How to treat neutropenia induced by chemotherapy or radiotherapy

Abstract

The present invention relates to a pharmaceutical composition comprising a protein complex, and its pharmaceutical applications for treating or preventing conditions characterized by impaired leukocyte production, such as neutropenia. The protein complex can also be formed by linking an immunoglobulin Fc region to a bioactive polypeptide via a non-peptide polymer linked to the immunoglobulin Fc region. [Solution] The present invention provides a method for treating chemotherapy-induced neutropenia in patients who require it by administering an effective amount of efrapegrastim to the patient, and a method for treating radiation-induced neutropenia in patients who require it by administering an effective amount of efrapegrastim to the patient.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition comprising a protein complex, and its pharmaceutical applications for treating or preventing conditions characterized by impaired leukocyte production, such as neutropenia. The protein complex can also be formed by linking an immunoglobulin Fc region to a bioactive polypeptide via a non-peptide polymer linked to the immunoglobulin Fc region. [Background technology]

[0002] Human granulocyte colony-stimulating factor (G-CSF) is a glucose-producing protein produced by matrix cells, macrophages, endothelial cells, fibroblasts, and mononuclear cells. G-CSF binds with high affinity to G-CSF receptors expressed on neutrophil progenitor cells in the bone marrow, inducing proliferation and differentiation of neutrophil progenitor cells into neutrophils that fight infection, without significant hematopoietic effects on other blood cell lineages. The use of recombinant G-CSF preparations is a well-established treatment for accelerating bone marrow recovery in patients with non-myeloid malignancies undergoing radiation or chemotherapy, preventing severe myelosuppression and associated complications, and reducing febrile neutropenia (FN).

[0003] Pegfilgrastim (Neulasta®; Amgen Inc.) is the most well-known PEGylated form of recombinant human G-CSF. Eflapegrastim is a sustained-release G-CSF developed to reduce not only the neutropenia complication associated with the use of myelosuppressive anticancer drugs, but also the severity and duration of severe neutropenia. Currently, the recommended dosage for both eflapegrastim (Rolontis®, HM10460A) and pegfilgrastim is administration the day after cytotoxic chemotherapy, which is generally necessary because patients who are weakened and in poor condition after chemotherapy often have to go to the hospital again. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2019 / 152530 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, in the treatment of neutropenia, there is a need to develop a same-day administration method for sustained-release G-CSF that provides similar or superior efficacy while alleviating the burden on patients. [Means for solving the problem]

[0006] In one embodiment, a method is provided for increasing absolute neutrophil count, granulocyte count in a subject eligible for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a stem cell donor in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent.

[0007] In another embodiment, a method is provided for treating or preventing a condition characterized by weakened leukocytosis in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent.

[0008] In yet another embodiment, the present invention provides a method for increasing absolute neutrophil count, granulocyte count in a bone marrow transplant-eligible subject, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a stem cell donor in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within 24 hours after the patient has received radiotherapy.

[0009] In yet another embodiment, a method is provided for treating or preventing a condition characterized by weakened leukocytosis in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has received radiotherapy.

[0010] In some specific cases, conditions characterized by weakened leukocytosis are selected from a group consisting of chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, reduced hematopoietic function, reduced immune function, decreased neutrophil count, reduced neutrophil recruitment, mobilization of peripheral hematopoietic progenitor cells, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, enhanced bone marrow engraftment during transplantation, enhanced bone marrow recovery during radiotherapy, compound-induced or chemotherapy-induced myeloplasmosis or myelosuppression, radiotherapy-induced myeloplasmosis or myelosuppression, and acquired immunodeficiency syndromes.

[0011] In some specific cases, the characteristic condition of weakened leukocytosis is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

[0012] In some specific cases, the method shortens the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia in patients who require it.

[0013] In some specific examples, the method includes administering an effective dose of efrapegrastim to the patient on the same day that the patient is administered chemotherapy or radiotherapy.

[0014] In some specific cases, an effective dose of efrapegrastim was administered to the patient, resulting in approximately 0.5 x 10⁻¹⁰ 9 The duration of absolute neutrophil counts below / L is shortened to approximately less than 6 hours, approximately less than 12 hours, or less than 24 hours.

[0015] In some specific cases, administration of an effective dose of efrapegrastim resulted in an increase in the absolute neutrophil count of approximately 0.5 x 10⁻¹⁴ in patients. 9 Prevent the value from falling below / L.

[0016] In some specific cases, when an effective dose of efrapegrastim is administered, the absolute neutrophil count of the patient is approximately 0.5 x 10⁻⁶. 9 / / From the first occurrence of less than L, approximately 1.5 x 10 9 The level may increase to over / L within approximately 4 days, 7 days, or 10 days. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows the results of chromatography of immunoglobulin Fc fragments obtained by cleaving immunoglobulin with papain. [Figure 2] This figure shows the SDS-PAGE results of purified immunoglobulin Fc fragments (M: molecular size marker, lane 1: IgG, lane 2: Fc). [Figure 3] This figure shows the effects of HM10460A and pegfilgrastim on absolute neutrophil count (ANC) after acute TC-induced neutropenia in normal SD rats (0 hours (A), +2 hours (B), +5 hours (C), and +24 hours (D) after chemotherapy). [Modes for carrying out the invention]

[0018] As generally described in this application, the disclosure provides a method for treating or preventing a condition characterized by increased absolute neutrophil count, granulocyte count, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production, or attenuated leukocyte production, in a patient in need of such treatment, and the method includes the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent or received radiotherapy.

[0019] definition To facilitate understanding of the present invention, numerous terms and phrases are defined below.

[0020] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Abbreviations used herein have their general meanings within the fields of chemistry and biology. Chemical structures and formulas described herein are to be interpreted according to the standard rules of chemical valence known in the field of chemistry.

[0021] Throughout the description, if compositions and kits are described as "having," "including," or "comprising" a particular component, or if processes and methods are described as "having," "including," or "comprising" a particular step, it is deemed that there exist compositions and kits of the present invention that are composed of additionally required components or composed of the components described, and that there exist methods and procedures of the present invention that are composed of additionally required steps or composed of the steps described.

[0022] In this application, if an element or component is included in and / or selected from the list of elements or components described, it should be understood that the element or component is either any one of the cited elements or components, or that the element or component may be selected from a group composed of two or more of the described elements or components.

[0023] Furthermore, the elements and / or features of the compositions or methods described herein should be understood to be combined in various ways, both explicitly and implicitly, without exceeding the spirit and scope of the invention. For example, where a particular compound is mentioned, that compound is also used in specific examples that are various compositions of the invention, and / or methods of the invention, unless understood differently from the context. That is, specific examples are described and illustrated within this application so as to make a clear and concise application, but it should be understood that the examples are intended to be combined and separated in various ways without exceeding the spirit and scope of the invention.

[0024] For example, it will be understood that all the features described and depicted herein can be applied to all aspects of the present invention described and depicted herein.

[0025] The articles "a" and "an" are used in this disclosure to refer to one or more (i.e., at least one) of the grammatical objects of the article, unless the context is inappropriate. For example, "element" means one element or one or more elements. The terms “and / or” are used in this disclosure to mean “and” or “or” unless otherwise expressly stated.

[0026] The expression "at least one of ~" should be understood, unless otherwise understood in context and usage, to include each of the objects listed after it, each of the cited objects, and any two or more diverse combinations of those objects individually. In relation to three or more listed objects, the expression "and / or" should be understood to have the same meaning, unless otherwise understood in context.

[0027] The use of the terms “include,” “incorporate,” “include,” “possess,” “possess,” “contain,” “contains,” “contains,” or “invites” (including their grammatical equivalents) should generally be understood as open and non-restrictive, without excluding, for example, additional unspecified elements or stages, unless otherwise specifically referred to or understood from context.

[0028] Where the term “about” is used before a quantitative value, the present invention also includes the specific quantitative value itself unless otherwise specifically referred to. As used in this application, the term “about” means a variation of ±10% from the expressed value, unless otherwise indicated or inferred from the context.

[0029] Throughout this specification, variables or parameters are described as groups or ranges. The descriptions are particularly intended to include all individual subcombinations of each member of such groups and ranges. For example, integers in the range of 0 to 40 specifically disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 individually, and integers in the range of 1 to 20 specifically disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 individually.

[0030] For example, the use of any example or exemplary language such as "~, etc." or "including" is merely for the purpose of further describing the invention as desirable and does not limit the scope of the invention unless requested. No language in the specification shall be construed as indicating that an unclaimed element is essential for the practice of the invention.

[0031] Generally, percentages specified for compositions are based on weight unless otherwise specified. Furthermore, if a variable is not defined, the previously stated definition of that variable applies.

[0032] The term "severe neutropenia" used in this application refers to 0.5x10 9 It is defined as neutropenia with an absolute neutrophil count of less than 1 / L. The terms "severe neutropenia" and "grade 4 neutropenia" are also used interchangeably.

[0033] The terms "pharmaceutical composition" or "pharmaceutical dosage form" used in this application refer to a combination of an active agent and an inactive or active carrier, which makes the composition particularly suitable for use in diagnosing or treating diseases in vivo or in vitro.

[0034] "Pharmacologically acceptable" means that it has been approved or will be approved by a federal or state regulatory agency of the United States, or by such agency of any country outside the United States, or that it is listed in the U.S. Pharmacopoeia or any other generally accepted pharmacopoeia for use in animals, particularly in humans.

[0035] As used in this application, "pharmaceutically acceptable excipients" refers to substances that aid in the administration of an activator to a subject and / or its absorption by the subject, and which may be included in the composition of the present invention without causing significant harmful toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline solutions (e.g., phosphate-buffered saline solutions), emulsions (e.g., oil-in-water or water-in-oil emulsions), lactate infusions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such formulations may be sterilized and, if desired, mixed with excipients such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, and / or aromatics that do not react adversely with the compounds of the present invention. For examples of such excipients, see Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0036] The “subjects” for which administration is considered include, but are not limited to, humans (e.g., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly)) and / or non-human animals, e.g., primates (e.g., Cynomorghus monkeys, Rhodeus ocellatus), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In specific examples, the subject is human. In specific examples, the subject is a non-human animal.

[0037] As used herein, “administer” means oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intravertebral administration, intracranial administration, intranasal administration, or subcutaneous administration, or implantation of a sustained-release device (e.g., a mini osmotic pump) into a subject. Such administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gingiva, nasal cavity, vagina, rectum, or percutaneous). Such parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intracutaneous, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other methods of delivery include, but are not limited to, the use of liposomal dosage forms, intravenous infusion, and transdermal patches. “Combined administration” means that the composition described herein is administered concurrently with, immediately before or immediately after, the administration of one or more additional therapies (e.g., anticancer agents, chemotherapy, radiotherapy, or treatment for neurodegenerative diseases). Efrapegrastim may be administered to patients alone or in combination with other drugs. This combination involves administering the compounds individually or in combination (one or more compounds or drugs), simultaneously or sequentially. Therefore, if desired, the formulation can be combined with other active substances (e.g., to reduce metabolic degradation).

[0038] The terms “disease,” “disorder,” and “condition” may be used interchangeably in this specification.

[0039] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to actions that reduce the severity of a disease, disorder, or condition, or delay or slow the progression of a disease, disorder, or condition, while the subject is suffering from a particular disease, disorder, or condition (e.g., “therapeutic treatment”), unless otherwise expressly stated.

[0040] Generally, an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response, for example, to treat upper urothelial carcinoma or non-muscle invasive bladder cancer. As will be understood by those skilled in the art, the effective amount of the compounds of the present disclosure will vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.

[0041] As used herein, the term "protein conjugate" or "conjugate" refers to a compound comprising one or more physiologically active polypeptides, one or more non-peptide polymers having reactive groups at both ends, and one or more immunoglobulin Fc fragments, wherein the three components are covalently linked. Also, to distinguish it from a "conjugate", a construct containing only two different molecules selected from a physiologically active polypeptide, a non-peptide polymer, and an immunoglobulin Fc fragment, and the two molecules being covalently bonded, is designated as a "complex".

[0042] As used herein, the term "immunoglobulin Fc fragment" refers to a protein that includes the constant region 2 (C H 2) and constant region 3 (C H 3) of the heavy chain of an immunoglobulin, but does not include the variable regions of the heavy and light chains of the immunoglobulin, the constant region 1 (C H 1) of the heavy chain, and the constant region 1 (C L 1) of the light chain. It also further includes the hinge region in the constant region of the heavy chain. In addition, the immunoglobulin Fc fragment of the present invention also includes part or all of the constant region 1 (C H 1) of the heavy chain variable region and / or the constant region 1 (C L 1) of the light chain, except for the variable regions of the heavy and light chains. Also, as long as it is substantially similar to the natural protein or has better physiological functions, the IgG Fc fragment is a fragment having deletions in relatively long portions in the amino acid sequences of C H 2 and / or C H 3. That is, the immunoglobulin Fc fragment of the present invention is 1) C H 1 domain, C H 2 domain, C H 3 domain, and C H4 domains, 2) C H 1 domain and C H 2 domains, 3) C H 1 domain and C H 3 domains, 4) C H 2 domains and C H 3 domains, 5) combinations of one or more domains and an immunoglobulin hinge region (or part of a hinge region), and 6) dimers of each domain of the heavy chain invariant region and the light chain invariant region.

[0043] In this specification, the term "deglycosylation" refers to the enzymatic removal of sugar moiety from an Fc fragment, and the term "aglycosylation" means that the Fc fragment is produced in a form that has not been saccharified by a prokaryote, preferably Escherichia coli.

[0044] As used herein, the term “combination” means that polypeptides coding single-chain immunoglobulin Fc regions of the same origin are linked to single-chain polypeptides of different origins to form a dimer or polymer. That is, the dimer or polymer may also be formed by two or more fragments selected from the group consisting of IgG1 Fc fragments, IgG2 Fc fragments, IgG3 Fc fragments, and IgG4 Fc fragments. As used herein, the term "hybrid" means that a single-stranded immunoglobulin Fc fragment contains sequences that encode two or more immunoglobulin Fc fragments of different origins.

[0045] As used herein, "non-peptide polymer" means a biocompatible polymer containing two or more repeating units linked to each other by covalent bonds excluding peptide bonds.

[0046] The terms "bioactive polypeptide," "bioactive protein," "active polypeptide," "polypeptide," or "protein drug" as used herein are interchangeable in their meaning and are characterized by being physiologically active forms that exhibit diverse in vivo physiological functions.

[0047] Efrapegrastim Rolontis (registered trademark), SPI-2012, HM10460A and 17,65 Efrapegrastim, known as SG-CSF, is a sustained-release granulocyte colony-stimulating factor (G-CSF) developed to reduce the severity and duration of severe neutropenia and complications of neutropenia associated with the use of myelosuppressive anticancer drugs or radiotherapy. Efrapegrastim consists of a recombinant human G-CSF analog (ef-G-CSF) linked to a dual-function polyethylene glycol linker and a recombinant fragment of the Fc region of human immunoglobulin G4 (IgG4). In a specific example, the recombinant human G-CSF analog (ef-G-CSF) differs from human G-CSF (SED ID NO:1) in that serine substitutions are present at positions 17 and 65 (SED ID NO:2). Theoretically, the Fc region of human IgG4 is considered to increase the serum half-life of ef-G-CSF.

[0048] ef-G-CSF is produced in the periplasmic membrane space by transformed Escherichia coli (E. coli) in a soluble form. Separately, the Fc fragment is produced as an inclusion body in transformed E. coli. ef-G-CSF and the Fc fragment are isolated independently and purified through a series of purification steps. The purified ef-G-CSF (SEQ ID NO: 2) and Fc fragments (SEQ ID NOs: 3 and 4) are linked via 3.4 kDa polyethylene glycol (PEG) molecules designed to have reactive groups at both ends. Efrapegrastim itself is a molecule obtained by linking the PEG linker at the N-terminus of both ef-G-CSF and the Fc fragment, respectively. The G-CSF analog is a 3.4 kDa polyethylene glycol analog (OHCCH2CH2(OCH2CH2)) with propylaldehyde end groups at both ends at the nitrogen atom of the N-terminal Thr residue. nIt is joined to OCH2CH2CHO) via reductive amination, forming a covalent bond. The resulting G-CSF-PEG complex is then ligated to the N-terminal Pro via reductive amination at the nitrogen of a recombinant Fc fragment mutant produced in E. coli, generating the final conjugate, efrapegrastim.

[0049] [ka]

[0050] In one embodiment, the present invention provides efrapegrastim for use in a method for increasing absolute neutrophil count, granulocyte count in subjects suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in donors in patients requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim to the patient within a period of less than 24 hours after administration of a chemotherapeutic agent.

[0051] In another embodiment, the present invention provides efrapegrastim for use in treating or preventing a condition characterized by weakened leukocytosis in a patient requiring such treatment, comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent.

[0052] In yet another embodiment, the present invention provides efrapegrastim for use in a method for increasing absolute neutrophil count, granulocyte count in subjects suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in donors in patients requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has received radiotherapy.

[0053] In yet another embodiment, the present invention provides efrapegrastim for use in treating or preventing a condition characterized by weakened leukocytosis in a patient requiring such treatment, comprising the step of administering an effective dose of efrapegrastim within 24 hours of the patient receiving radiotherapy.

[0054] The detailed explanations in the following sections on the treatment of chemotherapy-induced neutropenia and the treatment of radiotherapy-induced neutropenia also apply to efrapegrastim as described in this section.

[0055] Pharmaceutical composition In one embodiment, a pharmaceutical composition is provided for use in a method for increasing absolute neutrophil count, granulocyte count in subjects suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in donors in patients requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim to the patient within a period of less than 24 hours after administration of a chemotherapeutic agent, and comprising efrapegrastim and a pharmaceutically acceptable carrier.

[0056] In another embodiment, the present invention provides a pharmaceutical composition comprising efrapegrastim and a pharmaceutically acceptable carrier for use in treating or preventing a condition characterized by weakened leukocyte production in a patient requiring such treatment, the step of administering an effective dose of efrapegrastim to the patient within 24 hours after the administration of a chemotherapeutic agent.

[0057] In one embodiment, a pharmaceutical composition is provided for use in a method for increasing absolute neutrophil count, granulocyte count in subjects suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in donors in patients requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has received radiotherapy, and comprising efrapegrastim and a pharmaceutically acceptable carrier.

[0058] In yet another embodiment, the present invention provides a pharmaceutical composition comprising efrapegrastim and a pharmaceutically acceptable carrier for use in treating or preventing a condition characterized by weakened leukocyte production in a patient requiring such treatment, comprising the step of administering an effective dose of efrapegrastim within 24 hours after the patient has received radiotherapy.

[0059] In certain specific examples, the pharmaceutically acceptable carrier is phosphate-buffered saline. In some specific examples, the phosphate-buffered saline is Dulbecco's phosphate-buffered saline. In certain specific examples, the pharmaceutically acceptable carrier is citrate buffer.

[0060] The pharmaceutical compositions provided in this application can be administered by a variety of routes, including, but not limited to, oral (enteral), parenteral (by injection), rectal, transdermal, intradermal, intradural, subcutaneous (SC), intravenous (IV), intramuscular (IM), and intranasal administration. In some specific examples, the pharmaceutical compositions disclosed in this application are administered parenterally. In some specific examples, the pharmaceutical compositions disclosed in this application are administered subcutaneously.

[0061] The pharmaceutical compositions provided in this application are presented in unit dosage forms to facilitate precise administration. The term "unit dosage form" refers to a physically separated unit suitable for a single dose pathway for human subjects and other mammals, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect together with appropriate pharmaceutical excipients. Typical unit dosage forms include pre-filled and measured ampoules or syringes for liquid compositions, or, in the case of solid compositions, pills, tablets, capsules, etc.

[0062] In a specific example, the restrictive composition provided in this application is administered to the patient as a subcutaneous injection solution.

[0063] In specific examples, the compounds provided in this application may be administered as a single active agent or in combination with other active agents.

[0064] The descriptions of pharmaceutical compositions provided herein primarily relate to pharmaceutical compositions suitable for administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Variations of pharmaceutical compositions suitable for administration to humans are well known in order to provide compositions suitable for administration to a variety of animals, and ordinary veterinary pharmacologists can design and / or perform such variations in general experiments. General considerations relating to the dosage formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21. st Found in ed., Lippincott Williams & Wilkins, 2005.

[0065] The detailed descriptions in the following sections on the treatment of chemotherapy-induced neutropenia and the treatment of radiotherapy-induced neutropenia also apply to the pharmaceutical compositions described in this section.

[0066] Method of Use and Treatment Treatment of chemotherapy-induced neutropenia In one embodiment, a method is provided for increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor, the method comprising the step of administering an effective dose of efrapegrastim to a patient within a period of less than 24 hours after the administration of a chemotherapeutic agent.

[0067] In another embodiment, a method is provided for treating or preventing a condition characterized by weakened leukocyte production in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim to the patient within a period of less than 24 hours after the administration of a chemotherapeutic agent.

[0068] In some specific examples, the condition characterized by weakened leukocytosis is selected from the group consisting of chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, hematopoietic dysfunction, immunodeficiency, neutropenia, decreased neutrophil count, decreased neutrophil recruitment, peripheral blood progenitor cell recruitment, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, increased bone marrow engraftment during transplantation, increased bone marrow recovery during radiotherapy, chemical-induced or chemotherapy-induced myeloplasmosis or myelosuppressant-induced myeloplasmosis or myelosuppressant-induced myeloplasmosis or myelosuppressant-induced myeloplasmosis, and acquired immunodeficiency syndromes.

[0069] In one specific example, the condition is chemotherapy-induced neutropenia. In one specific example, the method can shorten the duration of chemotherapy-induced neutropenia in patients who require it.

[0070] In one specific example, the method includes administering an effective dose of efrapegrastim to the patient on the same day that the chemotherapeutic agent is administered.

[0071] In some specific cases, an effective dose of efrapegrastim was administered to the patient, resulting in approximately 0.5 x 10⁻¹⁰ 9 The duration of absolute neutrophil counts below 1 / L can be shortened to less than approximately 24 hours. Specifically, effective doses of efrapegrastim can shorten the duration of absolute neutrophil counts in patients by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts below 1 / L can be shortened to approximately 24 hours, approximately 12 hours, or approximately 8 hours. More specifically, an effective dose of efrapegrastim can reduce the duration of absolute neutrophil counts in patients by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts less than 1 / L can be shortened to approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In one specific example, an effective dose of efrapegrastim can reduce the duration of absolute neutrophil counts to approximately 0.5 x 10 in a patient. 9The duration of absolute neutrophil counts below 1 / L is shortened to less than approximately 24 hours. In one specific example, administration of an effective dose of efrapegrastim reduces the patient's neutrophil count by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts below 1 / L is shortened to less than approximately 12 hours. In one specific example, administration of an effective dose of efrapegrastim reduces the patient's neutrophil count by approximately 0.5 x 10⁻¹⁰. 9 This shortens the duration of absolute neutrophil counts below / L to less than approximately 8 hours.

[0072] In some specific cases, administration of an effective dose of efrapegrastim increased the patient's absolute neutrophil count by approximately 0.5 x 10⁻¹⁴. 9 This prevents the value from falling below / L.

[0073] In some specific cases, chemotherapy-induced neutropenia is characterized by an absolute neutrophil count of 0.5 x 10⁻⁶. 9 In severe neutropenia with a dose less than 10 / L, the patient's absolute neutrophil count increased to approximately 0.5 x 10⁻¹⁰ upon administration of an effective dose of efrapegrastim. 9 Within approximately 4 days, 7 days, or 10 days from the first occurrence of less than / L, approximately 1.5x10 9 It increases to more than / L. Specifically, the absolute neutrophil count of the patient increases by approximately 0.5 x 10⁻¹⁰. 9 From the first occurrence of less than / L, approximately 1.5 x 10 9 The time to recover to an absolute neutrophil count of 0.5x10⁻¹⁰ / L or higher is less than approximately 10 days, less than approximately 7 days, or less than approximately 4 days. In specific cases, chemotherapy-induced neutropenia is characterized by an absolute neutrophil count of 0.5x10⁻¹⁰ / L. 9 Severe neutropenia with a value of less than / L, and in patients, approximately 0.5 x 10 9 For absolute neutrophil counts less than / L, approximately 1.5 x 10 9 The time to recover to an absolute neutrophil count of 1 / L or more is approximately less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days.

[0074] In some specific examples, the method is intended to increase the absolute neutrophil count of patients who require it, by approximately 0.5 x 10⁻⁶. 9 From the absolute neutrophil count less than / L, approximately 1.5 x 109 The recovery time to an absolute neutrophil count of 10 days or more is less than approximately 10 days. Specifically, the patient's recovery time is approximately 0.5 x 10 9 From the absolute neutrophil count less than / L, approximately 1.5 x 10 9 The recovery time to an absolute neutrophil count of 10 / L or more is approximately 10 days, approximately 7 days, or less than approximately 4 days. In specific cases, in patients, approximately 0.5 x 10 9 From absolute neutrophil counts less than / L, approximately 1.5 x 10⁻¹⁰ neutrophils were observed in the patient. 9 The time it takes to recover to an absolute neutrophil count of 1 / L or more is approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or less than 10 days.

[0075] In one specific example, an effective dose of efrapegrastim is administered simultaneously with the chemotherapeutic agent.

[0076] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours after administration of the chemotherapy agent.

[0077] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours after the administration of the chemotherapy agent.

[0078] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 3 hours, or 5 hours after the administration of the chemotherapeutic agent.

[0079] In one specific example, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

[0080] In a specific example, the myelosuppressive chemotherapeutic agent is selected from the group consisting of docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

[0081] In specific cases, patients are receiving chemotherapy agents to treat a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin lymphoma.

[0082] In some specific cases, an effective dose of efrapegrastim is administered to the patient parenterally at a dose of approximately 2 mg to 18 mg of efrapegrastim. In one specific case, the dose is approximately 13.2 mg of efrapegrastim per day.

[0083] In specific cases, effective doses of efrapegrastim are approximately 2.0 mg to 5.0 mg, 5.0 mg to 15.0 mg, 7.0 mg to 15.0 mg, 9.0 mg to 15.0 mg, 11.0 mg to 15.0 mg, 13.0 mg to 15.0 mg, 5.0 mg to 13.0 mg, 5.0 mg to 11.0 mg, and 5.0 mg to This includes parenteral administration at doses of approximately 9.0 mg, approximately 5.0 mg to approximately 7.0 mg, approximately 7.0 mg to approximately 13.0 mg, approximately 7.0 mg to approximately 11.0 mg, approximately 7.0 mg to approximately 9.0 mg, approximately 9.0 mg to approximately 13.0 mg, approximately 9.0 mg to approximately 11.0 mg, approximately 11.0 mg to approximately 13.0 mg, or approximately 15.0 to approximately 18.0 mg of efrapegrastim.

[0084] In specific examples, an effective dose of efrapegrastim is administered parenterally in amounts of approximately 12.0 mg, 12.2 mg, 12.4 mg, 12.6 mg, 12.8 mg, 13.0 mg, 13.2 mg, 13.4 mg, 13.6 mg, 13.6 mg, 13.8 mg, or 14.0 mg of efrapegrastim. In specific examples, an effective dose of efrapegrastim is administered parenterally in amounts of approximately 13.2 mg of efrapegrastim.

[0085] Specifically, the dosage of efrapegrastim can be either administered as a single dose within 24 hours of chemotherapy administration, on the same day the chemotherapy agent is administered to the patient, or in one to five divided doses.

[0086] Treatment of radiation-induced neutropenia In one embodiment, a method is provided for increasing absolute neutrophil count, granulocyte count in a subject eligible for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a stem cell donor, the method comprising the step of administering an effective dose of efrapegrastim within a period of less than 24 hours after the patient has received radiotherapy.

[0087] In another embodiment, a method is provided for treating or preventing a condition characterized by weakened leukocytosis in a patient requiring such treatment, the method comprising the step of administering an effective dose of efrapegrastim to the patient within 24 hours after receiving radiotherapy.

[0088] In some specific cases, conditions characterized by weakened leukocytosis are selected from a group consisting of radiotherapy-induced neutropenia, reduced hematopoietic function, reduced immune function, decreased neutrophil count, reduced neutrophil recruitment, mobilization of peripheral hematopoietic cells, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, enhanced bone marrow engraftment during transplantation, enhanced bone marrow recovery in radiotherapy, radiotherapy-induced myeloplasticity or myelosuppressants, and acquired immunodeficiency syndromes.

[0089] In one specific example, the condition is radiotherapy-induced neutropenia. In one specific example, the method can shorten the duration of radiotherapy-induced neutropenia in patients who require it.

[0090] In one specific example, the method includes administering an effective dose of efrapegrastim on the same day that the patient receives radiotherapy.

[0091] In a specific example, the effective dose of efrapegrastim administered to the patient is approximately 0.5 x 10⁻¹⁰ 9 The duration of absolute neutrophil counts below 1 / L can be shortened to less than approximately 24 hours. Specifically, effective doses of efrapegrastim can shorten the duration of absolute neutrophil counts in patients by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts below 1 / L can be shortened to approximately 24 hours, approximately 12 hours, or approximately 8 hours. More specifically, an effective dose of efrapegrastim can shorten the duration of absolute neutrophil counts in patients by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts less than 1 / L can be shortened to approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In one specific example, an effective dose of efrapegrastim can reduce the duration of absolute neutrophil counts to approximately 0.5 x 10 in a patient. 9 The duration of absolute neutrophil counts below 1 / L is shortened to less than approximately 24 hours. In one specific example, administration of an effective dose of efrapegrastim reduces the patient's neutrophil count by approximately 0.5 x 10⁻¹⁰. 9 The duration of absolute neutrophil counts below 1 / L is shortened to less than approximately 12 hours. In one specific example, administration of an effective dose of efrapegrastim reduces the patient's neutrophil count by approximately 0.5 x 10⁻¹⁰. 9 This shortens the duration of absolute neutrophil counts below / L to less than approximately 8 hours.

[0092] In some specific cases, administration of an effective dose of efrapegrastim increased the patient's absolute neutrophil count by approximately 0.5 x 10⁻¹⁴. 9Prevent the value from falling below / L.

[0093] In some specific cases, radiotherapy-induced neutropenia is characterized by an absolute neutrophil count of 0.5 x 10⁻¹⁰. 9 In severe neutropenia with a dose less than 10 / L, the patient's absolute neutrophil count increased to approximately 0.5 x 10⁻¹⁰ upon administration of an effective dose of efrapegrastim. 9 From the first occurrence of less than / L, approximately 1.5 x 10 9 The absolute neutrophil count should be increased to 0.5x10⁻¹⁰ / L or higher within approximately 4 days, 7 days, or 10 days. Specifically, the patient's absolute neutrophil count should be increased to approximately 0.5x10⁻¹⁰ / L. 9 From the first occurrence of less than / L, approximately 1.5 x 10 9 The time to recover to an absolute neutrophil count of 0.5x10⁻¹⁰ / L or higher is approximately less than 4 days, less than 7 days, or less than 10 days. In specific cases, radiotherapy-induced neutropenia is characterized by an absolute neutrophil count of 0.5x10⁻¹⁰ / L. 9 The value is less than / L, and in patients, it is approximately 0.5 x 10 9 From the absolute neutrophil count less than / L, approximately 1.5 x 10 9 The time to recover to an absolute neutrophil count of 1 / L or more is approximately less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days.

[0094] In some specific examples, the method is intended to increase the absolute neutrophil count in patients who require it, and in patients, approximately 0.5 x 10 9 From absolute neutrophil counts less than / L, approximately 1.5 x 10⁻¹⁰ neutrophils were found in the patient. 9 The recovery time to an absolute neutrophil count of 10 / L or more is less than approximately 10 days. Specifically, in patients, approximately 0.5 x 10 9 From the absolute neutrophil count less than / L, approximately 1.5 x 10 9 The recovery time to an absolute neutrophil count of 10L or more is approximately less than 10 days, approximately less than 7 days, or approximately less than 4 days.

[0095] In a specific example, in the patient, approximately 0.5 x 10 9 From absolute neutrophil counts less than / L, approximately 1.5 x 10⁻¹⁰ neutrophils were found in the patient. 9The recovery time to an absolute neutrophil count of 1 / L or more is approximately less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, less than 9 days, or less than 10 days.

[0096] In one specific example, an effective dose of efrapegrastim is administered in conjunction with radiation therapy.

[0097] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours after radiotherapy.

[0098] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours after receiving radiotherapy.

[0099] In specific cases, an effective dose of efrapegrastim is administered within approximately 0.5 hours, 3 hours, or 5 hours after receiving radiotherapy.

[0100] In a specific example, the patient is receiving radiation therapy to treat a cancer selected from a group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin lymphoma.

[0101] In some specific cases, an effective dose of efrapegrastim is administered to the patient parenterally at a dose of approximately 2 mg to 18 mg of efrapegrastim. In one specific case, the dose is approximately 13.2 mg of efrapegrastim per day.

[0102] In specific cases, effective doses of efrapegrastim are approximately 2.0 mg to 5.0 mg, 5.0 mg to 15.0 mg, 7.0 mg to 15.0 mg, 9.0 mg to 15.0 mg, 11.0 mg to 15.0 mg, 13.0 mg to 15.0 mg, 5.0 mg to 13.0 mg, 5.0 mg to 11.0 mg, and 5.0 mg to This includes parenterally administering doses of efrapegrastim of approximately 9.0 mg, approximately 5.0 mg to approximately 7.0 mg, approximately 7.0 mg to approximately 13.0 mg, approximately 7.0 mg to approximately 11.0 mg, approximately 7.0 mg to approximately 9.0 mg, approximately 9.0 mg to approximately 13.0 mg, approximately 9.0 mg to approximately 11.0 mg, approximately 11.0 mg to approximately 13.0 mg, or approximately 15.0 mg to approximately 18.0 mg.

[0103] In specific examples, an effective dose of efrapegrastim is administered parenterally in doses of approximately 12.0 mg, 12.2 mg, 12.4 mg, 12.6 mg, 12.8 mg, 13.0 mg, 13.2 mg, 13.4 mg, 13.6 mg, 13.6 mg, 13.8 mg, or 14.0 mg of efrapegrastim. In specific examples, an effective dose of efrapegrastim is administered parenterally in doses of approximately 13.2 mg of efrapegrastim.

[0104] Specifically, the dosage of efrapegrastim can be selectively administered within 24 hours of the day of radiation therapy, either as a single dose on the day the patient received radiation therapy or in 1 to 5 divided doses. [Examples]

[0105] To further fully understand the disclosures described herein, the following examples are provided. The synthetic and biological examples described herein are provided to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed as limiting their scope in any way.

[0106] Example 1: Efrapegrastim ( 17,65 Manufacturing of SG-CSF-PEG-Fc Step 1: Production of immunoglobulin Fc fragments using immunoglobulins The immunoglobulin Fc fragments were manufactured as follows:

[0107] 200 mg of immunoglobulin G (IgG, Green Cross) with a molecular weight of 150 kDa, dissolved in 10 mM phosphate buffer, was treated with 2 mg of the protein hydrolase papain (Sigma), and the mixture was reacted at 37°C for 2 hours with gradual stirring.

[0108] After the enzymatic reaction, Superdex column chromatography, protein A column chromatography, and cation exchange resin column chromatography were sequentially performed to purify the generated immunoglobulin Fc fragments. Specifically, the reaction mixture was loaded onto a Superdex 200 column (Superdex 200, Pharmacia) equilibrated with 10 mM sodium phosphate buffer (PBS, pH 7.3), and eluted from the column at a flow rate of 1 ml / min using the same buffer. Unreacted immunoglobulin molecules (IgG) and F(ab')2, which have relatively larger molecular weights than the immunoglobulin Fc fragments, were removed first as they were eluted before the immunoglobulin Fc fragments. Fab fragments, which have similar molecular weights to the immunoglobulin Fc fragments, were removed by protein A column chromatography (Figure 1). Immunoglobulin Fc fragment-containing fractions eluted from a Superdex 200 column were loaded onto a Protein A column (Pharmacia) equilibrated with 20 mM phosphate buffer (pH 7.0) at a flow rate of 5 ml / min. The column was then washed with the same buffer to remove unbound proteins. Subsequently, the Protein A column was eluted with 100 mM sodium citrate buffer (Na citrate, pH 3.0) to obtain high-purity immunoglobulin Fc fragments. The purified Fc fraction was then finally purified using a cation exchange resin column (poly CAT, Poly LC). The column loaded with the Fc fraction was then eluted into a linear concentration gradient of 0.15 M–0.4 M sodium chloride in 10 mM acetate buffer (pH 4.5) to obtain a high-purity immunoglobulin Fc fraction. This high-purity immunoglobulin Fc fraction was analyzed by 10% SDS-PAGE (Lane 2 in Figure 2).

[0109] Stage 2: 17,65 Manufacturing of SG-CSF-PEG complex Human granulocyte colony-stimulating factor (HPG) was prepared by dissolving 3.4-kDa polyethylene glycol, ALD-PEG-ALD (Shearwater), which has aldehyde reactive groups at both ends, in 100 mM phosphate buffer at a concentration of 5 mg / ml. 17,65 SG-CSF (MW: 18.6kDa), 17,65SG-CSF:PEG was mixed in a molar ratio of 1:5. Sodium cyanoborohydride (NaCNBH3, Sigma), a reducing agent, was added to the mixture at a final concentration of 20 mM, and the mixture was reacted at 4°C for 3 hours with gentle stirring until the PEG was formed. 17,65 It was made to be linked to the amino terminus of SG-CSF. PEG and 17,65 To obtain a 1:1 complex with SG-CSF, size exclusion chromatography was performed on the reaction mixture using a Superdex® column (Pharmacia). 17,65 The SG-CSF-PEG complex was eluted using 10 mM potassium phosphate buffer (pH 6.0) as the elution buffer, and the PEG was not bound to it. 17,65 SG-CSF, unreacted PEG, and PEG 17,65 The dimeric byproduct linked to the SG-CSF molecule was removed. 17,65 The SG-CSF-PEG complex was concentrated at 5 mg / ml. This experiment provided the highest reactivity and produced the smallest amount of dimer-like byproducts. 17,65 The optimal reaction molar ratio of SG-CSF to PEG was found to be 1:5.

[0110] Stage 3: Manufacturing of 17,65S-G-CSF-PEG-Fc jointed material Purified in step 2 above 17,65 To ligate the SG-CSF-PEG complex to the N-terminus of the immunoglobulin Fc fragment, the immunoglobulin Fc fragment (approximately 53 kDa) prepared in step 1 was dissolved in 10 mM phosphate buffer in 1:1, 1:2, 1:4, and 1:8 ratios. 17,65 SG-CSF-PEG complex:Fc molar ratio, 17,65 The SG-CSF-PEG complex was mixed with the phosphate buffer solution. After adjusting the phosphate buffer concentration of the reaction mixture to 100 mM, the reducing agent NaCNBH3 was added to the reaction mixture at a final concentration of 20 mM, and the reaction was carried out at 4°C for 20 hours with gentle stirring. This experiment provided the highest reactivity and produced the fewest by-products, such as dimers. 17,65 It was found that the optimal reaction molar ratio of the SG-CSF-PEG complex to Fc is 1:2.

[0111] Stage 4: Separation and purification of the G-CSF-PEG-Fc composite. After the reaction in the above step 3, perform Superdex size exclusion chromatography on the reaction mixture to remove unreacted substances and by-products, and the generated 17,65 S-G-CSF-PEG-Fc protein foldamer was purified. The reaction mixture was concentrated and loaded onto a Superdex column, and then 10 mM phosphate buffer (pH 7.3) was passed through the column at a flow rate of 2.5 ml / min to remove unbound Fc and unreacted substances. Then, by column elution, 17,65 the S-G-CSF-PEG-Fc protein foldamer fraction was collected. The collected 17,65 S-G-CSF-PEG-Fc protein foldamer fraction contains a small amount of impurities, unreacted Fc, and interferon alpha dimer, so cation exchange chromatography was performed to remove the impurities. 17,65 The S-G-CSF-PEG-Fc protein foldamer fraction was loaded onto a poly CATLP column (poly LC) equilibrated with 10 mM sodium acetate (pH 4.5), and the column was eluted with 1 M NaCl using a 0 - 0.5 M linear concentration gradient of NaCl in 10 mM sodium acetate buffer (pH 4.5). Finally, an anion exchange column was used, 17,65 and the S-G-CSF-PEG-Fc protein foldamer was purified. 17,65 The S-G-CSF-PEG-Fc protein foldamer fraction was loaded onto a poly WAXLP column (poly LC) equilibrated with 10 mM Tris-HCl (pH 7.5), and the column was eluted with 1 M NaCl using a 0 - 0.3 M linear concentration gradient of NaCl in 10 mM Tris-HCl (pH 7.5) to separate the S-G-CSF-PEG-Fc protein foldamer in a high purity form. 17,65 The efficacy of eflapegrastim (HM10460A), a long-acting G-CSF analog, was compared with pegfilgrastim in a murine model of chemotherapy-induced neutropenia using various dosing regimens.

[0112] Example 2: Efficacy study of efrapegrastim using different dosage regimens in rats with docetaxel / cyclophosphamide-induced neutropenia. ​

[0113] In the following study, efrapegrastim was prepared basically as described in Example 1.

[0114] (i) Research material [Table 1]

[0115] [Table 2]

[0116] [Table 3]

[0117] Manufacturing of HM10460A solution for subcutaneous administration Preparation of a 61.8 μg / kg HM10460A solution for subcutaneous administration: 92.7 μL of HM10460A (6.0 mg / mL) stock solution was diluted with 7.3 μL of DPBS 1790.

[0118] Preparation of a 372.0 μg / kg HM10460A solution for subcutaneous administration: 558.0 μL of HM10460A (6.0 mg / mL) stock solution was diluted with 17442.0 μL of DPBS.

[0119] Preparation of a 496.0 μg / kg HM10460A solution for subcutaneous administration: 744.0 μL of HM10460A (6.0 mg / mL) stock solution was diluted with 17256.0 μL of DPBS.

[0120] The test substance was prepared based on the G-CSF protein dosage indicated on the drug label (HM10460A).

[0121] The HM10460A solution for subcutaneous administration was diluted with DPBS to a final dose concentration of 2 mL / kg.

[0122] Preparation of pegfilgrastim solution for subcutaneous administration Preparation of a 103.3 μg / kg pegfilgrastim solution for subcutaneous administration: A stock solution of pegfilgrastim (10 mg / mL) was diluted with 7.0 μL of DPBS 1790.

[0123] Preparation of a 620.0 μg / kg pegfilgrastim solution for subcutaneous administration: A stock solution of pegfilgrastim (10 mg / mL) was diluted with 17442 μL of DPBS.

[0124] Pegfilgrastim solution for subcutaneous administration was diluted with DPBS to a final dose concentration of 2 mL / kg.

[0125] Manufacturing of neutropenia-inducing drug solutions In rats, docetaxel / cyclophosphamide was administered at a 1 / 3 human equivalent dose (docetaxel 4 mg / kg and CPA 32 mg / kg) ("TC") to induce neutropenia.

[0126] Preparation of 32 mg / kg cyclophosphamide solution for subcutaneous administration: 2560.0 g of cyclophosphamide powder (CPA, Sigma, USA) was diluted with 80,000 μL of distilled water (DW) (Daihan, South Korea).

[0127] Preparation of 4 mg / kg docetaxel solution for subcutaneous administration: 29070.0 μL of Docetaxel inj. (Hanmi Pharmaceuticals, South Korea) (42.68 mg / mL) was diluted with 30930.0 μL of commercially available formulation buffer (FB) (distilled water (DW) with ethanol 127.4 mg / mL).

[0128] Docetaxel and cyclophosphamide solutions for subcutaneous administration were diluted in FB to a final dose concentration of 1 mL / kg. HM10460A and pegfilgrastim were diluted in DPBS to a final dose concentration of 2 mL / kg.

[0129] (ii) Method Testing system

[0130] [Table 4]

[0131] Animal management and identification

[0132] [Table 5]

[0133] Animal husbandry

[0134] [Table 6]

[0135] Dosage administration

[0136] [Table 7]

[0137] Group design and dose levels

[0138] [Table 8]

[0139] (iii) Observation and measurement body weight To calculate the appropriate volume dose, body weight was measured twice, on day -1 and day 0, before administration of TC and the test substance.

[0140] ANC Profile Blood from all animals was collected from the jugular vein 1 day prior to chemotherapy and analyzed for neutrophil count (NEUT#). This neutrophil count was used as the NEUT on day 0 prior to administration, and grouping was based on the NEUT on day 0. In addition, blood was collected once daily using a 26G 1mL syringe at 6 hours on day 0 after administration of the test substance, and for 8 days thereafter. A total of 0.2mL of blood was collected and placed in an automated hematological cell analyzer, Sysmex, XN1000-V (Sysmex corp., Japan) to confirm the ANC. Although the ANC is generally calculated from total WBC x (%Segs + %Bands), the ANC can also be calculated using the Sysmex system because the amount of neutrophils measured by the Sysmex system already includes neutrophil band types in the data.

[0141] Duration of the neutropenia profile The primary endpoint for this study was determined by the duration of neutropenia ("DN"), which was determined based on a cutoff value for neutrophil levels calculated using a normal vehicle (average overall neutrophil level).

[0142] (iv) Results ANC Profile The time course of neutrophil counts is illustrated in Figure 3. Neutrophil counts at 1 / 6 of the clinical dose (pegfilgrastim 103.3 μg / kg and HM10460A 61.8 μg / kg) peaked at 8 days and 5-6 days, respectively, after the start of drug administration, without any difference between therapies. Furthermore, neutrophil counts at the clinical dose (pegfilgrastim 620 μg / kg and HM10460A 372 μg / kg) peaked at 5-8 days and 6 days, respectively, after the start of drug administration for pegfilgrastim and HM10460A. In addition, the peak neutrophil count for the high dose of HM10460A (496 μg / kg) occurred between 6 and 7 days, without any change in administration method, across all time periods.

[0143] DN Profile The DN values ​​for HM10460A and pegfilgrastim administered 24 hours after chemotherapy at 1 / 6 clinical doses (HM10460A 61.8 μg / kg and pegfilgrastim 103.3 μg / kg) were determined to be 0.2 days and 1.8 days, respectively (Table 9). Shorter intervals between chemotherapy and the administration of the test substance (5 hours, 2 hours, and simultaneous administration) increased the DN of pegfilgrastim to 2.4 days. In contrast, only a slight increase was observed for HM10460A, at 0.6 days.

[0144] When administered at clinical doses (HM10460A 372 μg / kg and pegfilgrastim 620 μg / kg), the drug interactions (DNs) for HM10460A and pegfilgrastim administered 24 hours after chemotherapy were observed to be 0 and 0.2 days, respectively (Table 10). As the interval between chemotherapy and the administration of the test substance shortened (5 hours, 2 hours, and simultaneous administration), the DN for pegfilgrastim increased to 1.4 days. On the other hand, when HM10460A was administered, the DN increased only slightly to 0.6 days, as observed at 1 / 6 clinical dose.

[0145] High-dose HM10460A (496 μg / kg) showed similar profiles (0.2 days) regardless of administration time, except for the D0+2h dosage (Table 11).

[0146] [Table 9]

[0147] [Table 10]

[0148] [Table 11]

[0149] Example 3: Administration of efrapegrastim 0.5 hours after docetaxel / cyclophosphamide-induced neutropenia in a human. In patients with early-stage breast cancer, docetaxel 75 mg / m² is administered as part of institutional standard treatment ("SOC").2 IV, cyclophosphamide 600 mg / m2. 0.5 hours (±5 minutes) after the completion of the IV infusion, a fixed dose of efrapegrastim 13.2 mg / 0.6 mL (3.6 mg G-CSF) was administered subcutaneously.

[0150] Example 4: Administration of efrapegrastim 3 hours after docetaxel / cyclophosphamide-induced neutropenia in a human. In patients with early-stage breast cancer, a fixed dose of efrapegrastim 13.2 mg / 0.6 mL (3.6 mg G-CSF) was administered subcutaneously 3 hours (±15 minutes) after the completion of administration of docetaxel 75 mg / m2 IV and cyclophosphamide 600 mg / m2 IV (infusion time determined by institutional SOC).

[0151] Example 5: Administration of efrapegrastim 5 hours after docetaxel / cyclophosphamide-induced neutropenia in a human. In patients with early-stage breast cancer, a fixed dose of efrapegrastim 13.2 mg / 0.6 mL (3.6 mg G-CSF) was administered subcutaneously 5 hours (±15 minutes) after the completion of administration of docetaxel 75 mg / m2 IV and cyclophosphamide 600 mg / m2 IV (infusion time determined by institutional SOC).

[0152] Example 6: A study on the duration of severe neutropenia in breast cancer patients receiving docetaxel and cyclophosphamide, including on the same day as efrapegrastim administration and after various administration schedules. Grade 4 neutropenia (absolute neutrophil count (ANC) <0.5×10 9 The duration of the effect ( / L) was evaluated after one treatment cycle.

[0153] Furthermore, the following was evaluated:

[0154] ●Patients with grade 4 neutropenia in treatment cycle 1 (ANC<0.5×10 9 Ratio of / L) ● In treatment cycle 1, ANC ≥ 1.5 × 10 in severe neutropenia 9 Time to recover / L ● Incidence of Grade III Febrile Neutropenia in Treatment Cycle 1 (ANC < 1.0 × 10) 9 / L) and 1) Single temperature > 38.3°C (101.0°F) or 2) One of the following temperatures for more than one hour: ≥38.0°C (100.4°F) ●Pharmacokinetics (PK) of efrapegrastim in treatment cycle 1 ● During one treatment cycle, the incidence of neutropenic complications in patients, including the use of anti-infective agents due to neutropenia and hospitalization. ●Safety of Efrapegrastim Therapeutic Use ●Peripheral blood CD34 + number Using a fixed dose of 13.2 mg / 0.6 mL (3.6 mg G-CSF), efrapegrastim was administered subcutaneously (SC) at various time intervals to patients with early-stage breast cancer after administration of docetaxel and cyclophosphamide (TC).

[0155] Treatment cycle 1 On day 1 of cycle 1, after TC administration, and after the completion of TC administration, a fixed dose of efrapegrastim was administered at one of the following time points: 0.5 hours (±5 minutes), 3 hours (±15 minutes), and 5 hours (±5 minutes).

[0156] Prior to TC administration, patients may receive prophylactic medication as part of institutional standard care (SOC). On day 1 of each treatment cycle, intravenous (IV) administration of TC is as follows: ● Docetaxel 75 mg / m2 IV, infusion time determined by the institution's State of Care (SOC); ● Cyclophosphamide 600 mg / m2 IV, infusion time determined by the institution's State of Care (SOC); ●Dose changes for docetaxel and cyclophosphamide were not permitted during Cycle 1. Up to 45 patients were enrolled in the study and randomly assigned to one of the three efrapegrastim administration schedules listed above, using a 1:1:1 ratio.

[0157] Blood for CBC (complete blood count) and pharmacokinetic (PK) analysis was collected on the day before TC administration, and at 1 hour (±15 minutes), 3 hours (±15 minutes), 6 hours (±15 minutes), 8 hours (±15 minutes), 24 hours (±2 hours), 48 hours (±2 hours), 72 hours (±2 hours), 144 hours (7 days ±1 day), and 192 hours (9 days ±1 day) after pegfilgrastim administration, and on the day before TC administration (day 22) of cycle 2. CBC analysis was performed in the clinical laboratory.

[0158] Additional CBC samples Only in treatment cycle 1, CBC samples were collected daily from day 4 to day 10. On day 10, if the ANC was ≤ 1.0×10 9 / mL, CBC samples were collected daily until the ANC became ≥ 1.5×10 9 / mL.

[0159] Peripheral blood CD34 in cycle 1 + Peripheral blood CD34 + count samples were collected from day 2 to day 10.

[0160] Cycle 1 Safety Visit Treatment cycle 2: On day 1 (day 22), all required assessments / evaluations were performed prior to TC administration related to treatment cycle 2.

[0161] Intermediate safety evaluation In each pegfilgrastim dosing schedule, when the first 3 patients completed treatment cycle 1 of the study, safety assessments were performed (total 9 patients). Safety assessments included adverse events (AEs), ANC and white blood cell (WBC) counts, duration of severe neutropenia (DSN), and neutropenia complications (hospitalization due to neutropenia, febrile neutropenia, use of anti-infective agents).

[0162] In each efrapegrastim administration schedule, after completing safety evaluations for the first three patients, if no safety findings were found in any of the three efrapegrastim administration schedules, the patient was randomly enrolled in another efrapegrastim administration schedule. If the safety review determined that one or more efrapegrastim administration schedules had to be discontinued, all newly enrolled patients were further randomly assigned to subsequent efrapegrastim administration schedules.

[0163] Suspension rules Safety was evaluated in the first three patients at each efrapegrastim administration date during one treatment cycle. Additional enrollment for the efrapegrastim administration date was discontinued if any of the following criteria were met.

[0164] 1) Two out of three patients reported febrile neutropenia and / or all efrapegrastim-related grade IV adverse events during treatment cycle 1. 2) Two out of three patients reported grade 4 neutropenia, with DSN > 2 days. Safety was continuously monitored. If a total of three or more patients (cumulative across the cohort) developed febrile neutropenia (FN) after interim safety monitoring, enrollment in the cohort was discontinued.

[0165] Cycle 2~4 Efrapegrastim 13.2 mg / 0.6 mL (3.6 mg G-CSF) was administered within 24 hours after the completion of TC administration in all efrapegrastim administration schedules. Patients required ANC ≥ 1.5 × 10 to initiate each subsequent chemotherapy cycle. 9 / L and platelet count ≥ 100 × 10 9 The patient must have / L. For safety, patients were tracked. Each cycle was 21 days.

[0166] In treatment cycles 2-4, blood samples for CBC were collected before chemotherapy and on day 1 of each treatment cycle, based on the status of care (SOC) per cycle. The CBC was prepared at the end-of-study visit 35(±5) days after the last dose of the study treatment (TC or efrapegrastim).

[0167] Research period Screening period: up to 30 days Treatment period: Up to 4 treatment cycles (21 days per treatment cycle) Safety follow-up visit for treatment cycle 1: In treatment cycle 2, 1 day (22 days) before TC administration. End of research visit: 35 (±5) days after the last administration of the research treatment (TC or efrapegrastim). Inclusion criteria Patients must have the willingness and ability to provide written consent, adhere to the efrapegrastim administration schedule, blood collection schedule, and fulfill all other research requirements.

[0168] Patients must receive a new diagnosis for histologically confirmed early-stage breast cancer (ESBC), which is limited to surgically treatable stage I-IIIA breast cancer.

[0169] The patient must be a candidate for azvant TC chemotherapy or neoazvant TC chemotherapy.

[0170] The patient (male or female) must be 18 years of age or older.

[0171] The patient must have adequate hematological, renal, and hepatic function as defined below.

[0172] ●ANC≧1.5×10 9 / L ● Platelet count ≥ 100 × 10 9 / L ●Hemoglobin ≥ 10 g / dL ●Calculated creatinine clearance rate > 50 mL / min ●Total bilirubin ≤ 1.5 mg / dL ● Aspartate aminotransferase (AST) / serum glutamate oxaloacetate transaminase (SGOT) and alanine aminotransferase (ALT) / serum glutamate pyruvate transaminase (SGPT) ≤ 2.5 × ULN and alkaline phosphatase ≤ 2.0 × ULN The patient must have an ECOG (eastern cooperative oncology group) performance status of ≤ 2.

[0173] Efrapegrastim is supplied in a pre-filled, sterile, single-use syringe at a dose of 13.2 mg / 0.6 mL (3.6 mg G-CSF). Dosage changes of efrapegrastim are not permitted.

[0174] evaluation In treatment cycle 1, grade 4 neutropenia (ANC<0.5x10 9 The duration of / L was evaluated.

[0175] In treatment cycle 1, grade 4 neutropenia (ANC<0.5×10 9 The proportion of patients with / L status was evaluated.

[0176] In treatment cycle 1, severe neutropenia was observed with an ANC of ≥ 1.5 × 10 9 We evaluated the time it takes to recover to / L.

[0177] In treatment cycle 1, grade 3 febrile neutropenia (ANC < 1.0 × 10) 9 The incidence rate of / L), and We evaluated either a single temperature ≥ 38.3°C (101.0°F) or a sustained temperature ≥ 38.0°C (100.4°F) for one hour or longer.

[0178] The pharmacokinetics (PK) of efrapegrastim during treatment cycle 1 were evaluated.

[0179] During treatment cycle 1, the incidence of neutropenic complications in patients, including the use of anti-infective agents due to neutropenia and hospitalization, was evaluated.

[0180] Peripheral blood CD34 + The number was evaluated.

[0181] Pharmacokinetic evaluation Each patient received a fixed dose of efrapegrastim after starting chemotherapy on day 1, according to their respective efrapegrastim administration schedule. Blood samples for pharmacokinetic measurements and CBC were collected according to the following schedule.

[0182] Day 1 of Cycle 1 Before administration (before TC administration) 1, 3, 6, and 8 hours (±15 minutes) from the time of efrapegrastim administration. On day 1, the administration times for efrapegrastim were 24, 48, and 72 (±2 hours). From the time of efrapegrastim administration on day 1, 144 hours (day 7 ± 1 day) and 192 hours (day 9 ± 1 day). Day 1 of Cycle 2 Before TC administration Additional CBC samples In cycle 1 only, CBC was also collected daily from day 4 to day 10. On day 10, ANC was ≤ 1.0 × 10 9 If / L, then ANC is ≥ 1.5 × 10 9 CBC was collected daily until the level reached / L.

[0183] Peripheral blood CD34+ count in cycle 1 Peripheral blood CD34 + The number was collected daily from the 2nd to the 10th.

[0184] Safety evaluation Safety throughout the study was assessed through reported / induced adverse events, laboratory evaluations, and physical examinations.

[0185] Integration by reference This application references a variety of issued patents, published patent applications, journal articles, and other publications, all of which are hereby integrated as references. In the event of any conflict between the integrated references and this specification, the specification shall prevail. Any specific examples of the subject matter of this disclosure that belong to the prior art are expressly excluded from one or more claims. Such examples are considered to be well known to those skilled in the art and are therefore excluded even if not expressly indicated herein. Any specific examples of the subject matter of this disclosure may be excluded from any claim for any reason, regardless of whether or not they are related to the existence of the prior art.

[0186] Equal portions The present invention may be embodied in other specific forms without deviating from its spirit or essential features. Therefore, the aforementioned examples should be considered illustrative in all respects rather than limiting the invention described herein. Accordingly, the scope of the invention is expressed by the claims rather than by the foregoing description, and all modifications within the meaning and scope of the equivalents of the claims are intended to be included herein.

[0187] [Sequence Listing Free Text] Sequence ID: 1 TPLGP ASSLP QSFLL KCLEQ VRKIQ GDGAA LQEKL A TYKLC HPEEL VLLGH SLGIP WAPLS SCPSQ ALQLA GCLSQ LHSGL FLYQG LLQAL EGISP ELGPT LDTLQ LDVAD FATTI WQQME ELGMA PALQP TQGAM PAFAS AFQRR AGGVL VASHL QSFLE VSYRV LRHLA QP Sequence ID: 2 TPLGP ASSLP QSFLL KSLEQ VRKIQ GDGAA LQEKL CATYK LCHPE ELVLL GHSLG IPWAP LSSCSQALQ LAGCL SQLHS GLFLY QGLLQ ALEGI SPELG PTLDT LQLDV ADFAT TIWQQ MEELG MAPAL QPTQG AMPAF ASAFQ RRAGG VLVAS HLQSF LEVSY RVLRH LAQP Sequence ID: 3 PSCPAPEPFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLGK Sequence ID: 4 PSCPAPEPFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLLSLGK

Claims

1. A pharmaceutical composition comprising efrapegrastim for use in patients requiring treatment, for increasing the absolute neutrophil count, or for treating or preventing a condition characterized by weakened leukocytosis, selected from the group consisting of chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, neutropenia, and neutrophil mobilization impairment, A pharmaceutical composition in which efrapegrastim is administered in an effective amount simultaneously with the administration of a chemotherapy agent or radiotherapy within 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

2. The pharmaceutical composition according to claim 1, wherein the condition is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.

3. The pharmaceutical composition according to claim 2, wherein the efrapegrastim shortens the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia in patients requiring such treatment.

4. The pharmaceutical composition according to claim 1, wherein an effective amount of efrapegrastim is administered to the patient on the same day that the patient is administered a chemotherapy agent or undergoes radiotherapy.

5. An effective dose of efrapegrastim was administered to the patient at a dose of 0.5 x 10⁻⁶. ９ A pharmaceutical composition according to any one of claims 1 to 4, which shortens the duration of absolute neutrophil counts less than / L to less than 6 hours, less than 12 hours, or less than 24 hours.

6. An effective dose of efrapegrastim increased the patient's absolute neutrophil count by 0.5 x 10⁻¹⁴. ９ The pharmaceutical composition according to claim 5, which prevents the concentration from falling below / L.

7. An effective dose of efrapegrastim increased the patient's absolute neutrophil count by 0.5 x 10⁻⁶. ９ Within 4 days, 7 days, or 10 days from the first occurrence of less than / L, 1.5 x 10 ９ A pharmaceutical composition according to any one of claims 1 to 4, wherein the amount increases to / L or more.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein an effective amount of efrapegrastim is administered simultaneously with a chemotherapeutic agent or radiotherapy.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein an effective dose of efrapegrastim is administered within 0.5 hours, within 3 hours, or within 5 hours after administration of a chemotherapy agent or radiotherapy.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

11. The pharmaceutical composition according to claim 10, wherein the myelosuppressive chemotherapeutic agent is selected from the group consisting of docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the patient is receiving chemotherapy or radiotherapy to treat a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumor and non-Hodgkin lymphoma.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein administering an effective amount of efrapegrastim includes administering 2 mg to 18 mg of efrapegrastim parenterally.

14. The pharmaceutical composition according to claim 13, wherein administering an effective amount of efrapegrastim comprises administering 13.2 mg of efrapegrastim parenterally.

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