Methods of mitigating lung injury in combination with radiation exposure and / or radiation or radiomimetic therapy

Abstract

Methods and kits are described for alleviating lung damage in subjects who have been treated with targeted radiotherapy or otherwise exposed to radiation.In particular, an effective amount of a thrombopoietin mimetic (e.g., RWJ-800088 or romiplostim) is used to alleviate radiation-induced lung damage.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 344,285, filed May 20, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listing) This application contains a sequence listing, which has been submitted electronically via PatentCenter as an XML format sequence listing with the file name "004852-213WO1 Sequence Listing.xml" created on May 15, 2023, and has a size of 13,348 bytes. The sequence listing submitted via PatentCenter is a part of this specification and is incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates to a method and a kit for alleviating lung injury in a subject in need thereof. In particular, the present invention relates to a method comprising administering to a subject an effective amount of a thrombopoietin (TPO) mimetic, and a kit comprising a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharma- ceutically acceptable carrier. TPO can be administered alone or in combination with other active agents to promote beneficial effects. [Background technology]

[0004] Radiation therapy is an essential strategy for cancer treatment. Approximately 60-70% of patients with malignant tumors undergo radiation therapy, or radiotherapy, which can cure many tumors, and the cure rate of radiation therapy for early tongue cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, and cervical cancer is about 90% (Hogle WP, Semin Oncol Nurs, 22(4):212-220, (2006)). However, when killing tumors, radiation therapy can cause off-target effects on normal tissues (including non-cancerous tissues inside radiation shields and distant tissues such as bone marrow), which limits the effectiveness of radiation therapy. Measures have been attempted to improve the accuracy of radiation therapy, including updating the equipment, using radiosensitizers in combination, and combining radiation therapy with chemotherapy, but the results remain unsatisfactory. Normal tissues cannot tolerate radiation-induced toxicity, which prevents the use of higher doses of radiation therapy in clinical applications. Radiation-induced lung injury is the most common clinical complication after radiotherapy. In severe cases, it can endanger the life of patients, especially those with lung, esophageal, breast and mediastinal tumors.

[0005] Radiation-induced lung injury (RILI) includes radiation pneumonitis in the early stage and radiation pulmonary fibrosis in the later stage. Such damage not only impairs tumor control but also seriously affects the quality of life of patients. Respiratory failure is one of the major causes of death in RILI. In addition, local hypoxia, inflammatory response, angiogenesis, changes in the local microenvironment, and immunosuppression caused by RILI promote tumor recurrence, invasion, and metastasis (van den Brenk, HA et al, Br J Radiol, 47(558):p.332-336,(1974)). Therefore, it is particularly important to manage RILI in clinics. The lack of effective drugs leads to the empirical use of high doses of glucocorticoids and anti-inflammatory drugs. These treatments often not only fail to improve the therapeutic effect of radiation therapy, but also cause many side effects, such as immunosuppression. Furthermore, the immunosuppressive microenvironment of the lung increases the risk of tumor recurrence and metastasis. Therefore, there is a need to identify drugs that can prevent and / or treat RILI.

[0006] It is generally accepted that the tumor-killing effect of radiation therapy is due to radiation-induced DNA damage and the production of free radicals in tumor cells (Muruve DA et al, Nature, 452(7183):103-107, (2008)). Subsequently, DNA fragments and reactive oxygen species (ROS) induce inflammation, during which activated macrophages synthesize and secrete large amounts of inflammatory cytokines, such as TNF-α, IL-1β, IL-8, etc. High levels of TNF-α and fibronectin together can cause early acute pneumonia, which can also promote the proliferation of fibroblasts and, at the same time, stimulate fibroblasts to secrete excess collagen. Radiation-induced oxidative damage in pulmonary capillary endothelial cells includes DNA breaks, cell death and increased reactive oxygen species / reactive nitrogen species (ROS / RNS), leading to accumulation, transcription and upregulated activity of hypoxia-inducible factor (HTF) in tumor cells (Lerman, OZ, et al., Blood, 116(18):3669-3676, (2010)). Under hypoxia, vascular endothelial cells (ECs) produce large amounts of the chemokine stromal cell-derived factor (SDF), which binds to the chemokine receptor CXCR4 and recruits BMDCs to inflammatory lesions (Du, R., et al., Cancer Cell, 13(3):206-220, (2008)). Studies have shown that bone marrow-derived cells (BMDCs) are important for the formation and growth of tumor neovascularization. The changes in the microenvironment provide favorable conditions for tumor recurrence and metastasis.

[0007] Although existing drugs for the prevention and treatment of RILI have some protective effects, in many cases they do not work well and / or may inhibit the therapeutic effectiveness of radiation therapy. Therefore, there is a need for drugs that can prevent RILI without affecting the effectiveness of radiation therapy.

[0008] Due to the radiosensitivity of the lungs, considerable efforts have been made in the clinic to limit the volume of normal lung tissue exposed to radiation when treating lung tumors in order to adequately treat patients' cancers, but it may be impossible to avoid exposure of normal tissue to radiation and long-term lung toxicity may result. Furthermore, cases of accidental radiation exposure have been described, including patients who received unintentional chest irradiation during treatment of breast, lung and other cancers, and in some cases subsequent pulmonary complications led to the death of the patient. Such results highlight the important role played by the lung in both early and late radiation lethality. Thus, in addition to countermeasures against classically recognized components of acute radiation syndrome (ARS), such as neutropenia and thrombocytopenia, there is a growing recognition that agents that specifically target the lung response are also needed, especially in the context of total body irradiation (TBI), as may be expected as part of radiation incidence.

[0009] Radiation-induced pulmonary syndrome is a delayed fatal event from accidental or deliberate exposure to radiation in the case of nuclear accidents or terrorism. In the case of nuclear accidents or deliberate attacks resulting in mass exposure to ionizing radiation, victims need to be triaged according to the severity of acute radiation sickness. Radiation-induced bone marrow syndrome and gastrointestinal (GI) syndrome occur at lower doses of radiation and develop earlier than radiation-induced pulmonary syndrome. Although acute lung injury is not an early event compared to radiation-induced gastrointestinal and hematological disorders, successful treatment of gastrointestinal and hematological syndrome may not completely save the patient, since death from respiratory distress at later time points is always a problem. Furthermore, many victims at risk of developing chronic injury do not show symptoms for months to years after exposure. It is therefore necessary to develop effective treatment strategies for the development of symptomatic injury.

[0010] Two stages of radiation lung injury are described. Acute radiation pulmonary inflammation (pneumonitis) can occur several weeks to six months after radiation exposure. This stage can be life threatening if large volumes of the lung are affected. In late radiation-induced lung injury, which occurs several months to years after radiation exposure, the number of inflammatory cells decreases and collagen deposition occurs, leading to irreversible pulmonary fibrosis.

[0011] The present invention addresses the need to prevent and / or treat RILI. Summary of the Invention

[0012] It has now been discovered that thrombopoietin (TPO) mimetics can alleviate lung injury in subjects in need.For example, it has been found that TPO mimetics have a significant alleviating effect on targeted radiation-induced lung injury (RILI).It is expected that TPO mimetics can have a significant effect when used alone or together with the administration of other active agents.

[0013] Thrombopoietin (TPO) is a growth factor synthesized and secreted by the liver. In addition to acting as a growth factor that stimulates megakaryocyte proliferation and differentiation via the thrombopoietin receptor (TPO-R or c-Mpl), recombinant human TPO (rhTPO) has been shown to promote platelet activation, and hepatic endothelial cell proliferation and migration in vitro (Cardier et al., Blood, 1998, 91:923-929).

[0014] Accordingly, in one general aspect, the present application relates to a method of alleviating RILI in a subject in need thereof, the method comprising administering to the subject an effective amount of a thrombopoietin (TPO) mimetic, preferably, the TPO mimetic comprises the amino acid sequence of SEQ ID NO:1, and more preferably, the TPO mimetic is RWJ-800088 or romiplostim.

[0015] In certain embodiments, a TPO mimetic is administered to a subject in combination with another active agent. The TPO mimetic can be administered to a subject before, after, or simultaneously with the other active agent.

[0016] In certain embodiments, the subject in need of the treatment of the present application is a subject treated with radiation therapy, preferably targeted radiation therapy that may result in RILI, such as targeted radiation therapy for lung disease, pre-irradiation for bone marrow transplantation, or targeted radiation therapy for esophageal cancer. The TPO mimetic may be administered to the subject before, after, or simultaneously with radiation therapy. In certain embodiments, the TPO mimetic is administered to the subject at least about 7 days before to about 7 days after, preferably at least about 24 hours before to at least about 24 hours after, the subject is administered a dose of radiation. In certain embodiments, the TPO mimetic is administered to the subject 24 hours before to at least about 24 hours after the subject is administered a dose of radiation. In some embodiments, the TPO mimetic is administered 24 to 2 hours before or after the radiation administration. In other embodiments, the TPO mimetic is administered 1 minute to 2 hours before or after the radiation administration. In some embodiments, the TPOm is administered 2 to 24 hours after the radiation administration.

[0017] In certain embodiments, subjects are treated with targeted radiation, preferably to the lungs, at a dose of 5-70 Gray (Gy) in 1-10 fractions.

[0018] In certain embodiments, the effective amount of the TPO mimetic for humans is about 1 to about 10 μg / kg, more preferably about 3 μg / kg to about 5 μg / kg of the subject's body weight. In preferred embodiments, the effective amount of the TPO mimetic is about 1 μg / kg of the subject's body weight. In certain preferred embodiments, the effective amount of the TPO mimetic is about 3 μg / kg of the subject's body weight when administered subcutaneously or intravenously. The effective dose for humans may be determined after determining the effective dose for mice by dividing by 100 based on observed differences in potency between species, for example, where a 3 mg / kg dose in mice and a 0.003 mg / kg dose in humans both result in an approximately 3-fold transient increase in platelets.

[0019] In certain embodiments, an effective amount of a TPO mimetic is administered to a subject by intravenous, intramuscular, intradermal, or subcutaneous injection. In a preferred embodiment, the TPO mimetic is administered by subcutaneous injection.

[0020] In another general aspect, the present application relates to a kit for alleviating RILI in a subject in need thereof. The kit comprises a pharmaceutical composition for alleviating RILI, comprising an effective amount of a TPO mimetic and a pharma- ceutically acceptable carrier. Optionally, the kit further comprises administration with at least one additional therapeutic agent. Optionally, the kit further comprises a device or tool for administering the TPO mimetic to a subject. Preferably, the kit comprises a TPO mimetic having the amino acid sequence of SEQ ID NO: 1, more preferably a TPO mimetic of RWJ-800088 or romiplostim.

[0021] In another general aspect, the application relates to a method for treating radiation pneumonitis in a subject in need thereof, the method comprising administering to the subject a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO:1 in an effective amount for treating radiation pneumonitis, preferably, the TPO mimetic is RWJ-800088 or romiplostim.

[0022] In certain embodiments, the subject is treated with targeted radiation therapy for a pulmonary disease, or the subject is treated with preparative irradiation for a bone marrow transplant.

[0023] In certain embodiments, subjects are treated with targeted radiation therapy at a dose of 5-70 Gray (Gy) in 1-10 fractions.

[0024] In certain embodiments, the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

[0025] In certain embodiments, the TPO mimetic is administered to the subject 7 days to 7 days after, 2 days to 2 days after, 24 hours to 24 hours after, preferably about 2 hours to 24 hours before or after the subject receives a dose of radiation.

[0026] In certain embodiments, the TPO mimetic is administered to the subject 2 hours to 36 hours, or 1 day, before the subject receives a dose of radiation.

[0027] In certain embodiments, the TPO mimetic is administered to the subject by either intravenous, intramuscular, intradermal, or subcutaneous injection.

[0028] In certain embodiments, the TPO mimetic is administered to the subject by subcutaneous injection.

[0029] In certain embodiments, administration of an effective amount of a TPO mimetic results in at least one of a decrease in elevation of chemokine KC or a decrease in elevation of alveolar neutrophil infiltration in the subject.

[0030] In a particular embodiment, the TPO mimetic is RWJ-800088. [Brief description of the drawings]

[0031] The above summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] Figure 1 shows the effect of TPOm pretreatment (1 day prior to irradiation at 1 mg / kg body weight by subcutaneous injection) on chemokine (IL-1β, IL-8, TNFα, TGFβ, MCP1, MCP2, KC and MIP2) mRNA expression in the lungs of mice (C57BL / 6J males, 9-12 weeks old) 14 days after whole chest irradiation (WTI) at 16 Gy delivered by X-ray irradiator. Lung data are presented as fold change in chemokine expression normalized to Hprt1 expression (n=5 for naive, n=5 for vehicle (phosphate buffered saline) and TPOm pretreated). For comparison, expression in the naive group is designated as 1. Data are expressed as mean ± SE. *P<0.05 vs. vehicle-treated mice. [Figure 2A]Figure 2 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on immune cell infiltration and permeability in mouse lungs 14 days after WTI. Figure 2A shows mouse lung sections stained with hematoxylin and eosin (H&E) to visualize cell density and immune infiltration. 200X total magnification. [Figure 2B] Figure 2B shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on immune cell infiltration and permeability in mouse lungs 14 days after WTI. Figure 2B shows the amount of protein in bronchoalveolar lavage measured by bicinchoninic acid assay (BCA) (n=3 for naive, n=5 for vehicle and TPOm pretreatment). *P<0.05 vs. vehicle-treated mice. [Figure 3A] Figure 3 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on collagen deposition in mouse lungs 7 months after WTI. Figure 3A shows mouse lung sections stained with trichrome blue and visualized for collagen deposition. 200X total magnification. [Figure 3B] Figure 3B shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on collagen deposition in the lungs of mice 7 months after WTI. Figure 3B shows the percentage of tissue section area positive for collagen (n=3 for naive, n=5 for vehicle, and n=5 for TPOm pretreated). *P<0.05 vs. vehicle-treated mice. [Figure 4A]Figure 4 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on senescence gene mRNA and protein expression in mouse lungs 7 months after WTI. mRNA and protein levels were assessed by qPCR and Western blot, respectively. Figure 4A shows data presented as fold change in p21 and p16 mRNA expression normalized to Hprt1 expression (n=3 for naive, n=5 for vehicle and n=4 for TPOm pretreated). Data are expressed as mean ± SE. *P<0.05 vs. vehicle-treated mice. [Figure 4B] Figure 4 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on senescence gene mRNA and protein expression in mouse lungs 7 months after WTI. mRNA and protein levels were assessed by qPCR and Western blot, respectively. Figure 4B shows a representative blot of lung protein lysates. [Figure 4C] Figure 4 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on senescence gene mRNA and protein expression in mouse lungs 7 months after WTI. mRNA and protein levels were assessed by qPCR and Western blot, respectively. Figure 4C shows the plotted densitometry values ​​of p16 and p21 proteins (normalized to β-actin levels) (n=3 for naive, vehicle and TPOm pretreatment). For comparison, expression in the naive group is designated as 1. [Figure 5A] Figure 5 shows the effect of TPOm pretreatment (1 day before radiation at 0.3 mg / kg body weight by subcutaneous injection) on lung density in mice 7 months after WTI. Lung density was imaged by micro-CT and measured by micro-CT analysis software. Figure 5A shows representative micro-CT cross-sectional images for each treatment. [Figure 5B]Figure 5 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on lung density in mice 7 months after WTI. Lung density was imaged by microCT and measured by microCT analysis software. Figures 5B and 5C show longitudinal quantification of left lung density at 3, 5 and 7 months. (n=3 for naive, n=5 for vehicle and n=4 for TPOm pretreatment). *P<0.05 vs vehicle-treated mice. [Figure 6] Figure 1 shows the effect of TPOm pretreatment on 250-day survival of mice with sublethal X-ray irradiation (18 Gy) via WTI. Mice were given vehicle or TPOm injections (1 mg / kg) 1 day prior to radiation (1X) or 1 day prior to radiation and 7 days after radiation (2X) (n=10 for irradiated group, n=5 for naive). *P<0.05 vs. vehicle-treated mice. [Figure 7A] Figure 7 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on neutrophil infiltration in mice measured 7 months after WTI. Figure 7A shows quantification of chemokine KC protein in lung lysates measured by ELISA (n=5). [Figure 7B] Figure 7B shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on neutrophil infiltration in mice measured 7 months after WTI. Figure 7B shows a representative lung section from a mouse stained with myeloperoxidase (MPO). 400X total magnification. [Figure 7C] Figure 7 shows the effect of TPOm pretreatment (1 day prior to radiation at 0.3 mg / kg body weight by subcutaneous injection) on neutrophil infiltration in mice measured 7 months after WTI. Figure 7C shows quantification of MPO-positive infiltrates in the lungs measured by ELISA (n=5). Data are presented as mean±SE. *P<0.05, ***P<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure is based, at least in part, on the identification of thrombopoietin (TPO) mimetics as therapeutic agents for mitigating radiation-induced lung injury in a subject in need thereof. The TPO mimetics may be formulated and administered to a subject who has been exposed, is being exposed, or will be exposed to radiation to ameliorate radiation-induced lung injury.

[0033] Various publications, articles and patents are cited or described in the "Background" and throughout the specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If not, certain terms used herein have the meanings set forth herein.

[0035] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0036] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the term "about." Thus, numerical values ​​typically include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within the range, unless the context clearly indicates otherwise.

[0037] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0038] As used herein, it will be understood that the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include the stated element or elements, but not to exclude other elements or elements other than those, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or that are inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0039] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0040] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, is inclusive of any listed element or elements, but indicates that no additional element or elements are added to the specified method, structure, or composition.

[0041] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited element or group of elements, optionally including any recited element or group of elements that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0042] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, that is to be treated or has been treated by a method according to an embodiment of the present invention. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0043] The words "right", "left", "lower" and "upper" designate directions in the drawings to which reference is made.

[0044] It should also be understood that the terms "about," "approximately," "generally," "substantially," and the like, used herein when referring to dimensions or features of preferred inventive components, indicate that the described dimensions / features are not precise boundaries or parameters, and do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, and the like).

[0045] As used herein, the term "in combination" refers to the use of multiple therapeutic agents in the context of administering two or more therapeutic agents to a subject. The use of the term "in combination" does not limit the order in which therapies are administered to a subject. For example, a first therapeutic agent (e.g., a composition described herein) can be administered to a subject prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously, or subsequently (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent to a subject.

[0046] The term "RTLI", or "radiation-induced lung injury", as used herein, refers to acute responses during or within the first few weeks of radiation exposure or radiation therapy (RT), or late responses several months after radiation exposure or RT. Examples of radiation-induced lung injury (RILI) can include, but are not limited to, radiation-induced lung inflammation, collagen deposition in the lungs, pleural effusion, and pulmonary fibrosis.

[0047] Radiation therapy The term "TRT", or "targeted radiation therapy", as used herein, refers to therapy that uses ionizing radiation or radiomimetic agents that are preferentially targeted or localized to specific organs or parts of the body. It is commonly used as part of cancer treatment. TRT, such as targeted ionizing radiation therapy, is also sometimes called radiotherapy, radiotherapy, irradiation, or X-ray therapy. There are three main categories of targeted ionizing radiation therapy: external beam radiation therapy (EBRT or XRT), internal radiation therapy, and total body radioisotope therapy. Radiation may be given in several treatments to deliver the same or slightly higher doses, called fractionated radiation therapy. As used herein, the term "radiomimetic agent", or "radiomimetic chemical agent", refers to a chemical agent that produces effects similar to those of ionizing radiation when administered to a subject. Examples of such effects include DNA damage. Examples of radiomimetic chemical agents include, but are not limited to, etoposide, doxorubicin, carboplatin, and bleomycin. Radiomimetic chemical agents as described herein can be administered locally to a subject to allow for targeted application of the agent in a therapeutic fashion.

[0048] External beam radiation therapy (EBRT) uses a machine that directs high energy rays to the tumor from outside the body. Current radiation technology allows for precise delivery of external beam radiation therapy, such as targeted radiation therapy, which uses a computer to create a three-dimensional image of the tumor in order to target the tumor as precisely as possible and give it the highest possible dose of radiation while sparing as much normal tissue as possible. Examples of EBRT include, but are not limited to, stereotactic radiation therapy, image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), helical tomotherapy, proton beam radiation therapy, and intraoperative radiation therapy (IORT). Of these, stereotactic radiation therapy is a special type of external beam radiation therapy. It uses focused radiation beams that target well-defined tumors using highly detailed image scans. There are two types of stereotactic radiation therapy: stereotactic radiosurgery (SRS) is stereotactic radiation therapy for the brain or spinal cord, while stereotactic body radiation therapy (SBRT) refers to more precise targeted radiation therapy for internal organs such as the lungs.

[0049] Internal radiation, also called brachytherapy, involves placing radioactive implants in or near the tumor inside the body. It allows for a higher dose of radiation in a smaller area than may be possible with external radiation therapy. It uses a radioactive source, usually sealed in a small holder called an implant. Different types of implants may be called pellets, seeds, ribbons, wires, needles, capsules, balloons, or tubes. Some such examples of internal radiation are Y-90 SIR-sphere and / or Thera-Sphere.

[0050] Targeted stereotactic radioisotope therapy (SRT) is also called unsealed source radiation therapy. Targeted radiopharmaceuticals are used in SRT to treat certain types of cancers systemically, such as thyroid, bone, and prostate. These drugs are typically linked to targeting entities, such as monoclonal antibodies or cell-specific ligands, and can be administered orally or intravenously, and then travel through the body until they reach the desired target, where the drug accumulates in relatively high concentrations.

[0051] As used herein, "subject treated with targeted radiation therapy" refers to a subject who is undergoing targeted radiation treatment, which may be prior to, following, or concurrent with administration of a TPO mimetic.

[0052] TPO mimetics As used herein, "TPOm", "TPO mimetic", or "thrombopoietin mimetic" refers to a compound, including a peptide, that can bind to and activate the thrombopoietin receptor, or c-mpl. Preferably, in a TPO mimetic useful in the methods of the present application, the peptide that can bind to and activate the thrombopoietin receptor does not have significant homology with thrombopoietin (TPO). The lack of homology with TPO reduces the possibility of the generation of antibodies against endogenous TPO. Examples of such peptides useful in TPO mimetics include, but are not limited to, those described in U.S. Patent Publication Nos. 2003 / 0158116, 2005 / 0137133, 2006 / 0040866, 2006 / 0210542, 2007 / 0148091, 2008 / 0119384, U.S. Patent Nos. 5,869,451, 7,091,311, 7,615,533, 8,227,422, WO 2007 / 021572, WO 2007 / 094781, and WO 2009 / 148954, the entire contents of each of which are incorporated herein by reference. More preferably, in the TPO mimetics useful in the methods of the present application, a peptide capable of binding to and activating the thrombopoietin receptor is covalently linked to a moiety that improves one or more properties of the peptide. As non-limiting examples, the moiety can be a hydrophilic polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polylactic acid, and polyglycolic acid. The moiety can also be a polypeptide, such as an Fc region or albumin.

[0053] US Pat. Nos. 7,576,056 and 7,723,295 to Janssen Pharmaceutica NV disclose the use of TPO compounds to treat patients suffering from thrombocytopenia.

[0054] US Pat. Nos. 8,067,367 and 8,283,313 to Janssen Pharmaceutica NV disclose methods of providing hematopoietic stem cells to a subject comprising administering a TPO compound.

[0055] U.S. Patent No. 7,615,533 to Janssen Pharmaceutica NV discloses a method for preventing the development of anemia following a treatment selected from the group consisting of treatment with a cytotoxic agent, treatment with an antitumor agent, and treatment with radiation, comprising administering to a subject in need thereof an effective amount of a TPO compound.

[0056] U.S. Patent Application Publication No. 20200164039 to Janssen Pharmaceutica NV discloses a method of protecting vascular integrity in a subject exposed to targeted radiation therapy, comprising administering to the subject an effective amount of a TPO compound.

[0057] U.S. Patent Application Publication No. 20200237870, by Janssen Pharmaceutica NV and Rutgers, The State University, discloses a method for alleviating at least one toxic effect of vesicants and caustic gases in a subject in need thereof, comprising administering to the subject an effective amount of a TPO compound.

[0058] U.S. Patent Application Publication No. 20200237871 to Janssen Pharmaceutica NV and Montefiore Medical Center discloses a method for alleviating targeted radiation therapy-induced liver disease in a subject in need thereof, comprising administering to the subject an effective amount of a TPO compound.

[0059] U.S. Patent Application Publication No. 20200237872 to Janssen Pharmaceutica NV discloses methods of mitigating vascular injury, promoting organ and / or hematopoietic recovery, enhancing survival, and / or protecting against organ and hematopoietic injury in human subjects being or having been exposed to radiation.

[0060] Application No. 63 / 261,957 to Janssen Pharmaceutica and Montefiore Medical Center discloses a method for increasing production of at least one of hematopoietic progenitor cells, bone marrow progenitor cells, endothelial progenitor cells and endothelial cells in a non-irradiated subject comprising administering to the subject an effective amount of a TPO compound.

[0061] In a preferred embodiment, a TPO mimetic useful in the methods of the present application comprises a peptide having an amino acid sequence of IEGPTLRQXaaLAARYaa (SEQ ID NO: 1), where Xaa is tryptophan (W) or β-(2-naphthyl)alanine (referred to herein as "2-Nal") and Yaa is alanine (A) or sarcosine (referred to herein as "Sar"). Preferably, the peptide of SEQ ID NO: 1 is covalently linked to PEG or fused to an Fc domain.

[0062] In some embodiments, the TPO mimetic useful in the methods of the present application comprises a peptide of SEQ ID NO: 1 covalently attached to PEG, preferably PEG having an average molecular weight between about 5,000 and about 30,000 daltons. Preferably, the PEG is selected from the group consisting of monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycolamine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES) and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM). PEGylation of the peptide results in reduced clearance of the compound without loss of efficacy. See, e.g., U.S. Patent No. 7,576,056, the entire contents of which are incorporated herein by reference.

[0063] In a preferred embodiment, the TPO mimetic useful in the present invention is RWJ-800088 or a derivative thereof. As used herein, "RWJ-800088" refers to a 29-mer peptide having two identical 14-mers (SEQ ID NOs: 2 and 5) linked by a lysine amide residue, as follows:

[0064] [ka] RWJ-800088 refers to a 29-mer peptide having a methoxypoly(ethylene glycol) (MPEG), or a pharma- ceutically acceptable salt or ester thereof, covalently attached to each N-terminal isoleucine. Thus, RWJ-800088 is composed of two 14 amino acid peptide chains of SEQ ID NO: 1, where Xaa is 2-Nal and Yaa is Sar, linked by a lysine amide residue and each N-terminal isoleucine is linked to a methoxypolyethylene glycol (MPEG) chain. Thus, RWJ-800088 is (MPEG-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-(2-Nal)-Leu-Ala-Ala-Arg-(Sar)). 2 -Lys-NH 2 (SEQ ID NOs: 2 and 5), where (2-Nal) is β-(2-naphthyl)alanine, (Sar) is sarcosine, and MPEG is methoxypoly(ethylene glycol), or a pharma- ceutically acceptable salt or ester thereof. Preferably, MPEG has a molecular weight of about 20,000 daltons, or represents methoxypolyethylene glycol 20000.

[0065] In one embodiment, RWJ-800088 has the molecular structure of formula (I) (SEQ ID NOs: 2 and 5, respectively), or a pharma- ceutically acceptable salt or ester thereof:

[0066] [ka]

[0067] In a preferred embodiment, the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000, and RWJ-800088 has the following full chemical name: Methoxypolyethyleneglycol20000-propionyl-L-isoleucyl-L-glutamyl-glycyl-L-prolyl-L-threonyl-L-leucyl-L-arginyl-L-glutaminyl-L-2-naphthylalanyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-sarkosyl-Ne-(Methoxypolyethyleneglycol20000-propionyl-L-isoleucyl-L-glutamyl-glycyl-L-prolyl-L-threonyl-L-leucyl-L-arginyl-L-glutaminyl-L-2-naphthylalanyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-sarkosyl-)-lysinamide (SEQ ID NOs: 6 and 7), or a pharma-ceutically acceptable salt or ester thereof. The molecular weight of the peptide without PEG is 3,295 daltons, and the molecular weight of the peptide with two 20,000 dalton MPEG chains is approximately 43,295 daltons.

[0068] In some embodiments, the TPO mimetic useful in the method of the present application comprises a peptide of SEQ ID NO: 1 fused to an Fc domain. By fusing the peptide to an Fc domain, the peptide can be stabilized in vivo. See U.S. Patent No. 6,660,843, the entire contents of which are incorporated herein by reference.

[0069] In another preferred embodiment, the TPO mimetic useful in the present invention is romiplostim.As used herein, "romiplostim" refers to a fusion protein having an Fc domain linked to the N-terminal isoleucine of the peptide of SEQ ID NO:1, where Xaa is W and Yaa is A.In particular, romiplostim has the following amino acid sequence: MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGIEGPTLRQWLAARAGG GGGGGGIEGPTLRQWLAARA (SEQ ID NO: 4) It has the thrombopoietin receptor binding domain amino acid sequence IEGPTLRQWLAARA (SEQ ID NO:3).

[0070] Dosage and Administration In the present invention, the inventors have discovered that TPO mimetics have a significant mitigating effect on RILI. Thus, the method of the present invention includes administering an effective amount of a TPO mimetic to a subject in need thereof, thereby achieving one or more beneficial results (e.g., mitigating one or more RILI) in a subject in need thereof (e.g., a subject exposed to radiation or a subject treated with radiation therapy).

[0071] The TPO mimetic may be administered, for example, as an active ingredient of a pharmaceutical composition together with a pharmaceutical carrier or diluent. The TPO mimetic may be administered by oral, pulmonary, parenteral (intramuscular (IM), intraperitoneal (IP), intravenous (IV), or subcutaneous (SC) injection), inhalation (by fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual administration route, and may be formulated in a dosage form suitable for each administration route. Preferably, the TPO mimetic is administered by subcutaneous injection. For example, WO 1993 / 25221 (Bernstein et al.) discloses biodegradable polymer microspheres containing erythropoietin (EPO), which may be administered topically, locally, or systemically by parenteral or enteral administration, preferably by oral administration. WO 1994 / 17784 (Pitt et al.) discloses that EPO can be administered systemically via the pulmonary route and that such delivery provides a comparable level of therapeutic benefit compared to other methods of EPO administration. Similar compositions and methods can be used for administration of the TPO mimetics of the present disclosure.

[0072] The solid dosage form for oral administration includes capsules, tablets, pills, powders and granules.In such solid dosage form, active peptide compound is mixed with at least one pharmaceutically acceptable carrier, for example, sucrose, lactose or starch.Such dosage form can also contain additional substances other than inert diluent, for example, lubricant such as magnesium stearate, as is customary.In the case of capsules, tablets and pills, dosage form can also contain buffering agent.Tablets and pills can be further prepared with enteric coating.

[0073] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. In addition to such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

[0074] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. They can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the composition, by irradiating the composition, or by heating the composition. They can also be prepared using sterile water or some other sterile injectable medium immediately before use.

[0075] The administration of the TPO mimetic is typically intramuscular, subcutaneous, or intravenous. However, other modes of administration, such as dermal, intradermal, or nasal, can also be envisaged. Intramuscular administration of the TPO mimetic can be achieved by using a needle to inject a suspension of the TPO mimetic composition. Alternatives include using a needleless injection device to administer the composition (e.g., using Biojector™), or using a lyophilized powder of the TPO mimetic composition.

[0076] For intravenous, cutaneous or subcutaneous injection, the TPO mimetic composition can be in the form of parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art can prepare suitable solutions well, for example, using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactate Ringer's injection.If necessary, preservatives, stabilizers, buffers, antioxidants and / or other additives can be included.Slow-release preparations can also be used.

[0077] Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active TPO mimetic, excipients such as cocoa butter or suppository wax. Compositions for nasal or sublingual administration are also prepared using standard excipients well known in the art.

[0078] Typically, administration has a therapeutic and / or prophylactic purpose to alleviate radiation-induced lung damage in a subject before, during, or after radiation therapy. In therapeutic applications, the TPO mimetic composition is administered to a subject during or after exposure to radiation therapy, and the TPO mimetic composition is administered in an amount sufficient to cure or at least partially alleviate radiation-induced lung damage. In prophylactic applications, the TPO mimetic composition is administered to a subject susceptible to or at risk of developing RILI before exposure to radiation, and enhances lung function in a subject in need thereof after exposure to radiation. In each of these scenarios, the amount of the TPO mimetic composition depends on the condition and nature of the exposure (e.g., type of radiation therapy, dose and length of exposure), the subject's physical characteristics (e.g., height, weight, disease state, etc.), and the design of the treatment (e.g., TPOm alone or TPOm in combination with another therapeutic agent, etc.).

[0079] A pharma- ceutically acceptable composition containing a TPO mimetic is administered to a subject to increase alleviation of radiation-induced lung injury. An amount of the composition sufficient to alleviate disease is defined as an "effective dose" or "effective amount" of the composition.

[0080] The actual amount administered, and the rate and time course of administration, will depend on the nature and severity of the condition being treated. Treatment prescriptions, such as doses, are within the responsibility of general practitioners and other physicians or veterinarians in a veterinary setting, and typically take into account the category and dose of radiation therapy, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

[0081] The TPO mimetic may be administered in combination with targeted radiation therapy.

[0082] In certain embodiments, the TPO mimetic is administered to the subject 7 days before to 7 days after or 2 days before to 2 days after or simultaneously with the targeted radiotherapy. In certain embodiments, the TPO mimetic is administered to the subject 24 hours before to 24 hours after or simultaneously with the targeted radiotherapy. For example, the TPO mimetic is administered to the subject 24, 20, 16, 12, 8, 4, 2, 1, 0.5, or 0.1 hours (or any range expressed therebetween) before the targeted radiotherapy, or 24, 20, 16, 12, 8, 4, 2, 1, 0.5, or 0.1 hours (or any range expressed therebetween) after the targeted radiotherapy. Preferably, the TPO mimetic is administered to the subject about 24 to about 1 hour (or any range expressed therebetween) before the subject is administered the targeted radiotherapy.

[0083] Given the disclosure of this application and the knowledge in the art, any suitable dose of radiation may be used in the method of this application. Experiments may be performed with mice subjected to IR and TPOm to investigate the nature of radiation damage to the host. Animals from different cohorts may be sacrificed at different time points (1 day, 2 days, 3 days, 1 week, 3 weeks, 6 weeks, and 12 weeks), and lung sections may be stained with H&E for histopathological analysis. BrdU and TUNEL staining may be performed to investigate lung cell proliferation and apoptosis, respectively.

[0084] In certain embodiments, the targeted radiation therapy is targeted radiation to the lungs, preferably at a dose of 5-70 Gray (Gy), e.g., 5, 10, 20, 30, 40, 50, 60, or 70 Gy (or any range expressed therebetween), in 1-10 fractions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractions. In other embodiments, the targeted radiation therapy is preparatory IR, preferably low-dose IR, more preferably administered in the clinic using stereotactic radiosurgery (SRS) or 3-D conformal TRT (3-D CRT) techniques. In other embodiments, the targeted radiation therapy is partial lung irradiation, administered to a portion of the lungs using the latest technology of IMRT.

[0085] Any suitable effective amount of TPOm may be used in the methods of the present application. Such effective amounts may be determined using methods known in the art in light of the present disclosure. In certain embodiments, the effective amount of the TPO mimetic is about 1 to about 10 μg / kg, such as 1, 2, 3, 4, 5, or 6 μg / kg (or any range expressed therebetween) of the subject's body weight. In a preferred embodiment, the effective amount of the TPO mimetic is about 1 to about 3 μg / kg of the subject's body weight.

[0086] In certain embodiments, an effective amount of a TPO mimetic is administered to a subject by either intravenous, intramuscular, intradermal, or subcutaneous injection. In a preferred embodiment, the TPO mimetic is administered by subcutaneous injection.

[0087] Following preparation of the TPO mimetic and optional formulation of the TPO mimetic into a composition, the composition may be administered to an individual, particularly a human, or another primate. Administration may be to a human, or to another mammal, such as a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, horse, donkey, monkey, dog, or cat. Delivery to a non-human mammal need not be for therapeutic purposes, but may be for use in an experimental context.

[0088] The TPO mimetic compositions of the present application may be administered alone or in combination with other treatments or additional therapeutic agents, either simultaneously or sequentially, depending on the condition being treated.

[0089] The term "additional therapeutic agent," as used herein, refers to any compound or therapeutic agent that is known to exhibit or exhibits advantageous properties when administered together with a TPO mimetic in the methods of the present application.

[0090] If desired, the TPO mimetic composition of the present application can be provided in a kit, pack, or dispenser that can contain one or more unit dosage forms containing the active ingredient. For example, the kit can include a metal or plastic foil, such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration. The kit can further include at least one additional therapeutic agent or a device for mitigating toxic effects. The kit can further include an additional therapeutic agent, such as those described herein. The device included in the kit can be, for example, a container, a delivery vehicle, or an administration device.

[0091] Pulmonary function tests As used herein, the phrase "pulmonary function" refers to the functions of the lungs, including, but not limited to, protein synthesis and pulmonary metabolic functions.

[0092] Pulmonary function may be determined using methods known in the art in light of the present disclosure.

[0093] Embodiment The present invention also provides the following non-limiting embodiments.

[0094] Embodiment 1 is a method of alleviating radiation-induced lung injury (RILI) in a subject in need thereof, comprising administering to the subject an effective amount of a thrombopoietin (TPO) mimetic, wherein administering to the subject an effective amount of the TPO mimetic alleviates radiation-induced lung injury in the subject.

[0095] Embodiment 1(a) is the method of embodiment 1, wherein the TPO mimetic comprises a peptide having the amino acid sequence of SEQ ID NO:1.

[0096] Embodiment 1(b) is the method of embodiment 1(a), wherein the peptide has the amino acid sequence of SEQ ID NO:2.

[0097] Embodiment 1(c) is the method of embodiment 1(a) or 1(b), wherein the TPO mimetic further comprises a hydrophilic polymer covalently attached to the peptide.

[0098] Embodiment 1(d) is the method of embodiment 1(c), wherein the hydrophilic polymer is one of i) polyethylene glycol (PEG), ii) polypropylene glycol, iii) polylactic acid, or iv) polyglycolic acid.

[0099] Embodiment 1(e) is the method of embodiment 1(d), wherein the hydrophilic polymer is PEG.

[0100] Embodiment 1(f) is the method of embodiment 1(e), wherein PEG is any one of monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM).

[0101] Embodiment 1(g) is the method of embodiment 1(e), wherein the PEG is methoxypoly(ethylene glycol) (MPEG).

[0102] Embodiment 1(h) is the method of embodiment 1(g), wherein the TPO mimetic is RWJ-800088 having the molecular structure of formula (I) or a pharma- ceutically acceptable salt or ester thereof.

[0103] Embodiment 1(i) is the method of embodiment 1(h), wherein the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000.

[0104] Embodiment 1(j) is the method of embodiment 1(a), wherein the peptide has the amino acid sequence of SEQ ID NO:3.

[0105] Embodiment 1(k) is the method of embodiment 1(j), wherein the peptide is fused to the polypeptide.

[0106] Embodiment 1(l) is the method of embodiment 1(k), wherein the polypeptide is an Fc domain.

[0107] Embodiment 1(m) is the method of embodiment 1(l), wherein the TPO mimetic is romiplostim.

[0108] Embodiment 1(m)(l) is the method of embodiment 1(m), wherein the romiplostim comprises the amino acid sequence of SEQ ID NO:4.

[0109] Embodiment 2 is the method of any one of embodiments 1 to 1(m)(l), wherein the radiation-induced lung injury is any one or more of the following diseases: radiation-induced pulmonary inflammation, collagen deposition in the lungs, pleural effusion, and pulmonary fibrosis.

[0110] Embodiment 2(a) is the method of embodiment 2, wherein the radiation-induced lung injury is an elevation of a lung enzyme.

[0111] Embodiment 3 is the method of any one of Embodiments 1-1(m)(l), wherein the subject is treated with targeted radiation therapy of the pulmonary disease.

[0112] Embodiment 3(a) is the method of embodiment 3, wherein the subject is treated with stereotactic radiotherapy.

[0113] Embodiment 3(b) is the method of embodiment 3, wherein the subject is treated with transarterial chemoembolization (TACE).

[0114] Embodiment 3(c) is the method of embodiment 3, wherein the pulmonary disease is a lung tumor.

[0115] Embodiment 3(d) is the method of embodiment 3, wherein the pulmonary disease is pulmonary metastasis.

[0116] Embodiment 3(e) is the method of embodiment 3, wherein the pulmonary disease is lung cancer.

[0117] Embodiment 3(f) is the method of embodiment 3, wherein the pulmonary disease is a genetic disorder.

[0118] Embodiment 3(g) is the method of embodiment 3(e), wherein the genetic disorder results in a protein deficiency.

[0119] Embodiment 4 is the method of any one of embodiments 1-1(m)(l), wherein the subject is treated with preparative irradiation for bone marrow transplantation.

[0120] Embodiment 4 is the method of any one of embodiments 1 to 1(m)(l), wherein the TPO mimetic is administered to the subject at least about 2 days before to at least about 2 days after administering the dose of targeted radiation to the subject.

[0121] Embodiment 4(a) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject at least about 24 hours before to at least about 24 hours after administering the dose of targeted radiation to the subject.

[0122] Embodiment 4(b) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 24 hours to 2 hours after administering the dose of radiation to the subject.

[0123] Embodiment 4(c) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 24 hours prior to administering the dose of radiation to the subject.

[0124] Embodiment 4(d) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 23 hours prior to administering the dose of radiation to the subject.

[0125] Embodiment 4(d) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 22 hours prior to administering the dose of radiation to the subject.

[0126] Embodiment 4(e) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 21 hours prior to administering the dose of radiation to the subject.

[0127] Embodiment 4(f) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 20 hours prior to administering the dose of radiation to the subject.

[0128] Embodiment 4(g) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 19 hours prior to administering the dose of radiation to the subject.

[0129] Embodiment 4(h) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 18 hours prior to administering the dose of radiation to the subject.

[0130] Embodiment 4(i) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 17 hours prior to administering the dose of radiation to the subject.

[0131] Embodiment 4(j) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 16 hours prior to administering the dose of radiation to the subject.

[0132] Embodiment 4(k) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 15 hours prior to administering the dose of radiation to the subject.

[0133] Embodiment 4(l) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 14 hours prior to administering the dose of radiation to the subject.

[0134] Embodiment 4(m) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 13 hours prior to administering the dose of radiation to the subject.

[0135] Embodiment 4(n) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 12 hours prior to administering a dose of radiation to the subject.

[0136] Embodiment 4(o) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 11 hours prior to administering the dose of radiation to the subject.

[0137] Embodiment 4(p) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 10 hours prior to administering the dose of radiation to the subject.

[0138] Embodiment 4(q) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 9 hours prior to administering the dose of radiation to the subject.

[0139] Embodiment 4(r) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 8 hours prior to administering the dose of radiation to the subject.

[0140] Embodiment 4(s) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 7 hours prior to administering the dose of radiation to the subject.

[0141] Embodiment 4(t) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 6 hours prior to administering the dose of radiation to the subject.

[0142] Embodiment 4(u) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 5 hours prior to administering the dose of radiation to the subject.

[0143] Embodiment 4(v) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 4 hours prior to administering the dose of radiation to the subject.

[0144] Embodiment 4(w) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 3 hours prior to administering the dose of radiation to the subject.

[0145] Embodiment 4(x) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 2 hours prior to administering the dose of radiation to the subject.

[0146] Embodiment 4(y) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 1 hour prior to administering a dose of radiation to the subject.

[0147] Embodiment 4(z) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 30 minutes prior to administering the dose of radiation to the subject.

[0148] Embodiment 4(a)(i) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 15 minutes prior to administering the dose of radiation to the subject.

[0149] Embodiment 4(a)(ii) is the method of embodiment 4, wherein the TPO mimetic is administered to the subject 0.1 to 2 hours after administering the dose of radiation to the subject.

[0150] Embodiment 5 is the method of any one of embodiments 1 to 1(m)(l), wherein the dose of radiation is 5-70 Gray (Gy).

[0151] Embodiment 5(a) is the method of embodiment 5, wherein the dose of radiation is 10 Gray (Gy).

[0152] Embodiment 5(b) is the method of embodiment 5, wherein the dose of radiation is 20 Gray (Gy).

[0153] Embodiment 5(c) is the method of embodiment 5, wherein the dose of radiation is 30 Gray (Gy).

[0154] Embodiment 5(d) is the method of embodiment 5, wherein the dose of radiation is 40 Gray (Gy).

[0155] Embodiment 5(e) is the method of embodiment 5, wherein the dose of radiation is 50 Gray (Gy).

[0156] Embodiment 5(f) is the method of embodiment 5, wherein the dose of radiation is 60 Gray (Gy).

[0157] Embodiment 5(g) is the method of embodiment 5, wherein the dose of radiation is 70 Gray (Gy).

[0158] Embodiment 5(h) is the method of any one of embodiments 5-5(g), wherein the dose of radiation is administered to the subject in 1-10 fractions.

[0159] Embodiment 6 is the method of any one of Embodiments 1-1(m)(l), wherein the effective amount of the TPO mimetic is about 1 to about 10 μg / kg of the subject's body weight.

[0160] Embodiment 6(a) is the method of embodiment 6, wherein the effective amount of the TPO mimetic is about 1 to about 5 μg / kg of the subject's body weight.

[0161] Embodiment 6(b) is the method of embodiment 6, wherein the effective amount of the TPO mimetic is about 1 μg / kg of the subject's body weight.

[0162] Embodiment 6(c) is the method of embodiment 6, wherein the effective amount of the TPO mimetic is about 3 μg / kg of the subject's body weight.

[0163] Embodiment 6(d) is the method of embodiment 6, wherein the effective amount of the TPO mimetic is about 5 μg / kg of the subject's body weight.

[0164] Embodiment 7 is the method of any one of Embodiments 1-1(m)(l), wherein an effective amount of the TPO mimetic is administered to the subject by either intravenous, intramuscular, intradermal, or subcutaneous injection.

[0165] Embodiment 7(a) is the method of embodiment 7, wherein an effective amount of the TPO mimetic is administered to the subject by subcutaneous injection.

[0166] Embodiment 7(b) is the method of embodiment 7, wherein an effective amount of the TPO mimetic is administered to the subject by intravenous injection.

[0167] Embodiment 7(c) is the method of embodiment 7, wherein an effective amount of the TPO mimetic is administered to the subject by intramuscular injection.

[0168] Embodiment 7(d) is the method of embodiment 7, wherein an effective amount of the TPO mimetic is administered to the subject by intradermal injection.

[0169] Embodiment 8 is a kit for alleviating radiation-induced lung disease in a subject in need thereof according to the method of any one of Embodiments 1-1(m)(l), comprising a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharma- ceutically acceptable carrier.

[0170] Embodiment 8(a) is the kit of embodiment 8, further comprising at least one additional therapeutic agent or device for mitigating radiation-induced lung injury.

[0171] Embodiment 9 is a method of treating radiation pneumonitis in a subject in need thereof, comprising administering to the subject a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO: 1, preferably the TPO mimetic is RWJ-800088 or romiplostim, in an amount effective to treat radiation pneumonitis.Embodiment 9(a) is the method of embodiment 20, wherein the subject is treated with targeted radiation therapy for a lung disease or the subject is treated with preparatory irradiation for a bone marrow transplant.

[0172] Embodiment 9(b) is the method of embodiment 9(b), wherein the targeted radiotherapy is selected from the group consisting of stereotactic radiotherapy and transarterial chemoembolization (TACE).

[0173] Embodiment 9(d) is the method of embodiment 9(b) or (c), wherein the subject is treated with targeted radiation therapy at a dose of 5 to 70 Gray (Gy) in 1 to 10 fractions.

[0174] Embodiment 9(e) is the method of any one of embodiments 9-9(d), wherein the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

[0175] Embodiment 9(f) is the method of any one of embodiments 9-9(e), wherein the TPO mimetic is administered to the subject 7 days to 7 days after, 2 days to 2 days after, 24 hours to 24 hours after, preferably about 2 hours to 24 hours before or after the subject is administered a dose of radiation.

[0176] Embodiment 9(g) is the method of embodiment 9(f), wherein the TPO mimetic is administered to the subject 2 hours to 36 hours or 1 day before administering a dose of radiation to the subject.

[0177] Embodiment 9(h) is the method of any one of Embodiments 9-9(g), wherein the TPO mimetic is administered to the subject by either intravenous, intramuscular, intradermal, or subcutaneous injection.

[0178] Embodiment 9(i) is the method of embodiment 9(h), wherein the TPO mimetic is administered to the subject by subcutaneous injection.

[0179] Embodiment 9(j) is a method according to any one of embodiments 9 to 9(h), wherein administration of an effective amount of a TPO mimetic results in at least one of a reduction in elevation of chemokine KC or a reduction in elevation of alveolar neutrophil infiltration in the subject.

[0180] Embodiment 9(k) is the method of any one of embodiments 1-9, wherein the TPO mimetic is RWJ-800088. EXAMPLES

[0181] Example 1 Thrombopoietin mimetics provide radioprotection and prevent pulmonary fibrosis in mice following whole-thoracic irradiation. Radiotherapy is an important treatment for thoracic cancer, but ionizing radiation damage to lung tissue remains a dose-limiting factor. Radiation-induced lung injury (RILI) involves an acute inflammatory phase with high immune cell recruitment and infiltration, followed by a late fibrotic phase with excessive collagen deposition and cellular senescence. Currently, there are no approved drugs to treat RILI. TPOm has been shown to provide radioprotection in the bone marrow and survival benefit in mice after total body irradiation. We evaluated the efficacy of TPOm in reducing the acute and delayed effects of RILI.

[0182] Methods: C57BL / 6 male mice (9-12 weeks old) were subcutaneously administered TPOm RWJ-800088 (0.3 mg / kg) or PBS (vehicle) 1 day prior to whole thoracic irradiation (WTI) with 16 Gy X-rays. Mice were sacrificed 2 weeks and 7 months after WTI (n=5 / time point / group) and lung tissues were collected for various analyses. A group of mice exposed to 18 Gy WTI (n=10 / group) was also subjected to an 8-month survival study.

[0183] Results: Two weeks after WTI, mRNA expression of the proinflammatory chemokines MCP1 and KC in lungs of TPOm-treated mice was 39% and 37% lower, respectively, than vehicle (p<0.05). H&E staining showed higher cell density in vehicle compared to TPOm-treated mice. Furthermore, at this time point, TPOm-treated mice leaked 28% less protein into bronchoalveolar lavage fluid compared to vehicle (p<0.05). At 7 months, TPOm-treated mice had 31% less collagen deposition than vehicle as judged by Trichrome Blue staining (p<0.05). This result is complemented by microCT analysis showing 21% lower density in lungs of TPOm-treated mice compared to vehicle (p<0.05). The mRNA levels of the senescence marker p21 were also significantly 52% lower in TPOm-treated mice than in vehicle (p<0.05).In an 8-month survival study, TPOm-treated mice had a significantly delayed mortality rate and an increased survival rate from 0% in vehicle to 40% (p<0.01).

[0184] Conclusion: TPOm reduces vascular leakage, inflammation, fibrosis, and senescence, leading to improved survival in WTI mice. Thus, TPOm appears to be a potential regimen for treating RILI.

[0185] These results suggest that TPOm and romiplostim have a regenerative effect on the lung after IR.

[0186] Example 2 Thrombopoietin mimetics prevent neutrophil infiltration in mice after whole-thoracic irradiation. Neutrophils are innate immune cells that rapidly infiltrate tissues after injury. Higher neutrophil infiltration is associated with more severe inflammation. To properly evaluate the anti-inflammatory effect of TPOm on radiation pneumonitis, we measured neutrophil recruitment chemokines, KC, and alveolar neutrophil infiltration.

[0187] Methods: Mice were treated as described in Example 1. Whole lung lysates were prepared from left lung tissue at the time of harvest. KC ELISA kit (R&D Systems, #DY453) was used according to the manufacturer's instructions. For each sample, two replicates were measured for KC protein.

[0188] At harvest, right lungs were fixed in 10% formalin, then embedded and sectioned at 5 μm. Anti-myeloperoxidase (MPO) antibody was obtained from Abeam (Cambridge, MA, USA). Immunoreactivity was visualized by ImmPACT histochemistry (Vector Labs, Burlingame, CA, USA) with hematoxylin counterstaining. For quantification of MPO+ cells in lung tissue, images were analyzed with ImageJ software (National Institutes of Health). Stained cells were counted and positive staining was evaluated using the "particle analysis" module. Data were quantified from three fields of view, one section per animal and five animals per group.

[0189] Results: In parallel with mRNA expression, the protein expression of KC in the lungs was also significantly decreased in the TPOm (RWJ-800088) group to levels similar to those of naive mice (Figure 7A). As KC is the major recruitment chemokine of neutrophils, MPO staining was used to evaluate neutrophil infiltration. Lung sections from the vehicle group showed significantly higher numbers of alveolar neutrophil accumulation than mice treated with TPOm (Figure 7C).

[0190] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood therefore that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

1. A method for alleviating radiation-induced or radiomimetic-induced lung injury in a subject in need thereof, comprising administering to the subject an effective amount of a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO:

1.

2. 2. The method of claim 1, wherein the radiation-induced or radiomimetic agent-induced lung injury is any one or more of pulmonary inflammation (pneumonitis) and pulmonary fibrosis, radiation-induced lung inflammation, collagen deposition in the lungs, pleural effusion, and pulmonary fibrosis.

3. 2. The method of claim 1, wherein the TPO mimetic is selected from RWJ-800088 and romiplostim.

4. 10. The method of claim 1, wherein the subject is treated with targeted radiation therapy for a pulmonary disease.

5. 5. The method of claim 4, wherein the targeted radiation therapy is selected from the group consisting of stereotactic radiation therapy and transarterial chemoembolization (TACE).

6. 5. The method of claim 4, wherein the subject is treated with targeted radiation at a dose of 5 to 70 Gray (Gy) in 1 to 10 fractions.

7. The method of claim 1 , wherein the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

8. 10. The method of claim 1, wherein the subject is treated with radiation therapy for cancer or the subject is treated with preparatory irradiation for bone marrow transplantation.

9. 2. The method of claim 1, wherein the TPO mimetic is administered to the subject 7 to 7 days, 2 to 2 days, 24 to 24 hours, preferably about 2 to 24 hours before or after the subject receives a dose of radiation.

10. The method of claim 1 , wherein a therapeutically effective amount of the TPO mimetic is administered to the subject.

11. 10. The method of claim 1, wherein the TPO mimetic is administered to the subject by either intravenous, intramuscular, intradermal, or subcutaneous injection.

12. 2. The method of claim 1, wherein administration of the effective amount of the TPO mimetic results in at least one of an increase in lung volume of non-irradiated lung lobes and a decrease in elevation of circulating lung injury markers in the subject.

13. A kit for alleviating radiation-induced lung damage in a subject in need thereof, comprising a pharmaceutical composition comprising an effective amount of a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO:1, and a pharma- ceutical acceptable carrier.

14. 1. A method of treating a subject having radiation-induced lung injury, comprising administering to the subject after exposure a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO:1 (preferably, said TPO mimetic is RWI-800088 or romiplostim) in an amount effective to treat radiation-induced lung injury occurring at least 3 months after exposure to radiation.

15. 15. The method of claim 14, wherein the subject is a patient undergoing radiation therapy, a nuclear power plant worker, a nuclear warfare responder, or a subject exposed to high levels of radiation due to a nuclear accident, war, or terrorist attack.

16. 15. The method of claim 14, wherein the radiation-induced lung damage occurs at least 6 months after exposure to radiation.

17. 15. The method of claim 14, wherein the thrombopoietin (TPO) mimetic inhibits one or more of radiation-induced pulmonary inflammation, collagen deposition in the lungs, pleural effusion, and pulmonary fibrosis.

18. The method of claim 14 , wherein the subject is a human.

19. 15. The method of claim 14, wherein the radiation-induced toxicity is radiation-induced lung injury consisting of radiation pneumonitis early in radiation therapy and radiation pulmonary fibrosis late in radiation therapy.

20. A method for treating radiation pneumonitis in a subject in need thereof, comprising administering to the subject a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO:1 (preferably, the TPO mimetic is RWJ-800088 or romiplostim) in an amount effective to treat radiation pneumonitis.

21. 21. The method of claim 20, wherein the subject is treated with targeted radiation therapy for a pulmonary disease or the subject is treated with preparative irradiation for a bone marrow transplant.

22. 22. The method of claim 21, wherein the targeted radiation therapy is selected from the group consisting of stereotactic radiation therapy and transarterial chemoembolization (TACE).

23. 23. The method of claim 21 or 22, wherein the subject is treated with the targeted radiation therapy at a dose of 5 to 70 Gray (Gy) in 1 to 10 fractions.

24. The method according to any one of claims 20 to 23, wherein the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

25. 25. The method of any one of claims 20 to 24, wherein the TPO mimetic is administered to the subject 7 to 7 days, 2 to 2 days, 24 to 24 hours, preferably about 2 to 24 hours before or after the subject receives a dose of radiation.

26. 26. The method of claim 25, wherein the TPO mimetic is administered to the subject 2 to 36 hours or 1 day before the subject receives a dose of radiation.

27. The method of any one of claims 20 to 26, wherein the TPO mimetic is administered to the subject by either intravenous, intramuscular, intradermal, or subcutaneous injection.

28. 28. The method of claim 27, wherein the TPO mimetic is administered to the subject by subcutaneous injection.

29. The method of any one of claims 20-28, wherein said administration of said effective amount of said TPO mimetic results in at least one of a reduced elevation of chemokine KC or a reduced elevation of alveolar neutrophil infiltration in said subject.

30. The method of any one of claims 1 to 29, wherein the TPO mimetic is RWJ-800088.

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