RU2841262C2 - Compositions for preventing or treating acute radiation sickness - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to use 1-palmitoyl-2-linoleoyl-3-acetylglycerola (PLAG) for treating or preventing acute radiation sickness in a subject also relates to use of 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG) for treatment subject exposed to adverse ionizing radiation, also refers to set for use in the treatment or prevention of acute radiation disease in subject including: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for using PLAG for treating or preventing acute radiation sickness (ARS) in a subject, also refers to set for use in the treatment or prevention of excessive effect of ionizing radiation, including: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for using PLAG for treating or preventing excessive exposure to ionizing radiation, also relates to a set for use in treating or preventing one or more subsyndromes of acute radiation sickness including: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for using PLAG for treating or preventing one or more sub-syndromes of acute radiation sickness.

EFFECT: group of inventions provides the induction of an increase in the levels of various immune cells, which were reduced by radiation on the ARS study models, in a significant increase in the survival rate of mice after irradiation, in the positive effect of PLAG on each symptom (reducing the level of mucositis and increasing the number of neutrophils in the blood), which affects the survival rate in the animal model of acute radiation sickness, and on the recovery of skin damage in the animals exposed to radiation.

12 cl, 8 tbl, 11 ex, 66 dwg

Description

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/691,604, filed June 28, 2018, and Korean Patent Application No. 10-2017-0162404, filed November 30, 2017, the contents of which are incorporated herein in their entirety for all purposes.

Field of technology to which the invention relates

In one aspect, methods and compositions comprising PLAG (1-palmitoyl-3-linoleoyl-3-acetylglycerol) are provided for the prevention, treatment, modulation, or amelioration of acute radiation sickness (ARS).

Prior art

Acute radiation sickness (ARS) is a disease that occurs when a large part of the human body is exposed to radiation; it is a dangerous disease that eventually leads to death by destroying the immune, hematopoietic, nervous and/or gastrointestinal systems. A nuclear explosion and a nuclear accident can result in radiation exposure that can cause acute radiation sickness.

The mainstay of treatment for patients exposed to radiation is the prevention and treatment of infection. Clinically, adjuvant therapy such as cytokines, antibiotics, and blood transfusions are given early after irradiation, and as the duration of neutropenia in patients increases, the risk of secondary infection also increases.

Thus, it is necessary to have new methods of treating acute radiation sickness.

Statement of the essence of the invention

In one aspect, new methods for the treatment and prevention of acute radiation sickness are provided that include PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol).

In another aspect, new methods are provided for treating a subject who has been exposed to ionizing radiation (especially adverse effects such as unintentional and/or non-therapeutic exposure, and/or exposure to excessive ionizing radiation, including gamma radiation), which comprises administering to the subject an effective amount of PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol).

In yet another additional aspect, methods of treating and preventing one or more subsyndromes of acute radiation sickness are provided, which comprise the use of PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol). Subsyndromes of ARS include hematopoietic, gastrointestinal, cutaneous and/or neurovascular.

In another aspect, methods are proposed for the treatment and prevention of hematopoietic (bone marrow) syndrome of acute radiation sickness, gastrointestinal syndrome of acute radiation sickness, cutaneous syndrome of acute radiation sickness, cardiovascular syndrome of acute radiation sickness, and/or acute radiation sickness of the central nervous system (CNS).

In an additional aspect, methods for the treatment and prevention of radiation-induced coagulopathy are proposed.

PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) can attenuate, prevent and/or treat the decrease in immune cells such as leukocytes, neutrophils and lymphocytes caused by radiation, and alleviate inflammatory diseases such as oral mucositis, and further improve the survival of subjects exposed to radiation.

PLAG may be administered to an individual for the first time even after a long period of time has passed since the individual was exposed to harmful radiation, such as gamma radiation. That is, the individual may receive therapeutic benefit, including increased survival time, even after such delayed treatment. See, for example, the in vivo results outlined in the following examples. This may be significant since the first treatment of an individual exposed to radiation may often be delayed.

Thus, in some aspects, PLAG is first administered (the first dose to the individual) within 3, 6, 12, 18, 24, 36, 48, 60, or 72 hours after the individual has been exposed to harmful radiation (including gamma radiation). In certain aspects, PLAG is first administered (the first dose to the individual) between 3 and 12, 18, 48, or 72 hours, or between 6 and 18, 24, or 48 hours after the individual has been exposed to harmful radiation (including gamma radiation). In certain aspects, the harmful radiation effect may be exposure to ionizing radiation, such as gamma radiation, from 1 Gy to 8 Gy or more for from 1 second to 30, 60, or 120 seconds or more.

In a further aspect, a pharmaceutical composition comprising PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) is provided for the prevention or treatment of acute radiation sickness.

Also provided is a pharmaceutical composition comprising PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) for the prevention or treatment of an individual exposed to ionizing radiation, in particular an individual exposed to excess ionizing radiation.

In another aspect, a functional health food or dietary supplement is provided that contains PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) as an active ingredient for the prevention or amelioration of acute radiation sickness.

In yet another further aspect, kits are provided for use in the treatment or prevention of acute radiation sickness, or in the treatment or prevention of exposure to excessive ionizing radiation.

Suitable kits of the invention may comprise (1) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); and (2) instructions for using PLAG to treat or prevent acute radiation sickness (ARS) in a subject or to treat or prevent exposure to excessive ionizing radiation. Preferably, the kit will contain a therapeutically effective amount of PLAG. The instructions may be in written form, including in the form of a product label.

The terms PLAG, EC-18 and 1-palmitoyl-2-linoleoyl-3-acetylglycerol are used interchangeably in this application and refer to the same compound.

In further aspects, a compound of Formula 1, as set forth below, may be used in the methods, compositions and kits of the invention, in particular for treating a subject suffering from acute radiation sickness or exposed to adverse ionizing radiation.

Other aspects are covered below.

Brief description of the drawings

The patent or application file shall contain at least one drawing executed in colour. Copies of this patent or patent application publication with colour drawings shall be provided by the Office upon request and payment of the required fee.

Figure 1 presents a schematic diagram of the experimental design related to radiation-induced acute radiation sickness in an animal model.

Figures 2A-2C show the blood levels of leukocytes (Figure 2A), neutrophils (Figure 2B), and lymphocytes (Figure 2C) after administration of PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) in an animal model of acute radiation sickness.

Figure 3 shows survival following PLAG administration in animal models of acute radiation sickness associated with oral mucositis.

Figure 4 shows the mucositis status after PLAG administration in an animal model of acute radiation sickness.

Figure 5 shows the estimated mucositis score in PLAG (EC-18)-treated and untreated animals in an animal model of acute radiation sickness associated with oral mucositis.

Figures 6A-6B show the blood neutrophil count after PLAG administration in an animal model of acute radiation sickness with oral mucositis at 7 days (Figure 6A) and 10 days (Figure 6B).

Figure 7A is a schematic diagram of EC-18 treatment (30 days) in an animal model of radiation-induced acute radiation syndrome. Figure 7B shows the survival of mice (BALB/c, 11 weeks) treated or not with EC-18 (250 mg/kg/day, p.o.) with total body irradiation (6.5 Gy). The survival rate in mice treated with EC-18 was improved compared to untreated (no EC-18) mice.

Figure 8 shows the survival of mice (BALB/c, 9 weeks) treated (n = 20) or not treated (n = 20) with EC-18 (250 mg/kg/day, p.o.) with total body irradiation (6.5 Gy) according to the schedule in Figure 7A. The survival of mice treated with EC-18 was improved compared to untreated (no EC-18) mice.

Figure 9 shows survival curves of mice (BALB/c, 9 weeks old) receiving total body irradiation (2, 4, and 6 Gy) without EC-18 treatment and with EC-18 treatment (250 mg/kg/day, PO), with total body irradiation at 6 Gy according to the schedule in Figure 7A.

Figure 10A is a schematic diagram of EC-18 treatment (17 days) in an animal model of radiation-induced acute radiation syndrome. Figure 10B shows the survival of mice (BALB/c, 9 weeks) treated or not with EC-18 (250 mg/kg/day, PO) with lethal body irradiation (7 Gy). The survival rate of EC-18-treated mice receiving lethal irradiation is improved until approximately day 15 compared to untreated (no EC-18) mice.

Figure 11 shows the survival of mice (BALB/c, 9 weeks) treated or not with EC-18 (250 mg/kg/day, p.o.) with lethal total body irradiation (8 Gy). The survival rate of mice treated with EC-18 is improved until approximately day 14 compared to untreated (no EC-18) mice, as plotted in Figure 10A.

Figure 12A is a schematic diagram of EC-18 treatment (12 days) in an animal model of radiation-induced acute radiation syndrome. Figure 12B shows the survival of mice (BALB/c, 9 weeks) treated or not with EC-18 (250 mg/kg/day PO) with lethal total body irradiation (10 Gy). The survival rate of irradiated mice treated with EC-18 is improved until approximately day 9 compared to untreated (no EC-18) mice according to the diagram in Figure 12A.

Figure 13A is a schematic diagram of EC-18 treatment (17 days) in an animal model of radiation-induced acute radiation syndrome. Figure 13B shows skin erythema in mice (BALB/c, 9 weeks old) treated or not with EC-18 (250 mg/kg/day orally) with lethal total body irradiation (8 Gy).

Figure 14A is a schematic diagram of EC-18 treatment (30 days) in an animal model of radiation-induced acute radiation syndrome. Figure 14B shows skin erythema in mice (BALB/c, 9 weeks old) treated or not with EC-18 (250 mg/kg/day orally) with total body irradiation (6.5 Gy).

Figure 15 shows skin erythema in mice (BALB/c, 11 weeks old) treated or not with EC-18 (250 mg/kg/day orally) with total body irradiation (6.5 Gy) according to the scheme in Figure 14A.

Figure 16 illustrates the schematic mechanism of action of EC-18 in radiation-induced neutropenia (RIN), which shows that EC-18 promotes the removal of DAMP (damage-associated molecular pattern) in a short period of time by inhibiting the continuous release of neutrophils from blood vessels.

Figure 17A shows the schematic diagram of the test for the effect of EC-18 on antiapoptosis in HaCaT cells. 5-fluorouracil and γ-irradiation induced damage to HaCaT.

Figure 17B shows the number of cells in the experiments depicted in Figure 17A according to 5-fluorouracil dose, irradiation, and PLAG (EC-18) treatment.

Figure 17C shows the frequency of early apoptosis (%) in the experiments shown in Figure 17A according to the amount of 5-fluorouracil, irradiation, and PLAG (EC-18) treatment.

Figure 17D shows the frequency of early apoptosis (%) in the experiments shown in Figure 17A at 1 ng/mL 5-fluorouracil, as a function of irradiation and PLAG (EC-18) treatment.

Figure 17E shows the frequency of early apoptosis (%) in the experiments depicted in Figure 17A at 10 ng/mL 5-fluorouracil as a function of irradiation and PLAG (EC-18) treatment.

Figure 17F shows the frequency of early apoptosis (%) in the experiments depicted in Figure 17A at 100 ng/mL 5-fluorouracil, as a function of irradiation and PLAG (EC-18) treatment.

Figure 18A shows the frequency of late apoptosis (%) in the experiments depicted in Figure 17A according to the amount of 5-fluorouracil, irradiation, and PLAG (EC-18) treatment.

Figure 18B shows the frequency of late apoptosis (%) in the experiments depicted in Figure 17A at 1 ng/mL 5-fluorouracil, as a function of irradiation and PLAG (EC-18) treatment.

Figure 18C shows the frequency of late apoptosis (%) in the experiments depicted in Figure 17A at 10 ng/mL 5-fluorouracil, depending on irradiation and PLAG (EC-18) treatment.

Figure 18B shows the frequency of late apoptosis (%) in the experiments depicted in Figure 17A at 100 ng/mL 5-fluorouracil, depending on irradiation and PLAG (EC-18) treatment.

Figure 19A illustrates a schematic diagram of testing the effect of EC-18 on intracellular ROS (reactive oxygen species) expression in HaCaT cells under 5-fluorouracil and γ-irradiation-induced HaCaT injury.

Figure 19B shows the results of ROS measurement in HaCaT cells exposed to 7 Gy γ-radiation after EC-18 treatment after irradiation or without treatment.

Figure 19C shows the number of HaCaT cells against ROS expression when cells were exposed to 7 Gy γ-irradiation, after EC-18 treatment after irradiation or without treatment (upper panel: 6 hours after exposure, lower panel: 24 hours after exposure).

Figure 20 illustrates a diagram for an exemplary experiment to test survival of EC-18 (PLAG) treatment in an animal model of acute radiation sickness induced by 6.5 Gy γ-rays (BALB/c mice, 11 weeks).

Figure 21 shows survival curves for BALB/c mice (n = 20 per group) not exposed to total body irradiation (control), exposed to 6.5 Gy of γ-radiation (whole body; untreated), and treated with EC-18 starting on Day 1 (24 hours post-irradiation) or Day 2 (48 hours post-irradiation).

Figure 22 shows the body weight of BALB/c mice that were not exposed to 6.5 Gy of γ-radiation and exposed to 6.5 Gy of γ-radiation (whole body), with EC-18 treatment at 24 or 48 hours after irradiation.

Figure 23 shows a schematic diagram of an experimental setup to test radiation-induced coagulopathy in a mouse model.

Figure 24 shows a diagram for an exemplary experiment including three groups (5-FU only, 5-FU + PLAG 125 mg/kg, 5-FU + PLAG 250 mg/kg) to test the time kinetics of neutrophils in 5-FU (100 mg/kg)-induced neutropenia.

Figure 25 shows in vitro experiments comparing the effects of EC-18 and G-CSF (granulocyte colony stimulating factor) by Western blotting.

Figure 26 is a table illustrating a comparison of EC-18 with G-CSF.

Figure 27 shows a diagram of the survival times of irradiated patients and the various ARS subsyndromes.

Figure 28A shows the survival of irradiated mice at each γ-radiation dose over 30 days of observation. Figure 28B shows the dose-response ratio (DRR) using probit models of BALB/c mice exposed to different doses of γ-radiation.

Figure 29 shows normalized changes in body weight of irradiated mice exposed to different doses of γ-radiation.

Figures 30A-B show the therapeutic effect of EC-18 administration in γ-ray-induced ARS. Figure 30A shows survival and Figure 30B shows body weight loss of mice irradiated with 6.11 Gy of γ-rays. *p < 0.05, **p < 0.01, ***p < 0.005, for 6.11 Gy vs. 6.11 Gy + EC-18 250 mg/kg.

Figures 31A-F show complete blood count parameters with differential white blood cell count in mice exposed to 6.11 Gy of γ radiation. Figures 31A-31F show white blood cell count (WBC; Figure 31A), absolute neutrophil count (ANC; Figure 31B), monocyte count (Figure 31C), absolute lymphocyte count (ALC; Figure 31D), platelet count (PLT; Figure 31E), and red blood cell count (RBC; Figure 31F), respectively. n = 5 mice per group.

Figures 32A-J show the therapeutic effect of EC-18 administration on complete blood count parameters with differential in mice exposed to 6.11 Gy of γ-radiation. Figures 32A-32E show the time course of white blood cell count (WBC; Figure 32A), absolute neutrophil count (ANC; Figure 32B), absolute lymphocyte count (ALC; Figure 32C), platelet count (PLT; Figure 32D), and red blood cell count (RBC; Figure 32E) over 15 days, respectively. Figures 32F-32J show the effect of EC-18 administration dose on WBC (Figure 32F), ANC (Figure 32G), ALC (Figure 32H), PLT (Figure 32I), and RBC (Figure 32J) at day 15, respectively; ns – non-significant differences; *p<0.05, **p<0.01, ***p<0.005.

Detailed description of the invention

I. Definitions

The abbreviations used in this application have their usual meanings in the fields of chemistry and biology. The chemical structures and formulas set forth in this application are constructed in accordance with the standard rules of chemical valency known in the field of chemistry.

As used in this application, the term "isomers" refers to compounds having the same number and type of atoms and hence the same molecular weight, but differing with respect to the structural arrangement or configuration of the atoms.

As used in this application, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and that are readily convertible from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds disclosed herein may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the invention.

The singular terms used herein denote one or more elements. For example, the singular forms are intended to also include the plural forms unless the context clearly indicates otherwise. It will be further understood that the terms "comprise," "include," "have," etc., when used in this description specify the presence of the claimed features, regions, integers, steps, processes, operations, elements, and/or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, processes, operations, elements, components, and/or combinations thereof.

"Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that facilitates the administration of the active agent and its absorption by the subject and can be included in the compositions of the present invention without causing significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, saline solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and colorants, and the like. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffers, colorants and/or flavoring substances and the like, which do not cause undesirable reactions with the compounds of the invention. The person skilled in the art will understand that other pharmaceutical auxiliary substances are useful in the present invention.

"Treatment" and "treatment" as used herein include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administration step may consist of a single administration or may include a series of administrations. The duration of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the compositions used in the treatment, or a combination thereof. It should also be understood that the effective dosage of the agent used for treatment or prophylaxis may increase or decrease during a given treatment or prophylaxis regimen. Changes in dosage may occur and be evident in standard diagnostic tests known in the art. In some cases, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a period of time sufficient to treat the patient. The term "treatment" and combinations thereof may include prevention of injury, pathology, condition, or disease. In some embodiments, treatment is prophylaxis. In some embodiments, treatment does not include prophylaxis.

The term "prevent" refers to reducing the occurrence of symptoms of a disease in a patient. As noted above, prevention may be complete (e.g., no detectable symptoms) or partial, such that fewer symptoms are observed than would occur without treatment.

The term "modulate" is used according to its simple, ordinary meaning and refers to the act of changing or varying one or more properties. "Modulation" refers to the process of changing or varying one or more properties. For example, a disease modulator reduces a symptom, cause, or characteristic of a target disease, such as ARS and its subsyndromes.

The terms "patient," "subject," "patient in need thereof," and "subject in need thereof" are used interchangeably herein and refer to a living organism suffering from or susceptible to a disease or condition that can be treated by administering a pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient or subject is a human.

An "effective amount" is an amount sufficient for a compound to achieve its intended purpose compared to the absence of the compound (e.g., to achieve the effect for which it is administered, to treat a disease, to decrease enzyme activity, to increase enzyme activity, to decrease the catabolic activity of an enzyme, or to reduce one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to facilitate the treatment, prevention, or reduction of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount". "Reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) means a decrease in the severity or frequency of the symptom(s) or the elimination of the symptom(s). A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, will produce the intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or the symptoms thereof. The full prophylactic effect is not necessarily manifested by a single dose, but may only be manifested after a series of doses. Thus, a prophylactically effective amount may be administered by one or more administrations. "Activity-reducing amount" as used herein refers to the amount of antagonist required to reduce enzyme activity compared to the absence of the antagonist. The term "function-impairing amount" as used herein refers to the amount of antagonist required to impair the function of an enzyme or protein compared to the absence of the antagonist. The exact amounts will depend on the purpose of treatment and will be determined by one skilled in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

A therapeutically effective amount of PLAG may be initially determined from cell culture assays. Target concentrations will be those concentrations of the active compound(s) that the methods described herein are capable of providing when measured using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for use in humans can also be determined in animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring the effectiveness of the compounds and adjusting the dosage upward or downward as described above. Adjusting the dose to achieve maximum effectiveness in humans based on the methods described above and other methods is within the capabilities of one of ordinary skill in the art.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a therapeutic agent sufficient to reduce a disorder as described above. For example, for a given parameter, a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%. 90%, or at least 100%. Therapeutic efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect, compared to a control.

Dosages may vary depending on the needs of the patient and the compound used. The dose administered to the patient in the context of the present invention should be sufficient to produce a positive therapeutic response in the patient over time. The size of the dose will also be determined by the presence, nature and extent of any side effects. Determining the correct dosage for a particular situation is within the competence of the practitioner. As a rule, treatment is initiated with smaller doses that are less than the optimal dose of the compound. Thereafter, the dosage is increased in small increments until the optimal effect is achieved under the particular circumstances. The amounts and intervals of dosage can be individually adjusted to provide levels of the compound administered that are effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is proportionate to the severity of the human pathological condition.

Using the principles presented in this application, it is possible to plan an effective prophylactic or therapeutic treatment regimen that does not cause significant toxicity and at the same time is effective in treating the clinical symptoms exhibited by a particular patient. This planning should include careful selection of the active compound, taking into account such factors as the potency of the compound, relative bioavailability, patient body weight, the presence and severity of side effects, the preferred route of administration, and the toxicity profile of the selected agent.

"Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that facilitates the administration of the active agent and its absorption by a subject and can be included in the compositions of the present invention without causing significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, saline solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and colorants, and the like. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing the osmotic pressure, buffers, colorants and/or flavoring substances and the like, which do not cause undesirable reactions with the compounds of the invention. A person skilled in the art will understand that other pharmaceutical auxiliary substances can be used in the present invention.

The term "preparation" is intended to include a composition of the active compound with an encapsulating material as a carrier providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier which is thus associated with it. Similarly, wafers and lozenges are included. Tablets, powders, capsules, pills, wafers and lozenges may be used as solid dosage forms suitable for oral administration.

As used herein, the term "administration" means oral administration, suppository administration, topical application, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a slow release device such as a mini-osmotic pump, into a subject. Administration by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal), is suitable for the preparation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

The compositions disclosed in this application can be delivered transdermally, topically, in the form of applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, wafers, gels, syrups, gruels, suspensions, etc., suitable for ingestion by a patient. Solid form preparations include powders, tablets, pills, capsules, wafers, suppositories and dispersible granules. Liquid form preparations include solutions, suspensions and emulsions, for example, solutions in water or in water/propylene glycol. The compositions of the present invention can further include components to provide sustained release and/or comfort. Such components include high molecular weight anionic mucomimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in U.S. Patent Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are hereby incorporated by reference in their entireties for all purposes. The compositions disclosed herein can also be delivered as microspheres for sustained release in the body. For example, the microspheres can be administered by intradermal injection of drug-containing microspheres that are slowly released subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7: 623-645, 1995); as biodegradable and injectable gel formulations (see, for example, Gao Pharm. Res. 12: 857-863, 1995) or as microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49: 669-674, 1997). In another embodiment, the formulations of the compositions of the present invention can be delivered using liposomes that fuse with the cell membrane or are capable of being endocytosed, i.e., using receptor ligands attached to the liposome that bind to cell surface membrane protein receptors, resulting in endocytosis. Using liposomes, especially when the surface of the liposomes carries receptor ligands specific for target cells or otherwise preferentially directed to a particular organ, it is possible to direct the delivery of the compositions of the present invention to target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989.) The compositions can also be delivered as nanoparticles.

Pharmaceutical compositions may include compositions in which the PLAG compound is contained in a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, among other things, on the condition being treated. When administered in ways to treat a disease, such compositions will contain an amount of the active ingredient effective to achieve the desired result, such as modulating the activity of a target molecule and/or reducing, eliminating, or slowing the progression of disease symptoms.

The dosage and frequency (single or multiple doses) of administration to a mammal may vary depending on a variety of factors, such as whether the mammal suffers from another disease and the route of administration; the height, age, sex, health condition, body weight, body mass index and diet of the patient; the nature and extent of the symptoms of the disease being treated, the type of concomitant treatment, complications from the disease being treated, or other related health problems. Other therapeutic regimens or agents may be used in combination with the methods and compounds of the present invention. Adjustment and manipulation of the prescribed dosages (e.g., frequency and duration) are within the skill of the art.

"Disease," "disorder," or "condition" refers to the state of health or well-being of a patient or subject that may be treated with the compounds or methods provided herein. As used herein, the term "acute radiation sickness" or "ARS" refers to a disease or disorder associated with radiation toxicity or radiation illness (e.g., acute radiation sickness (ARS), including hematopoietic (bone marrow) acute radiation syndrome, gastrointestinal acute radiation syndrome, cutaneous acute radiation syndrome, cardiovascular acute radiation syndrome, and/or central nervous system (CNS) acute radiation syndrome). The term "acute radiation sickness" or "ARS" may also include radiation-induced coagulopathy. In some embodiments, the disease is acute radiation sickness (ARS). In some embodiments, ARS occurs in a subject upon exposure to radiation of about 0.1 Gy (or 10 rads) or more, about 0.2 Gy (or 20 rads) or more, about 0.3 Gy (or 30 rads) or more, about 0.4 Gy (or 40 rad) or more, about 0.5 Gy (or 50 rad) or more, about 0.6 Gy (or 60 rad) or more, about 0.7 Gy (or 70 rad) or more, about 0.8 Gy (or 80 rad) or more, about 0.9 Gy (or 90 rad) or more, about 1 Gy (or 100 rad) or more, about 2.0 Gy (or 200 rad), about 3.0 Gy (or 300 rad), or about 4.0 Gy (or 400 rad) or more.

In some embodiments, ARS may occur in a subject upon exposure to radiation (including gamma radiation) of from about 1 Gy (or 100 rad) to about 8 Gy (or 800 rad) for any varying periods of time, such as at least 1, 2, 5, 10, 30, 60, 80, 120, 180, 240, or 300 seconds.

In some embodiments, the methods and compositions disclosed herein for treating a subject that has been exposed to ionizing radiation (particularly, has been exposed to excessive ionizing radiation, including gamma radiation) may comprise using or administering to the subject an effective amount of PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) in a manner suitable when the subject is exposed to radiation of from about 1 Gy (or 100 rads) to 8 Gy (or 800 rads) or more for any varying periods of time, such as at least 1, 2, 5, 10, 30, 60, 80, 120, 180, 240, or 300 seconds.

In some embodiments, the disease or disorder comprises cutaneous radiation syndrome, such as skin lesions, erythema, altered sensation, itching, swelling, blistering, scaling, ulcers, necrosis, hair loss, onycholysis, and the like. In some embodiments, the disease or disorder comprises a decrease in neutrophils or an infection caused by a decrease in the number of white blood cells. In some embodiments, the disease or disorder comprises hemorrhage. In some embodiments, the disease or disorder comprises diarrhea. In some embodiments, the disease or disorder comprises dehydration or electrolyte imbalance. In some embodiments, the disease or disorder comprises seizures and/or coma.

As discussed, in some embodiments, the methods and compositions disclosed herein are used to treat a subject that has been exposed to ionizing radiation. In some aspects, the exposure may be unintentional or accidental. In further aspects, the radiation will not be used for therapeutic purposes, such as conventional radiation will not be radiotherapy that can be used to treat cancer or other therapy. In further aspects, at least a substantial portion (e.g., an entire limb and/or torso and/or an entire head region) of the subject may be exposed to radiation.

As described herein, any one or more of such unintentional, incidental, non-therapeutic and/or significant effects on a body part may be referred to as "adverse" exposure.

II. Compositions

As discussed, one aspect of the present invention provides a therapeutic pharmaceutical composition for the prevention or treatment of acute radiation syndrome, comprising PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) (also referred to herein as EC-18) as an active ingredient.

As used in this application, the term "acute radiation sickness" is an acute illness caused by radiation exposure to the whole body or a significant portion of the body; acute radiation sickness is also known as radiation toxicity or radiation syndrome. Radiation is the energy released when an unstable nucleus is transformed into another nucleus. When radiation passes through the body, the radiation energy is absorbed and can cause ionization in the tissue. At this time, _H2O is ionized and can transform the subject's DNA.

Clinical characteristics of acute radiation sickness include hematopoietic syndrome, gastrointestinal syndrome, cardiovascular syndrome, and neurovascular syndrome.

As described above, acute radiation sickness can damage radiation-sensitive systems such as the immune, hematopoietic and gastrointestinal systems, and lead to death. Therefore, the most important factor to be considered for the preventive or therapeutic effect on acute radiation sickness is to increase the survival of subjects exposed to radiation. In the present invention, acute radiation sickness can be any of the symptoms of hematopoietic syndrome, gastrointestinal syndrome, cardiovascular syndrome and neurovascular syndrome, but at the same time, acute radiation sickness is not limited to the above symptoms. The composition of the present invention is effective in preventing, treating or alleviating the above symptoms and ultimately improves the survival of subjects exposed to radiation.

Acute radiation sickness typically progresses through four clinical stages: prodromal phase, latent phase, manifest phase, and recovery or death. Depending on the amount of radiation absorbed, symptoms may appear within hours or weeks. The prodromal phase usually begins within 48 hours of exposure but may last up to 6 days after exposure, and symptoms may include nausea, vomiting, fatigue, autonomic instability, and loss of consciousness. The latent phase may last from days to weeks depending on the degree of radiation exposure, and clinical symptoms may not appear completely or partially. However, symptoms such as lymphopenia, granulocytopenia, and myelogenous deficiency may occur at this stage. Symptoms of the manifest phase may be delayed for several weeks. Symptoms of the manifest phase may include hematopoietic syndrome, gastrointestinal syndrome, cardiovascular syndrome, and neurovascular syndrome, depending on the degree of radiation exposure. Patients exposed to radiation can go through all four stages within a few hours and die within a short period of time.

Among acute radiation sickness, a hematopoietic syndrome caused, induced, or associated with a radiation dose of approximately 0.7-10 Gy, a gastrointestinal syndrome caused, induced, or associated with a radiation dose of approximately 10-30 Gy, and a cardiovascular/neurovascular syndrome caused, induced, or associated with a radiation dose of approximately 50 Gy can be distinguished. In exemplary embodiments of the present invention, acute radiation syndrome includes syndromes caused, induced, or caused by a radiation dose of about 0.1 to 100 Gy, about 0.1 to 80 Gy, about 0.1 to 70 Gy, about 0.1 to 60 Gy, about 0.1 to 50 Gy, or about 0.7 or 50 Gy, and may be caused, induced, or caused by a radiation dose of about 0.1 Gy, about 0.5 Gy, about 0.7 Gy, about 1.0 Gy, about 2.0 Gy, about 3.0 Gy, or about 4.0 Gy or more that can result in death in a particularly exposed subject (Reports of Practical Oncology and Radiotherapy, 2011, 16 (4): 123-130), but is not limited thereto.

In further exemplary embodiments, acute radiation sickness includes syndromes caused, induced, or caused by a radiation dose of from about 1 Gy (or 100 rad) to about 8 Gy (or 800 rad) over any various periods of time, such as at least 1, 2, 5, 10, 30, 60, 80, 120, 180, 240, or 300 seconds.

As used in this application, the term "prevention" means any action that inhibits or delays the onset, spread, or recurrence of a specified disorder or disease, such as acute radiation sickness, upon administration of a compound or composition as described herein. As used in this application, the term "treatment" means any action that improves or alleviates the symptoms of a specified disorder or disease, such as acute radiation sickness, following administration of a compound or composition as described herein.

In certain aspects, the compound administered to the subject has the following Formula 1:

Formula 1,

where R ₁ and R ₂ independently represent a fatty acid residue with 14-20 carbon atoms.

Glycerol derivatives of Formula I above are sometimes referred to in this application as monoacetyldiacylglycerols (MDAGs). The fatty acid moiety refers to the acyl moiety resulting from the formation of an ester bond upon reaction of the fatty acid and the alcohol. Non-limiting examples of R ₁ and R ₂ thus include palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, arachidonoyl, etc. Preferred combinations of R ₁ and R ₂ (R ₁ / R 2) include oleoyl / palmitoyl, palmitoyl / oleoyl, palmitoyl / linoleoyl, palmitoyl / linolenoyl, palmitoyl / arachidonoyl, palmitoyl / stearoyl, palmitoyl / palmitoyl, oleoyl / stearoyl, linoleoyl / palmitoyl, linoleoyl / stearoyl, stearoyl / linoleoyl, stearoyl / oleoyl, myristoyl / linoleoyl, myristoyl / oleoyl, etc. In optical activity, the monoacetyldiacylglycerol derivatives of Formula 1 may be (R)-form, (S)-form or racemic mixture, and may include stereoisomers thereof. In certain aspects, in compounds wherein the substituents R ₁ and/or R ₂ are unsaturated fatty acid residues, one or more double bonds that are suitably present may have a cis configuration. In other aspects, one or more double bonds of the substituents R ₁ and/or R ₂ may be present in a trans configuration. In certain aspects, such one or more double bonds will only be present in a cis configuration.

In a certain preferred aspect, the compound administered to the subject is PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) having the structure of the following chemical Formula 2 (a compound which is also sometimes referred to herein as EC-18):

In addition, in the present invention, PLAG may include all of the above-mentioned compounds of Chemical Formula 2 and other obvious derivatives from chemical transformations known in the industry. For example, addition and substitution reactions for increasing the stability of the compound or for preparing the compound may be carried out in a range that does not affect the pharmacological effect of PLAG, and all of these chemical derivatives are included in the scope of the present invention.

PLAG is known to have a therapeutic effect on neutropenia, thrombocytopenia, and mucositis caused by cancer chemotherapy. However, the effectiveness of PLAG in the prevention or treatment of acute radiation syndrome, including the ability to improve survival in people with acute radiation syndrome, has not been reported.

In an exemplary embodiment, it was found that PLAG significantly increases the number of leukocytes, neutrophils and lymphocytes in the blood of subjects exposed to radiation (Table 1 and Figure 2) and improves mucositis in an animal model of severe acute radiation sickness, where oral mucositis was induced by chemoradiotherapy (Figures 4 and 5) and increases the number of neutrophils in the blood (Figure 6), and as a result, PLAG significantly increases the survival of the study subjects (Figure 3). Therefore, the composition of the present invention has an excellent effect as a therapeutic pharmaceutical composition for the prevention or treatment of acute radiation sickness.

The therapeutic pharmaceutical composition of the present invention can be administered within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 12 hours, within 16 hours, within 20 hours, within 30 hours, within 40 hours or within 48 hours after irradiation, but not limited thereto, as discussed above.

In addition, the above composition can be used as a single therapeutic agent, or can be used in combination with another drug known to be effective in the treatment of acute radiation sickness. For example, the above composition can be used with one or more therapeutic agents including proteins, small molecule drugs, nucleic acids or the like. For example, the composition can be used with a therapeutic agent including granulocyte colony-stimulating factor (G-CSF), but the use is not limited thereto. In addition, the above composition can be used together with analgesics, antiulcer agents, antidiarrheals, antibiotics, antipyretics, nutritional supplements and antioxidants that can help in the prevention or treatment of acute radiation sickness.

The term "use" in the present invention means administering the therapeutic pharmaceutical composition of the present invention to a patient by any suitable route, and the route of use of the composition of the present invention can be carried out by various routes, both orally and non-orally. The therapeutic pharmaceutical composition of the present invention can be prepared in the form of various compositions depending on the routes of use.

The frequency of application of the composition of the present invention is not particularly limited, but it may be applied once a day or several times a day in divided doses.

The therapeutic pharmaceutical composition of the present invention can be used as a single drug and can be used as a combination drug containing another drug, and can be prepared using a pharmaceutically acceptable carrier, excipient or diluent to obtain a single dose or a multi-dose container.

The term "pharmaceutical composition" in the context of the present description means a composition prepared for the purpose of preventing or treating diseases, and which can be formulated in various forms according to conventional methods. For example, it can be formulated in the form of oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated in a form for external use, in the form of suppositories and sterilized injection solutions.

In addition, the pharmaceutical composition of the present invention can be prepared with an additional pharmaceutically acceptable carrier for each formulation. The term "pharmaceutically acceptable carrier" used in the present application may refer to a carrier or diluent that does not stimulate the body and does not inhibit the biological activity and characteristic of the administered compound. The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the industrial field and pharmaceutically acceptable can be used.

Saline solution, sterilized water, intravenous fluids, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol are non-limiting examples of carriers used. These carriers can be used alone or in combination of two or more of them. The carrier can include a non-natural carrier. If necessary, other commonly used additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. The carrier can be composed of a diluent, a dispersant, a surfactant, a binder, a lubricant for preparing an injection solution such as an aqueous solution, a suspension, an emulsion, and pills, capsules, granules or tablets and the like.

In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of PLAG. The term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and is typically in the range of about 0.001 to 5000 mg/kg, preferably about 0.05 to 1000 mg/kg, which can be administered once a day or several times a day in divided dosages. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient will depend on the nature and degree of response to be achieved, the particular composition, including the use of other agents, the age, body weight, sex and diet of the patient, the time of administration, the route of administration and the proportion of the composition, the duration of treatment, drugs used or administered together with the particular composition, and similar compounds well known in the medical industry.

As already mentioned, kits are also provided. For example, in this aspect, the PLAG compound can be suitably packaged in suitable containers labeled, for example, for use as a therapy for treating a subject suffering from acute radiation sickness or a subsyndrome thereof, or exposed to excessive ionizing radiation (e.g., gamma radiation). The containers can include the PLAG compound or composition and one or more of a suitable stabilizer, a carrier molecule, and/or the like, depending on the intended use. In other embodiments, the kit further comprises one or more therapeutic agents that alleviate some of the symptoms or secondary infections or disorders that can be associated with acute radiation sickness or a subsyndrome thereof, or with exposure to excessive ionizing radiation (e.g., gamma radiation). Accordingly, packaged products (e.g., sterile containers containing one or more compositions described herein and packaged for storage, shipment, or sale in concentrated form or ready-to-use concentrations) and kits comprising a PLAG compound and instructions for use are also within the scope of the invention. A product may include a container (e.g., a vial, jar, bottle, bag, or the like) containing a PLAG compound or composition. In addition, an article or kit may further include, for example, packaging materials, instructions for use, syringes, delivery devices for treating or monitoring a condition for which prevention or treatment is required.

The product may also contain accompanying information (e.g., a printed label or insert, or other medium describing the use of the product (e.g., an audio or video cassette)). The accompanying information may be associated with the container (e.g., attached to the container) and may describe the manner in which the compositions are to be used (e.g., frequency and route of administration), directions, and other uses. The compositions may be ready for administration (e.g., present in appropriate dosage units) and may include one or more additional pharmaceutically acceptable adjuvants, carriers, or other diluents, and/or an additional therapeutic agent. Alternatively, the compositions, for example, may be presented in concentrated form with a diluent and instructions for dilution.

Another aspect of the present invention is a functional health food composition for the prevention or amelioration of acute radiation sickness, comprising PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) as an active ingredient.

In the present invention, the term "improvement" means all actions that at least reduce the degree of a symptom associated with the condition being treated. In this case, the food health functional composition can be used simultaneously or separately with a drug for treatment before or after the onset of a disease for the prevention or relief of acute radiation sickness.

PLAG does not exhibit significant toxicity to cells and has an improving effect on acute radiation syndrome, so PLAG can be prepared and taken in the form of a food-based functional health composition.

Functional food is the same term as food for special health uses (FoSHU). It refers to products that have been processed in such a way that the biological control function is more effective in addition to the nutritional value. The food can be prepared in various forms such as tablets, capsules, powders, granules, liquids, rings, etc., to achieve a beneficial effect in terms of skin regeneration.

For this purpose, the content level of PLAG contained in the health functional food is not specifically limited, but may be from 0.01 to 100 mass%, in particular from 1 to 80 mass%, based on the total mass of the health functional food.

The health functional food composition of the present invention may also contain a pharmaceutically acceptable carrier.

There is no particular limitation on the types of health functional foods including the PLAG of the present invention, and examples thereof include drinks, chewing gum, tea, vitamin complex, health food supplements and the like. Other ingredients that do not affect the effect of alleviating acute radiation sickness may be added to the food, and their type is not particularly limited. For example, various plant extracts, dietary acceptable food supplements or other natural carbohydrates may be added as an additional ingredient.

The food additive described above is added to obtain the health functional food of each composition, and it can be appropriately selected and used by a person skilled in the relevant technical field. For example, various nutritional additives, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjuster, stabilizer, preservative, glycerin, alcohol, gas-forming agent used in carbonated drink, and the like can be used, but their types are not limited to the above.

In addition, the above-described health functional food may contain additional ingredients that are commonly used in food to improve the smell, taste, appearance, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, the food may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu), and the like. It may also contain amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the described health functional food may include one or more preservatives (such as potassium sorbate, sodium benzoate, salicylic acid and sodium dehydroacetate), bactericides (such as bleaching powder and high-strength bleaching powder, sodium hypochlorite), antioxidants (butyl hydroxyanilide (BHA), butyl hydroxytoluene (BHT), etc.), dyes (such as aniline pigments), color-forming agents (such as sodium nitrite and sodium acetates), bleaching agents (sodium sulfite), seasonings (such as MSG, monosodium glutamate), sweeteners (such as dulcin, cyclamate, sodium saccharin), flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen-D-tartrate), fortifier, emulsifiers, thickeners, encapsulating agents, gum bases, inhibitors foaming, solvent, improver, etc. The above additives are selected depending on the type of food and used in the appropriate quantity.

The health functional food composition of the present invention can be produced by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the industry. In addition, unlike a conventional medicine, the health functional food can have an advantage, for example, since there can be no side effect when used for a long time, and better tolerability is noted.

The pharmaceutical composition can be prepared and administered in a wide variety of dosage forms. The described compounds can be administered orally, rectally, or by injection (e.g., intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally).

For preparing pharmaceutical compositions from the compounds described herein, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, wafers, suppositories, and dispersible granules. A solid carrier may be one or more substances that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrants, or an encapsulating material.

In powders, the carrier may be a finely divided solid mixed with the finely divided active ingredient. In tablets, the active ingredient may be mixed with a carrier having the necessary binding properties in suitable proportions and compressed to the required shape and size.

Powders and tablets preferably contain from 5% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting wax, cocoa butter and the like. The term "preparation" is intended to include a composition of the active compound with an encapsulating material as a carrier providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier which is thus associated with it. Likewise, wafers and lozenges are included. Tablets, powders, capsules, pills, wafers and lozenges can be used as solid dosage forms suitable for oral administration.

To prepare suppositories, a low-melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is dispersed uniformly in it, for example by stirring. The melted homogeneous mixture is then poured into molds of convenient size, allowed to cool and thereby harden.

Liquid forms of preparations include solutions, suspensions, and emulsions, such as aqueous solutions or water/propylene glycol solutions. For parenteral administration, liquid preparations may be prepared as a solution in aqueous polyethylene glycol.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers and thickeners, as required. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active component in water with viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.

Also included are solid forms of preparations which are intended to be converted, shortly before use, into liquid preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active ingredient, colouring agents, flavourings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents and the like.

The pharmaceutical preparation is preferably in a unit dosage form. In such a form, the preparation is divided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, the package containing individual quantities of the preparation, such as packaged tablets, capsules, and powders in vials or ampoules. In addition, the unit dosage form may be a capsule, tablet, cachet, or lozenge, or it may be an appropriate quantity of any of these in packaged form.

The amount of active ingredient in a single dose preparation may vary or be adjusted from 0.1 mg to 10,000 mg depending on the specific application and the content of the active ingredient. The composition may also contain other compatible therapeutic agents, if necessary.

Some compounds may have limited solubility in water and, therefore, may require a surfactant or other suitable cosolvent in the composition. Such cosolvents include Polysorbate 20, 60, and 80; Pluronic F-68, F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Such cosolvents are typically used in an amount of from about 0.01% to about 2% by weight. Viscosity greater than that of simple aqueous solutions may be necessary to reduce variability in dosing of the formulations, to reduce physical separation of the components of a suspension or emulsion of the formulation, and/or to improve other properties of the formulation. Such viscosity increasing agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, chondroitin sulfate and its salts, hyaluronic acid and its salts, and combinations thereof. Such agents are typically used in an amount of about 0.01% by weight to 2% by weight.

The pharmaceutical compositions may further include components to provide sustained release and/or comfort. Such components include high molecular weight anionic mucomimetic polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in U.S. Patent Nos. 4,911,920, 5,403,841, 5,212,162, and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.

The pharmaceutical composition may be intended for intravenous use. The pharmaceutically acceptable excipient may include buffers to adjust the pH to the desired range for intravenous use. Many buffers are known, including inorganic acid salts such as phosphate, borate, and sulfate.

Effective dosages

The pharmaceutical composition may include compositions in which the active ingredient is contained in a therapeutically effective amount, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, among other things, on the condition being treated.

The dosage and frequency (single or multiple doses) of the compounds administered may vary depending on a variety of factors, including the route of administration; the recipient's height, age, sex, health, body weight, body mass index, and diet; the nature and extent of the symptoms of the disease being treated; the presence of other diseases or other health problems; the type of concomitant treatment; and complications from any disease or treatment regimen. Other therapeutic regimens or agents may be used in combination with the methods and compounds disclosed herein.

Dosages may vary depending on the requirements of the subject and the compound used. The dose administered to a subject, in the context of the pharmaceutical compositions provided herein, should be sufficient to provide a beneficial therapeutic response in the subject over time. The size of the dose will also be determined by the presence, nature, and extent of any adverse effects. Typically, treatment is initiated with lower doses that are less than the optimal dose of the compound. The dosage is then increased in small increments until the optimal effect is achieved under the given circumstances.

Dosage amounts and intervals may be individually adjusted to provide levels of administered compounds that are effective for the specific clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's pathological condition.

Using the teachings presented in this application, it is possible to plan an effective prophylactic or therapeutic treatment regimen that does not cause significant toxicity and yet is fully effective in treating the clinical symptoms exhibited by a particular patient. This planning should include careful selection of the active compound, taking into account such factors as the potency of the compound, relative bioavailability, patient body weight, the presence and severity of adverse side effects, the preferred route of administration, and the toxicity profile of the selected agent.

Toxicity

The relationship between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between the LD ₅₀ (the amount of compound that causes death in 50% of the population) and the ED ₅₀ (the amount of compound that produces an effect in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from cell culture assays and/or animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of plasma concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration employed. See, for example, Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.1, pl, 1975. The exact composition, route of administration, and dosage may be selected by the individual physician taking into account the patient's condition and the particular method in which the compound is used.

When parenteral administration is necessary or desirable, particularly suitable mixtures for the compounds included in the pharmaceutical composition may be injectables, sterile solutions, oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous dextrose solutions, saline, purified water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene block copolymers, and the like. Ampoules are convenient unit doses. Pharmaceutical additives suitable for use in the pharmaceutical compositions provided herein may include those described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, the teachings of both of which documents are hereby incorporated by reference.

III. Treatment methods

Another aspect of the present invention is a method for preventing or treating acute radiation sickness, comprising the step of administering PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) to a subject.

The definition of PLAG (also referred to in this application as EC-18) and acute radiation syndrome is given above.

Since PLAG has a prophylactic and therapeutic effect on acute radiation sickness, it is possible to prevent or treat acute radiation sickness by administering a composition containing PLAG to an individual.

PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) has an excellent effect in the prevention and treatment of acute radiation sickness by increasing the survival rate of subjects exposed to radiation. Accordingly, the pharmaceutical composition and the food health functional composition containing PLAG as an active ingredient of the present invention can be effectively used for the prevention, treatment or alleviation of acute radiation sickness.

IV. Examples

Although the foregoing section has been described in some detail for purposes of illustration and example for clarity of understanding, it will be apparent to those skilled in the art that certain minor changes and modifications will be practiced in light of the foregoing. Accordingly, the description and examples should not be construed as limiting the scope of any invention set forth in this application.

All references cited in this application, including patent applications and publications, are incorporated herein by reference in their entirety.

Example 1: Effect of PLAG (1-palmitoyl-2-linoleoyl-3-acetylglycerol) on the reduction of immune cells in the blood in an animal model of acute radiation sickness (ARS).

BALB/c mice (9-week-old male) were obtained from Koatech (Pyeongtaek, Republic of Korea) and maintained in a specific pathogen-free (SPF) medium to establish an animal model of acute radiation sickness. To evaluate the effect of PLAG on the reduction of immune cells by radiation, the following three groups were used for this study: (1) normal control group (positive control); (2) radiation therapy - radiation-induced leukopenia (RIL) group (negative control group), and (3) RIL + PLAG treatment group (experimental group).

Specifically, mice in the RIL and RIL + PLAG groups were exposed to whole body irradiation at 1 Gy (100 rad = 1.06 min). For the RIL + PLAG treatment group, 50 mg/kg/day PLAG (Enzychem Lifesciences Co., Daejeon, Republic of Korea) was orally administered daily for 4 consecutive days after irradiation. The normal control group was not exposed to irradiation. On the 5th day after irradiation, blood samples were collected, and then a complete blood count (CBC) was performed using a Mindray BC-5300 automatic hematology analyzer (Shenzhen Mindray Bio-medical Electronics, China) to count the numbers of leukocytes, neutrophils, and lymphocytes in the blood (Figure 1).

As a result, in the RIL + PLAG group, the level of leukocytes, neutrophils and lymphocytes in the blood was 63% (2.18 ± 0.31 vs. 3.56 ± 0.84), 34% (0.87 ± 0.04 vs. 1.17 ± 0.35) and 85% ± 0.25 vs. 2.2 ± 0.42) (Table 1 and Figure 2), increasing statistically significantly.

Table 1

Normal control
(n=3) RIL
(n=3) RIL + PLUG
(n=3) Leukocytes (10 ³ /μl) 7.76 ± 1.96 2.18 ± 0.31 3.56 ± 0.84 Neutrophils (10 ³ /μl) 1.32 ± 0.53 0.87 ± 0.04 1.17 ± 0.35 Lymphocytes (10 ³ /µl) 6.05 ± 1.36 1.19 ± 0.25 2.2 ± 0.42

Therefore, it is evident from the above results that PLAG exhibits the effect of inducing an increase in the levels of various immune cells that were reduced by radiation exposure in the ARS study model.

Example 2: Effect of PLAG on survival in an animal model of acute radiation sickness (ARS).

In order to further confirm the effect of PLAG on acute radiation sickness, an animal model of radiation-induced oral mucositis was developed and tested as an animal model of acute radiation sickness with more severe conditions than those in Example 1 above. First, BALB/c mice (female, 8 weeks old) were obtained from Koatech (Republic of Korea) and maintained in a specific pathogen-free (SPF) medium to establish a model of radiation-induced oral mucositis. In this example, the first group with induced oral mucositis (negative control) was compared with the second group where PLAG was orally administered to the animals with induced oral mucositis.

Specifically, mice were exposed to 1 Gy of whole body gamma irradiation and 0.2 cm scratches were made on the tongue of mice at 0, 7, 10, and 16 days with the same force and depth using an 18-gauge needle. On day 2 after irradiation, 5-fluorouracil (50 mg/kg/day) was administered intraperitoneally and PLAG was administered orally at 250 mg/kg/day for 18 days (Table 2).

Table 2

Induced oral mucositis group Group with induced oral mucositis + PLGA application Number of subjects 7 (females) 7 (females) Scratch on the tongue
(0, 2, 10 and 16 days) 0.2 cm 0.2 cm Gamma irradiation (Day 2) 1 Gr 1 Gr Dosage/day Phosphate buffer solution PLAG (250 mg/kg) Method of administration/day Orally
0 or 18 days Orally
0 or 18 days Complete blood count/calendar 7th and 10th day 7th and 10th day

As a result, the survival rate in the oral mucositis-induced group was only 28% (2/7, 72%) on day 18, while there was 85% (6/7, 15% (Figure 3)) survival in the oral mucositis-induced group treated with PLAG. These results indicate that PLAG treatment significantly improved the survival rate of mice despite the decrease in hematopoietic cells from gamma irradiation and 5-fluorouracil treatment, as well as the increased risk of infection caused by oral wounds with tongue scratch. This also suggests that PLAG has excellent preventive and therapeutic effects.

Example 3: Effects of PLAG on each symptom affecting survival in an animal model of acute radiation sickness.

In the animal model used in Example 2 above, the level of oral mucositis and the number of neutrophils in the blood were determined.

The oral mucositis rate was determined by counting the ulceration and swelling of the tongue and the incidence of wound atrophy, and calculating the oral mucositis score for a total of 5 observers with data blinding. On the 7th and 10th days, blood was collected in the experimental groups, and the number of neutrophils in the blood was measured by performing a complete blood count (CBC) using a Mindray BC-5300 automatic hematology analyzer (Shenzhen Mindray Bio-medical Electronics, China).

As a result, visually confirmed oral mucositis levels were significantly reduced in the PLAG-treated group compared to the non-PLAG-treated group, with less ulceration and tongue swelling and a lower incidence of wound atrophy (Figure 4). The oral mucositis score was also significantly reduced (Figure 5). At days 7 and 10, the blood neutrophil count was reduced by approximately 95% in the radiation oral mucositis group at 10 days, but the neutrophil count was significantly increased in the daily PLAG-treated group (Figure 6).

Example 4: Improving Survival

To evaluate the effect of PLAG on survival in animal models exposed to radiation, the following experimental groups were used, as shown in Table 3, and the results are presented in Figure 7B and Figures 8-12. As discussed in the Examples above, the first group of untreated (negative control) mice with ARS induction after irradiation was compared to the second group of treated mice with ARS induction that received PLAG after irradiation.

Table 3

Experimental groups Figure 7B • Balb/c: 11 weeks, males (n=10) or females (n=10)
• γ-irradiation: 6.5 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 8 • Balb/c: 9 weeks, males (n=10) or females (n=10)
• γ-irradiation: 6.5 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 9 • Balb/c: 9 weeks, males (n=5) or females (n=5)
• γ-irradiation: 2, 4, 6 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation (2 Gy)
2) group that did not receive treatment after irradiation (4 Gy)
3) group that did not receive treatment after irradiation (6 Gy)
4) group receiving EC-18 after irradiation (6 Gy) Figure 10B • Balb/c: 9 weeks, males (n=5) or females (n=5)
• γ-irradiation: 7 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 11 • Balb/c: 9 weeks, males (n=5) or females (n=5)
• γ-irradiation: 8 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 12 • Balb/c: 9 weeks, males (n=2) or females (n=2)
• γ-irradiation: 10 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation

Example 5: Treatment of erythema

To evaluate the effect of PLAG on skin damage repair in animals exposed to radiation, the following experimental groups were used as shown in Table 4 and the results are shown in Figures 13-15. As discussed in the Examples above, the first group of untreated mice (negative control) after irradiation was compared to the second group of mice treated with PLAG after irradiation.

Table 4

Experimental group Figure 13B • Balb/c: 9 weeks, males (n=5) or females (n=5)
• γ-irradiation: 8 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 14B • Balb/c: 9 weeks, males (n=10) or females (n=10)
• Γ-irradiation: 6.5 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation Figure 15 • Balb/c: 11 weeks, males (n=10) or females (n=10)
• γ-irradiation: 6.5 Gy (100 rad = 1.06 min), total irradiation
• EC-18: 250 mg/kg/day (oral)
• Group: 1) group that did not receive treatment after irradiation
2) group receiving EC-18 after irradiation

Example 6: Effect on antiapoptosis of HaCaT cells.

To evaluate the effects of PLAG on irradiated HaCaT cells, the following experimental groups were used and the results are shown in Figures 17B-18D.

• HaCaT: human keratinocytes

• PLAG: pre-treatment with 100 µg/ml for 1 hour

• 5-fluorouracil: 1, 10, 100 ng/ml

• Gamma radiation: 7 Gy

• check apoptosis after 24 hours

• Cell counting after trypan blue staining

• Apoptosis (annexin V + 7-amino-actinomycin D)

Example 7. Effect on reactive oxygen species (ROS) of HaCaT cells.

To evaluate the effect of PLAG on irradiated HaCaT cells, the experimental groups as in Example 6 were used and the results are shown in Figures 19A-19C.

Example 8: Improving Survival

Figure 20 is an exemplary graph for testing the survival rates of treatment in an animal model of acute radiation sickness induced by 6.5 Gy γ-irradiation (mice, BALB/c, 11 weeks). Figure 21 shows the results of the survival curves of BALB/c mice exposed to 6.5 Gy γ-irradiation with daily treatment with EC-18 after irradiation. NC represents the untreated control without irradiation; 6.5 Gy is the survival of mice exposed to 6.5 Gy γ-irradiation; 6.5 Gy + PLAG (+ 1d) is the survival of mice exposed to 6.5 Gy γ-irradiation and treated with PLAG for 1 day; and 6.5 Gy + PLAG (+ 2d) – survival of mice exposed to 6.5 Gy γ-irradiation and treated with PLAG for 2 days. Figure 22 shows the changes in body weight of BALB/c mice in Figure 21.

Various assays can be established to measure survival after γ-irradiation in animal models. For example, Figure 23 depicts an exemplary diagram for an exemplary survival testing experiment in mouse models. Figure 24 depicts an exemplary diagram for an exemplary experiment including three groups (5-FU only, 5-FU + PLAG 125 mg/kg, 5-FU + PLAG 250 mg/kg) to test the time kinetics of neutrophils in 5-fluorouracil (100 mg/kg)-induced neutropenia.

Example 9. PLAG effects measured in vitro (Western blot).

The effect of EC-18 compared to G-CSF on cells after γ-irradiation was measured by Western blotting (Figure 25). The experiment allows (i) to show the correlation between EGFR (epidermal growth factor receptor) activity and abnormal growth and induction of metastasis of breast cancer cells in TAN medium; and (ii) to confirm the mechanism of inhibitory effect of PLAG treatment in this condition. In fact, it was confirmed that EGFR activity was increased in neutrophil-stimulated or G-CSF-activated neutrophils groups, however, EGFR activity (EGFR phosphorylation) was decreased in PLAG-treated group. Figure 26 allows to show the effects of PLAG compared to G-CSF.

Example 10: Survival time for different types of radiation sickness.

Figure 27 presents a diagram of the survival times of patients exposed to radiation and the various ARS syndromes.

Example 11: EC-18 for the treatment of acute radiation sickness

We investigated the effectiveness of EC-18 for the development of medical countermeasures against acute radiation sickness (ARS) by analyzing mortality and morbidity caused by ionizing radiation.

Materials and methods

Animals

Specific-pathogen-free (SP-FR) male and female BALB/c mice (10 weeks old) were obtained from Koatech Co. (Pyongtaek, Republic of Korea). After receipt, mice were housed at 5 animals per cage in a pathogen-free room and acclimatized for 1 week under constant temperature and normal light cycles. All animals were fed a standard mouse chow diet with water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Biology and Biotechnology, and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the Korean National Laws on the Humane Treatment of Animals.

Determination of the lethal dose (LD) of total body γ-irradiation (TBI)

Whole-body irradiation of animals was performed using a gamma irradiator (JL Shepherd & Associates, San Fernando, USA) with a ⁶⁰ Co source (exposure rate 0.833 Gy/min). For γ-irradiation survival experiments, mice were grouped into 20 mice (10 males and 10 females) per experimental group; 6.0 Gy, 6.2 Gy, 6.4 Gy, and 6.5 Gy. Animal survival and body weight were recorded daily for 30 days.

Creation of a mouse model of acute radiation sickness (ARS) induced by γ-irradiation

For survival experiments following an LD70/30 dose of 6.11 Gy of γ-irradiation (TBI, ⁶⁰ Co, 0.833 Gy/min), mice were divided into 20 mice (10 males and 10 females) per experimental group; γ-irradiation only group (positive control), γ-irradiation with EC-18 10 mg/kg group, γ-irradiation with EC-18 50 mg/kg group, and γ-irradiation with EC-18 250 mg/kg group. EC-18 (Enzychem Lifesciences, Jaecheon, Republic of Korea) was suspended in PBS and administered orally once daily starting from day 1 after irradiation. PBS was administered to the positive control group. Survival and body weight of animals were recorded daily for 30 days.

For the time course analysis of hematopoietic ARS syndrome (H-ARS), female mice were divided into 5 experimental groups: γ-irradiation only (positive control) and γ-irradiation with EC-18 250 mg/kg group. For the dose effect analysis of EC-18 in H-ARS, mice were divided into 8 mice (5 males and 3 females) per experimental group: γ-irradiation only (positive control), γ-irradiation with EC-18 50 mg/kg group, γ-irradiation with EC-18 100 mg/kg group, γ-irradiation with EC-18 250 mg/kg group, and γ-irradiation with EC-18 100 mg/kg group. Whole blood was collected from the orbital sinuses using EDTA-free capillary tubes (Kimble Chase Life Science and Research Products LLC, FL, USA) and collection tubes containing K3E-K3EDTA (Greiner Bio-One International, Kremsmünster, Austria). Blood cells were counted and classified by complete blood count (CBC) using a Mindray BC-5000 auto-hematology analyzer (Shenzhen Mindray Biomedical Electronics, Guangdong Sheng, China). Animal blood cell values were recorded daily for 30 days.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Paired Mantel-Cox log-rank test for survival curve was used to assess the significance of survival and compare the duration of neutropenia between the control and EC-18 treated groups. Median survival was calculated as the sum of survival times for all mice/total number of mice. One-way ANOVA test was used to compare statistical differences between more than two groups. All other statistical analyses were performed using paired Student's t-test and p-values < 0.05 were considered statistically significant.

Results

Dose-response ratio (DRR) and LDXX/30

Radiation dose is a significant predictor of mortality and morbidity with increasing dose. Figure 28A shows the survival of BALB/c mice exposed to different doses of ^60Co γ-irradiation. Dose of 6.0, 6.2, 6.4, and 6.5 Gy resulted in 60, 80, 100, and 100% mortality, respectively, after 30 days of observation. Figure 28B shows the dose-response ratio (DRR) using probit models. The mean survival times of deaths for each radiation dose group were 15.30 ± 4.98; 13.69 ± 3.26; 14.15 ± 3.48, and 15.85 ± 4.42 days, respectively (Table 5). Thirty-day survival was calculated at each radiation dose and plotted as the percentage of mortality on the y-axis. Based on the probit model, LD30/30, LD50/30, LD70/30, and LD95/30 were determined with 95% confidence intervals for each dose (Table 6). The LD70/30 was found to be 6.11 Gy, and this parameter was used to evaluate several biological parameters (e.g., survival, weight loss, and hematopoietic cell loss) in subsequent studies.

Table 5. Mortality of BALB/c mice after 30 days of γ-irradiation.

Radiation dose (Gy) Mortality Survival time of the deceased (days) Mean ± SD Median 6.00 12/20 (60%) 15.30 ± 4.98 15.50 6.20 16/20 (80%) 13.69 ± 3.26 13.50 6.40 20/20 (100%) 14.15 ± 3.48 14.00 6.50 20/20 (100%) 15.85 ± 4.42 14.50

Table 6. Estimated radiation dose in BALB/c mice after γ-irradiation.

LD XX/30 LD calculated (Gy) Lower 95% CI (Gy) Upper 95% CI (Gy) LD30/30 5.31 4.98 5.56 LD50/30 5.79 5.59 5.96 LD70/30 6.11 5.98 6.22 LD95/30 6.39 6.30 6.48

Gamma radiation also caused a significant decrease in body weight of irradiated mice (Figure 29). It was difficult to find a strict relationship between radiation dose and body weight loss at the initial stage (0-10 days), but body weights of the lower dose groups (6.0 and 6.2 Gy) were restored to the normal range by the end of the observation period.

Therapeutic effect of EC-18 administration on survival, mean lifespan and body weight of irradiated mice with LD70/30 under general irradiation.

We then investigated the therapeutic effect of EC-18 on the survival of irradiated mice with a total irradiation LD70/30 (6.11 Gy) during the 30-day observation period. The survival rate of the positive control group was 20%, whereas the survival rates of the groups treated with EC-18 10, 50, and 250 mg/kg were 20, 40, and 80%, respectively (Figure 30A). Moreover, EC-18 significantly increased the mean survival time of irradiated mice in a dose-dependent manner (Table 7). The corresponding mean survival time of the groups treated with EC-18 10, 50, and 250 mg/kg was 19.3, 22.3, and 28.2 days, compared to 17.9 days in the positive control group.

Table 7. Effect of EC-18 administration dose on survival and average lifespan of irradiated mice.

Number of surviving mice/total number Survival Average life expectancy Average survival, days Log rank test,
p* Control 16/20 80% 28.2 30 <0.0001 EC-18 10 mg/kg 8/20 40% 22.3 20 0.0464 EC-18 50 mg/kg 4/20 20% 19.3 17 0.4425 EC-18 250 mg/kg 4/20 20% 17.9 15

EC-18 administration also effectively prevented the severe body weight loss induced by γ-irradiation (Figure 30B). Specifically, the EC-18 group at a dose of 250 mg/kg significantly reduced the γ-irradiation-induced body weight loss from day 18 post-irradiation to the end of the experiment, compared with the positive control group. In addition, the number of mice experiencing 20% of the baseline body weight loss decreased dramatically as the EC-18 dose increased (Table 8).

Table 8. Dose-dependent effect of EC-18 administration on body weight loss in irradiated mice.

Weight loss ≥10% Weight loss ≥20% n % n % Control 16 80 8 40 EC-18 10 mg/kg 17 85 14 70 EC-18 50 mg/kg 11 55 7 35 EC-18 250 mg/kg 3 15 3 15

Based on the observations in this study, we conclude that EC-18 has therapeutic potential to improve survival and prevent weight loss in γ-ray-induced ARS.

Therapeutic effect of EC-18 administration in γ-radiation-induced hematological syndrome ARS

A single session of whole-body γ-irradiation (6.11 Gy) rapidly depleted all hematopoietic cell types including absolute neutrophil count (ANC), monocyte count, absolute lymphocyte count (ALC), platelet count (PLT), and red blood cell (RBC) count within 3 days post-irradiation (Figure 31). The mean first-day neutropenia was 2.8 ± 0.45 days, and the duration of the neutropenic state was 18.0 ± 1.41 days. All individuals in the irradiated group experienced severe neutropenia (ANC < 100 cells/μL), and the duration of severe neutropenia was 16 ± 0.00. The mean trough ANC after γ-irradiation was 0.0±0.00 cells/μL, and the time to recovery to ANC≥500 or 1000 cells/μL was 27±1.41 days. Moreover, γ-irradiation caused a decrease in PLT by more than 90% within 7 days after irradiation, which began to recover at 26 days after irradiation. RBC gradually decreased and began to recover at 22 days after irradiation.

The therapeutic effect of EC-18 administration in ARS hematopoietic syndrome was then evaluated. EC-18 administration significantly attenuated γ-irradiation-induced depletion of white blood cell (WBC), absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) in irradiated mice (Figures 32A-J). Groups of BALB/c mice (n = 5 females for Figures 32A-32E and n = 8, 5 males, 3 females for Figures 32F-32J) were exposed to 6.11 Gy of γ-irradiation and orally administered various doses of EC-18 once daily until day 15 or left untreated. Figures 32A-32E show the time course of white blood cell count (WBC; Figure 32A), absolute neutrophil count (ANC; Figure 32B), absolute lymphocyte count (ALC; Figure 32C), platelet count (PLT; Figure 32D), and red blood cell count (RBC; Figure 32E) over 15 days, respectively. Figures 32F-32J show the effect of EC-18 administration dose on WBC (Figure 32F), ANC (Figure 32G), ALC (Figure 32H), PLT (Figure 32I), and BRC (Figure 32J) at day 15, respectively. ns, non-significant differences; *p < 0.05, **p < 0.01, ***p < 0.005.

EC-18 administration significantly attenuated the γ-irradiation-induced decrease in ANC. The mean first day of neutropenia (ANC < 500 cells/μL) in the control and EC-18 groups were 1.8 ± 1.09 and 2.2 ± 1.09 days (2-sided P value = 0.62), respectively. Although EC-18 did not protect irradiated mice from severe neutropenia, it effectively reduced the duration of severe neutropenia from 13.0 days to 7.2 ± 1.79 days. Furthermore, EC-18 significantly increased the mean nadir ANC level after γ-irradiation from 4.0 ± 5.48 cells/μL to 20.0 ± 10.00 cells/μL (2-sided P value = 0.035). Administration of EC-18 attenuated the decrease in PLT and RBC induced by whole body irradiation (Figures 32D, 32E and 32I, 32J). Based on our observations, we concluded that EC-18 may have therapeutic potential to attenuate the decrease in blood cell counts in γ-ray-induced ARS.

It is understood that the examples and embodiments described in this application are intended for illustrative purposes only and that various modifications or changes in light thereof will occur to those skilled in the art and are intended to be included within the spirit and scope of this application and the scope of the claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (18)

1. The use of 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG) for the treatment or prophylaxis of acute radiation sickness in a subject. 2. Use of 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG) for the treatment of a subject exposed to adverse ionizing radiation. 3. The use according to claim 1 or 2, wherein the subject suffers from or is susceptible to hematopoietic acute radiation syndrome, radiation coagulopathy, gastrointestinal acute radiation syndrome, cardiovascular acute radiation syndrome, or central nervous system (CNS) acute radiation syndrome, or a combination thereof. 4. The use according to any one of claims 1-3, wherein 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG) is first administered to the subject within 36 hours after radiation exposure. 5. Use according to any one of paragraphs 1-4, wherein PLAG is administered with granulocyte colony-stimulating factor (G-CSF). 6. The use according to any one of claims 1-5, wherein PLAG is administered with one or more selected from the group consisting of analgesics, antiulcer agents, antidiarrheals, antibiotics, antipyretics, nutritional supplements and antioxidants. 7. A kit for use in the treatment or prevention of acute radiation sickness in a subject, comprising: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for the use of PLAG for the treatment or prevention of acute radiation sickness (ARS) in a subject. 8. A kit for use in the treatment or prevention of excessive exposure to ionizing radiation, comprising: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for the use of PLAG for the treatment or prevention of excessive exposure to ionizing radiation. 9. A kit for use in the treatment or prevention of one or more subsyndromes of acute radiation sickness, comprising: (a) 1-palmitoyl-2-linoleoyl-3-acetylglycerol (PLAG); (b) instructions for the use of PLAG for the treatment or prophylaxis of one or more subsyndromes of acute radiation sickness. 10. A kit according to any one of claims 7-9, containing a therapeutically effective amount of PLAG. 11. A kit according to any of paragraphs 7-10, containing written instructions for the use of PLAG. 12. A kit according to any one of paragraphs 7-11, wherein the instructions are a product label.

Images