Methods for mitigating lung injury associated with exposure to radiation and / or radiation or quasi-radiation therapy

Abstract

Methods and kits for reducing lung injury in a subject treated with targeted radiation therapy or otherwise exposed to radiation are described. In particular, an effective amount of a thrombopoietin mimetic, such as RWJ-800088 or romisctin, is used to mitigate radiation-induced lung injury.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 344,285, filed on May 20, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference sequence listing submitted electronically

[0004] This application contains a sequence listing, which is or will be submitted electronically via PatentCenter in an XML formatted sequence listing, with the file name "004852-213WO1 Sequence Listing.xml", created on May 15, 2023, and with a size of 13,348 bytes. The sequence listing submitted via PatentCenter is or will be a part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to methods and kits for reducing lung injury in subjects in need thereof. Specifically, the present invention relates to methods comprising administering to a subject an effective amount of a thrombopoietin (TPO) mimetic, and a kit containing a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier. TPO can be administered alone or in combination with other active agents to promote beneficial effects. Background Art

[0006] Radiotherapy is an indispensable strategy for treating cancer. About 60%-70% of patients with malignant tumors receive radiotherapy or radiotherapy, which can cure many tumors, and the cure rate of radiotherapy 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, radiotherapy may cause off-target effects on normal tissues (including non-cancerous tissues and distal tissues within the radiation shield, such as bone marrow), which limits the efficacy of radiotherapy. Although various measures have been tried, including updating equipment to improve radiotherapy accuracy, the coordinated use of radiosensitizers, and the combination of radiotherapy and chemotherapy, the results are still unsatisfactory. Normal tissues cannot tolerate radiation-induced toxicity, which prevents the use of higher doses of radiotherapy in clinical applications. Radiation-induced lung injury is the most common clinical complication after radiotherapy. In severe cases, it can be life-threatening for patients, especially those with lung, esophageal, breast, and mediastinal tumors.

[0007] Radiation-induced lung injury (RILI) includes early radiation pneumonitis and late radiation pulmonary fibrosis. This damage not only undermines tumor control, but also seriously affects the patient's quality of life. Respiratory failure is one of the main causes of death in RILI. In addition, local hypoxia, inflammatory response, angiogenesis, local microenvironment changes and immunosuppression caused by RILI will promote tumor recurrence, invasion and metastasis [van den Brenk, HA et al., Br J Radiol, 47 (558): pp. 332-336, (1974)]. Therefore, it is particularly important to manage RILI in clinical practice. The lack of effective drugs has led to the empirical use of high-dose glucocorticoids and anti-inflammatory drugs. These measures usually not only fail to improve the therapeutic effect of radiotherapy, but also cause many side effects, such as immunosuppression. In addition, the immunosuppressive microenvironment of the lung promotes the risk of tumor recurrence and metastasis. Therefore, it is necessary to identify agents that can prevent and / or treat RILI.

[0008] It is generally believed that the tumoricidal effect of radiotherapy 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) trigger 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 initial acute pneumonia, which can also promote the proliferation of fibroblasts and stimulate fibroblasts to secrete excessive collagen. Radiation-induced oxidative damage in pulmonary capillary endothelial cells, including DNA fragmentation, cell death and an increase in reactive oxygen species / reactive nitrogen species (ROS / RNS), causes the accumulation, transcription and upregulation of hypoxia-inducible factor (HTF) activity in tumor cells [Lerman, OZ et al., Blood, 116 (18): 3669-3676, (2010)]. Under hypoxia, endothelial cells (EC) produce a large amount of chemokine stromal cell-derived factor (SDF), which binds to the chemokine receptor CXCR4 and recruits BMDC to inflammatory lesions [Du, R. et al., Cancer Cell, 13 (3): 206-220, (2008)]. Studies have shown that bone marrow-derived cells (BMDC) are crucial for the formation and growth of tumor neovascularization. Changes in the microenvironment provide favorable conditions for tumor recurrence and metastasis.

[0009] Although existing drugs for the prevention and treatment of RILI have some protective effects, in many cases they do not work adequately and / or may inhibit the therapeutic efficacy of radiotherapy. Therefore, there is a need for drugs that can prevent RILI without affecting the efficacy of radiotherapy.

[0010] Although considerable effort has been made clinically to limit the volume of normal lung tissue exposed to radiation when treating lung tumors due to the radiosensitivity of the lung, it is impossible to avoid exposure of normal tissue to radiation in order to adequately treat a patient's cancer and long-term pulmonary toxicity may occur. In addition, cases of accidental radiation exposure have been described, including patients who received unintended chest irradiation during treatment of breast, lung, and other cancers; in some cases, subsequent pulmonary complications resulted in patient death. Such outcomes emphasize the critical role of the lung in both early and late radiation mortality. Therefore, there is increasing recognition that, in addition to countermeasures against the typical recognized components of acute radiation syndrome (ARS) (such as neutropenia and thrombocytopenia), agents that specifically target lung responses are needed, particularly in the setting of total body irradiation (TBI), such as might be expected as part of radiation delivery.

[0011] Radiation-induced pulmonary syndrome is a delayed lethal event resulting from accidental or intentional exposure to irradiation in the context of a nuclear accident or terrorism. In the event of a nuclear accident or deliberate attack that exposes a large population to ionizing radiation, it will be necessary to classify the victims according to the severity of acute radiation illness. Radiation-induced bone marrow syndrome and gastrointestinal (GI) syndrome occur at lower doses of radiation and have an earlier onset 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 the gastrointestinal and hematological syndromes may not completely rescue the patient, as mortality due to respiratory distress at later time points is always a problem. In addition, many victims at risk for developing chronic injury will not develop symptoms for months to years after exposure. Therefore, it is necessary to develop therapeutic strategies that are effective against the onset of symptomatic injury.

[0012] Two stages of radiation-induced lung injury have been described. Acute radiation-induced lung disease (pneumonitis) can occur weeks to 6 months after irradiation. This stage can be life-threatening if a large area of ​​the lung is affected. In late radiation-induced lung injury, which occurs months to years after irradiation, the number of inflammatory cells decreases and collagen deposition occurs, leading to irreversible lung fibrosis.

[0013] The present invention addresses the need for preventing and / or treating RILI. Summary of the invention

[0014] It has now been found that thrombopoietin (TPO) mimetics can reduce lung injury in subjects in need thereof. For example, TPO mimetics were found to have a significant mitigating effect on targeted radiation-induced lung injury (RILI). It is expected that TPO mimetics can have a significant effect if used alone or in conjunction with the administration of other active agents.

[0015] Thrombopoietin (TPO) is a growth factor synthesized and secreted by the liver. In addition to acting as a humoral growth factor that stimulates the proliferation and differentiation of megakaryocytes through the thrombopoietin receptor (TPO-R or c-Mpl), recombinant human TPO (rhTPO) has been shown to promote platelet activation and liver endothelial cell growth and migration in vitro (Cardier et al., Blood, 1998, 91: 923-929).

[0016] Therefore, in one general aspect, the present application relates to a method for reducing 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, more preferably, the TPO mimetic is RWJ-800088 or romiplostim.

[0017] 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.

[0018] 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, which can cause RILI, such as targeted radiation therapy for lung disease, preparatory irradiation for bone marrow transplantation, or targeted radiation therapy for esophageal cancer. TPO mimetics can be applied to the subject before, after, or simultaneously with radiation therapy. In certain embodiments, TPO mimetics are applied to the subject at least about 7 days before to about 7 days after a dose of radiation is applied to the subject, preferably at least about 24 hours before to at least about 24 hours after. In certain embodiments, TPO mimetics are applied to the subject 24 hours before a dose of radiation is applied to the subject. In some embodiments, TPO mimetics are applied to the subject 24 hours before or after a dose of radiation. In other embodiments, TPO mimetics are applied 1 minute to 2 hours before or after a dose of radiation. In some embodiments, TPOm is applied 2 hours to 24 hours after the radiation dose.

[0019] In certain embodiments, the subject is treated with targeted radiation, preferably to the lungs, at a dose of 5 Gray (Gy) to 70 Gy in 1 to 10 fractions.

[0020] In certain embodiments, the effective amount of a TPO mimetic for a human is about 1 μg / kg to about 10 μg / kg of subject body weight, more preferably about 3 μg / kg to about 5 μg / kg of subject body weight. In a preferred embodiment, the effective amount of a TPO mimetic is about 1 μg / kg of subject body weight. In certain preferred embodiments, when administered subcutaneously or intravenously, the effective amount of a TPO mimetic is about 3 μg / kg of subject body weight. Based on the observed differences in efficacy between species, after determining the effective dose for mice, the effective dose for humans can be determined by dividing by 100, where, for example, a dose of 3 mg / kg in mice and a dose of 0.003 mg / kg in humans both produce a transient increase in platelets of approximately 3x.

[0021] In certain embodiments, an effective amount of a TPO mimetic is administered to a subject by intravenous, intramuscular, or subcutaneous injection. In a preferred embodiment, the TPO mimetic is administered by subcutaneous injection.

[0022] 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, the pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier. Optionally, the kit also comprises administration with at least one additional therapeutic agent. Optionally, the kit also comprises a device or tool for administering the TPO mimetic to the subject. Preferably, the kit comprises a TPO mimetic having an amino acid sequence of SEQ ID NO: 1, more preferably a TPO mimetic of RWJ-800088 or romiplostim.

[0023] In another general aspect, the present 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 amount effective to treat radiation pneumonitis, preferably the TPO mimetic is RWJ-800088 or romiplostim.

[0024] In certain embodiments, the subject is treated with targeted radiation therapy for lung disease, or the subject is treated with preparatory irradiation for bone marrow transplantation.

[0025] In certain embodiments, the subject is treated with targeted radiation therapy at a dose of 5 Gray (Gy) to 70 Gy in 1 to 10 fractions.

[0026] In certain embodiments, the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

[0027] In certain embodiments, the TPO mimetic is administered to the subject: 7 days before to 7 days after; 2 days before to 2 days after; 24 hours before to 24 hours after, preferably about 2 hours to 24 hours before or after, a dose of radiation is administered to the subject.

[0028] In certain embodiments, the TPO mimetic is administered to the subject 2 hours to 36 hours or 1 day prior to administering a dose of radiation to the subject.

[0029] In certain embodiments, the TPO mimetic is administered to the subject by any of intravenous, intramuscular, intradermal, or subcutaneous injection.

[0030] In certain embodiments, the TPO mimetic is administered to the subject by subcutaneous injection.

[0031] In certain embodiments, administration of an effective amount of a TPO mimetic produces at least one of the following results: a reduction in elevation of the chemokine KC or a reduction in elevation of alveolar neutrophil infiltration in the subject.

[0032] In certain embodiments, the TPO mimetic is RWJ-800088. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The foregoing summary and the following detailed description of preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0034] Figure 1 The effect of TPOm pretreatment (1 day before irradiation, at 1 mg / kg body weight, by subcutaneous injection) on the mRNA expression of chemokines (IL-1β, IL-8, TNFα, TGFβ, MCP1, MCP2, KC and MIP2) in the lungs of mice (C57BL / 6J males, 9-12 weeks old) 14 days after whole chest irradiation (WTI) of 16Gy delivered by an x-ray irradiator is shown. Lung data are expressed as the change fold of chemokine expression normalized to Hprt1 expression (for original, n=5; for vehicle (phosphate buffered saline) and TPOm pretreatment, n=5). For comparison, the expression in the original group is designated as 1. Data are shown as mean ± SE. *P<0.05 relative to vehicle-treated mice.

[0035] Figure 2A-2B Shown are the effects of TPOm pretreatment (0.3 mg / kg body weight, injected subcutaneously, 1 day before irradiation) on immune cell infiltration and permeability in the lungs of mice 14 days after WTI. Figure 2AShown is a mouse lung section stained with hematoxylin and eosin (H&E) to observe cellularity and immune infiltration. 200X total magnification. Figure 2B Shown are protein amounts in bronchoalveolar lavage fluid measured by bicinchoninic acid assay (BCA) (n=3 for naive; n=5 for vehicle and TPOm pre-treatment).*P<0.05 relative to vehicle-treated mice.

[0036] Figure 3A-3B Shown is the effect of TPOm pretreatment (0.3 mg / kg body weight, injected subcutaneously 1 day before irradiation) on collagen deposition in the lungs of mice 7 months after WTI. Figure 3A Mouse lung sections stained with trichrome blue and visualizing collagen deposition are shown. 200X total magnification. Figure 3B The percentage of collagen-positive tissue section area is shown (n=3 for naive; n=5 for vehicle; and n=5 for TPOm pre-treatment). *P<0.05 relative to vehicle-treated mice.

[0037] Figure 4A-4C The effect of TPOm pretreatment (0.3 mg / kg body weight, injected subcutaneously 1 day before irradiation) on the mRNA and protein expression of senescence genes in the lungs of mice 7 months after WTI is shown. The mRNA and protein levels were evaluated by qPCR and Western blot, respectively. Figure 4A Data are shown 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 pre-treatment). Data are shown as mean±SE. *P<0.05 relative to vehicle-treated mice. Figure 4B Representative blots of lung protein lysates are shown. Figure 4C Density values ​​of p16 and p21 proteins plotted (normalized to β-actin levels) are shown (n=3 for naive, vehicle and TPOm pre-treatment). For comparison, expression in the naive group was assigned 1.

[0038] Figure 5A - Figure 5C shows the effect of TPOm pretreatment (0.3 mg / kg body weight, injected subcutaneously 1 day before irradiation) on lung density in mice 7 months after WTI. Lung density was measured by micro-CT imaging and by micro-CT analysis software. Figure 5A Representative micro-CT cross-sectional images of each treatment are shown. Figure 5B5C shows longitudinal quantification of left lung density at months 3, 5, and 7. (n=3 for naive; n=5 for vehicle; and n=4 for TPOm pre-treatment). *P<0.05 relative to vehicle-treated mice.

[0039] Figure 6 The effect of TPOm pretreatment on 250-day survival of mice at more lethal doses of X-ray radiation (18 Gy) via WTI is shown. Mice were administered vehicle or TPOm injections (1 mg / kg) 1 day before irradiation (1X) or 1 day before irradiation and 7 days after irradiation (2X) (n=10 for irradiated groups; n=5 for naive). *P<0.05 relative to vehicle-treated mice.

[0040] Figure 7A-7C Shown is the effect of TPOm pretreatment (0.3 mg / kg body weight, injected subcutaneously 1 day before irradiation) on neutrophil infiltration in mice measured 7 months after WTI. Fig. 7A Quantification of chemokine KC protein in lung lysates measured by ELISA is shown (n=5). Figure 7B Representative lung sections from mice stained with myeloperoxidase (MPO) are shown. 400X total magnification. Figure 7C Quantification of MPO-positive infiltrates in the lung measured by ELISA is shown (n=5). Data are shown as mean±SE. *P<0.05, ***P<0.0001. DETAILED DESCRIPTION

[0041] The present disclosure is based, at least in part, on the identification of thrombopoietin (TPO) mimetics as therapeutic agents for reducing radiation-induced lung injury in subjects in need thereof. TPO mimetics can be formulated and administered to subjects who have been, are, or will be exposed to radiation to reduce radiation-induced lung injury.

[0042] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0043] 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 the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0044] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0045] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include ± 10% of the 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). Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0046] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0047] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0048] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0049] As used herein, the term "consisting of" used throughout the specification and claims means including any listed integer or group of integers, but additional integers or groups of integers cannot be added to the specified method, structure, or composition.

[0050] As used herein, the term "consisting essentially of" used throughout the specification and claims means including any recited integer or group of integers, and optionally including any recited integer or group of integers that does not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2111.03.

[0051] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, that will be 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.

[0052] The words "right," "left," "lower," and "upper" designate directions in the drawings to which reference will be made.

[0053] It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, the use of "about," "approximately," "substantially," and similar terms herein indicates that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without changing the least significant digits.

[0054] As used herein, in the context of two or more therapies being applied to a subject, the term "combination" refers to the use of more than one therapy. The use of the term "combination" does not limit the order in which the therapy is applied to a subject. For example, the first therapy (e.g., compositions as described herein) can be applied to a subject before the second therapy (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 ago), at the same time, or afterwards (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 later) is applied.

[0055] As used herein, the term "RTLI" or "radiation-induced lung injury" refers to an acute reaction during or within the first few weeks of radiation exposure or radiation therapy (RT), or as a late reaction several months after radiation exposure or RT. Examples of radiation-induced lung injury (RILI) may include, but are not limited to, radiation-induced lung inflammation, collagen deposition in the lungs, multiple fluid perfusions, and lung fibrosis.

[0056] Radiation therapy

[0057] As used herein, the term "TRT" or "targeted radiation therapy" refers to a therapy using ionizing radiation or a pseudo-radiant that preferentially targets or locates a specific organ or part of the body. It is commonly used as part of cancer treatment. TRT, such as targeted ionizing radiation therapy, is sometimes also referred to as radiation therapy, radiotherapy, irradiation or x-ray therapy. Targeted ionizing radiation therapy is mainly divided into three types: external beam radiation therapy (EBRT or XRT), internal radiation therapy, and whole body radioactive isotope therapy. Radiation can be divided into several treatments to deliver the same or slightly higher doses, which is called fractionated radiation therapy. As used herein, the term "pseudo-radiant" or "pseudo-radiation chemotherapeutic" refers to a chemical agent that produces an effect similar to ionizing radiation when administered to a subject. Examples of such effects include DNA damage. Examples of pseudo-radiation chemotherapeutic agents include, but should not be limited to, etoposide, doxorubicin, carboplatin, and bleomycin. Pseudo-radiation chemotherapeutic agents, such as those described herein, can be locally administered to a subject to allow targeted application of the agent in a therapeutic manner.

[0058] External beam radiation therapy (EBRT) uses a machine that guides high-energy rays from outside the body into the tumor. Current radiation technology allows accurate delivery of external beam radiation therapy, such as targeted radiation therapy, which uses a computer to create a 3-dimensional picture of the tumor, so as to target the tumor as accurately as possible and give it the highest possible dose of radiation, while protecting normal tissue as much as possible. Examples of EBRT include, but are not limited to, stereotactic radiation therapy, image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), spiral tomography, proton beam radiation therapy, and intraoperative radiation therapy (IORT). Among them, stereotactic radiation is a special type of external beam radiation therapy. It uses a focused radiation beam, using extremely detailed imaging scans to target a well-defined tumor. There are two types of stereotactic radiation irradiation: stereotactic radiation surgery (SRS) is a stereotactic radiation therapy for the brain or spine, and stereotactic body radiation therapy (SBRT) refers to a more accurate targeted radiation therapy for organs (such as lungs) in the body.

[0059] Internal radiation is also known as brachytherapy, where a radioactive implant is placed in or near a tumor in the body. It allows for higher doses of radiation in a smaller area than external radiation therapy. It uses a radiation source that is usually sealed in a small stent called an implant. Different types of implants may be called pellets, seeds, ribbons, wires, needles, capsules, balloons, or tubes. Several such examples of internal radiation are Y-90SIR balls and / or Thera balls.

[0060] Targeted whole body radioisotope therapy (SRT) is also called unsealed source radiotherapy. Targeted radioactive drugs are used in SRT to treat certain types of cancers, such as thyroid, bone and prostate. These drugs, usually connected to a targeting entity (such as a monoclonal antibody or a cell-specific ligand), can be administered orally or placed in a vein; they then travel through the body until they reach the desired target, where the drug will accumulate in relatively high concentrations.

[0061] As used herein, a "subject treated with targeted radiation therapy" refers to a subject undergoing targeted radiation therapy, and the treatment may be performed before, after, or simultaneously with the administration of a TPO mimetic.

[0062] TPO Simulants

[0063] As used herein, "TPOm", "TPO mimetics" or "thrombopoietin mimetics" refers to compounds comprising peptides capable of binding to and activating thrombopoietin receptors or c-mpl. Preferably, in the TPO mimetics useful in the present invention, the peptides capable of binding to and activating thrombopoietin receptors have no significant homology to thrombopoietin (TPO). The lack of homology to TPO reduces the likelihood of producing antibodies against endogenous TPO. Examples of such peptides that can be used for TPO mimetics include, but are not limited to, U.S. Publication Nos. 2003 / 0158116, 2005 / 0137133, 2006 / 0040866, 2006 / 0210542, 2007 / 0148091, 2008 / 0119384; U.S. Pat. Nos. 5,869,451, 7,091,311, 7,615,533, 8,227,422; those described in International Patent Publications WO2007 / 021572, WO2007 / 094781, and WO2009 / 148954, the entire contents of which are incorporated herein by reference. More preferably, in the TPO mimetics that can be used for the present invention, the peptide that is capable of binding to and activating the thrombopoietin receptor is covalently linked to a portion that improves one or more properties of the peptide. As a non-limiting example, 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.

[0064] US Patent Nos. 7,576,056 and 7,723,295 to Janssen Pharmaceutica NV disclose the use of TPO compounds to treat patients suffering from thrombocytopenia.

[0065] US Patent Nos. 8,067,367 and 8,283,313 to Janssen Pharmaceutica NV disclose a method of providing hematopoietic stem cells to a subject comprising administering a TPO compound.

[0066] US Patent No. 7,615,533 to Janssen Pharmaceutica NV discloses a method of preventing the development of anemia following treatment selected from the group consisting of treatment with a cytotoxic agent, treatment with an anti-tumor agent, and treatment with radiation, the method comprising administering to a subject in need thereof an effective amount of a TPO compound.

[0067] U.S. Published Application No. 20200164039 to Janssen Pharmaceutica NV discloses a method of protecting vascular integrity in a subject exposed to targeted radiation therapy, the method comprising administering to the subject an effective amount of a TPO compound.

[0068] U.S. Published Application No. 20200237870 to Janssen Pharmaceutica NV and Rutgers, The State University discloses a method of mitigating the toxic effects of at least one of a vesicant and a corrosive gas in a subject in need thereof, the method comprising administering to the subject an effective amount of a TPO compound.

[0069] U.S. Published Application No. 20200237871 to Janssen Pharmaceutica NV and Montefiore Medical Center discloses a method of alleviating targeted radiation therapy-induced liver disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a TPO compound.

[0070] U.S. Published Application No. 20200237872 to Janssen Pharmaceutica NV discloses a method of reducing vascular damage, promoting organ and / or hematopoietic recovery, improving survival, and / or preventing organ and hematopoietic damage in a human subject exposed to or having been exposed to radiation.

[0071] Application Serial No. 63 / 261,957 to Janssen Pharmaceutica and Montefiore Medical Center discloses a method of increasing the production of at least one of hematopoietic progenitor cells, myeloid progenitor cells, endothelial progenitor cells, and endothelial cells in a non-irradiated subject, the method comprising administering to the subject an effective amount of a TPO compound.

[0072] In a preferred embodiment, TPO mimetics useful in the present invention include peptides having the following amino acid sequence: IEGPTLRQXaaLAARYaa (SEQ ID NO: 1), wherein 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.

[0073] In some embodiments, TPO mimetics useful in the present invention include peptides of SEQ ID NO: 1 covalently linked to PEG, preferably PEG having an average molecular weight between about 5,000 daltons and about 30,000 daltons. Preferably, PEG is selected from the group consisting of monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trisulfonate (MePEG-TRES) and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM). PEGylation of peptides results in reduced clearance of compounds without loss of potency. See, e.g., U.S. Pat. No. 7,576,056, the entire contents of which are incorporated herein by reference.

[0074] 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) connected by the following lysinamide residues:

[0075]

[0076] and having a methoxypoly(ethylene glycol) (MPEG) covalently linked to each N-terminal isoleucine, or a pharmaceutically acceptable salt or ester thereof. Thus, RWJ-800088 consists of two 14 amino acid peptide chains of SEQ ID NO: 1, wherein Xaa is 2-Nal and Yaa is Sar, connected by a lysinamide residue, and each N-terminal isoleucine is linked to a methoxypoly(ethylene glycol) (MPEG) chain. Thus, RWJ-800088 has an abbreviated molecular structure of (MPEG-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-(2-Nal)-Leu-Ala-Ala-Arg-(Sar))2-Lys-NH2 (SEQ ID NOs: 2 and 5); wherein (2-Nal) is β-(2-naphthyl)alanine, (Sar) is sarcosine, and MPEG is methoxypoly(ethylene glycol), or a pharmaceutically acceptable salt or ester thereof. Preferably, MPEG has a molecular weight of about 20,000 Daltons or stands for methoxypolyethylene glycol 20000.

[0077] In one embodiment, RWJ-800088 has a molecular structure of Formula (I) (SEQ ID NOs 2 and 5, respectively), or a pharmaceutically acceptable salt or ester thereof:

[0078]

[0079] In a preferred embodiment, the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000, and RWJ-800088 has the following full chemical name:

[0080] Methoxypolyethylene glycol 20000-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-(methoxypolyethylene glycol 20000-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 NO: 6 and 7), or a pharmaceutically 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.

[0081] In some embodiments, TPO mimetics useful in the present invention comprise a peptide of SEQ ID NO: 1 fused to an Fc domain. Fusion of the peptide to the Fc domain can stabilize the peptide in vivo. See, e.g., U.S. Pat. No. 6,660,843, the entire contents of which are incorporated herein by reference.

[0082] In another preferred embodiment, the TPO mimetic that can be used 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, wherein Xaa is W and Yaa is A. Specifically, romiplostim has the following amino acid sequence:

[0083] MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

[0084] PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE

[0085] YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

[0086] KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

[0087] GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGIEGPTLRQWLAARAGG

[0088] GGGGGGIEGPTLRQWLAARA (SEQ ID NO:4),

[0089] It has a thrombopoietin receptor binding domain amino acid sequence of IEGPTLRQWLAARA (SEQ ID NO: 3).

[0090] Dosage and Administration

[0091] In the present invention, the inventors of the present invention have found that TPO mimetics have a significant mitigating effect on RILI. Therefore, the method of the present invention comprises administering an effective amount of TPO mimetics to a subject in need thereof, thereby achieving one or more beneficial results, such as mitigating one or more RILI, in a subject in need thereof (e.g., a subject exposed to radiation or treated with radiation therapy).

[0092] TPO mimetics can be used, for example, as an active ingredient of a pharmaceutical composition in combination with a pharmaceutical carrier or diluent. TPO mimetics can be administered orally, pulmonary, parenterally (intramuscular (IM), intraperitoneal (IP), intravenously (IV) or subcutaneously (SC) injection), inhaled (via fine powder preparations), transdermally, nasally, vaginally, rectally or sublingually in a dosage form suitable for each administration route. Preferably, TPO mimetics are administered by subcutaneous injection. For example, International Publication WO1993 / 25221 (Bernstein et al.) discloses biodegradable polymer microspheres containing erythropoietin (EPO), which can be administered parenterally or enterally, preferably orally, to the body surface, topically or systemically. WO1994 / 17784 (Pitt et al.) discloses that EPO can be administered systemically via a pulmonary route, and that this delivery produces a considerable level of therapeutic benefit compared to other EPO administration methods. Similar compositions and methods can be used to administer TPO mimetics of the present disclosure.

[0093] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active peptide compound is mixed with at least one pharmaceutically acceptable carrier (such as sucrose, lactose or starch). As in common practice, this dosage form may also include additional substances different from inert diluents, such as lubricants (e.g., magnesium stearate). For capsules, tablets and pills, the dosage form may also include a buffer. In addition, tablets and pills with enteric coatings may be prepared.

[0094] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, wherein elixirs contain inert diluents commonly used in the art, such as water. In addition to such inert diluents, the composition may also include adjuvants, such as wetting agents, emulsifying agents and suspending agents, as well as sweeteners, flavoring agents, and aromatics.

[0095] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers 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 may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. They may be sterilized, for example, by filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by radiating the composition or by heating the composition. They may also be prepared with sterile water or other sterile injection media immediately before use.

[0096] Administration of TPO mimetics is typically intramuscular, subcutaneous, or intravenous. However, other modes of administration are also contemplated, such as dermal, intradermal, or intranasal administration. Intramuscular administration of the TPO mimetic may be achieved by injecting a suspension of the TPO mimetic composition using a needle. An alternative method is to administer the composition using a needle-free injection device (e.g., using a Biojector TM ) or a lyophilized powder of the TPO mimetic composition.

[0097] For intravenous, cutaneous or subcutaneous injection, the TPO mimetic composition can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity and stability. One skilled in the art is fully capable of preparing a suitable solution using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed. Sustained release formulations may also be used.

[0098] 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 a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

[0099] Typically, administration will have therapeutic and / or preventive purposes to reduce radiation-induced lung injury in a subject before, during or after radiation therapy. In therapeutic applications, a 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 provide relief from radiation-induced lung injury. In preventive applications, a TPO mimetic composition is administered to a subject susceptible to RILI or at risk of developing RILI before exposure to radiation, and the lung function of a subject in need after exposure to radiation is enhanced. In each of these situations, the amount of the TPO mimetic composition will depend on the state and nature of exposure (e.g., the type of radiation therapy, the dose and duration of exposure), the physical characteristics of the subject (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.).

[0100] Administration of a pharmaceutically acceptable composition containing a TPO mimetic to a subject results in alleviation of radiation-induced lung injury. The amount of the composition sufficient to alleviate the disease is defined as an "effective dose" or "effective amount" of the composition.

[0101] The actual amount of application, and the rate of application and time course will depend on the nature and severity of the disease being treated. Treatment prescriptions (e.g., dosage decisions, etc.) are the responsibility of general practitioners and other doctors, or in the case of veterinary medicine, by veterinarians, and generally consider the type and dosage of radiation therapy, the condition of individual patients, delivery sites, methods of application, and other factors known to doctors. Examples of these techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., 1980.

[0102] TPO mimetics can be administered in conjunction with targeted radiation therapy.

[0103] In certain embodiments, TPO mimetics are applied to a subject 7 days before to 7 days after targeted radiation therapy or 2 days before to 2 days after or simultaneously. In certain embodiments, TPO mimetics are applied to a subject 24 hours before to 24 hours after targeted radiation therapy or simultaneously. For example, TPO mimetics are applied to a subject 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 0.5 hour or 0.1 hour (or any range represented therein) before targeted radiation therapy, or 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 0.5 hour or 0.1 hour (or any range represented therein) after targeted radiation therapy. Preferably, TPO mimetics are applied to a subject about 24 to about 1 hour (or any range represented therein) before targeted radiation therapy is applied to a subject.

[0104] In view of the disclosure in the application and the knowledge in the art, any suitable dose of radiation can be used in the method of the application. In order to study the nature of radiation damage to the host, experiments can also be carried out with mice receiving IR and TPOm. Animals from different groups can be killed at different time points (1 day, 2 days, 3 days, 1 week, 3 weeks, 6 weeks and 12 weeks), and lung sections can be stained with H&E for histopathological analysis. BrdU and TUNEL staining can be carried out to check lung cell proliferation and apoptosis respectively.

[0105] In certain embodiments, targeted radiation therapy is targeted radiation to the lung, preferably at a dose of 5 Gy (Gy)-70 Gy, such as 5 Gy, 10 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy or 70 Gy (or any range expressed therein), divided into 1 to 10 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. In other embodiments, targeted radiation therapy is preparatory IR, preferably lower dose IR, more preferably administered using stereotactic radiation surgery (SRS) or 3-D conformal TRT (3-D CRT) technology in the clinic. In other embodiments, targeted radiation therapy is partial lung irradiation administered to parts of the lung using modern IMRT technology.

[0106] Any suitable effective amount of TPOm can be used in the methods of the present application. In view of the present disclosure, such effective amounts can be determined using methods known in the art. In certain embodiments, the effective amount of a TPO mimetic is from about 1 μg / kg to about 10 μg / kg of subject body weight, such as 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg or 6 μg / kg of subject body weight (or any range expressed therein). In a preferred embodiment, the effective amount of a TPO mimetic is from about 1 μg / kg to about 3 μg / kg of subject body weight.

[0107] In certain embodiments, an effective amount of a TPO mimetic is administered to a subject by any of intravenous, intramuscular, intradermal, or subcutaneous injection. In a preferred embodiment, the TPO mimetic is administered by subcutaneous injection.

[0108] After the TPO mimetic is produced and optionally formulated into a composition, these compositions can be administered to an individual, particularly a human or another primate. Administration can be to a human or another mammal, such as a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, horse, cow, donkey, monkey, dog or cat. Delivery to a non-human mammal need not be for therapeutic purposes, but can be used in an experimental setting.

[0109] The TPO mimetic compositions of the invention may be administered alone or in combination (either simultaneously or sequentially) with other treatments or additional therapeutic agents, depending on the condition to be treated.

[0110] As used herein, the term "additional therapeutic agent" refers to any compound or therapeutic agent known or demonstrated to have beneficial properties when administered with a TPO mimetic in the methods of the present application.

[0111] If desired, the TPO mimetic composition can be present in a kit, package or dispenser, which 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, package or dispenser can be accompanied by instructions for administration. The kit can also include at least one additional therapeutic agent or a device for mitigating toxic effects. The kit can also include additional therapeutic agents, such as those described herein. The device included in the kit can be, for example, a container, a delivery vehicle or an administration device.

[0112] Lung function tests

[0113] As used herein, the phrase "pulmonary function" refers to the function of the lungs, including but not limited to protein synthesis and pulmonary metabolic function.

[0114] In view of the present disclosure, lung function can be determined using methods known in the art.

[0115] Implementation

[0116] The present invention also provides the following non-limiting embodiments.

[0117] Embodiment 1 is a method for reducing radiation-induced lung injury (RILI) in a subject in need thereof, the method comprising: administering an effective amount of a thrombopoietin (TPO) mimetic to the subject, wherein administering the effective amount of the TPO mimetic to the subject reduces the radiation-induced lung injury in the subject.

[0118] Embodiment 1(a) is a method according to embodiment 1, wherein the TPO mimetic comprises a peptide having an amino acid sequence of SEQ ID NO:1.

[0119] Embodiment 1(b) is a method according to embodiment 1(a), wherein the peptide has the amino acid sequence of SEQ ID NO:2.

[0120] Embodiment 1(c) is a method according to embodiment 1(a) or 1(b), wherein the TPO mimetic further comprises a hydrophilic polymer covalently linked to the peptide.

[0121] Embodiment 1(d) is a method according to embodiment 1(c), wherein the hydrophilic polymer is any one of the following: i) polyethylene glycol (PEG), ii) polypropylene glycol, iii) polylactic acid, or iv) polyglycolic acid.

[0122] Embodiment 1(e) is a method according to embodiment 1(d), wherein the hydrophilic polymer is PEG.

[0123] Embodiment 1(f) is a method according to embodiment 1(e), wherein the PEG is any one of the following: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trisulfonate (MePEG-TRES) or monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0124] Embodiment 1(g) is a method according to embodiment 1(e), wherein the PEG is methoxypoly(ethylene glycol) (MPEG).

[0125] Embodiment 1(h) is a method according to embodiment 1(g), wherein the TPO mimetic is RWJ-800088 having the structure of Formula (I), or a pharmaceutically acceptable salt or ester thereof.

[0126] Embodiment 1(i) is a method according to embodiment 1(h), wherein the MPEG in the RWJ-800088 is methoxypolyethylene glycol 20000.

[0127] Embodiment 1(j) is a method according to embodiment 1(a), wherein the peptide has the amino acid sequence of SEQ ID NO:3.

[0128] Embodiment 1(k) is a method according to embodiment 1(j), wherein the peptide is fused to a polypeptide.

[0129] Embodiment 1(l) is a method according to embodiment 1(k), wherein the polypeptide is an Fc domain.

[0130] Embodiment 1(m) is a method according to embodiment 1(l), wherein the TPO mimetic is romiplostim.

[0131] Embodiment 1(m)(l) is a method according to embodiment 1(m), wherein romiplostim comprises the amino acid sequence of SEQ ID NO:4.

[0132] Embodiment 2 is a method according to 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 lung inflammation, collagen deposition in the lungs, multiple fluid perfusions, and lung fibrosis.

[0133] Embodiment 2(a) is a method according to embodiment 2, wherein the radiation-induced lung injury is elevated lung enzymes.

[0134] Embodiment 3 is a method according to any one of Embodiments 1 to 1(m)(l), wherein the subject is treated with targeted radiation therapy for lung disease.

[0135] Embodiment 3(a) is a method according to Embodiment 3, wherein the subject is treated with stereotactic radiation therapy.

[0136] Embodiment 3(b) is a method according to Embodiment 3, wherein the subject is treated with transarterial chemoembolization (TACE).

[0137] Embodiment 3(c) is a method according to Embodiment 3, wherein the lung disease is a lung tumor.

[0138] Embodiment 3(d) is a method according to Embodiment 3, wherein the lung disease is lung metastasis.

[0139] Embodiment 3(e) is a method according to Embodiment 3, wherein the lung disease is lung cancer.

[0140] Embodiment 3(f) is a method according to Embodiment 3, wherein the lung disease is a genetic disorder.

[0141] Embodiment 3(g) is a method according to embodiment 3(e), wherein the genetic disorder results in a protein deficiency.

[0142] Embodiment 4 is a method according to any one of Embodiments 1 to 1(m)(l), wherein the subject is treated with preparatory irradiation for bone marrow transplantation.

[0143] Embodiment 4 is a method according to any one of Embodiments 1 to 1(m)(1), wherein the TPO mimetic is administered to the subject at least about 2 days before to at least about 2 days after administering a dose of targeted radiation to the subject.

[0144] Embodiment 4(a) is a method according to 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 a dose of targeted radiation to the subject.

[0145] Embodiment 4(b) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject between 24 hours and 2 hours after administering the dose of radiation to the subject.

[0146] Embodiment 4(c) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 24 hours prior to administering the dose of radiation to the subject.

[0147] Embodiment 4(d) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 23 hours prior to administering the dose of radiation to the subject.

[0148] Embodiment 4(d) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 22 hours prior to administering the dose of radiation to the subject.

[0149] Embodiment 4(e) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 21 hours prior to administering the dose of radiation to the subject.

[0150] Embodiment 4(f) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 20 hours prior to administering the dose of radiation to the subject.

[0151] Embodiment 4(g) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 19 hours prior to administering the dose of radiation to the subject.

[0152] Embodiment 4(h) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 18 hours prior to administering the dose of radiation to the subject.

[0153] Embodiment 4(i) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 17 hours prior to administering the dose of radiation to the subject.

[0154] Embodiment 4(j) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 16 hours prior to administering the dose of radiation to the subject.

[0155] Embodiment 4(k) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 15 hours prior to administering the dose of radiation to the subject.

[0156] Embodiment 4(l) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 14 hours prior to administering the dose of radiation to the subject.

[0157] Embodiment 4(m) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 13 hours prior to administering the dose of radiation to the subject.

[0158] Embodiment 4(n) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 12 hours prior to administering the dose of radiation to the subject.

[0159] Embodiment 4(o) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 11 hours prior to administering the dose of radiation to the subject.

[0160] Embodiment 4(p) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 10 hours prior to administering the dose of radiation to the subject.

[0161] Embodiment 4(q) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 9 hours prior to administering the dose of radiation to the subject.

[0162] Embodiment 4(r) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 8 hours prior to administering the dose of radiation to the subject.

[0163] Embodiment 4(s) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 7 hours prior to administering the dose of radiation to the subject.

[0164] Embodiment 4(t) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 6 hours prior to administering the dose of radiation to the subject.

[0165] Embodiment 4(u) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 5 hours prior to administering the dose of radiation to the subject.

[0166] Embodiment 4(v) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 4 hours prior to administering the dose of radiation to the subject.

[0167] Embodiment 4(w) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 3 hours prior to administering the dose of radiation to the subject.

[0168] Embodiment 4(x) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 2 hours prior to administering the dose of radiation to the subject.

[0169] Embodiment 4(y) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 1 hour prior to administering the dose of radiation to the subject.

[0170] Embodiment 4(z) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 30 minutes prior to administering the dose of radiation to the subject.

[0171] Embodiment 4(a)(i) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 15 minutes prior to administering the dose of radiation to the subject.

[0172] Embodiment 4(a)(ii) is a method according to Embodiment 4, wherein the TPO mimetic is administered to the subject 0.1 hour to 2 hours after administering the dose of radiation to the subject.

[0173] Embodiment 5 is a method according to any one of embodiments 1 to 1(m)(l), wherein the dose of radiation is 5 Gray (Gy) to 70 Gray.

[0174] Embodiment 5(a) is a method according to embodiment 5, wherein the dose of radiation is 10 Gray (Gy).

[0175] Embodiment 5(b) is a method according to embodiment 5, wherein the dose of radiation is 20 Gray (Gy).

[0176] Embodiment 5(c) is a method according to embodiment 5, wherein the dose of radiation is 30 Gray (Gy).

[0177] Embodiment 5(d) is a method according to embodiment 5, wherein the dose of radiation is 40 Gray (Gy).

[0178] Embodiment 5(e) is a method according to embodiment 5, wherein the dose of radiation is 50 Gray (Gy).

[0179] Embodiment 5(f) is a method according to embodiment 5, wherein the dose of radiation is 60 Gray (Gy).

[0180] Embodiment 5(g) is a method according to embodiment 5, wherein the dose of radiation is 70 Gray (Gy).

[0181] Embodiment 5(h) is a method according to any one of Embodiments 5 to 5(g), wherein said dose of said radiation is administered to said subject in 1 to 10 fractions.

[0182] Embodiment 6 is a method according to any one of embodiments 1 to 1(m)(1), wherein the effective amount of the TPO mimetic is from about 1 μg / kg to about 10 μg / kg of the subject's body weight.

[0183] Embodiment 6(a) is a method according to embodiment 6, wherein the effective amount of the TPO mimetic is from about 1 μg / kg to about 5 μg / kg body weight of the subject.

[0184] Embodiment 6(b) is a method according to embodiment 6, wherein the effective amount of the TPO mimetic is about 1 μg / kg body weight of the subject.

[0185] Embodiment 6(c) is a method according to embodiment 6, wherein the effective amount of the TPO mimetic is about 3 μg / kg body weight of the subject.

[0186] Embodiment 6(d) is a method according to Embodiment 6, wherein the effective amount of the TPO mimetic is about 5 μg / kg body weight of the subject.

[0187] Embodiment 7 is a method according to any one of embodiments 1 to 1(m)(1), wherein the effective amount of the TPO mimetic is administered to the subject by any of intravenous, intramuscular, intradermal, or subcutaneous injection.

[0188] Embodiment 7(a) is a method according to Embodiment 7, wherein said effective amount of said TPO mimetic is administered to said subject by subcutaneous injection.

[0189] Embodiment 7(b) is a method according to Embodiment 7, wherein said effective amount of said TPO mimetic is administered to said subject by intravenous injection.

[0190] Embodiment 7(c) is a method according to embodiment 7, wherein said effective amount of said TPO mimetic is administered to said subject by intramuscular injection.

[0191] Embodiment 7(d) is a method according to Embodiment 7, wherein said effective amount of said TPO mimetic is administered to said subject by intradermal injection.

[0192] Embodiment 8 is a kit for reducing radiation-induced lung disease in a subject in need thereof according to any one of embodiments 1 to 1(m)(l), the kit comprising a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier.

[0193] Embodiment 8(a) is a kit according to Embodiment 8, further comprising at least one additional therapeutic agent or device for reducing the radiation-induced lung injury.

[0194] Embodiment 9 is 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 amount effective to treat the radiation pneumonitis, preferably the TPO mimetic is RWJ-800088 or romiplostim. Embodiment 9(a) is a method according to embodiment 20, wherein the subject is treated with targeted radiation therapy for lung disease, or the subject is treated with preparatory radiation for bone marrow transplantation.

[0195] Embodiment 9(b) is a method according to embodiment 9(b), wherein the targeted radiation therapy is selected from the group consisting of: stereotactic radiation therapy and transarterial chemoembolization (TACE).

[0196] Embodiment 9(d) is a method according to embodiment 9(b) or 9(c), wherein the subject is treated with targeted radiation therapy at a dose of 5 Gray (Gy) to 70 Gray in 1 to 10 fractions.

[0197] Embodiment 9(e) is a method according to any one of Embodiments 9 to 9(d), wherein the subject is treated for a lung tumor or lung metastasis, preferably lung cancer.

[0198] Embodiment 9(f) is a method according to any one of embodiments 9 to 9(e), wherein the TPO mimetic is administered to the subject at a time of: 7 days before to 7 days after; 2 days before to 2 days after; 24 hours before to 24 hours after, preferably about 2 hours to 24 hours before, after a dose of radiation is administered to the subject.

[0199] Embodiment 9(g) is a method according to embodiment 9(f), wherein the TPO mimetic is administered to the subject 2 hours to 36 hours or 1 day prior to administering a dose of radiation to the subject.

[0200] Embodiment 9(h) is a method according to any one of embodiments 9 to 9(g), wherein the TPO mimetic is administered to the subject by any of intravenous, intramuscular, intradermal, or subcutaneous injection.

[0201] Embodiment 9(i) is a method according to embodiment 9(h), wherein the TPO mimetic is administered to the subject by subcutaneous injection.

[0202] Embodiment 9(j) is a method according to any one of embodiments 9 to 9(h), wherein said administration of said effective amount of said TPO mimetic produces at least one of the following results: a decrease in the increase in chemokine KC or a decrease in the increase in alveolar neutrophil infiltration in said subject.

[0203] Embodiment 9(k) is a method according to any one of Embodiments 1 to 9, wherein the TPO mimetic is RWJ-800088.

[0204] Example

[0205] Example 1

[0206] Thrombopoietin mimics provide radioprotection and prevent lung fibrosis in mice after whole-thoracic irradiation

[0207] Although radiation therapy is an important treatment modality for thoracic cancer, 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. There are currently no approved drugs for the treatment of RILI. TPOm has been shown to provide radiation protection in the bone marrow and provide survival benefits in mice after whole-body irradiation. The inventors of this case evaluated the efficacy of TPOm in reducing the acute and delayed effects of RILI.

[0208] Methods: TPOm RWJ-800088 (0.3 mg / kg) or PBS (vehicle) was administered subcutaneously to C57BL / 6 male mice (9-12 weeks old) 1 day before whole chest radiation (WTI) of 16 Gy delivered by x-ray. Mice were sacrificed 2 weeks and 7 months after WTI (n=5 / time point / group) to collect lung tissue for various analyses. Groups of mice exposed to 18 Gy WTI (n=10 / group) were also subjected to an 8-month survival study.

[0209] Results: At 2 weeks after WTI, mRNA expression of proinflammatory chemokines MCP1 and KC in the lungs of TPOm-treated mice was 39% and 37% lower than that of the vehicle, respectively (p<0.05). H&E staining showed higher cellularity in the vehicle compared to TPOm-treated mice. In addition, at this time point, TPOm-treated mice leaked 28% less protein into the bronchoalveolar lavage fluid compared to the vehicle (p<0.05). At 7 months, collagen deposition in mice treated with TPOm was 31% lower than that in the vehicle as judged by trichrome blue staining (p<0.05). This result was confirmed by microCT analysis, which showed that the density in the lungs of mice treated with TPOm was 21% lower than that of the vehicle (p<0.05). The mRNA levels of the aging marker p21 in TPOm-treated mice were also significantly lower by 52% (p<0.05) compared to the vehicle. In the 8-month survival study, the death of mice treated with TPOm was significantly delayed and the survival rate increased from 0% in vehicle to 40% (p<0.01).

[0210] Conclusion: TPOm reduced vascular leakage, inflammation, fibrosis, and senescence, leading to improved survival in WTI mice. Therefore, TPOm appears to be a potential treatment for RILI.

[0211] These results suggest that TPOm and romiplostim have a regenerative effect on the lung after IR.

[0212] Example 2

[0213] Thrombopoietin mimetics prevent neutrophil infiltration in mice after whole-thoracic irradiation

[0214] Neutrophils are innate immune cells that acutely infiltrate into tissues after injury. Higher neutrophil infiltration is associated with more severe inflammation. To properly evaluate the anti-inflammatory effect of TPOm on radiation pneumonitis, neutrophil recruitment chemokine KC and alveolar neutrophil infiltration were measured.

[0215] Methods: Mice were treated as described in Example 1. Whole lung lysates were prepared from left lung tissue at harvest. KC ELISA kit (R&D Systems, #DY453) was used according to the manufacturer's instructions. For each sample, 2 replicates of KC protein were measured.

[0216] At harvest, the right lung was fixed with 10% formalin, then embedded and sliced ​​at 5 um. Anti-myeloperoxidase (MPO) antibodies were obtained from Abeam (Cambridge, MA, USA). Counterstained with hematoxylin, immunoreactivity was observed by ImmPAC Thistochemistry (Vector Labs, Burlingame, CA, USA). To quantify MPO+ cells in lung tissue, images were analyzed with ImageJ software (National Institutes of Health); stained cells were counted using the "Analyze Particles" module to assess positive staining. Data were quantified from 3 fields of view of 1 slice per animal and 5 animals per group.

[0217] Results: In parallel with mRNA expression, in the TPOm(RWJ-800088) group, KC protein expression in the lung was also significantly reduced to a level similar to that of the original ( Fig. 7A Since KC is the main recruiting chemokine for neutrophils, MPO staining was used to assess neutrophil infiltration. Lung sections of the vehicle group showed a significantly greater number of alveolar neutrophil accumulation compared to mice treated with TPOm ( Figure 7C ).

[0218] It will be appreciated by those skilled in the art that modifications may be made to the described embodiments without departing from the broad inventive concept of the present invention. Therefore, it will be appreciated that the present invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined in this specification.

Claims

1. A method of reducing radiation-induced or radiation-mimicking agent-induced lung injury in a subject in need thereof, the method comprising: An effective amount of a thrombopoietin (TPO) mimetic comprising the amino acid sequence of SEQ ID NO: 1 is administered to the subject.

2. The method of claim 1, wherein the radiation-induced or radiation-mimicking agent-induced lung injury is any one or more of the following: lung lesions (pneumonia) and lung fibrosis, radiation-induced lung inflammation, collagen deposition in the lungs, multiple fluid perfusions, and lung fibrosis.

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

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

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. The method of claim 4, wherein the subject is treated with targeted radiation at a dose of 5 Gray (Gy) to 70 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. 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. The method of claim 1, wherein the TPO mimetic is administered to the subject: 7 days before to 7 days after; 2 days before to 2 days after; 24 hours before to 24 hours after, preferably about 2 hours to 24 hours before or after, a dose of radiation is administered to the subject.

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

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

12. The method of claim 1, wherein said administering of said effective amount of said TPO mimetic produces at least one of: an increase in vital capacity of a non-irradiated lung lobe of said subject and a decrease in elevation of circulating markers of lung injury.

13. A kit for reducing radiation-induced lung injury in a subject in need thereof, the kit 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 pharmaceutically acceptable carrier.

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

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

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

17. The method of claim 14, wherein the thrombopoietin (TPO) mimetic inhibits one or more of the following: radiation-induced lung inflammation, collagen deposition in the lung, multiple fluid perfusions, and lung fibrosis.

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

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

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

21. The method of claim 20, wherein the subject is treated with targeted radiation therapy for lung disease, or the subject is treated with preparatory irradiation for bone marrow transplantation.

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. The method of claim 21 or 22, wherein the subject is treated with targeted radiation therapy at a dose of 5 Gray (Gy) to 70 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. The method of any one of claims 20 to 24, wherein the TPO mimetic is administered to the subject: 7 days before to 7 days after; 2 days before to 2 days after; 24 hours before to 24 hours after, preferably about 2 hours to 24 hours before or after, a dose of radiation is administered to the subject.

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

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

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 to 28, wherein the administration of the effective amount of the TPO mimetic produces at least one of the following results: a reduction in the increase in chemokine KC or a reduction in the increase in alveolar neutrophil infiltration in the subject.

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

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