Methods for reducing the toxic effects of blistering agents and corrosive gases

Abstract

The present invention relates to methods and kits for reducing the toxic effects of at least one of a blistering agent and a corrosive gas in a subject in need thereof. Specifically, an effective amount of a thrombopoietin (TPO) mimetic, such as RWJ‑800088 or romiplostim, is used to reduce the toxic effects of a blistering agent or a corrosive gas.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 62 / 796,754, filed January 25, 2019, the contents of which are incorporated herein by reference in their entirety.

[0003] References to electronically submitted sequence listings

[0004] This application contains a sequence listing, which was electronically submitted via EFS-Web in ASCII format. The file name is "688097.0953 / 457WO Sequence Listing," the creation date is January 23, 2020, and the size is approximately 3.6 kb. The sequence listing submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present application relates to methods and kits for reducing the toxic effects of at least one of a vesicant and a corrosive gas in a subject in need thereof. In particular, the present application relates to methods comprising administering to a subject an effective amount of a thrombopoietin (TPO) mimetic, and kits comprising a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier. Background Art

[0006] Inhalation exposure to vesicant and corrosive gases is known to induce acute lung injury, characterized by edema, microhemorrhages, intracellular influx, and endothelial / epithelial cell damage. In a small number of documented human exposures, the doses of toxic gases are high enough to cause death within a short period of time. However, in the majority of survivors of exposure events, the debilitating acute effects gradually subside, accompanied by the progression of profibrotic processes, leading to irreversible pulmonary fibrosis and causing long-term morbidity.

[0007] While personal protective equipment (i.e., respirators) can be used to block exposure, and specialized neutralizers are being developed to neutralize these active substances after they are released into the environment, more is needed to mitigate the toxic effects of these substances immediately after human exposure.

[0008] For example, U.S. Patent Application Publication No. 2003 / 0083321 discloses compositions and methods for minimizing or avoiding the deleterious effects of blistering agents, such as mustard gas and other agents that cause blistering reactions. The compositions comprise at least one matrix metalloproteinase inhibitor, preferably in combination with at least one protease inhibitor.

[0009] International Patent Application Publication No. WO 2014028509 discloses a method for treating a subject with bone marrow damage caused by exposure to mustard gas and nitrogen mustard. The method involves administering a c-Mpl receptor agonist, such as a thrombopoietin (TPO) mimetic. International Patent Application Publication No. WO 2008 / 086025 also discloses a method for treating respiratory diseases by administering a compound having erythropoietin activity or thrombopoietin (TPO) activity.

[0010] TPO is known to regulate platelet levels by binding to c-Mpl on megakaryocytes (stimulating platelet maturation) and existing platelets (providing negative feedback) (Mitchell and Bussell, Semin. Hematol. 52(1):46-52 (2015)). TPO may also act directly on the vascular system by binding to c-Mpl receptors located on vascular endothelial cells (Langer et al., J. Mol. Cell Cardiol. 47(2):315-25 (2009)). Several studies have shown that thrombopoietin has direct vascular protective effects in animal models of doxorubicin-mediated cardiovascular injury (Chan et al., Eur. J. Heart Fail. 13(4):366-76 (2011)), cardiovascular ischemia-reperfusion injury (Baker et al., Cardiovasc. Res. 77(1):44-53 (2008)), and stroke (Zhou et al., J. Cereb Blood Flow Metab. 31(3):924-33 (2011)). However, recombinant human TPO (rhTPO) is not a viable treatment because it induces cross-reactive antibodies against endogenous TPO, leading to chronic thrombocytopenia (Li et al., Blood 98(12):3241-8 (2001)).

[0011] New methods are needed to mitigate the toxic effects of vesicants and corrosive gases following human exposure. Summary of the Invention

[0012] Thrombopoietin (TPO) mimetics have been shown to significantly mitigate a number of blister-induced injury endpoints, many of which are related to the function of TPO mimetics as vascular protectants.

[0013] Therefore, in one general aspect, the present application relates to a method for reducing the toxic effects of at least one of a blistering agent and a corrosive gas in a subject exposed to the blistering agent or the corrosive gas, wherein the method comprises administering to a subject in need thereof 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.

[0014] In certain embodiments, the subject is in need of mitigation of the toxic effects of a vesicant. The vesicant can be, for example, selected from the group consisting of rectified mustard, mustard, mustard / Lewisite, mustard / T, nitrogen mustard, sesquimustard, sulfur mustard, phosgene oxime, cantharidin, and furanocoumarins.

[0015] In certain embodiments, the subject needs to mitigate the toxic effects of a corrosive gas. For example, the corrosive gas can be selected from hydrogen sulfide, hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen cyanide, arsine, phosphine, nitric oxide, nitrogen dioxide, sulfur dioxide, ozone, chlorine, methylamine, and ammonia.

[0016] In certain embodiments, the toxic effect is one or more selected from the group consisting of vascular damage, microbleeding, cellular influx, pulmonary fibrosis, mortality, and morbidity, wherein the toxic effect is caused by at least one of a vesicant and a corrosive gas. Preferably, the toxic effect is pulmonary microbleeding.

[0017] The pathogenesis of acute lung injury and acute respiratory distress syndrome is associated with the generation of reactive oxygen species, which are similar to those produced in the lungs by vesicants and chemical poisons. Similar to chemical injury, under pathological conditions such as acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), excessive generation of oxygen free radicals leads to increased endothelial permeability, loss of vascular microvascular connectivity integrity, and solute and fluid migration within the alveolar cavity. Therefore, the present application provides a method for reducing the toxic effects of oxidative stress in a subject in need thereof, the method comprising administering to the subject an effective amount of a TPO mimetic. The present application also provides a method for reducing the toxic effects of endothelial injury in a subject, comprising administering to the subject an effective amount of a TPO mimetic. Another embodiment provided herein is a method for reducing the toxic effects of oxidative stress and endothelial injury, comprising administering to a subject an effective amount of a TPO mimetic. The provided method for oxidative stress or endothelial injury comprises administering to a subject in need thereof an effective amount of a TPO mimetic, and in certain embodiments, the TPO mimetic may comprise the amino acid sequence of SEQ ID NO: 1. Alternatively, the TPO mimetic may be RWJ-800088. The TPO mimetic administered may also be romiplostim.

[0018] As described herein, the TPO mimetics, such as RWJ-800088 or romiplostim, can be administered to a subject to mitigate toxic effects associated with exposure to chemical blistering agents or corrosive gases.

[0019] In another aspect, the present application relates to a kit for reducing the toxic effects of at least one of a blistering agent and a corrosive gas in a subject exposed to the blistering agent or the corrosive gas. The kit comprises a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier, as well as at least one other therapeutic agent or device for reducing blistering agent damage. Optionally, the kit further comprises a means for administering the TPO mimetic to the subject. Preferably, the kit comprises a TPO mimetic comprising the amino acid sequence of SEQ ID NO: 1, more preferably, the TPO mimetic is RWJ-800088 or romiplostim. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above summary of the invention, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that this application is not limited to the precise embodiments shown in the drawings.

[0021] Figure 1A and 1B The effects of TPOm treatment on bleeding, as assessed by optical density of bronchoalveolar lavage (BAL) and white blood cell counts in BAL, are shown. In this study, groups of male Wistar rats received nitrogen mustard (NM). Six hours later, the rats were treated with vehicle or TPOm. On day 3, the rats were scanned with MRI using an FSE (T2-weighted) sequence to characterize the extent of pulmonary edema caused by NM exposure. After imaging, the rats were euthanized and lung lavage was performed with PBS. The levels of red blood cells (RBCs) and white blood cells (WBCs) in the Bronchoalveolar lavage (BAL) fluid, as well as the concentration of the fibrogenic factor, transforming growth factor beta 1 (TGF-1b), were determined using standard methods. Figure 1A Shows the RBC content, Figure 1B The WBC content is shown, indicating that the administration of TPOm reduced the pulmonary microbleeding and WBC influx into the air spaces of the lungs induced by NM exposure. Figure 1C The concentration of TGF-1b in BAL fluid after NM+ / -TPOm treatment is shown, indicating that TPOm alleviated the increase of TGF-1b caused by NM (p<0.05).

[0022] Figures 2A-2CThe vasoprotective effects of romiplostim treatment were observed in the bronchoalveolar lavage (BAL) cellular fraction in parallel with TPOm. In this study, groups of male Wistar rats received nitrogen mustard (NM). Six hours later, the rats were treated with vehicle, romiplostim, or TPOm. On day 4, the relative levels of red blood cells in the BAL were visualized by digital imaging of the BAL ( Figure 2A ), by measuring the absorbance of the sample at 540 nm ( Figure 2B ) were quantified by blood cell counting ( Figure 2C ) to measure the number of white blood cells (WBC). Figure 2A The red color of the BAL (indicating erythrocyte content) is shown (dark gray in B and W), and 2B shows the optical density difference, also indicating erythrocyte content. Figure 2C The WBC content is shown, indicating that both romiplostim and TPOm administration can reduce pulmonary microbleeding and the influx of WBC into the air spaces of the lungs caused by NM exposure.

[0023] Figures 3A-3C The vasoprotective effects of TPOm were demonstrated in postmortem gross organ pathology. In this study, rats were euthanized, the chest cavity was surgically opened, and the descending blood vessels were severed. Blood outside the collapsed lung was gently flushed with phosphate-buffered saline (PBS) and then blotted with a wet surgical pad. Digital photography of the lungs was performed without perfusing the pulmonary vasculature with PBS. Figure 3A lungs of rats in the vehicle control group, reflecting the blood in the pulmonary vessels; Figure 3B Shown is a rat lung exposed to NM, showing numerous sites of sub-plural microhemorrhages (arrows) with reduced surrounding perfusion. Figure 3C The severity and number of microbleeding sites were significantly reduced compared with those in rats treated with NM alone ( Figure 3B These results suggest that TPOm can prevent severe vascular damage associated with the progression of microhemorrhage and cellular influx after exposure to NM.

[0024] Figure 4 The effect of TPOm on NM-induced pulmonary edema was shown, as measured by T2-weighted magnetic resonance imaging, showing the progression and recovery of pulmonary edema at 3, 42, and 70 days after NM exposure. The mean edema volume in the NM / TPOm-treated group, which received a single dose of TPOm at 6 hours after exposure, increased from 1736 mm to 70 days after NM exposure. 3 Reduced to 1592 and 1387 mm 3, but this effect was not statistically significant. At later time points, TPOm was administered again on day 7 or on days 7 and 14. On days 42 and 70, the mean pulmonary edema volume in rats treated with TPOm was lower compared with the test group exposed only to NM (p = 0.0104 for the group administered TPOm at 6 hours and 7 days; p = 0.0770 for the group administered TPOm at 6 hours, 7 days, and 14 days), indicating that TPOm accelerated the resolution of edema.

[0025] Figure 5A and 5B The effects of TPOm on NM-induced pulmonary fibrosis were shown. In a long-term chronic study investigating the effects of TPOm on the progression of pulmonary fibrosis, rats received multiple doses of TPOm (2 or 3 doses) after NM vesicant treatment. MRI examinations were performed periodically over a period of time following exposure to assess the level of pulmonary edema. After 70 days of exposure, the rat lungs were inflated and fixed in situ to maintain native lung capacity. The lungs were then excised, fixed overnight in formalin, and imaged by ex vivo microCT equilibration with phosphotungstic acid (PTA), a microCT radiographic contrast agent with a high binding affinity for collagen. The PTA treatment method enabled quantification and visualization of the progression of pulmonary fibrosis in two and three dimensions. After microCT scanning, the lungs were transferred to 70% ethanol for 4 days to remove the phosphotungstic acid (PTA) and undergo histological evaluation. Figure 5A The data presented show that TPOm significantly reduced many elements of fibrotic conditions in the lungs exposed to NM (fibrosis scores of 2.11 and 1.89 in the TPOm-treated group, respectively, compared to 4.33 in the vehicle control group). 5B shows a grayscale CT image representing lung radiodensity after PTA treatment, indicating that TPOm reduced the number and altered the distribution of collagen-containing fibrotic lesions, which appear as dotted light gray foci throughout the lungs, in the lungs of rats exposed to NM.

[0026] Figure 6 To demonstrate the effects of NM-induced mortality, groups of rats were treated with a single dose of NM at the estimated LD50. The animals were housed in normal shoebox-sized housing and weighed daily and observed frequently throughout the study. Figure 6 The study showed that administering TPOm after exposure to NM prolonged life and reduced mortality. The NM / Veh-treated group experienced greater weight loss than the NM / TPOm-treated group. TPOm treatment significantly protected against weight loss (5% to 7%, ~20 grams), suggesting that the drug has an ameliorative effect on morbidity. DETAILED DESCRIPTION

[0027] The present invention is based, at least in part, on the identification of thrombopoietin (TPO) mimetics as therapeutic agents for mitigating the toxic effects of at least one of a blistering agent and a corrosive gas in a subject in need thereof. TPO mimetics can be formulated and administered to a subject who is or will be exposed to a blistering agent or a corrosive gas to protect against the toxic effects.

[0028] Various publications, articles, and patents are cited or described in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like 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 materials constitute part of the prior art with respect to any invention disclosed or claimed.

[0029] General Terms and Definitions

[0030] 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 set forth in the specification.

[0031] 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.

[0032] 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 any case. Thus, numerical values ​​generally include ±10% of the stated numerical 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, unless the context clearly indicates otherwise, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of numerical values ​​within the range.

[0033] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein by mere routine experimentation. Such equivalents are intended to be encompassed within the present invention.

[0034] As used herein, the terms "comprises," "comprising," "having," or "containing," or any other variation thereof, will be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of 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 in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly indicated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, situation A or B is satisfied by any 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).

[0035] As used herein, the term "and / or" in conjunction with multiple listed elements should be understood to cover both individual and combined options. For example, where two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any of these options should be understood to fall within this meaning and therefore meet the requirements of the term "and / or" as used herein. The concurrent applicability of more than one of the multiple options should also be understood to be within its meaning and therefore meet the requirements of the term "and / or."

[0036] As used herein, the term "consisting of, or variations such as "consisting of," used throughout the specification and claims means that any stated integer or group of integers is included, but additional integers or groups of integers may not be added to the specified method, structure, or composition.

[0037] As used herein, the term "consisting essentially of, or variations such as "consisting essentially of," as used throughout the specification and claims, means that any recited integer or group of integers is included and optionally substituting any recited integer or group of integers that does not materially alter the basic or novel property of the specified method, structure, or composition. See MPEP §2111.03.

[0038] As used herein, "subject" refers to any animal, preferably a mammal, and most preferably a human, that is to 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, and the like, more preferably humans.

[0039] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings relative to objects to which reference is made.

[0040] It should also be understood that when referring to dimensions or features of components of the preferred invention, the terms "about," "approximately," "generally," "substantially," and similar terms used herein indicate the dimensions / features being described, not the strict boundaries or parameters, and do not exclude minor variations that are functionally identical or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include the least significant digits not being altered using mathematical and industrial principles generally recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0041] As used herein, the term "toxic effect" refers to acute and / or chronic toxicity of a vesicant or corrosive gas in a subject following exposure to the vesicant or corrosive gas. The term "toxic effect" may include, but is not limited to, edema, vascular damage, microbleeding (e.g., pulmonary microbleeding), cellular influx, endothelial / epithelial cell damage, or pulmonary fibrosis. The term "toxic effect" may also refer to mortality or morbidity.

[0042] As used herein, a "blistering agent" refers to a toxic chemical compound that typically causes long-lasting damage to tissues that come into contact with or around the compound. Blisters can cause severe pain and irritation to the skin, eyes, and mucous membranes. Examples of blistering agents include, but are not limited to, rectified mustard, mustard gas, Lewisite, mustard / Lewisite, mustard / T, nitrogen mustard, sesquimustard, sulfur mustard, phosgene oxime, cantharidin, or furanocoumarins. Blisters also include blistering agents, which are so named because they can cause severe chemical burns, resulting in painful blisters on the patient's body.

[0043] Most vesicants fall into three categories: sulfur mustard, which is a sulfur-containing agent that includes mustard gas; nitrogen mustard, an agent similar to sulfur mustard but based on nitrogen rather than sulfur; and Lewisite, an early vesicant developed during World War I but not used. Phosgene oxime, also known as urticant (itching agent), is also included among vesicants. Although the term vesicant is often used to describe large-scale burns caused by chemical leaks or chemical warfare agents, some naturally occurring substances, such as cantharidin, are also vesicants (blistering agents). Furocoumarins, another naturally occurring vesicant, can also cause blistering indirectly, for example, by greatly increasing skin photosensitivity.

[0044] "Corrosive gases," as used herein, are gases that, upon contact, cause burns or corrosion to human tissue through chemical reactions. Examples of corrosive gases include, but are not limited to, hydrogen sulfide, hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen cyanide, arsine, phosphine, nitric oxide, nitrogen dioxide, sulfur dioxide, ozone, chlorine, methylamine, and ammonia. Corrosive gases can have the ability to burn or corrode human tissue through chemical reactions. There are generally three types of corrosive gases: acidic gases, alkaline gases, and oxidizing gases.

[0045] As used herein, the term "other therapeutic agent" refers to any compound or therapeutic agent known to have or exhibit beneficial properties when administered with TPO or a TPO mimetic. Examples of such agents include, but are not limited to, analgesics, antimicrobials, other TPO mimetics, other cytokines, soluble Mpl receptors, hematopoietic factors, interleukins, growth factors or antibodies, and chemotherapeutic agents. The other cytokine can be stem cell factor (SCF), interleukin 3 (IL-3), or Flt-3 ligand. Ku et al., Blood, 87:4544-4551 (1996); Sitnicka et al., Blood, 87:4998-5005 (1996). In particular, the other therapeutic agent can be atropine, atropine sulfate, homatropine, a narcotic analgesic, or N-acetylcysteine ​​(NAC).

[0046] TPO simulants

[0047] As used herein, "TPOm," "TPO mimetic," or "thrombopoietin mimetic" refers to a compound comprising a peptide capable of binding to and activating the thrombopoietin receptor. Preferably, in the TPO mimetic useful in the present invention, the peptide capable of binding to and activating the thrombopoietin receptor has no significant homology to thrombopoietin (TPO). This lack of homology to TPO reduces the likelihood of developing antibodies to TPO. Examples of such peptides that can be used in TPO mimetics include, but are not limited to, those disclosed in U.S. 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; International Patent Publication Nos. 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 in the present invention, the peptide that is 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.

[0048] In a preferred embodiment, the TPO mimetic useful in the present invention comprises a peptide comprising the following amino acid sequence:

[0049]

[0050] 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.

[0051] In some embodiments, the TPO mimetic useful in the present invention comprises a peptide of SEQ ID NO: 1 covalently linked to PEG, preferably a PEG having an average molecular weight of about 5,000 to about 30,000 daltons. Preferably, the PEG is selected from monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-trifluoroethanesulfonate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). PEGylation of the peptide reduces the clearance rate of the compound without reducing efficacy. See, for example, U.S. Patent No. 7,576,056, the entire contents of which are incorporated herein by reference.

[0052] 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 the following 29-membered peptide having two identical 14-membered chains connected by a lysinamide residue (SEQ ID NO: 2):

[0053]

[0054] And having methoxy polyethylene glycol (MPEG) covalently linked to each N-terminal isoleucine, or a pharmaceutically acceptable salt or ester thereof. Therefore, RWJ-800088 comprises two 14 amino acid peptide chains of SEQ ID NO: 1 linked by lysinamide residues, wherein Xaa is 2-Nal and Yaa is Sar, and each N-terminal isoleucine is linked to a methoxy polyethylene glycol (MPEG) chain. Therefore, the simplified molecular structure of RWJ-800088 is (MPEG-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-(2-Nal)-Leu-Ala-Ala-Arg-(Sar))2-Lys-NH2; wherein (2-Nal) is β-(2-naphthyl)alanine, (Sar) is sarcosine, and MPEG is methoxy polyethylene 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.

[0055] In one embodiment, RWJ-800088 has a molecular structure of Formula (I), or a pharmaceutically acceptable salt or ester thereof:

[0056]

[0057] In a preferred embodiment, the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000, and the full chemical name of RWJ-800088 is: Methoxypolyethylene glycol 20000-Propanyl-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-glutamyl-L-2-naphthylalanyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-sarkosyl-)-lysinamide, or a pharmaceutically acceptable salt or ester thereof. The molecular weight of the peptide without PEG is 3,295 Daltons, while the molecular weight of the peptide with two 20,000 Dalton MPEG chains is approximately 43,295 Daltons.

[0058] In some embodiments, a TPO mimetic useful in the present invention comprises 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, for example, U.S. Patent No. 6,660,843, the entire contents of which are incorporated herein by reference.

[0059] In another preferred embodiment, the TPO mimetic useful in the present invention is romiplostim. As used herein, "romiplostim" refers to a fusion protein comprising 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:

[0060]

[0061] It has the amino acid sequence of the thrombopoietin receptor binding domain

[0062]

[0063] Dosage and administration

[0064] In the present invention, the inventors have discovered that TPO mimetics significantly mitigate the toxic effects of vesicants or corrosive gases in humans. Therefore, the methods of the present invention comprise administering an effective amount of a TPO mimetic to a subject in need thereof, thereby mitigating one or more acute and chronic endpoints of NM-induced lung injury in the subject in need thereof, such as vascular damage, pulmonary hemorrhage, influx of inflammatory cells into lung air spaces, accumulation of profibrotic cytokines in the lung, progression of pulmonary fibrosis, vesicant-induced edema, and vesicant-induced mortality and morbidity. The subject in need thereof, such as a subject exposed to a vesicant or a subject exposed to a corrosive gas, is administered a TPO mimetic.

[0065] TPO mimetics can be administered, for example, as the active ingredient of a pharmaceutical composition together with a pharmaceutical carrier or diluent. TPO mimetics can be administered orally, pulmonary, parenterally (intramuscular (IM), intraperitoneal (IP), intravenously (IV) or subcutaneously (SC)), by inhalation (via micropowder formulations), transdermal, nasal, vaginal, rectal or sublingual routes of administration, in dosage forms suitable for each route of administration. For example, International Publication No. WO1993 / 25221 (Bernstein et al.) discloses biodegradable polymer microspheres containing erythropoietin (EPO), which can be administered parenterally or enterally, preferably orally, and can be administered topically, locally or systemically. WO1994 / 17784 (Pitt et al.) discloses that EPO can be administered systemically via the pulmonary route, and that this mode of delivery can produce a considerable level of therapeutic benefit compared to other methods of administering EPO. Similar compositions and methods can be used for the administration of TPO mimetics of the present invention.

[0066] 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. Such dosage forms may also conventionally include substances other than inert diluents, such as lubricants, such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage form may also contain a buffering agent. Tablets and pills may also be prepared with an enteric coating.

[0067] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, and inert diluents commonly used in the art (e.g., 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 flavoring agents.

[0068] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous or non-aqueous vehicles are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil and corn oil), gelatin, and injectable organic esters (e.g., ethyl oleate). Such dosage forms may also include adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. They may be sterilized by, for example, filtering through a bacterial retention filter, including a sterilant, irradiating the composition, or heating the composition. They may also be prepared with sterile water or some other sterile injection media prior to use.

[0069] TPO mimetics are typically administered intramuscularly, subcutaneously, or intravenously. However, other routes of administration are also contemplated, such as transdermal, intradermal, or nasal administration. Intramuscular injection of TPO mimetics can be achieved by injecting a suspension of the TPO mimetic composition using a needle. Alternatively, a needle-free injection device can be used to administer the composition (e.g., using a Biojector™) or a lyophilized powder of the TPO mimetic composition.

[0070] For intravenous, transdermal or subcutaneous injection, or injection into the affected area, the TPO mimetic composition can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has an appropriate pH, isotonicity, and stability. Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles (e.g., 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.

[0071] 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 using standard excipients well known in the art.

[0072] Typically, administration is for therapeutic and / or prophylactic purposes, namely to mitigate the toxic effects of the blistering agent or corrosive gas in a subject before, during, or after exposure to the blistering agent or corrosive gas. In therapeutic applications, the TPO mimetic composition is administered to a subject during or after exposure to the blistering agent or corrosive gas, and the amount of the TPO mimetic composition administered is sufficient to cure or at least partially mitigate the toxic effects. In prophylactic applications, the TPO mimetic composition is administered to a subject susceptible to or at risk of exposure to the blistering agent or corrosive gas before exposure to the blistering agent or corrosive gas. In each case, the amount of the TPO mimetic composition depends on the state and nature of the exposure (e.g., the type and concentration of the blistering agent or corrosive gas, the duration of exposure), and the subject's physical characteristics (e.g., height, weight, etc.).

[0073] A pharmaceutically acceptable composition comprising a TPO mimetic is administered to a subject to reduce the toxic effects of a vesicant or toxic gas in the subject. An amount of the composition sufficient to reduce the toxic effects is defined as an "effective dose" or "effective amount" of the composition.

[0074] The effective amount of the TPO mimetic administered to a subject in the methods provided herein can vary depending on the subject. The literature documents species differences in the effectiveness of other TPO mimetics due to differences in receptor affinity (Erickson-Miller CL, et al. "Discovery and characterization of a selective, non-peptidyl thrombopoietin receptor agonist," Exp. Hematol., 2005; 33: 85-93 and Nakamura T, et al. "A novel non-peptidyl human c-Mpl activator stimulates human megakaryopoiesis and thrombopoiesis," Blood. 2006; 107: 4300-7). Therefore, in some embodiments, the effective amount of the TPO mimetic can be 0.1 μg to 6 μg / kg subject body weight. In another embodiment, the effective amount of the TPO mimetic can be 2.25 to 4 μg / kg subject body weight. For some subjects, the effective amount of the TPO mimetic can be 0.1 to 6 mg / kg subject body weight. In another embodiment, the effective amount of a TPO mimetic can be 2.25 to 4 mg / kg of subject body weight. For other subjects, the effective amount of a TPO mimetic can be 6 to 60 mg / kg of subject body weight. In another embodiment, the effective amount of a TPO mimetic can be 20 to 40 mg / kg of subject body weight.

[0075] The actual dosage, rate of administration, and time course will depend on the nature and severity of the condition being treated. The prescription of treatment, such as the determination of dosage, is the responsibility of general practitioners and other physicians, or in the context of veterinary medicine, the responsibility of vesicants, and generally takes into account the type and concentration of the vesicant or corrosive gas, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980. Based on the dosage ranges described herein, the specific dosage examples provided, and taking into account that different species may require dosage adjustments based on receptor sensitivity, those skilled in the art will be able to determine the effective dosage for the subject.

[0076] In certain embodiments, a single dose of an effective amount of a TPO mimetic is administered to a subject.

[0077] In certain embodiments, multiple doses of an effective amount of a TPO mimetic are administered to a subject.

[0078] After the TPO mimetic is prepared and optionally formulated into a composition, the composition can be administered to a subject, particularly a human or other primate. Administration can be to a human or another mammal, such as a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog, or cat. Delivery to non-human mammals need not be for therapeutic purposes, but can be used in an experimental setting, for example, to study the mechanism by which vascular integrity is protected by administration of a TPO mimetic.

[0079] Depending on the condition to be treated, the TPO mimetic compositions of the present invention can be administered alone or in combination with other therapies, either simultaneously or sequentially.

[0080] If desired, the TPO mimetic composition can be presented in a kit, package, or dispenser that can contain one or more unit dosage forms containing the active ingredient. The kit can, for example, comprise metal or plastic foil, such as a blister pack. The kit, package, or dispenser can be accompanied by instructions for administration. The kit can further contain at least one other therapeutic agent or device for mitigating toxic effects.

[0081] The other therapeutic agents included in the kit are any compounds or therapeutic agents known or shown to have advantageous properties when administered with TPO or a TPO mimetic. Examples of such agents include, but are not limited to, analgesics, antimicrobials, other TPO mimetics, other cytokines, soluble Mpl receptors, hematopoietic factors, interleukins, growth factors or antibodies, and chemotherapeutic agents. Other cytokines may include stem cell factor (SCF), interleukin 3 (IL-3), or Flt-3 ligand (Ku et al., Blood, 87:4544-4551 (1996); Sitnicka et al., Blood, 87:4998-5005 (1996)).

[0082] The device included in the kit can be, for example, a container, a delivery vehicle, or an administration device.

[0083] Implementation Plan

[0084] The present invention also provides the following non-limiting embodiments.

[0085] Embodiment 1 is a method for reducing toxic effects,

[0086] (i) the toxic effect is a toxic effect of at least one of a vesicant and a corrosive gas in a subject in need thereof,

[0087] The method comprises administering to the subject an effective amount of a thrombopoietin (TPO) mimetic, wherein the TPO mimetic is administered to the subject in an effective amount to mitigate a toxic effect of at least one of a vesicant and a corrosive gas; or

[0088] (ii) the toxic effect is a toxic effect of oxidative stress or endothelial damage in a subject in need thereof,

[0089] The method comprises administering to the subject an effective amount of a TPO mimetic, wherein the TPO mimetic is administered to the subject in an amount effective to reduce the toxic effects of oxidative stress or endothelial damage.

[0090] Embodiment 1(a) is a method as described in embodiment 1, wherein the TPO mimetic comprises a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0091] Embodiment 1(b) is a method as described in embodiment 1(a), wherein the peptide comprises the amino acid sequence of SEQ ID NO:2.

[0092] Embodiment 1(c) is the method of embodiment 1(a) or 1(b), wherein the TPO mimetic further comprises a hydrophilic polymer covalently linked to the peptide.

[0093] Embodiment 1(d) is a method as described in embodiment 1(c), wherein the hydrophilic polymer is any one of: i) polyethylene glycol (PEG), ii) polypropylene glycol, iii) polylactic acid or iv) polyglycolic acid.

[0094] Embodiment 1(e) is a method as described in embodiment 1(d), wherein the hydrophilic polymer is PEG.

[0095] Embodiment 1(f) is a method as described in embodiment 1(e), wherein PEG is any one 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-trifluoroethanesulfonate (MePEG-TRES) or monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0096] Embodiment 1(g) is a method as described in embodiment 1(e), wherein the PEG is methoxypolyethylene glycol (MPEG).

[0097] Embodiment 1(h) is a method as described in embodiment 1(g), wherein the TPO mimetic is RWJ-800088 having the molecular structure of formula (I).

[0098] Embodiment 1(h)(1) is a method as described in embodiment 1(g), wherein the TPO mimetic is RWJ-800088 having a molecular structure of formula (I) or a pharmaceutically acceptable salt or ester thereof.

[0099] Embodiment 1(i) is a method as described in embodiment 1(h) or 1(h)(1), wherein the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000.

[0100] Embodiment 1(j) is a method as described in embodiment 1(a), wherein the peptide comprises the amino acid sequence of SEQ ID NO:3.

[0101] Embodiment 1(k) is a method as described in embodiment 1(j), wherein the peptide is fused to a polypeptide.

[0102] Embodiment 1(1) is a method as described in embodiment 1(k), wherein the polypeptide is an Fc domain.

[0103] Embodiment 1(m) is a method as described in embodiment 1(l), wherein the TPO mimetic is romiplostim.

[0104] Embodiment 1(m)(1) is a method as described in embodiment 1(m), wherein romiplostim comprises the amino acid sequence of SEQ ID NO:4.

[0105] Embodiment 2 is the method of any one of embodiments 1-1(m)(1), wherein an effective amount of the TPO mimetic is administered to the subject prior to exposing the subject to at least one of the blistering agent and the corrosive gas.

[0106] Embodiment 3 is the method of any one of embodiments 1-1(m)(1), wherein the TPO mimetic is administered to the subject after the subject is exposed to at least one of the vesicant and the corrosive gas.

[0107] Embodiment 4 is the method of any one of embodiments 1-1(m)(1), wherein the TPO mimetic is administered to the subject while the subject is exposed to at least one of the blistering agent and a corrosive gas.

[0108] Embodiment 5 is a method as described in any one of embodiments 1 to 4, wherein the blistering agent is selected from the group consisting of rectified mustard, mustard, Lewisite, mustard / Lewisite, mustard / T, nitrogen mustard, sesquimustard, sulfur mustard, phosgene oxime, cantharidin, and furanocoumarins.

[0109] Embodiment 6 is a method as described in any one of embodiments 1-5, wherein the corrosive gas is selected from hydrogen sulfide, hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen cyanide, arsine, phosphine, nitric oxide, nitrogen dioxide, sulfur dioxide, ozone, chlorine, methylamine and ammonia.

[0110] Embodiment 7 is the method of any one of embodiments 1 to 6, wherein the subject is in need of mitigation of the toxic effects of at least one of a blistering agent and a corrosive gas, wherein the blistering agent is selected from the group consisting of rectified mustard, mustard, Lewisite, mustard / Lewisite, mustard / T, nitrogen mustard, sesquimustard, sulfur mustard, phosgene oxime, cantharidin, and furanocoumarins; and wherein the corrosive gas is selected from the group consisting of hydrogen sulfide, hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen cyanide, arsine, phosphine, nitric oxide, nitrogen dioxide, sulfur dioxide, ozone, chlorine, methylamine, and ammonia.

[0111] Embodiment 7(a) is a method as described in embodiment 7, wherein the subject is in need of mitigation of a toxic effect of at least one vesicant.

[0112] Embodiment 7(b) is a method as described in embodiment 7, wherein the subject is in need of mitigation of the toxic effects of at least one corrosive gas.

[0113] Embodiment 7(c) is a method as described in embodiment 7, wherein the subject is in need of mitigation of the toxic effects of at least two vesicants.

[0114] Embodiment 7(d) is a method as described in embodiment 7, wherein the subject is in need of mitigation of the toxic effects of at least two corrosive gases.

[0115] Embodiment 7(e) is the method of embodiment 7, wherein the subject is in need of mitigation of the toxic effects of at least three or more vesicants.

[0116] Embodiment 7(f) is a method as described in embodiment 7, wherein the subject is in need of mitigation of the toxic effects of at least three or more corrosive gases.

[0117] Embodiment 8 is the method of any one of embodiments 1 to 7, wherein the effective amount of the TPO mimetic is based on the subject's body weight.

[0118] Embodiment 8(a) is a method as described in any one of embodiments 1-7, wherein the effective amount of the TPO mimetic is 0.1 to 6 μg / kg body weight of the subject.

[0119] Embodiment 8(b) is a method as described in any one of embodiments 1-7, wherein the effective amount of the TPO mimetic is 2.25 to 4 μg / kg body weight of the subject.

[0120] Embodiment 8(c) is a method as described in any one of embodiments 1-7, wherein the effective amount of the TPO mimetic is 0.1 to 6 mg / kg body weight of the subject.

[0121] Embodiment 8(d) is a method as described in any one of embodiments 1-7, wherein the effective amount of the TPO mimetic is 2.25 to 4 mg / kg body weight of the subject.

[0122] Embodiment 8(e) is a method as described in any one of embodiments 1-7, wherein the effective amount of the TPO mimetic is 6 to 60 mg / kg body weight of the subject.

[0123] Embodiment 9 is a method as described in any of embodiments 1-8(a), wherein the toxic effect is one or more selected from vascular damage, pulmonary hemorrhage, influx of inflammatory cells into lung air spaces, accumulation of profibrotic cytokines in the lung, pulmonary fibrosis, pulmonary edema, mortality and morbidity, and wherein the toxic effect is caused by at least one of a vesicant and a corrosive gas.

[0124] Embodiment 9(a) is a method as described in embodiment 9, wherein the toxic effect is vascular damage.

[0125] Embodiment 9(b) is a method as described in embodiment 9, wherein the toxic effect is pulmonary hemorrhage, preferably pulmonary microhemorrhage.

[0126] Embodiment 9(c) is a method as described in embodiment 9, wherein the toxic effect is an influx of inflammatory cells into the lung air spaces.

[0127] Embodiment 9(d) is a method as described in embodiment 9, wherein the toxic effect is the accumulation of profibrotic cytokines in the lung.

[0128] Embodiment 9(e) is a method as described in embodiment 9, wherein the toxic effect is pulmonary fibrosis.

[0129] Embodiment 9(f) is a method as described in embodiment 9, wherein the toxic effect is pulmonary edema.

[0130] Embodiment 9(g) is a method as described in embodiment 9, wherein the toxic effect is mortality caused by at least one of a blistering agent and a corrosive gas.

[0131] Embodiment 9(h) is a method as described in embodiment 9, wherein the toxic effect is morbidity caused by at least one of a blistering agent and a corrosive gas.

[0132] Embodiment 10 is the method of any one of embodiments 1-9(h), wherein the effective amount of the TPO mimetic is administered to the subject by any of intravenous, intramuscular, intradermal, or subcutaneous injection.

[0133] Embodiment 10(a) is a method as described in embodiment 10, wherein the effective amount of the TPO mimetic is administered to the subject by subcutaneous injection.

[0134] Embodiment 10(b) is a method according to embodiment 10, wherein the effective amount of the TPO mimetic is administered to the subject by intravenous injection.

[0135] Embodiment 10(c) is a method according to embodiment 10, wherein the effective amount of the TPO mimetic is administered to the subject by intramuscular injection.

[0136] Embodiment 10(d) is a method as described in embodiment 10, wherein the effective amount of the TPO mimetic is administered to the subject by intradermal injection.

[0137] Embodiment 11 is a kit for reducing the toxic effects of at least one of a vesicant and a corrosive gas in a subject in need thereof, comprising a pharmaceutical composition comprising an effective amount of a TPO mimetic and a pharmaceutically acceptable carrier.

[0138] Embodiment 11(a) is a kit according to embodiment 11, wherein the TPO mimetic comprises a peptide comprising the amino acid sequence of SEQ ID NO:1.

[0139] Embodiment 11(b) is a kit according to embodiment 11, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2.

[0140] Embodiment 11(c) is a kit as described in embodiment 11(a) or 11(b), wherein the TPO mimetic further comprises a hydrophilic polymer covalently linked to the peptide.

[0141] Embodiment 11(d) is a kit according to embodiment 11, wherein the hydrophilic polymer is any one of polyethylene glycol (PEG), polypropylene glycol, polylactic acid and polyglycolic acid.

[0142] Embodiment 11(e) is a kit as described in embodiment 11(d), wherein the hydrophilic polymer is PEG.

[0143] Embodiment 11(f) is a kit as described in embodiment 11(e), wherein PEG is any one of monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-trifluoroethanesulfonate (MePEG-TRES) or monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0144] Embodiment 11(g) is a kit as described in embodiment 11(e), wherein the PEG is methoxypolyethylene glycol (MPEG).

[0145] Embodiment 11(h) is a kit as described in embodiment 11(g), wherein the TPO mimetic is RWJ-800088 having the molecular structure of formula (I) or a pharmaceutically acceptable salt or ester thereof.

[0146] Embodiment 11(i) is a kit according to embodiment 11(h), wherein the MPEG in RWJ-800088 is methoxypolyethylene glycol 20000.

[0147] Embodiment 11(j) is a kit as described in embodiment 11(a), wherein the peptide comprises the amino acid sequence of SEQ ID NO:3.

[0148] Embodiment 11(k) is a kit as described in embodiment 11(j), wherein the peptide is fused to a polypeptide.

[0149] Embodiment 11(1) is a kit as described in embodiment 11(k), wherein the polypeptide is an Fc domain.

[0150] Embodiment 11(m) is a kit as described in embodiment 11(l), wherein the TPO mimetic is romiplostim.

[0151] Embodiment 11(m)(1) is a kit as described in embodiment 11(m), wherein romiplostim comprises the amino acid sequence of SEQ ID NO:4.

[0152] Embodiment 12 is a kit as described in any of embodiments 11-11(m)(1), further comprising at least one additional therapeutic agent or device for mitigating toxic effects.

[0153] Embodiment 13 is a kit according to embodiment 12, wherein the other therapeutic agent is selected from analgesics, antibacterial drugs, other TPO mimetics, other cytokines, soluble Mpl receptors, hematopoietic factors, interleukins, growth factors or antibodies and chemotherapeutic agents.

[0154] Example

[0155] Example 1 Effect of TPOm on NM-induced lung injury

[0156] Materials and methods

[0157] Animals: Male Wistar rats (300-320 g) were divided into two groups: Nm / Veh and Nm / TPOm.

[0158] Nitrogen Mustard Exposure: This study used mechlorethamine, a non-gaseous nitrogen mustard (NM), as a surrogate for sulfur mustard to allow for vesicant exposure in experimental animals and minimize the risk to researchers. It is anticipated that the information gained in these NM studies will be directly applicable to guiding the design and definition of endpoints of interest in studies involving sulfur mustard gas exposure. Specifically, groups of male Wistar rats (300-320 g) were instilled with 0.125 mg / kg of the sulfur mustard surrogate, nitrogen mustard, via tracheal instillation on day 0.

[0159] Synthesis and Treatment of TPOm: TPOm was synthesized by Janssen Pharmaceuticals according to literature methods (see, e.g., U.S. Patent No. 7,576,056). TPOm was reduced in sterile saline, sterile filtered, aliquoted, and stored at -20°C until use. Six hours after exposure to nitrogen mustard (NM), a single dose of TPOm (0.3 mg / kg) was administered subcutaneously. This dose was chosen based on its efficacy in whole-body irradiation studies in rats: administration of TPOm at multiple time points resulted in a significant survival benefit compared to placebo.

[0160] Magnetic resonance imaging (MRI) measurements: On day 3, rats were scanned with MRI using FSE (T2-weighted) sequences to characterize the extent of pulmonary edema induced by NM exposure.

[0161] Bronchoalveolar lavage (BAL) measurements: After MRI scanning, rats were euthanized and their lungs were lavaged with PBS in divided doses (3 × 5 ml). The red blood cell (RBC), white blood cell (WBC) and transforming growth factor beta 1 (TGF-1b) contents in the bronchoalveolar lavage (BAL) were measured using standard methods. Specifically, multiple aliquots of lavage fluid were collected from each rat, and the red blood cell content was recorded by digital photography. The BAL specimens were gently mixed, and the white blood cell count in the specimens was determined by standard trypan blue exclusion and blood cell counting. BAL samples were collected from each treatment group, centrifuged, and the TGF-1b concentration in the cell-free supernatant was determined by TGF-1b ELISA.

[0162] Gross organ pathology measurements: Rats were euthanized, and the chest cavity was surgically opened and the descending blood vessels severed. Blood outside the collapsed lung was gently flushed with phosphate-buffered saline (PBS) and then absorbed with a wet surgical pad. Without perfusing the pulmonary vasculature with PBS, the lungs were digitally photographed.

[0163] result

[0164] TPOm and romiplostim had protective effects on microbleeding and cellular influx after exposure to NM.

[0165] The vasoprotective effect of TPOm treatment (0.3 mg / kg, 6 h after NM-instillation) was observed in the bronchoalveolar lavage (BAL) cellular fraction. Figure 1A , optical density determination) and WBC ( Figure 1B , blood cell count, p < 0.05) showed that TPOm administration could reduce pulmonary microbleeding and WBC influx into the lung air chamber caused by NM exposure. TPOm could also alleviate the increase in TGF-1b concentration in BAL fluid caused by NM ( Figure 1C , p<0.05).

[0166] In parallel with TPOm, vasoprotective effects of romiplostim treatment (0.1 & 1.0 mg / kg, 6 h after NM-instillation) were observed in BAL cellular fractions. Figure 2A - visual inspection and 2B- optical density) and leukocyte count ( Figure 2C ) assay showed that both romiplostim and TPOm administration reduced NM exposure-induced pulmonary microbleeding and leukocyte influx into the lung air spaces.

[0167] The vascular protective effect of TPOm was also observed in the gross organ pathology of autopsy. Figures 3A-3CAs shown, autopsy showed that the lungs of vehicle control rats were uniform in color, reflecting the uniform color of the blood contained in the pulmonary vasculature, while the lungs of rats exposed to NM showed a large number of sub-plural microbleeds (arrows) surrounded by pale areas of reduced perfusion. Although the rats exposed to NM showed a mottled appearance after receiving TPOm, the severity and number of microbleeds were significantly reduced compared to rats treated with NM alone. These results indicate that TPOm can prevent the severe vascular damage associated with the progression of microbleeds and cellular influx after exposure to NM.

[0168] Effects of TPOm on pulmonary edema after exposure to NM

[0169] A single dose of TPOm (0.3 mg / kg) was administered 6 hours after NM exposure, and the effect of TPOm on pulmonary edema was evaluated on day 3 after NM exposure; multiple doses of TPOm were administered 6 hours and 7 days after NM exposure; or multiple doses of TPOm were administered 6 hours, 7 days, and 14 days after NM exposure. On day 3 after NM exposure, the mean edema volume in the NM / TPOm-treated group, which received a single dose of TPOm at 6 hours after exposure, increased from 1736 mm to 1736 mm in the NM-treated group. 3 Reduced to 1592 and 1387 mm 3 , but this effect was not statistically significant. At later time points, TPOm was administered again on day 7 or on days 7 and 14. On days 42 and 70, the mean pulmonary edema volume in rats treated with TPOm was lower compared with the test group exposed only to NM (p = 0.0104 for the group administered TPOm at 6 hours and 7 days; p = 0.0770 for the group administered TPOm at 6 hours, 7 days, and 14 days), indicating that TPOm accelerated the resolution of edema.

[0170] Example 2 Observation of the effect of TPOm on NM-induced pulmonary fibrosis on day 42 and / or day 70

[0171] Materials and methods

[0172] Animals: Male Wistar rats (300-320 g) were divided into two groups: Nm / Veh and Nm / TPOm.

[0173] Nitrogen mustard exposure: In this study, groups of male Wistar rats were instilled with 0.125 mg / kg of nitrogen mustard, a sulfur mustard substitute, via tracheal instillation on day 0.

[0174] Synthesis of TPOm and TPOm Treatment: TPOm was synthesized by Janssen Pharmaceuticals according to literature methods (see, e.g., U.S. Patent No. 7,576,056). TPOm was reduced in sterile saline, sterile filtered, aliquoted, and stored at -20°C until use. In this extended study, rats received multiple doses of TPOm (0.3 mg / kg) at two different dosing schedules (6 hours, 6 hours, and 7 days vs. 6 hours, 7 days, and 14 days) after treatment with NM vesicle to investigate the effect of TPOm on the progression of pulmonary fibrosis.

[0175] Magnetic resonance imaging (MRI) measurements: MRI examinations were performed regularly over a period of time following exposure to assess the extent of pulmonary edema. Seventy days after exposure, the rats' lungs were fixed in situ with air inflated to maintain natural lung capacity. The lungs were then excised, fixed overnight in formalin, equilibrated with phosphotungstic acid (PTA), and subjected to ex vivo micro-CT imaging. PTA is a micro-CT radiographic contrast agent with a high binding affinity for collagen. This PTA treatment method enables the quantification and visualization of the progression of pulmonary fibrosis in two and three dimensions. A multi-parameter scoring system was used to score the fibrosis of each lung based on the number and distribution of fibrotic and radiodense foci present in ex vivo three-dimensional CT images of lungs treated with the collagen-specific contrast agent phosphotungstic acid (PTA). In this example, radiodense fibrotic tissue was observed throughout the normal lung parenchyma (light gray), demonstrating the amount and distribution of pulmonary fibrosis.

[0176] Long-term protective effect of TPOm on NM-induced pulmonary fibrosis

[0177] The results of this study indicate that TPOm affects the endpoints of lung fibrosis induced by NM. Using a multi-parameter scoring system developed for the assessment of lung fibrosis, it was shown that TPOm significantly reduced the fibrotic conditions of lungs exposed to NM in many aspects ( Figure 5A Furthermore, CT images reflecting the radiodensity of PTA-treated lungs showed that TPOm reduced the number and altered the distribution of collagen-containing fibrotic lesions in the lungs of rats exposed to NM ( Figure 5B ).

[0178] Example 3 Effect of TPOm on Mortality Caused by NM

[0179] Materials and methods

[0180] Animals: Male Wistar rats (300-320 g) were divided into two groups: Nm / Veh and Nm / TPOm.

[0181] Nitrogen mustard exposure: A series of preliminary studies were conducted to determine the dose range of nitrogen mustard (NM) that causes mortality in Wistar rats over a 2-week period. These studies demonstrated a very steep dose-response for NM-induced mortality, with an estimated LD50-LD70 range of 1.23-3.75 mg / kg. Based on the severity observed systematically in these dose-finding studies, a 1.7 mg / kg dose of NM was selected for subsequent studies to investigate the effects of TPOm on NM-induced mortality. In this study, groups of male Wistar rats (10 / group) were treated with mechlorethamine, a sulfur mustard surrogate, via intratracheal instillation on day 0 at an estimated LD50 of 1.7 mg / kg. The animals were then returned to their normal shoebox-sized housing. They were weighed daily and observed frequently throughout the study.

[0182] Synthesis and Treatment of TPOm: TPOm was synthesized by Janssen Pharmaceuticals according to literature methods (see, e.g., U.S. Patent No. 7,576,056). TPOm was reduced in sterile saline, sterile filtered, aliquoted, and stored at -20°C until use. Six hours after exposure to nitrogen mustard (NM), a single dose of TPOm (0.3 mg / kg) was administered subcutaneously.

[0183] Mortality and body weight measurements: Dead animals were recorded, moribund rats were euthanized and recorded as dead, and the data were used to construct a survival rate graph ( Figure 6 Daily body weight was also affected, with a difference of 5%-7% (~20 g) in body weight loss between the NM / Veh and NM / TPOm treated groups.

[0184] result

[0185] Protective effect of TPOm on mortality induced by NM

[0186] The results are as follows Figure 6 As shown. From the survival rate curve ( Figure 6 ) clearly exceeded the inferred LD50 for NM expected for this study. This is likely due to the steepness of the dose response, which leads to variability in LD50 determinations. Although this study was not powered to resolve significance, it is important to note that TPOm administration 6 hours after NM exposure was found to prolong life and reduce mortality. Furthermore, the NM / Veh group experienced greater weight loss than the NM / TPOm-treated group, and the protective effect of TPOm-related weight loss was significant (5%-7%, ~20 grams). These results suggest that TPOm ameliorates vesicular agent-induced mortality and reduces morbidity by preventing exposure-induced weight loss.

[0187] Those skilled in the art will appreciate that changes may be made to the above embodiments without departing from the broad inventive concept thereof. Therefore, it should be understood 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 by this specification. Sequence Listing <110> Janssen Pharmaceuticals Ltd. <120> Methods for mitigating the toxic effects of blistering agents and corrosive gases <130> 688097.0953 / 457WO <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> X <222> (9)..(9) <223> Tryptophan or beta-(2-naphthyl)alanine <220> <221> X <222> (14)..(14) <223> Alanine or creatine <400> 1 Ile Glu Gly Pro Thr Leu Arg Gln Xaa Leu Ala Ala Arg Xaa 1 5 10 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> X <222> (9)..(9) <223> beta-(2-naphthyl)alanine <220> <221> X <222> (14)..(14) <223> Sarcosine <400> 2 Ile Glu Gly Pro Thr Leu Arg Gln Xaa Leu Ala Ala Arg Xaa 1 5 10 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala 1 5 10 <210> 4 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Recombinant peptide <400> 4 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg 225 230 235 240 Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile 245 250 255 Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala 260 265

Claims

1. Use of a thrombopoietin (TPO) mimetic in the preparation of a medicament for reducing the toxic effects of nitrogen mustard in a subject, the thrombopoietin mimetic comprising the amino acid sequence of SEQ ID NO: 1, wherein The thrombopoietin mimetic is RWJ-800088 or romiplostim; The toxic effect is pulmonary fibrosis caused by the nitrogen mustard.

2. The use of claim 1, wherein the toxic effect further comprises vascular damage caused by the nitrogen mustard.

3. The use according to claim 1, wherein the toxic effect further comprises pulmonary microhemorrhage caused by the nitrogen mustard.

4. The use of claim 1, wherein the toxic effects further comprise mortality or morbidity caused by the nitrogen mustard.

5. The use according to any one of claims 1 to 4, wherein the effective amount of the TPO mimetic is administered to the subject by intravenous injection, intramuscular injection, intradermal injection or subcutaneous injection.

6. Use of a kit for preparing a medicament for reducing the toxic effects of nitrogen mustard in a subject in need thereof, wherein the kit comprises a pharmaceutical composition comprising an effective amount of a thrombopoietin (TPO) mimetic and a pharmaceutically acceptable carrier, wherein the TPO mimetic comprises the amino acid sequence of SEQ ID NO: 1, wherein, The thrombopoietin mimetic is RWJ-800088 or romiplostim, The toxic effect is pulmonary fibrosis caused by the nitrogen mustard; Optionally, the kit further comprises a means for administering the TPO mimetic to the subject.

7. The use of claim 6, wherein the kit further comprises at least one other therapeutic agent or device for alleviating the toxic effects; wherein the other therapeutic agent is selected from analgesics, antibacterial drugs, stem cell factors, Flt-3 ligands, hematopoietic factors, interleukins, growth factors, antibodies and chemotherapeutic agents.

8. The use of claim 7, wherein the other therapeutic agent is selected from atropine, atropine sulfate, homatropine, narcotic analgesics and N-acetylcysteine.

9. The use according to claim 7, wherein the other therapeutic agent is interleukin-3.

10. The use according to claim 6, wherein the toxic effect further comprises vascular damage caused by the nitrogen mustard.

11. The use according to claim 6, wherein the toxic effect further comprises pulmonary microhemorrhage caused by the nitrogen mustard.

12. The use of claim 6, wherein the toxic effects further comprise mortality or morbidity caused by the nitrogen mustard.

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