leucine (ie, one or more atoms of leucine are replaced with another atom or functional group). The chemical structure of leucine is shown below.

Unless otherwise stated, the amounts of leucine and trileucine provided herein are provided as a weight percentage (wt % or w/w %) of the formulation. Since dry powder formulations contain substantially less water, the weight component of dry powder formulations is therefore the dry weight percentage of the final formulation.

In an exemplary embodiment, the dry powder formulation comprises about 10% to about 45% leucine (w/w%), or about 20% to about 45% leucine (w/w%). . In some embodiments, the dry powder formulation comprises about 40% by weight (w/w%) leucine.

In an exemplary embodiment, the dry powder formulation comprises about 2% to about 20% by weight (w/w%) trileucine. In some embodiments, the dry powder formulation comprises about 20% by weight (w/w%) trileucine.

In embodiments of formulations that include leucine, trileucine, and active agents, leucine and trileucine provide improved compacted bulk density properties as described herein, as well as allow for improved storage and delivery. The desired ratio range is maintained to provide the desired microparticle properties. In some embodiments, the weight ratio of leucine and trileucine in the microparticle, ie, leucine:trileucine, is about 0.01:1 to about 20:1. In preferred embodiments, the weight ratio of leucine:trileucine in the microparticles is less than about 4:1. Further increasing the amount of trileucine present in the formulation is likely to have beneficial effects regarding moisture fastness and maintenance of particle morphology during scale-up.

Unless otherwise specified, ratios described herein refer to weight percent (w/w - also referred to as "weight ratio" or w/w%), i.e., the weight of leucine in the formulation described herein. : Expressed as the weight of trileucine. This ratio is determined by preparing the desired mg/mL concentrations of leucine and trileucine in the raw material and then drying to remove the raw material solvent, so that the starting concentration ratio (expressed in mg/mL) of leucine: This is accomplished by providing atomized microparticles that maintain the final weight ratio of trileucine.

Exemplary weight percentages of leucine and trileucine that can be utilized in dry powder formulations to achieve these ratios are described herein. Suitably, the dry powder formulation comprises about 5% to about 15% leucine (w/w%) and about 1% to about 5% trileucine (w/w%). In some embodiments, the dry powder formulation comprises about 8% to about 11% leucine (w/w%) and about 2% to about 4% trileucine (w/w%). , in some preferred embodiments, the dry powder formulation comprises about 10% leucine and about 2.6% trileucine (w/w%).

The use of a combination of leucine and trileucine in a dry powder formulation allows for the preparation of microparticles while providing the desired stability compared to dry powder formulations containing only one of these components. This makes it possible to reduce the total amount of leucine and/or trileucine required for. This helps overcome the solubility limitations of trileucine and leucine and provides higher throughput.

An exemplary process for preparing a dry powder formulation according to embodiments herein may be performed as follows. The liquid raw material containing the desired final ingredients of the dry powder formulation is reduced to a fine mist using an atomizer. The mist is then dried as described herein. The sprayed droplets initially contain components dissolved as droplets. As the droplets dry, different components of the formulation begin to become saturated and precipitate at varying rates. As described herein, a shell begins to form around the outer surface of the microparticles of the dry powder formulation. The shell preferably includes leucine and trileucine components on the outer surface of the shell. Note that although leucine and trileucine are preferentially located on the outer surface of the microparticle, small amounts of leucine and trileucine can also be found throughout the microparticle. In embodiments, higher concentrations of leucine and trileucine are preferably found at or near the surface of the microparticle rather than near the center of the microparticle. The presence of leucine and trileucine on the surface of microparticles can be analyzed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). In some embodiments, the core of the microparticle contains a substantial amount of active agent, preferably in amorphous form, along with other excipient components as described herein. As used herein, a "substantial amount" of active agent means that at least about 60% of the active agent (i.e., of the total active agent in the formulation) is located at or near the center of the microparticle; Preferably, at least about 70%, more preferably at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and in embodiments from about 95% to 100%. of the active agent is located at or near the center of the microparticle.

Buffers Exemplary buffers that may be included in the dry powder formulation include various phosphate buffers, citrate buffers (such as sodium citrate), histidine buffers, glycine buffers, acetate buffers, and tartrate buffers. buffering agents, as well as combinations of such buffering agents. Preferably the buffer is a phosphate buffer. Most preferably, the dry powder formulation comprises potassium dihydrogen phosphate and anhydrous disodium phosphate. In some embodiments, the dry powder formulation may include about 0.01% to 5% w/w buffer salt. In some embodiments, the dry powder formulation may include about 0.01% to 3% w/w buffer salt.

Buffers also provide control of the pH of the dry powder formulation, suitably from about pH 5 to about pH 8, such as from about pH 5 to about pH 6, from about pH 5.5 to about pH 6.5, from about pH 6 to about pH 7, Maintain a pH of about pH 6.5 to about pH 7.5, or about pH 7 to about pH 8.

In compositions and formulations that "consist essentially" of the recited ingredients, such compositions and formulations do not substantially affect the essential and novel properties of the recited ingredients and the claimed formulation. Contains ingredients that do not give Ingredients that do not substantially affect the fundamental and novel properties of the claimed formulation are those that do not limit the ability of leucine and trileucine to stabilize the dry powder formulation. Suitably, compositions and formulations consisting essentially of the listed ingredients specifically exclude other amino acids or tripeptide amino acids, but may include additional sugars, buffers, etc.

Tonicity Agents In some embodiments, the dry powder formulations described herein include a tonicity agent, such as sodium chloride. In some embodiments, the dry powder formulation comprises 0-15% w/w sodium chloride. Suitably, the dry powder formulation contains about 0.4-15% w/w sodium chloride.

Microparticle Size The microparticles that make up the dry powder formulations described herein, when provided in aerosol form, preferably have a specified mass median aerodynamic diameter (MMAD).

As used herein, "mass median aerodynamic diameter" or "MMAD" is a measure of the aerodynamic size of dispersed microparticles. Aerodynamic diameter is used to describe an aerosolized powder in terms of its settling behavior and is the diameter of a unit density sphere that has the same settling velocity in air as a microparticle. Aerodynamic diameter encompasses particle shape, density, and physical size of a microparticle. As used herein, MMAD, unless otherwise specified, refers to the midpoint or median of the aerodynamic particle size distribution of an aerosolized powder as determined by cascade impact. Preferably, the microparticles of the dry powder formulations provided herein are at least 1 μm or larger, more preferably about 1 μm to about 10 μm, about 2 μm to about 8 μm, about 2 μm to about 7 μm, about 2 μm to about 6 μm. , about 2 μm to about 5 μm, about 2 μm to about 4 μm, about 2 μm to about 3 μm, about 3 μm to about 4 μm, about 2 μm, or about 3 μm.

Suitably, the fine particle fraction (fraction of particles emitted from an inhalation device of a dry powder formulation with an aerodynamic particle diameter of less than 5 μm) as described herein is ≧50%, more preferably ≧60 %. This fine particle fraction (FPF) contributes to low device retention with less than 20%, preferably less than 15%, less than 10%, or less than 5% of the dry powder formulation remaining in the device after delivery to the patient. obtain.

Exemplary Dry Powder Formulation Some exemplary formulations are shown in the table below.

Accordingly, in some preferred embodiments, the dry powder formulation of the present invention comprises an anti-IL-4 receptor alpha (IL -4Rα) lipocalin mutein. In some embodiments, the dry powder formulation contains a buffering agent, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate, and/or a tonic agent such as sodium chloride. further comprising an agent.

Thus, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose, leucine, and trileucine, the formulation comprising a nominal delivered dose of the lipocalin mutein of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg when the formulation is administered to a subject by oral inhalation. , or a variant or fragment thereof, the formulation being administered to the subject by oral inhalation, the formulation comprising a buffer, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or as a phosphate buffer. or further includes a tensioning agent such as anhydrous disodium phosphate and/or sodium chloride.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises the amino acid sequence anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose, leucine, and trileucine, the formulation comprising a nominal dosage of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg or about 30 mg when administered to a subject by oral inhalation. A delivered dose of the lipocalin mutein, or a variant or fragment thereof, is provided, and the formulation includes a buffering agent, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffering agent. It further includes a buffering agent and/or a tensioning agent such as sodium chloride.

Thus, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose, leucine, and trileucine, the formulation having a nominal delivered dose of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg when the formulation is administered to a subject by oral inhalation. The lipocalin muteins, or variants or fragments thereof, are provided, and the formulation comprises a buffer, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate, and/or chloride. Buffers such as sodium may further be included.

In another preferred embodiment, the dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and leucine. include. In some embodiments, the dry powder formulation contains a buffering agent, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate, and/or a tonic agent such as sodium chloride. further comprising an agent.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and leucine, the formulation comprises a nominal delivered dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of the lipocalin mutein, or variant or variant thereof, when the formulation is administered to a subject by oral inhalation. The fragments are provided and the formulation is buffered with a buffer, such as a phosphate buffer, e.g. potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffer, and/or a tension agent such as sodium chloride. further comprising an agent.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and leucine, the formulation delivers a nominal delivery of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg or about 30 mg when administered to the subject by oral inhalation. A dose of the above lipocalin mutein, or a variant or fragment thereof, is provided, the formulation comprising a buffer, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffer. and/or tensioning agents such as sodium chloride.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and leucine, the formulation comprises a nominal delivered dose of the lipocalin mutein of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg when the formulation is administered to the subject by oral inhalation. , or a variant or fragment thereof, the formulation comprising a buffer, e.g. a phosphate buffer, e.g. potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffer, and/or or may further include a tensioning agent such as sodium chloride.

In another preferred embodiment, the dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and trileucine. including. In some embodiments, the dry powder formulation contains a buffering agent, e.g., a phosphate buffer, e.g., potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffering agent, and/or a tonicity agent such as sodium chloride. further including.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and trileucine, the formulation having a nominal dosage of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg or about 30 mg when administered to the subject by oral inhalation. A delivered dose of the lipocalin mutein, or a variant or fragment thereof, is provided, and the formulation includes a buffering agent, such as a phosphate buffer, such as potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffering agent. Buffers and/or tensioning agents such as sodium chloride may further be included.

Accordingly, in some embodiments, a dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and trileucine, the formulation comprising a nominal delivered dose of the lipocalin of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg when the formulation is administered to the subject by oral inhalation. A mutein, or a variant or fragment thereof, is provided, the formulation comprising a buffer, such as a phosphate buffer, for example potassium dihydrogen phosphate and/or anhydrous disodium phosphate as a buffer; or may further include a tensioning agent such as sodium chloride.

Each of the individual components may be present in the amounts and/or ratios described herein.

In some embodiments, the dry powder formulation of the invention may be as shown in one of Tables 5-16 below, where AZD1402 is an anti-IL comprising the amino acid sequence set forth in SEQ ID NO:1. -4 receptor alpha (IL-4Rα) lipocalin mutein, or a fragment or variant thereof.

Accordingly, in some preferred embodiments, the dry powder formulation of the invention comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose. , including leucine and trileucine. In some embodiments, the dry powder formulation further comprises a buffering agent, such as a phosphate buffer.

In another preferred embodiment, the dry powder formulation comprises an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof, trehalose and leucine. In some embodiments, the dry powder formulation further comprises a buffering agent, such as a phosphate buffer.

Dry powder formulations containing lipocalin muteins may be administered alone or in combination with other treatments, either simultaneously or sequentially. For example, lipocalin muteins may be administered in combination with corticosteroids, such as inhaled corticosteroids (ICS), long-acting beta2 agonists (LABA), and/or long-acting muscarinic antagonists (LAMA). . In some embodiments, lipocalin muteins may be administered in combination with low or moderate dose ICS, inhaled LABA and/or inhaled LAMA. Low, medium and high daily doses of ICS are described in the Pocket Guide for Asthma Management and Prevention, Global Initiative for Asthma, Updated 2020.

Systemic Exposure As used herein, “systemic exposure” means that a substantial portion of the inhaled lipocalin mutein enters the circulatory system and, optionally, the whole body can be affected by the lipocalin mutein. . Systemic exposure may mean that the amount of lipocalin mutein that enters the circulation is quantifiable. Systemic exposure can be equivalent to a quantifiable concentration of lipocalin mutein entering the bloodstream. This exposure can be expressed by the blood (serum, plasma, or whole blood) concentration of the lipocalin mutein, which can be measured over time and recorded by a series of parameters including the area under the curve (AUC). Systemic exposure to lipocalin muteins can also affect biomarkers, the levels of which can be directly correlated to lipocalin mutein concentration and thus systemic exposure. The term "quantifiable" or "detectable" when used in connection with systemic exposure, exposure expressed by blood (serum, plasma or whole blood) concentrations of lipocalin muteins, or as otherwise known in the art. exposure as expressed by the level of a biomarker measurable by one or more analytical methods. Such analytical methods include, but are not limited to, chromatographic methods such as ELISA, competitive ELISA, fluorescence titration, calorimetry, mass spectrometry (MS), and high performance liquid chromatography (HPLC). It is also understood that measurements made using such analytical methods are associated with detection limits, such as instrument detection limits, method detection limits, and quantitation limits.

Pharmacokinetic Properties The results presented herein demonstrate that administration of a nominal delivered dose of 10 mg of lipocalin mutein to the lungs by inhalation of a dry powder formulation, as administration of a nominal delivered dose of 18 mg of lipocalin mutein by inhalation of a nebulized formulation, Shown to provide equivalent systemic exposure of lipocalin muteins. Systemic exposure in this context was measured by C _max , AUC, and AUC _last as defined in Table 69. Thus, administration of lipocalin muteins by inhalation of a dry powder formulation advantageously achieves systemic exposure levels approximately twice as high as doses administered by inhalation of a nebulized formulation. Similarly, systemic exposure to lipocalin muteins, as measured by C _max , AUC, and AUC _last , was greater after administration of a nominally delivered dose of 30 mg lipocalin mutein to the lungs by inhalation of the dry powder formulation than by nebulization into the lungs. It was approximately 73-78% lower after administration of a nominal delivered dose of 18 mg of lipocalin mutein via inhalation of the formulation or after administration of a nominal delivered dose of 10 mg of lipocalin mutein to the lung via inhalation of the dry powder formulation.

Unless the context requires otherwise, the terms "comprise" and "include" and "comprises" and "including" are used throughout this specification, including the following claims. Variations such as "comprising" and "including" mean the inclusion of a given integer or step, or group of integers or steps, but not any other integer or step, or integer or step. It will be understood that this does not imply any exclusion of the group.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Please note that. Thus, for example, reference to a "lipocalin mutein" includes one or more lipocalin muteins.

As used herein, the term "and/or" means "and," "or," and "all or any other of the elements connected by the above term." It includes the meaning of "combination".

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, the term also includes specific numbers; for example, "about twenty" includes twenty.

As used herein, the term "at least about" includes a specific number, eg, "at least about six" includes six.

The features disclosed in the foregoing description, or in the claims below, or in the accompanying drawings, may be found in the specific form or means for performing the disclosed functions or methods for obtaining the disclosed results. or expressed in terms of steps, and may be utilized to realize the invention in its various forms, individually or in any combination of such features, as necessary.

Although the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.

For the avoidance of doubt, any theoretical explanation provided herein is provided for the purpose of improving the understanding of the reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

Example 1 - Aerosol Performance Characteristics of 85% W/W AZD1402 and Buffer Salt Formulation The following example evaluates the aerosol performance of a formulation containing AZD1402 and buffer salt in a dry powder inhaler device. The aerosol performance outputs listed in Table 19 were tested with formulations at the following ratios of AZD1402: 5.2:1 or higher (NLT) of PBS. In all Examples 1-15, all product performance characterization was completed using the Monodose RS01 device with size 3 capsules. Next Generation Impactor (NGI) analysis was performed with an air flow rate of 60 L/min.

AZD1402 was originally in a liquid formulation at a concentration of 50 mg/ml NLT. A stock solution was prepared by diluting the AZD1402 solution with water at a total solids concentration of 50 mg/mL. The feed solution was spray dried using an outlet temperature of 65°C, feed rate, 1.85 ml/min, atomizer air, 2150 liters/hour, and drying gas flow, 23 kg/hour. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 19.

A cascade impact test was conducted in accordance with United States Pharmacopeia Chapter 601 (USP <601>) to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impactor device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 20 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

In all Examples 1-15, enzyme-linked immunosorbent assay (ELISA) is used to demonstrate the binding activity of AZD1402 to the designated target, the IL-4 receptor. The method is an antibody capture, antigen excess assay and relies on AZD1402 being immobilized on a 96-well plate coated with the antigen, recombinant human sIL-4 alpha receptor. Immobilized AZD1402 is detected by adding a primary anti-AZD1402 antibody followed by a secondary antibody labeled with HRP enzyme. HRP enzymes catalyze the reaction of substrates to produce colored products. The amount of colored product is directly proportional to the amount of secondary antibody present, which in turn is directly proportional to the amount of AZD1402 captured by the antigen. Standards of AZD1402 at various known concentrations are incubated with excess antigen to construct a calibration curve. The resulting log concentration versus absorbance response is plotted with a four-parameter logistic (4PL) curve fit.

Results are summarized in Table 20, including 12 month stability of capsules stored individually in sealed aluminum pouches.

Example 2 - Aerosol Performance Characteristics of 2 W/W % AZD1402 in Trehalose Formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 22 were tested with formulations having a drug loading of 2% w/w and a proportion of AZD1402. NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total solids concentration of 40-50 mg/mL by first dissolving trehalose in water and then adding it to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 22.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 20 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

The results of the aerosol analysis are summarized in Table 23. This formulation failed to comply with stability due to initial results showing unsatisfactory properties.

Example 3 - Aerosol Performance Characteristics of 60 W/W % AZD1402 in Trehalose Formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 25 were tested with formulations having a drug loading of 60% w/w and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total solids concentration of 40-50 mg/mL by first dissolving trehalose in water and then adding it to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 25.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 20 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 26.

Example 4 - Aerosol performance characteristics of 2 W/W % AZD1402 in a trehalose-leucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose and leucine in a dry powder inhaler device. The aerosol performance outputs listed in Table 28 were tested with formulations having a drug loading of 2 w/w% and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 28.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis, including the 6 month stability of capsules stored individually in sealed aluminum pouches, are summarized in Table 29.

Example 5 - Aerosol performance characteristics of 60 W/W% AZD1402 in a leucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 31 were tested with formulations having a drug loading of 60% w/w and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 40-50 mg/mL solids by first dissolving trileucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65°C, feed rate of 6.0 ml/min, atomizer air of 6.5 kg/hr, and drying gas flow of 40 kg/hr. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation. This formulation had sufficient chemical and physical stability, including bioactivity, and aerosol performance during storage, at a time when the source of trileucine was in question.

Data from the physical powder property attributes are listed in Table 31.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 20 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 20 mg of a spray-dried powder formulation was dispersed from a dry powder inhaler device at a rate of 60 L/min (corresponding to a 4 kPa pressure drop in the inhaler) according to USP methodology. Airflow velocity was delivered to the impinger. Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis, including the 12 month stability of capsules stored individually in sealed aluminum pouches, are summarized in Table 32.

Example 6 - Aerosol performance characteristics of 60 W/W % AZD1402 in a trehalose-leucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 34 were tested with formulations having a drug loading of 60% w/w and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution at a total concentration of 40 mg/mL solids. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 34.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 40 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to USP methods. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 40 mg of a spray-dried powder formulation was dispersed from a dry powder inhaler device at a rate of 60 L/min (corresponding to a 4 kPa pressure drop in the inhaler) according to USP methodology. Airflow velocity was delivered to the impinger. Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 35.

Example 7 - Aerosol performance characteristics of 2W/W% AZD1402 in a trehalose-leucine-trileucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 37 were tested with formulations having a drug loading of 2 w/w% and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve improved physical robustness such as acceptable moisture content, particle properties, and moisture resistance in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 37.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 38.

Example 8 - Aerosol performance characteristics of 60 W/W% AZD1402 in a trehalose-leucine-trileucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 40 were tested with formulations having a drug loading of 60% w/w and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution at a total concentration of 40 mg/mL solids. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve improved physical robustness such as acceptable moisture content, particle properties, and moisture resistance in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 40.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 40 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to USP methods. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 40 mg of a spray-dried powder formulation was dispersed from a dry powder inhaler device at a rate of 60 L/min (corresponding to a 4 kPa pressure drop in the inhaler) according to USP methodology. Airflow velocity was delivered to the impinger. Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 41.

Example 9 - Aerosol performance characteristics of 11 W/W% AZD1402 in a trehalose-leucine-trileucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 43 were tested with formulations having a drug loading of 11 w/w% and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65°C, feed rate of 6.0 ml/min, atomizer air of 9.5 kg/hr, and drying gas flow of 40 kg/hr. Parameters were selected to achieve acceptable particle properties and improved physical robustness, such as maintaining moisture tolerance and biological activity of the dry powder formulation for inhalation purposes. The formulation exhibited improved physical robustness such as good aerosol performance, moisture resistance, biological activity, and was physically and chemically stable throughout the storage period.

Data from the physical powder property attributes are listed in Table 43.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis, including the 6 month stability of capsules stored individually in sealed aluminum pouches, are summarized in Table 44.

Example 10 - Aerosol performance characteristics of 33 W/W% AZD1402 in a trehalose-leucine-trileucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 46 were tested with formulations having a drug loading of 33 w/w% and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65°C, feed rate of 6.0 ml/min, atomizer air of 9.5 kg/hr, and drying gas flow of 40 kg/hr. Parameters were selected to achieve acceptable moisture content and particle characteristics in order to maintain bioactivity of the dry powder formulation intended for inhalation. The formulation exhibited improved physical robustness such as good aerosol performance, moisture resistance, biological activity, and was physically and chemically stable throughout the storage period.

Data from the physical powder property attributes are listed in Table 46.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 47.

Example 11 - Aerosol performance characteristics of 60 W/W% AZD1402 in a trehalose-leucine-trileucine formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 49 were tested with formulations having a drug loading of 60% w/w and the following proportions of AZD1402: NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared by first dissolving leucine, trileucine and trehalose in water and then adding to the AZD1402 solution at a total concentration of 50 mg/mL solids. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 6.0 ml/min, atomizer air of 9.5 kg/hr, and a drying gas flow of 40 kg/hr. Parameters were selected to achieve improved physical robustness such as acceptable water, particle properties, and moisture resistance in order to maintain biological activity of the dry powder formulation intended for inhalation. The formulation exhibited improved physical robustness such as good aerosol performance, moisture resistance, biological activity, and was physically and chemically stable throughout the storage period.

Data from the physical powder property attributes are listed in Table 49.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 18.5 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at an airflow rate of 60 L/min according to the USP method. It was delivered to NGI. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 18.5 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a 4 kPa pressure drop in the inhaler) according to USP method. ) was delivered to the impinger at an airflow rate of Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis including 6 month stability of capsules stored individually in sealed aluminum pouches are summarized in Table 50.

Example 12-3 Aerosol Performance Characteristics of 5W/W% AZD1402 in a W/W% Trileucine Formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 52 were tested with formulations having a drug loading of 5% w/w and a proportion of AZD1402. NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving trileucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve improved physical robustness such as acceptable moisture content, particle properties, and moisture resistance in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 52.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis, including the two-month stability of capsules stored individually in sealed aluminum pouches, are summarized in Table 53.

Example 13 - Aerosol Performance Characteristics of 5W/W% AZD1402 in a 5W/W% Trileucine Formulation The following example evaluates the aerosol performance of a formulation containing trehalose in a dry powder inhaler device. The aerosol performance outputs listed in Table 55 were tested with formulations having a drug loading of 5% w/w and a proportion of AZD1402. NLT5.2:1 PBS

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving trileucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65° C., a feed rate of 2.0 ml/min, atomizer air of 2150 liters/hour, and a drying gas flow of 23 kg/hour. Parameters were selected to achieve improved physical robustness such as acceptable moisture content, particle properties, and moisture resistance in order to maintain bioactivity of the dry powder formulation intended for inhalation.

Data from the physical powder property attributes are listed in Table 55.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of the aerosol analysis, including the two-month stability of capsules stored individually in sealed aluminum pouches, are summarized in Table 56.

Example 14 - Aerosol performance characteristics of 10 W/W % AZD1402 in a trehalose-leucine-trileucine formulation and histidine buffer The following example demonstrates the aerosol performance of a formulation containing trehalose, leucine, and trileucine in a dry powder inhaler device. Evaluate performance. The aerosol performance outputs listed in Table 58 were tested with formulations having a drug loading of 10% w/w and a proportion of AZD1402. NLT4.1:1 histidine.

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65°C, feed rate of 6.0 ml/min, atomizer air of 9.5 kg/hr, and drying gas flow of 40 kg/hr. Parameters were selected to achieve acceptable particle properties and improved physical robustness, such as maintaining moisture tolerance and biological activity of the dry powder formulation for inhalation purposes. The formulation exhibited improved physical robustness such as good aerosol performance, moisture resistance, biological activity, and was physically and chemically stable throughout the storage period.

Data from the physical powder property attributes are listed in Table 58.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device and NGI at an airflow rate of 60 L/min according to the USP method. Delivered to. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 10 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a pressure drop of 4 kPa in the inhaler) according to the USP method. was delivered to the impinger at an airflow rate of . Samples from the impinger are collected and assayed for AZD1402 content.

The results of aerosol analysis of capsules individually stored in sealed aluminum pouches are summarized in Table 59.

Example 15 - Aerosol performance characteristics of 50 W/W % AZD1402 in a trehalose-leucine-trileucine formulation and histidine buffer The following example demonstrates the aerosol performance of a formulation containing trehalose, leucine, and trileucine in a dry powder inhaler device. Evaluate performance. The aerosol performance outputs listed in Table 61 were tested with formulations having a drug loading of 50% w/w and a proportion of AZD1402. NLT4.1:1 histidine.

AZD1402 was initially in a liquid formulation at a concentration of 50 mg/mL NLT. A stock solution was prepared with a total concentration of 50 mg/mL solids by first dissolving leucine and trehalose in water and then adding to the AZD1402 solution. The feed solution was spray dried using an outlet temperature of 65°C, feed rate of 6.0 ml/min, atomizer air of 9.5 kg/hr, and drying gas flow of 40 kg/hr. Parameters were selected to achieve acceptable particle properties and improved physical robustness, such as maintaining moisture tolerance and biological activity of the dry powder formulation for inhalation purposes. The formulation exhibited improved physical robustness such as good aerosol performance, moisture resistance, biological activity, and was physically and chemically stable throughout the storage period.

Data from the physical powder property attributes are listed in Table 61.

A cascade impact test was conducted according to USP <601> to determine the aerosol performance of the spray-dried formulation when delivered from a dry powder inhaler device. The cascade impact device used was the Next Generation Impactor (NGI; USP 41, Chapter <601>). For the aerosol measurements performed in this example, one size 3 HPMC capsule containing 18.5 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at an airflow rate of 60 L/min according to the USP method. It was delivered to NGI. Samples from each stage of NGI were collected and assayed for AZD1402 content.

Delivered dose testing of the spray-dried formulation when delivered from the Monodose dry powder inhaler device was performed according to USP <601> using different dose collection units. The powder aerosol is collected in a glass device, an impeder, equipped with an expandable inlet for stiffness and a sintered glass filter (pore size 40-100 μm) (also Hugosson S, et al, Pharm Forum 1993; 19). No. 3): 5458-5466). In this example, one size 3 HPMC capsule containing 18.5 mg of spray-dried powder formulation was dispersed from a dry powder inhaler device at 60 L/min (corresponding to a 4 kPa pressure drop in the inhaler) according to USP method. ) was delivered to the impinger at an airflow rate of Samples from the impinger are collected and assayed for AZD1402 content.

The results of aerosol analysis of capsules individually stored in sealed aluminum pouches are summarized in Table 62.

Example 16. A Randomized, Open-Label, 3-Period, 3-Treatment, Crossover Study to Evaluate the Pharmacokinetic Effects of Inhalation Devices and Formulation After a Single Inhaled Dose of AZD1402 in Healthy Subjects 2.1 Study and Dose Rationale This study evaluated the effect of an inhaled powder (delivered via a Plastiape Monodose inhaler) on the PK properties of AZD1402 and compared with the nebulized solution (delivered via an InnoSpire Go) administered in previously conducted studies. ) was intended to be compared with The test results provide information regarding the PK profile of the test material used for further clinical development. An open-label design was chosen as PK assessment was the primary objective. This study was conducted in healthy subjects to minimize the effects of intercurrent disease conditions or medications on study measurements. Subjects received all three test products in random order.

The InnoSpire Go nebulizer nominal dose of 18 mg (Test Product A) has been safe and well-tolerated in studies conducted to date, clinically relevant for future development programs, and measurable for up to 18 hours. was selected because it provided a suitable serum concentration.

A Plastiape Monodose dry powder inhaler (DPI) nominal delivery dose of 10 mg (Treatment B) was selected to deliver an equivalent pulmonary dose to Treatment A (based on calculated nebulizer and DPI inhalation device efficiency) of 9 mg. To evaluate the dose proportionality of the inhalation formulation and Plastiape Monodose inhaler device, a Plastiape Monodose inhaler nominal delivery dose of 30 mg (Test Product C) was selected.

The selected dose is well below the nominal delivered dose of the InnoSpire Go nebulizer, which is up to 160 mg, and was administered to healthy subjects in a single escalating dose study and was safe and well-tolerated (NCT03384290) .

2.2 Overall Study Design and Plan: Description This study was a randomized, open-label, three-period, three-treatment, single-dose, single-center, cross-over study. In this study, 18 healthy male and female subjects were randomized to ensure at least 12 subjects were evaluable. Subjects were considered evaluable if they completed all three treatment periods without significant protocol deviations. Each subject received all three doses of IMP. No placebo was used in this study.

Subjects were randomized to one of six treatment sequences and underwent three single-dose trials.
The products of AZD1402 are listed below.
- Test Product A: AZD1402 nebulizer solution at a nominal dose of 18 mg, administered via an InnoSpire Go nebulizer.
- Test Product B: AZD1402 inhalation powder at a nominal delivery dose of 10 mg administered via the Plastiape Monodose inhaler.
- Test Product C: AZD1402 inhalation powder at a nominal delivery dose of 30 mg administered via the Plastiape Monodose inhaler.

This study includes:
- Screening period up to 28 days before the first dose of AZD1402.
- Three treatment periods in which subjects reside in the clinical unit from the day before administering AZD1402 (Day-1) until at least 48 hours after dosing, and are discharged on day 3.
-Follow-up visit 10-12 days after the last dose of AZD1402 during treatment period 3.

Each treatment period was separated by a minimum washout period of 5 days between doses. All subjects signed the ICF before participating in specific study-related procedures. Subjects attended a screening visit within 28 days prior to receiving their first dose of AZD1402. If eligible, you returned to Treatment Period 1 when you took your baseline assessment and received one of the three study products in randomized order. For each treatment period, subjects received a single dose of IMP on the morning of day 1 and underwent further evaluation over a 48-hour period post-dose.

The research flowchart is shown in Figure 1.

Examples of the ingredients used to make the 12 mg capsules (12 mg/MD) of this study are listed in Table 1 above.

2.3 Subject Dose Selection and Timing During each treatment period, subjects received a single inhaled dose of AZD1402. Inhalation of AZD1402 as a nebulizer solution via an InnoSpire Go nebulizer for test product A (Treatment A) and via a Plastiape Monodose inhaler for test products B (Treatment B) and Test Products C (Treatment C). Administered as a powder.

Doses were administered after an overnight fast of at least 10 hours. Subjects were allowed to drink water to prevent dehydration up to 1 hour prior to IMP administration. Water was provided ad libitum starting 1 hour after IMP administration, and a light breakfast was provided 2 hours after IMP administration. All subjects received study product in randomized order.

2.4 Subject Placement A total of 18 subjects were randomly assigned to one of six treatment groups as planned. All randomized subjects received a single dose of all three investigational medicinal products (IMPs) (ie, AZD1402 Treatments A, B, and C) during three treatment periods. Each treatment period was separated by a minimum washout period of 5 days between doses, as planned.

Each of the 18 randomized subjects completed all three treatment periods. No subjects withdrew from the study or discontinued treatment.

2.5 Demographics All subjects included in this study were male. The majority of subjects were Caucasian (77.8%).

No differences related to subject demographic characteristics were observed across the six treatment sequences at baseline. Differences in mean age between treatment sequences were not considered significant due to the small number of subjects in the study and likewise within each treatment sequence.

2.6 Analysis of Pharmacokinetic Data 2.6.1 Serum Concentrations Each subject and time point of AZD1402 after a single inhaled dose via the InnoSpire Go nebulizer (18 mg delivered dose) or Plastiape Monodose inhaler (10 mg or 30 mg delivered dose) Summary statistics of the serum concentrations of .

Geometric mean serum AZD1402 concentration versus time profiles are presented in FIG. 2 (linear scale) and FIG. 3 (semi-logarithmic scale). If the actual collection time of a sample deviated by more than 10% from the nominal time, the results were excluded from summary statistics and geometric mean plots.

2.6.2 Serum Pharmacokinetic Parameters of AZD1402 Summary statistics of serum AZD1402 PK parameters are presented in Table 64. Statistical analysis of important PK parameters is presented in Tables 65 and 66.

One profile from Period 1 E0001133, Treatment C had a positive pre-dose concentration of 1.54 ng/mL. This concentration was less than 5% Cmax and was excluded from the PK analysis.

For two profiles, the PK parameters were not calculable or were limited: E0001133, Treatment A was completely below the limit of quantification (BLQ) throughout the sampling interval and therefore the PK parameters were not calculable , E0001143, Treatment A had only one quantifiable concentration and therefore only Cmax and tmax could be calculated.

For the six profiles, AUC, AUC/D, CL/F, Vz/F and MRT values were excluded from the summary statistics due to extrapolation of AUC greater than 20%. E0001110, Treatment B; E0001114, Treatments A and B; E0001116, Treatments B and C; and E0001118, Treatment B. One profile (E0001135, Treatment C) adjusted R square < _0.8 ^{t 1/2} λz and the t ^1/2 _λz dependent variable was excluded from summary statistics.

3.00 to 3.50 hours after inhalation of AZD1402 via either a nebulizer (Treatment A, 18 mg delivered dose) or a Plastiape Monodose inhaler (Treatment B, 10 mg delivered dose or Treatment C, 30 mg delivered dose). Sustained and relatively long absorption was observed with similar median tmax. Individual tmax ranged from 1.98 to 8.27 h, and the ranges were similar across all three treatments.

After reaching Cmax, serum concentrations _of AZD1402 decreased in an essentially monophasic manner, with similar mean t ^1/2 λz values across all three groups. 6.919 hours, 7.457 hours and 6.912 hours for treatments A, B and C respectively. The regression analysis applied to the data had a generally good fit (adjusted R-square >0.8) and appeared to adequately reflect the decrease in concentration.

The t ^1/2 λz of most _of the profiles after treatments A and B and three profiles after treatment C were calculated over a period of less than three times the obtained half-life. No data were excluded due to half-life as _the profile was generally monophasic and t ^1/2 λz across all three groups was consistent.

The mean CL/F and Vz/F values for Treatment B and Treatment C using the Plastiape Monodose inhaler were similar, but higher after administration via the InnoSpire Go nebulizer (Treatment A). These data, combined with the higher dose normalized exposure after treatments B and C, are consistent with the lower bioavailability of AZD1402 when dosed as the Innospire Go formulation.

Intersubject variability in AZD1402 was moderate to high for AUC (48.79%, 35.84%, and 62.74% for treatments A, B, and C, respectively) and high for AUClast (48.79%, 35.84%, and 62.74% for treatments A, B, and C, respectively). (51.86%, 57.71%, 68.69%, respectively) and high in Cmax (101.1%, 47.76%, 67.82% for treatments A, B, and C, respectively).

Exposure to AZD1402 was similar between treatments A and B, as judged by Cmax and AUC, with the mean AUClast for treatment A being slightly higher than the mean AUClast for treatment B. Through inferential statistical analysis, comparing between treatments A and B, the geometric LS mean ratios of Cmax and AUC are 105.45% and 102.39%, respectively, and the geometric LS mean ratio of AUClast is 122 It was shown that it was .21%. For all three parameters, the 90% CI of the geometric LS mean contained 100%.

Exposure (Cmax, AUC, and AUClast) after treatment A was approximately 27% of the exposure after treatment C: Geometric LS mean ratio (90% CI) for Cmax, AUC, and AUClast, respectively. , 27.47% (18.90, 39.94), 27.09% (20.25, 36.24), and 26.50% (19.40, 36.20).

Exposure (Cmax, AUC, and AUClast) after treatment B was approximately 22-26% of the exposure after treatment C: the geometric LS mean ratios (90% CI) were For AUClast, they were 26.05% (18.03, 37.64), 26.46% (19.72, 35.50), and 21.68% (16.04, 29.32).

For the evaluation of dose proportionality for the two dry powder treatments B and C, the slope estimates for Cmax and AUC are 1.196 and 1.259, respectively, and the corresponding 90% CIs include units of 1 and are approximately dose proportional. suggested an increase in However, the slope estimate for AUClast was slightly above 1, 1.368 (90% CI: 1.222, 1.614).

AUC = area under the serum concentration-time curve extrapolated from time zero to infinity, AUC/D = area under the serum concentration-time curve extrapolated from time zero to infinity divided by dose, AUClast = time zero area under the serum concentration curve from time zero to the last quantifiable concentration, AUClast/D = area under the serum concentration curve from time zero to the last quantifiable concentration divided by dose, Cmax = maximum observed Serum concentration, Cmax/D = maximum observed serum concentration divided by dose, tmax = time to reach maximum observed concentration, ^t1 / ₂ λz = terminal slope of the semi-log concentration time curve (λz) related λz = terminal elimination rate constant, MRT = mean residence time of unchanged drug in the circulatory system from zero to infinity, CL/F = total body clearance of drug from the apparent serum after extravascular administration , Vz/F = apparent volume of distribution during the terminal phase after extravascular administration.

2.6.3 Pharmacokinetic Conclusions Stable absorption was observed after a single administration of AZD1402 nebulizer solution via InnoSpire Go nebulizer or inhalation powder via Plastiape Monodose inhaler, median tmax was 3.00 The average t1/2λz was approximately 7 hours and was similar between treatments.
- Systemic exposure to AZD1402 was comparable between the InnoSpire Go nebulizer with a nominal delivered dose of 18 mg and the Plastiape Monodose inhaler with a nominal delivered dose of 10 mg, as judged by Cmax, AUC and AUClast.
Systemic exposure to AZD1402 after administration via an InnoSpire Go nebulizer with a nominal delivered dose of 18 mg or a Plastiape Monodose inhaler with a nominal delivered dose of 10 mg, as determined by Cmax, AUC, and AUClast, was 30 mg nominal The delivered dose was approximately 73%-78% lower than the systemic exposure following administration via the Plastiape Monodose inhaler.
- The Cmax and AUC of AZD1402 after a single inhalation of the dry powder via the Plastiape Monodose inhaler increased significantly and dose-proportionally over nominally delivered doses of 10-30 mg.

2.7 Safety Conclusions Table 67 summarizes the AEs reported with the three single-dose treatments: A (18 mg once daily), B (10 mg once daily) and C ( 30 mg once daily), and total.

A total of 13 subjects (72.2% of 18 treated subjects) experienced at least one TEAE during the study. The most frequently reported TEAEs (>2 subjects) by preferred term were cough (2 subjects reported episodes during treatment A, 1 subject reported episodes during treatment B, and 1 subject reported episodes during treatment C); (4 subjects who reported episodes during treatment) and headache (2 subjects who reported episodes during treatment A and treatment B, and 1 subject who reported an episode during treatment C).

The majority of subjects (72.2%) experienced mild TEAEs. The remaining subjects experienced moderate TEAEs: 2 subjects (11.1%) during Treatment A, 3 subjects (16.7%) during Treatment B, and 4 subjects (22%) during Treatment C. .2%).

One subject was a healthy volunteer with a past history of childhood asthma, ongoing allergies to dust mites, cats, and tree bark, and a history of respiratory inflammation during treatment period 3 after inhalation of Treatment C. An unexpected serious adverse reaction (SUSAR) was suspected (AZD1402 inhalation powder, nominal dose 30 mg). The subject's predicted decrease in FEV1 and C-reactive protein levels rose outside normal limits, and both returned to pre-dose levels when the subject recovered 21 days after SUSAR onset. This SUSAR was of moderate intensity and was considered by the investigator to be related to IMP.

Overall, the results of this study indicate that AZD1402 administered by inhalation in DPI form is safe in healthy volunteers and merits further testing in patients with pulmonary diseases, such as asthma.

2.8 Discussion AZD1402 is a recombinant, monospecific Anticalin® (modified lipocalin) protein that targets interleukin-4 receptor alpha (IL-4Rα) and is not regulated by standard therapy. It is being developed as an inhaled therapy for the treatment of moderate to severe persistent asthma in patients with no symptoms. This was a randomized, open-label, 3-period, 3-treatment crossover study to evaluate the effect of the inhaler device and formulation on PK after a single inhaled dose of AZD1402 in healthy subjects. . This study evaluated the effect of an inhaled powder (delivered via the Plastiape Monodose inhaler) at two different doses on the PK properties of AZD1402 and a nebulized solution (delivered via the Plastiape Monodose inhaler) administered in clinical studies conducted to date. The aim was to compare the

Exposure to AZD1402 was similar between treatments A and B, as judged by Cmax and AUC, with the average AUClast for treatment A being slightly higher than treatment B. Inferential statistical analysis shows that compared between treatments A and B, the geometric LS mean ratios of Cmax and AUC are 105.45% and 102.39%, respectively, and the geometric LS mean ratio of AUClast is 122%. .21%. For all three parameters, the 90% CI of the geometric LS mean contained 100%.

Drug exposure (Cmax, AUC, and AUClast) after treatment A was approximately 27% after treatment C: for Cmax, AUC, and AUClast, the geometric LS mean ratios (90% CI) were, respectively. They were 27.47% (18.90, 39.94), 27.09% (20.25, 36.24), and 26.50% (19.40, 36.20).

Drug exposure (Cmax, AUC, and AUClast) after treatment B was approximately 22-26% after treatment C: Geometric LS mean ratios (90% CI) for Cmax, AUC, and AUClast They were 26.05% (18.03, 37.64), 26.46% (19.72, 35.50), and 21.68% (16.04, 29.32), respectively.

Evaluation of the dose proportionality of the two dry powder treatments B and C shows that the slope estimates for Cmax and AUC are 1.196 and 1.259, respectively, and the corresponding 90% Ci contains 1 unit and is approximately dose proportional. suggested an increase in However, the slope estimate for AUClast was slightly above 1, 1.368 (90% CI: 1.222, 1.614).

The selected doses (InnoSpire Go nebulizer nominal delivered dose 18 mg [Treatment A], Plastiape Monodose inhaler nominal delivered dose 10 mg [Treatment B], and Plastiape Monodose inhaler nominal delivered dose 30 mg [Treatment C]) are SAD It was safe and well-tolerated, well below the nominal delivered dose of the InnoSpire Go nebulizer up to 160 mg administered to healthy subjects in the study (PRS-060-PCS_06_17).

2.10 Conclusions AZD1402 was steadily absorbed after a single dose of nebulized solution via the InnoSpire Go nebulizer or inhaled powder via the Plastiape Monodose inhaler. Median Tmax ranged from 3.00 to 3.50 hours, and mean t1/2λz was approximately 7 hours and was similar between treatments.
- Systemic exposure to AZD1402 was comparable between the InnoSpire Go nebulizer with a nominal delivered dose of 18 mg and the Plastiape Monodose inhaler with a nominal delivered dose of 10 mg.
- Systemic exposure to AZD1402 using the InnoSpire Go nebulizer with a nominal delivered dose of 18 mg or the Plastiape Monodose inhaler with a nominal delivered dose of 10 mg was lower than that using the Plastiape Monodose inhaler with a nominal delivered dose of 30 mg. The Cmax and AUC of AZD1402 after a single inhalation of the dry powder via the Plastiape Monodose inhaler increased significantly and dose-proportionally with nominal delivered doses ranging from 10 to 30 mg.
- Overall, the safety of AZD1402 administered by inhalation in DPI form merits further testing in patients with pulmonary diseases, such as asthma.

Example 17 Two Parts to Evaluate the Efficacy and Safety of Three Inhaled Dose Levels of AZD1402 Administered as a Dry Powder Twice a Day for 4 Weeks in Adults with Asthma of Moderate Dose Inhaled Corticosteroids This is a Phase IIa, randomized, double-blind, placebo-controlled, dose-ranging, multicenter study to evaluate the efficacy and safety of inhaled AZD1402. This is a two-part study. Part 1 was a lead-in study of each dose level to evaluate safety and pharmacokinetics (PK) in an asthma patient population controlled with intermediate-dose inhaled corticosteroids (ICS) long-acting beta agonists (LABAs). (lead-in) cohort, progressing in Part 2 to adults with uncontrolled asthma on intermediate-dose ICS-LABA. Part 2 will be initiated for each dose level following the assessment of safety and PK at the relevant dose level in Part 1. The total study period for each participant in both Parts 1 and 2 was approximately 3.5 months, with a 2-week screening period, 4-week run-in period, 4-week treatment period, and 4-week follow-up period. be.

Part 1 of the study was randomized, double-blind, placebo-controlled, and conducted in parallel for two lower dose levels (Part 1a), followed by an open-label safety review and a safety review. Escalation to the highest dose (Part 1b) is made depending on the results of

Part 1a will be randomized 1:1:1 to receive one of two lower AZD1402 dry powder inhaler (DPI) doses (1 or 3 mg nominal delivered dose) or placebo in parallel. Consists of participants. Part 1b will consist of 15 participants who will be randomized 2:1 to receive the highest dose of AZD1402 DPI (10 mg nominal delivered dose) or placebo.

Examples of ingredients that can be used to make 1 mg, 3 mg and 10 mg capsules for this study are listed in Tables 2, 3 and 4 above.

Part 1a Lead-in Cohort - Nominal delivered dose of AZD1402 oral inhalation 1 mg twice daily (BID) via DPI
AZD1402 oral inhalation 3 mg nominal delivered dose via DPI BID
・Placebo oral inhalation BID via DPI

Part 1b Lead-in Cohort Nominal delivered dose of AZD1402 oral inhalation 10 mg via DPI BID
・Placebo oral inhalation BID via DPI

Part 2 will be randomized, double-blind, placebo-controlled and will include 360 participants and will evaluate 3 inhaled dose levels of AZD1402 compared to placebo.

Apart from scheduled clinic visits, part 2 of the study, 4 weeks of dosing, will take place at home. Part 2a, which includes two lower dose levels (1 mg and 3 mg nominal delivered doses), will be initiated in parallel after the open-label safety review of Part 1a. The higher dose, the 10 mg nominal delivered dose (Part 2b), will be included in Part 2 after the open-label review of Part 1b, depending on the results of the safety review. When Part 2b begins, all three dose levels (and placebo) will be run in parallel.

Part 2 includes:
AZD1402 oral inhalation 1 mg nominal delivered dose via DPI BID
AZD1402 oral inhalation 3 mg nominal delivered dose via DPI BID
AZD1402 oral inhalation 10 mg nominal delivered dose via DPI BID
・Placebo oral inhalation BID via DPI

Example 18 - Exposure profile after once-daily dosing of AZD1402 To predict the exposure profile after once-daily dosing of AZD1402, single-dose study NCT03384290 (described in Example 2 of WO2020/200960) Intravenous (i.v.) exposure data from 2000 were fitted to a two-compartment pharmacokinetic model using Nonmem (version 7.3.0). Subsequently, the inhalation rate of inhaled AZD1402 through the lungs was determined in studies NCT03384290 (a single ascending dose (SAD) study described in Example 2 of WO2020/200960), NCT03574805 (WO2020/200960), described in Example 16 above. 200960), and inhalation exposure data from NCT03921268. The final model was used to simulate PK serum exposure following once and twice daily administration of various doses. Figure 4 shows that a five-fold higher once daily nominal delivered dose (5 mg) is required to achieve the same daily minimum serum concentration as the twice daily nominal delivered dose of 1 mg. . The once-daily dose is 5 times higher (or 2.5 times the total daily dose) than the twice-daily dose, as also summarized in the table below for other doses: It is predicted that this will be necessary.

REFERENCES A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations of these references are provided below. Each of these references is incorporated herein in its entirety.
Pervaiz, S. , &Brew, K. (1987) FASEB J. 1,209-214
Flower, D. R. (1996) Biochem. J. 318, 1-14
Flower, D. R. et al. (2000) Biochim. Biophys. Acta1482, 9-24
Skerra, A. (2000) Biochim. Biophys. Acta1482, 337-350
You, J. , et al. (2010) Electrophoresis 31, 1853-1861
Skerra, A. (2001) Rev. Mol. Biotechnol. 74, 257-275
Schlehuber, S. , and Skerra, A. (2002) Biophys. Chem. 96, 213-228
Redl, B. (2000) Biochim. Biophys. Acta1482;241-248
Glasgow, B. J. et al. (1995) Curr. Eye Res. 14,363-372
Gasymov, O. K. et al. (1999) Biochim. Biophys. Acta1433, 307-320
Breustedt, D. A. et al. (2005) J. Biol. Chem. 280,1,484-493
Altschul, et al. (1997) Nucleic Acids Res. 25, 3389-3402
Altschul, et al. (1990) J. Mol. Biol. 215, 403-410
Smith, et al. (1981) J. Mol. Biol. 147, 195-197
Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85(8): 2444-2448
Roundhal, et al. , 2016, Current concepts of severe asthma. J. Clin. Invest. 126, 2394-2403
The Pocket Guide for Asthma Management and Prevention, Global Initiative for Asthma, Updated2020

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The following numbered sections describing aspects of the invention are part of the description of the invention.
1. 1. A method for treating asthma in a human subject, the method comprising: a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1; administering to said subject by inhalation a dry powder formulation comprising a variant or fragment, wherein a nominal delivered dose of about 0.1 mg to about 30 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. said method, wherein said method is administered.

2. The method of clause 1, wherein a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg is administered to the subject by oral inhalation.

3. A method for treating asthma in a human subject, the method comprising: a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1; administering to said subject by inhalation a dry powder formulation comprising a variant or fragment, wherein a nominal metered dose of about 0.2 mg to about 40 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. said method, wherein said method is administered.

4. A nominal metered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.55 mg, about 0.6 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg is administered to the subject. The method according to clause 3, wherein the method is administered.

5. The method of any one of the preceding clauses, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject at least once a day.

6. 6. The method of clause 5, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject twice a day.

7. The nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg.

8. A total nominal delivered dose of said lipocalin mutein, or variant or fragment thereof, of about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg per day is administered to said subject. the method of.

9. The nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 0.55 mg, about 0.6 mg, about 1.1 mg, about The method of clause 6, wherein the amount is 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg.

10. A total nominal metered dose of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg per day of said lipocalin mutein, or a variant thereof or 7. The method of clause 5 or 6, wherein the fragment is administered to said subject.

11. A method according to any one of the preceding clauses, wherein said formulation comprises a plurality of microparticles, said microparticles comprising said lipocalin mutein, or a variant or fragment thereof.

12. A method according to any one of the preceding clauses, wherein the dry powder formulation comprises trehalose.

13. A method according to any one of the preceding clauses, wherein the dry powder formulation comprises leucine.

14. 14. The method of clause 12 or 13, wherein the dry powder formulation comprises trileucine.

15. 15. The method of clause 14, wherein the dry powder formulation comprises trehalose, leucine, and trileucine.

16. A dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject, comprising: The method comprises administering the dry powder formulation by inhalation, wherein a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg is administered to the subject by oral inhalation, and the lipocalin mutein, or variant or fragment thereof, is administered to the subject by oral inhalation; The dry powder formulation, wherein the IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

17. The use according to clause 16, wherein a nominal delivered dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. Dry powder formulation for.

18. A dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject, comprising: The method comprises administering the dry powder formulation by inhalation, wherein a nominal metered dose of about 0.2 mg to about 40 mg of the lipocalin mutein, or a variant or fragment thereof, is administered to the subject by oral inhalation, The dry powder formulation, wherein the IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

19. A nominal metered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.55 mg, about 0.6 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg is administered to the subject. 19. A dry powder formulation for the use according to clause 18, which is administered.

20. A dry powder formulation for use according to any one of clauses 16 to 19, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject at least once a day.

21. 21. A dry powder formulation for use according to clause 20, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject twice a day.

22. The nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily is about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of a dry powder formulation for use according to clause 21.

23. A total nominal delivered dose of about 1 mg, about 2 mg, about 6 mg, about 20 mg, or about 60 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject per day. Dry powder formulation for use.

24. The nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day is about 0.55 mg, about 0.6 mg, about 1.1 mg, about Dry powder for use according to clause 21, wherein 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject twice a day. formulation.

25. A total nominal metered dose of about 1.1 mg, about 1.2 mg, about 2.2 mg, about 6.6 mg, about 22.2 mg, about 24 mg, or about 72 mg per day of said lipocalin mutein, or variant thereof, or Dry powder formulation for use according to clause 20 or 21, wherein the fragment is administered to said subject.

26. Dry powder formulation for use according to any one of clauses 16 to 25, wherein said formulation comprises a plurality of microparticles, said microparticles comprising said lipocalin mutein, or a variant or fragment thereof.

27. Dry powder formulation for use according to any one of clauses 16 to 26, wherein said dry powder formulation comprises trehalose.

28. Dry powder formulation for use according to any one of clauses 16 to 27, wherein said dry powder formulation comprises leucine.

29. 29. A dry powder formulation for use according to clause 27 or 28, wherein said dry powder formulation comprises trileucine.

30. A dry powder formulation for use according to clause 29, wherein said dry powder formulation comprises trehalose, leucine, and trileucine.

31. A dry powder formulation, said formulation comprising an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

32. 32. The dry powder formulation of clause 31, wherein the formulation comprises trehalose.

33. 33. The dry powder formulation according to clause 31 or 32, wherein the dry powder formulation comprises leucine.

34. 34. The dry powder formulation according to clause 32 or 33, wherein the dry powder formulation comprises trileucine.

35. 35. The dry powder formulation of clause 34, wherein the dry powder formulation comprises trehalose, leucine, and trileucine.

36. Any of clauses 31-35, wherein the formulation provides a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg when the formulation is administered to the subject by oral inhalation. 2. The dry powder preparation according to item 1.

37. When the formulation is administered to the subject by oral inhalation, the formulation delivers a nominal delivered dose of about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, or about 30 mg of the lipocalin mutein, or variant or fragment thereof. 37. A dry powder formulation according to clause 36, which provides.

38. Any one of clauses 31-37, wherein said formulation comprises a nominally metered dose of said lipocalin mutein, or a variant or fragment thereof, of about 0.2 mg to about 40 mg for administration to said subject by oral inhalation. Dry powder formulation as described.

39. The formulation has a nominal weight of about 0.55 mg, about 0.6 mg, about 1.1 mg, about 3.3 mg, about 11.1 mg, about 12 mg, or about 36 mg for administration to the subject by oral inhalation. 39. A dry powder formulation according to clause 38, comprising a dose of said lipocalin mutein, or variant or fragment thereof.

40. A dry powder formulation according to any one of clauses 31 to 39, wherein the formulation comprises a plurality of microparticles, the microparticles comprising the lipocalin mutein, or a variant or fragment thereof.

41. 41. A method for treating asthma in a human subject, said method comprising administering a dry powder formulation according to any one of clauses 31-40 by oral inhalation.

42. A dry powder formulation according to any one of clauses 31 to 40 for use in a method for treating asthma in a human subject, said method comprising administering said dry powder formulation to said subject by oral inhalation. said dry powder formulation, comprising administering said dry powder formulation.

43. Use of a dry powder formulation according to any one of clauses 31 to 40 for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of asthma in a human subject, wherein said treatment is carried out by oral inhalation. Said use comprising administering said dry powder formulation.

Claims (46)

1. A method for treating asthma in a human subject, the method comprising: a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1; administering to said subject by inhalation a dry powder formulation comprising a variant or fragment, wherein a nominal delivered dose of about 0.1 mg to about 30 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. said method, wherein said method is administered. A nominal delivered dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg of said lipocalin mutein, or variant or fragment thereof, is delivered to said subject by oral inhalation. 2. The method of claim 1, wherein the method is administered to 1. A method for treating asthma in a human subject, the method comprising: a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1; administering to said subject by inhalation a dry powder formulation comprising a variant or fragment, wherein a nominal metered dose of about 0.2 mg to about 40 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject by oral inhalation. said method, wherein said method is administered. about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 4. The method of claim 3, wherein a nominal metered dose of 36 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. 12. The method of any one of the preceding claims, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject at least once a day. 6. The method of claim 5, wherein the lipocalin mutein, or variant or fragment thereof, is administered to the subject twice a day. The nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily may be about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 7. The method of claim 6, wherein the amount is 5 mg, about 6 mg, about 10 mg, or about 30 mg. A total nominal delivered dose of about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 8 mg, about 10 mg, about 12 mg, about 20 mg, or about 60 mg of the lipocalin mutein, or variant or fragment thereof, per day is delivered to the subject. 7. The method of claim 5 or 6, wherein the method is administered. The nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day may be about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2. 7. The method of claim 6, wherein the amount is 2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 36 mg. about 1.1 mg, about 1.2 mg, about 2.2 mg, about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22.2 mg, about 24 mg, or per day. 7. The method of claim 5 or 6, wherein a total nominal metered dose of about 72 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. 2. A nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg is administered to the subject once per day. The method described in. 11. The method of any one of the preceding claims, wherein the formulation comprises a plurality of microparticles, the microparticles comprising the lipocalin mutein, or a variant or fragment thereof. 5. The method of any one of the preceding claims, wherein the dry powder formulation comprises trehalose. 6. The method of any one of the preceding claims, wherein the dry powder formulation comprises leucine. 15. The method of claim 13 or 14, wherein the dry powder formulation comprises trileucine. 16. The method of claim 15, wherein the dry powder formulation comprises trehalose, leucine, and trileucine. A dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject, comprising: The method comprises administering the dry powder formulation by inhalation, wherein a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg is administered to the subject by oral inhalation, and the lipocalin mutein, or variant or fragment thereof, is administered to the subject by oral inhalation; The dry powder formulation, wherein the IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof. A nominal delivered dose of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg of said lipocalin mutein, or variant or fragment thereof, is delivered to said subject by oral inhalation. 18. A dry powder formulation for use according to claim 17, administered to. A dry powder formulation comprising a therapeutically effective amount of an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein, or a variant or fragment thereof, for use in a method for treating asthma in a human subject, comprising: The method comprises administering the dry powder formulation by inhalation, wherein a nominal metered dose of about 0.2 mg to about 40 mg of the lipocalin mutein, or a variant or fragment thereof, is administered to the subject by oral inhalation, The dry powder formulation, wherein the IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof. about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 20. A dry powder formulation for use according to claim 19, wherein a nominal metered dose of 36 mg of said lipocalin mutein, or variant or fragment thereof, is administered to said subject. A dry powder formulation for use according to any one of claims 17 to 20, wherein said lipocalin mutein, or variant or fragment thereof, is administered to said subject at least once a day. 22. A dry powder formulation for use according to claim 21, wherein the lipocalin mutein, or variant or fragment thereof, is administered to the subject twice a day. The nominal delivered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice daily may be about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 23. A dry powder formulation for use according to claim 22, which is 5 mg, about 6 mg, about 10 mg, or about 30 mg. A total nominal delivered dose of about 1 mg, about 2 mg, about 4 mg, about 6 mg, about 10 mg, about 12 mg, about 20 mg, or about 60 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject per day. , a dry powder formulation for use according to claim 21 or 22. The nominal metered dose for each dose of the lipocalin mutein, or variant or fragment thereof, administered to the subject twice a day may be about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2. 2 mg, about 3.3 mg, about 4.4 mg, about 5.5 mg, about 6.6 mg, about 11.1 mg, about 12 mg, or about 36 mg of the lipocalin mutein, or variant or fragment thereof, twice a day; 23. A dry powder formulation for use according to claim 22, which is administered to said subject. about 1.1 mg, about 1.2 mg, about 2.2 mg, about 4.4 mg, about 6.6 mg, about 8.8 mg, about 11 mg, about 13.2 mg, about 22.2 mg, about 24 mg, or per day. 23. A dry powder formulation for use according to claim 21 or 22, wherein a total nominal metered dose of about 72 mg of the lipocalin mutein, or variant or fragment thereof, is administered to the subject. 10. A nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg is administered to the subject once per day. 18. Dry powder formulation for use according to item 17. A dry powder formulation for use according to any one of claims 17 to 27, wherein said formulation comprises a plurality of microparticles, said microparticles comprising said lipocalin mutein, or a variant or fragment thereof. A dry powder formulation for use according to any one of claims 17 to 28, wherein the dry powder formulation comprises trehalose. A dry powder formulation for use according to any one of claims 17 to 29, wherein the dry powder formulation comprises leucine. 31. A dry powder formulation for use according to claim 29 or 30, wherein the dry powder formulation comprises trileucine. 32. A dry powder formulation for use according to claim 31, wherein said dry powder formulation comprises trehalose, leucine, and trileucine. A dry powder formulation, said formulation comprising an anti-IL-4 receptor alpha (IL-4Rα) lipocalin mutein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof. 34. The dry powder formulation of claim 33, wherein the formulation comprises trehalose. 35. A dry powder formulation according to claim 33 or 34, wherein the dry powder formulation comprises leucine. 36. A dry powder formulation according to claim 34 or 35, wherein the dry powder formulation comprises trileucine. 37. The dry powder formulation of claim 36, wherein the dry powder formulation comprises trehalose, leucine, and trileucine. Any of claim 3337, wherein the formulation provides a nominal delivered dose of the lipocalin mutein, or variant or fragment thereof, of about 0.1 mg to about 30 mg when the formulation is administered to the subject by oral inhalation. Dry powder formulation according to item 1. When the formulation is administered to the subject by oral inhalation, the formulation may have a nominal dosage of about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 10 mg, or about 30 mg. 39. The dry powder formulation of claim 38, providing a delivered dose of the lipocalin mutein, or variant or fragment thereof. When the formulation is administered to the subject by oral inhalation, the formulation contains a nominal delivered dose of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg of the lipocalin mutein, or 39. A dry powder formulation according to claim 38, providing a variant or fragment. Any one of claims 33-40, wherein the formulation comprises a nominally metered dose of the lipocalin mutein, or a variant or fragment thereof, of about 0.2 mg to about 40 mg for administration to the subject by oral inhalation. Dry powder formulation as described in. The formulation is about 0.55 mg, about 0.6 mg, about 1.1 mg, about 2.2 mg, about 3.3 mg, 4.4 mg, about 5.5 mg, for administration to the subject by oral inhalation. 42. The dry powder formulation of claim 41, comprising a nominal metered dose of the lipocalin mutein, or variant or fragment thereof, of about 6.6 mg, about 11.1 mg, about 12 mg, or about 36 mg. 43. A dry powder formulation according to any one of claims 33 to 42, wherein the formulation comprises a plurality of microparticles, the microparticles comprising the lipocalin mutein, or a variant or fragment thereof. 44. A method for treating asthma in a human subject, said method comprising administering a dry powder formulation according to any one of claims 33 to 43 by oral inhalation. 44. A dry powder formulation according to any one of claims 33 to 43 for use in a method for treating asthma in a human subject, wherein said method comprises administering said dry powder formulation to said subject by oral inhalation. said dry powder formulation comprising administering. 44. Use of a dry powder formulation according to any one of claims 33 to 43 for the manufacture of a medicament for the therapeutic and/or prophylactic treatment of asthma in a human subject, wherein said treatment is performed by oral inhalation. said use comprising administering said dry powder formulation by.