Compositions Comprising 15-HEPE and Methods of Treating or Preventing Cancer and Neurologic Disease

The present disclosure provides compositions comprising 15-HEPE and methods of using same for treating and/or preventing cancer and neurological diseases in a subject in need thereof.

FIELD

The present disclosure provides compositions, formulations and methods of treating or preventing cancer and neurologic diseases by administering a pharmaceutical composition comprising 15-hydroxyeicosapentaenoic acid (also referred to as 15-HEPE) or a derivative thereof to a subject in need thereof.

BACKGROUND

Apoptosis is a genetically regulated and tightly controlled process involved in eliminating defective cells from tissues. Under normal conditions, the rate of apoptosis and cellular proliferation are in balance, thereby maintaining homeostasis. Under certain disease states, apoptosis can be upregulated, creating an imbalance that creates to tissue damage and loss of function. Caspases are a group of cysteine proteases critical for apoptosis of eukaryotic cells. Activation of the caspase cascade, if completed, leads to activation of the terminal effector caspase-3—the terminal effector caspase.

SUMMARY

In some embodiments, the present disclosure provides methods of treating and/or preventing cancer in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a derivative thereof or a composition comprising 15-HEPE or a derivative thereof. In some embodiments, the cancer is selected from carcinoma, CNS cancer, liver cancer, hepatocellular carcinoma, skin cancer, prostate cancer, breast cancer and lung cancer.

In some embodiments, the present disclosure provides methods of treating and/or preventing a neurological disorder in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a derivative thereof or a composition comprising 15-HEPE or a derivative thereof. In some embodiments, the neurological disorder is selected from catalepsy, epilepsy, encephalitis, meningitis, migraine, Huntington's, Alzheimer's, Parkinson's, and multiple sclerosis.

In some embodiments, the present disclosure provides uses of 15-HEPE in the manufacture of a medicament for treating and/or preventing cancer or a neurological disorder in a subject.

In some embodiments, the present disclosure provides a method of promoting normal cell proliferation in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides q method of preventing cell death in an organ in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of reducing cancerous tumor proliferation in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of delaying tumor growth in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides method of inhibiting caspase-3 activity in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of sensitizing cancer cells to radiation therapy in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE and concurrently or thereafter treating cancer cells in the subject with radiation.

Other features and advantages of the technology disclosed herein will be apparent from the following detailed description.

DETAILED DESCRIPTION

The present disclosure provides compositions comprising 15-HEPE, and methods of using same for treating and/or preventing cancer or a neurological disease in a subject in need thereof.

15-Hydroxy-eicosa-5,8,11,13,17-pentaenoic acid is a functionalized fatty acid having the general structure shown in Formula (I).

15-HEPE can be synthesized from eicosapentaenoic acid (EPA) according to methods known in the art. As used herein, the term “15-HEPE” may refer to 15-HEPE in its free acid form (e.g, 15-hydroxy-eicosa-5,8,11,13,17-pentaenoic acid; R═R′═H in Formula (I)) and/or a derivative thereof, such as a pharmaceutically acceptable ester (R≠H), a conjugate, or a salt (R is an ion) consistent with Formula (I), or mixtures of any of the foregoing. A derivative of 15-HEPE (e.g., R≠H and/or R′≠H) may be used instead. In some embodiments, the 15-HEPE is used in the free acid form (i.e., R═H). Alternatively, pharmaceutically acceptable esters or salts of 15-HEPE are used in certain embodiments of the present disclosure. In some embodiments, the 15-HEPE is in the form of a C1-4alkyl ester such as methyl ester (R═CH3) or ethyl ester (R═CH2CH3) form. 15-HEPE is a chiral molecule and may be used in the 15(S)- or 15(R)-enantiomeric form, in an enantiomerically enriched form, or as a racemic mixture. Used herein, “15-HEPE” includes all such forms, with no limitation as to stereospecificity. In another embodiment, the 15-HEPE comprises the 15(S) form: 15(S)-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid, or a derivative thereof. In some embodiments, the 15-HEPE may be used in the form of the ethyl ester.

As used herein, “EPA” refers to eicosa-5,8,11,14,17-pentaenoic acid, also known as 20:5n-3, an omega-3 fatty acid. EPA is readily obtainable through commercial sources.

Accordingly, in one aspect of the present disclosure, a method of treating and/or preventing cancer or a neurological disease in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition comprising 15-HEPE.

The present disclosure provides 15-HEPE, or a composition comprising 15-HEPE, for use in the treatment and/or prevention of cancer or a neurological disease.

The present disclosure provides a use of 15-HEPE, or a composition comprising 15-HEPE, in the manufacture of a medicament for treating and/or preventing cancer or a neurological disease.

The present disclosure also provides formulations of 15-HEPE and formulations comprising 15-HEPE and methods of using these formulations for treating and/or preventing cancer or a neurological disorder.

The present disclosure further provides a pharmaceutical composition for oral delivery, comprising 15-HEPE. That composition may comprise a pharmaceutically acceptable excipient. The 15-HEPE may be in any form as discussed herein. The 15-HEPE may be present from about 50 mg to about 3000 mg.

In one embodiment, compositions of the present disclosure comprise 15-HEPE as an active ingredient. 15-HEPE is the abbreviation for 15-hydroxyeicosapentaenoic acid, a compound that can be synthesized via methods known in the art, such as exposure of eicospentaenoic acid to the enzyme 15-lipoxygenase. As used herein, the term “15-HEPE” refers to 15-HEPE in its free acid form (e.g., 15-hydroxyeicosapentaenoic acid) and/or a pharmaceutically acceptable ester, conjugate or salt thereof, or mixtures of any of the foregoing. A derivative of 15-HEPE may be used instead, though this does not include any derivative compound missing the hydroxy group of 15-HEPE. The term “pharmaceutically acceptable” in the present context means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components of the composition.

In one embodiment, the 15-HEPE is in the form of an ester (also referred to herein as E-15-HEPE, 15-HEPE EE, or ethyl-15-HEPE). In another embodiment, the 15-HEPE comprises a C1-C5alkyl ester of 15-HEPE. In another embodiment, the 15-HEPE comprises 15-HEPE methyl ester, 15-HEPE propyl ester, or 15-HEPE butyl ester. In still another embodiment, the 15-HEPE comprises the optically active 15(S)-Hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid. This isomer may be used in any of the forms discussed above.

In another embodiment, the 15-HEPE comprises lithium 15-HEPE, mono, di-or triglyceride 15-HEPE or any other ester or salt of 15-HEPE, or the free acid form of 15-HEPE.

In various embodiments, the present disclosure provides pharmaceutical compositions, for example orally deliverable compositions, comprising 15-HEPE. In one embodiment, the compositions comprise a therapeutically effective amount of 15-HEPE. In one embodiment, the pharmaceutical composition comprises about 0.1% to about 99%, about 1% to about 95%, about 5% to about 90% by weight of 15-HEPE.

In one embodiment, the pharmaceutical composition comprises about at least about 70%, at least about 80% or at least about 90%, by weight, of 15-HEPE. In one embodiment, the pharmaceutical composition comprises at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%, by weight of 15-HEPE.

In one embodiment, 15-HEPE present in a composition of the present disclosure comprises at least 90% by weight 15-HEPE. 15-HEPE compositions can comprise even higher purity 15-HEPE, for example at least 95% by weight 15-HEPE or at least 97% by weight 15-HEPE, wherein the 15-HEPE is any form of 15-HEPE as set forth herein. The purity of 15-HEPE can further be defined (e.g. impurity profile) by any of the descriptions of 15-HEPE provided herein.

Above are discussed the amounts of the 15-HEPE in the pharmaceutical composition and their purity. The nature of the essential fatty acids and their synthesis is such that the 15-HEPE composition may include moieties from other essential fatty acids in the essential fatty acid metabolic cascade.

In one embodiment, a composition of the present disclosure contains not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, or not more than about 0.5%, by weight of other omega-3 fatty acids including alpha linolenic acid, stearidonic acid, docosahexaenoic acid (DHA) or derivatives thereof. In other embodiments there is substantially no, or no such other omega-3 fatty acids present.

In another embodiment, 15-HEPE represents at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%, by weight, of all fatty acids present in a composition of the present disclosure.

There may be present some residual eicosapentaenoic acid from the synthesis of the 15-HEPE. There may be not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, or not more than about 0.5%, by weight EPA. Alternatively, there is substantially no, or no, EPA in a form which has not been modified to the hydroxyl-form.

Dosage Forms

A composition for use in accordance with the disclosure can be formulated as one or more dosage units. The terms “dose unit” and “dosage unit” herein refer to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.

In some embodiments, compositions of the present disclosure are in the form of orally deliverable dosage forms or units. Non-limiting examples of suitable dosage forms include tablets (e.g. suspension tablets, bite suspension tablets, rapid dispersion tablets, chewable tablets, etc), caplets, capsules (e.g. a soft or a hard gelatin capsule or HPMC capsule), lozenges, sachets, cachets, troches, pellets, suspension, elixirs, syrups or any other solid dosage form reasonably adapted for oral administration. The terms “oral delivery” and “oral administration” herein include any form of delivery wherein the agent or composition is placed in the mouth of the subject under treatment, whether swallowed or not. This therefore includes buccal and sublingual administration, as well as esophageal administration.

Alternatively, compositions of the present disclosure can also be formulated for rectal, topical, or parenteral (e.g. subcutaneous, intramuscular, intravenous and intradermal or infusion) delivery.

In discussing the amount of 15-HEPE in a composition of the present disclosure, this may be split over several dosage forms. There is a limit as to the size for oral administration. If a subject is to be administered up to 4 g 15-HEPE a day, this may be by up to 4 capsules, each providing 1 g 15-HEPE, or up to 16 capsules, each providing 0.25 g 15-HEPE, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 capsules per day, each providing about 0.25 g to about 1 g of 15-HEPE.

Compositions of the present disclosure can be in the form of liquid dosage forms or dose units to be imbibed directly or they can be mixed with food or beverage prior to ingestion. Non-limiting examples of suitable liquid dosage forms include solutions, suspensions, elixirs, syrups, liquid aerosol formulations, and the like.

In another embodiment, compositions of the present disclosure comprise one or more pharmaceutically acceptable excipients. The term “pharmaceutically acceptable excipient” herein means any substance, not itself a therapeutic agent, used as a carrier or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition. By way of example only, a pharmaceutical composition according to the present disclosure may comprise one or more of: antioxidants, surfactants, preservatives, flavouring agents, co-solvents, viscosity aids, suspension aids, and lipophilic phases.

Therapeutic Methods

The compositions and formulations disclosed herein may be used in the treatment of cancer. In one embodiment the cancer is associated with an o rgan or tissue associated with the CNS, a lung, a liver, a heart, a kidney, a bowel, a stomach, one or more eyes, mediastinum, bone marrow, retroperitoneaum, skin, an intestine, a joint, a reproductive organ, a prostate, a breast or a combination thereof. In some embodiments, the 15-HEPE is administered to reduce tumor proliferation or delay tumor growth.

In some embodiments, the 15-HEPE is administered to promote normal cell proliferation.

In some embodiments, the 15-HEPE is administered to radiosensitize cancer cells, and can be used a radiosensitizer in radiation therapy.

In some embodiments, the composition is orally administered.

In some embodiments, the 15-HEPE is the only active ingredient in the composition. In other embodiments, the composition further comprises an additional agent for affecting the fibrosis therapy.

In some embodiments of the methods disclosed herein, the 15-HEPE is the only active ingredient in the composition. In some embodiments, the composition further comprises an additional agent for affecting the cancer or CNS disease.

In some embodiments, the method further comprises identifying the subject as having cancer or CNS disease before administering the composition comprising 15-HEPE. In some embodiments, the method further comprises identifying the subject as having an increased risk of developing cancer or CNS disease before administering the composition comprising 15-HEPE. In some embodiments, the step of identifying comprises screening for a genetic mutation in a nucleic acid molecule associated with the subject. In some embodiments, the step of identifying comprises obtaining an analysis of blood and/or serum associated with the subject. In some embodiments, the step of identifying comprises examining a tissue associated with the subject. In some embodiments, the step of examining comprises analyzing a histological tissue sample (e.g., a biopsy) associated with the subject.

In some embodiments, the present disclosure provides uses of 15-HEPE in the manufacture of a medicament for treating and/or preventing cancer or a neurological disorder in a subject.

In some embodiments, the present disclosure provides a method of promoting normal cell proliferation in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of preventing cell death in an organ in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of reducing cancerous tumor proliferation in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of delaying tumor growth in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of inhibiting caspase-3 activity in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE.

In some embodiments, the present disclosure provides a method of sensitizing cancer cells to radiation therapy in a subject in need thereof, the method comprising administering to the subject 15-HEPE or a composition comprising 15-HEPE and concurrently or thereafter treating cancer cells in the subject with radiation.

In one embodiment, the method comprises administering a pharmaceutical composition as disclosed herein to a subject once per day, twice per day, three times per day, or more than three times per day.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

While the present disclosure is capable of being embodied in various forms, the present description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the present disclosure, and is not intended to limit the technology disclosed herein to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the technology disclosed herein in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” In this manner, slight variations from a stated value can be used to achieve substantially the same results as the stated value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values recited as well as any ranges that can be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that can be formed by dividing a recited numeric value into any other recited numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios can be unambiguously derived from the numerical values presented herein and in all instances such ratios, ranges, and ranges of ratios represent various embodiments of the present disclosure.

As used herein, “treating” or “treatment” of a disease, disorder, or condition includes at least partially: (1) preventing the disease, disorder, or condition, i.e. causing the clinical symptoms of the disease, disorder, or condition not to develop in a mammal that is exposed to or predisposed to the disease, disorder, or condition but does not yet experience or display symptoms of the disease, disorder, or condition; (2) inhibiting the disease, disorder, or condition, i.e., arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (3) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms. The term “prevention” in relation to a given disease or disorder means: preventing the onset of disease development if none had occurred, preventing the disease or disorder from occurring in a subject that may be predisposed to the disorder or disease but has not yet been diagnosed as having the disorder or disease, and/or preventing further disease/disorder development if already present.

An “effective amount,” as used herein, refers to the amount of an active composition that is required to confer a therapeutic effect on the subject. A “therapeutically effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, in some embodiments, an “effective amount” for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. In some embodiments, an appropriate “effective amount” in any individual case is determined using techniques, such as a dose escalation study. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. In other embodiments, an “effective amount” of a compound disclosed herein, such as a compound of Formula (I), is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. In other embodiments, it is understood that “an effect amount” or “a therapeutically effective amount” varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. The term “pharmaceutically acceptable” in the present context means that the substance in question does not produce unacceptable toxicity to the subject or interaction with other components of the composition.

Without further description, it is believed that one of ordinary skill in the art may, using the preceding description and the following illustrative examples, make and utilize the agents of the present disclosure and practice the claimed methods. The following working examples are provided to facilitate the practice of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLE

The potential inhibitory effect of compounds 15(S)-HEPE EE and EPA EE was evaluated on staurosporine-induced caspase activation in normal human dermal fibroblasts (NHDF). More specifically, at the end of the treatments with the test compounds in staurosporine-activated NHDF, the caspase 3/7 activity was measured after cell lysis using a specific fluorogenic substrate.

Test compounds are shown in Table 1.

Fibroblasts were seeded in 24-well plates and cultured for 24 hours in culture medium. The medium was then removed and replaced by assay medium containing or not (control) the test compounds and cells were pre-incubated for 24 hours. After pre-incubation, the medium was replaced with assay medium containing the inducer (staurosporine at 100 nM) and containing or not (control) the test compounds. The cells were then incubated for 24 hours. Non-stimulated controls (without inducer) were performed in parallel. All experimental conditions were performed in n=3.

At the end of incubation, the cell layers were lysed for caspase 3/7 activity measurement and protein quantification was also performed on other dedicated culture plates performed in parallel with identical treatments. Enzyme activity assay and protein quantification assay were performed in separated plates due to the fact that DTT used in the lysis buffer for caspase activity interferes with protein quantification.

For caspase 3/7 activity measurement, cell layers were first detached using trypsin treatment and transferred in microtubes before performing the cell lysis.

The positive control was recombinant human active caspase-3 (BD Biosciences, ref. 556471), tested at 50 ng/ml.

Cell lysates of fibroblasts, were either treated or not (controls) with the test compounds for 48 hours, and with staurosporine during the last 24 hours of incubation (except for the non-stimulated control) with the following buffers and reagents:

Cell lysis was achieved by adding 100 μl of lysis buffer/microtube on the cell pellets and a freeze/thaw cycle.

Ac-DEVD-AMC is a synthetic tetrapeptide fluorogenic substrate for caspase-3 and caspase-7 and contains the amino acid sequence of the PARP cleavage site at Asp-216. Thus the tetrapeptide substrate can be used to identify and quantify the caspase-3/7 activity. Enzymes cleave the tetrapeptide between D and AMC resulting in the release of fluorogenic AMC, which can be quantified with a spectrofluorometer.

Cell lysates (30 μl/well) or recombinant human active caspase-3 were incubated in assay buffer, in presence of the substrate, for 3 hours at 37° C. under agitation (final volume 100 μl). The specificity of the reaction was evaluated by testing a stimulated control condition in presence of the caspase 3 specific inhibitor Ac-DEVD-CHO (0.1 μM). In parallel, an additional control (assay buffer containing the substrate) was also performed to determine the background signal.

At the end of incubation, the fluorescence intensity emitted by the reaction product (cleaved Ac-DEVD-AMC) was measured at wavelengths λex380 nm and λem440 nm using a spectrofluorimeter (SPECTRAmax® Gemini, Molecular Devices).

The results of the MTT reduction assay and the observation of the cell layers are shown in Tables 2 and 3, below.

In presence of the positive control (recombinant human active caspase 3, tested at 50 ng/ml), the detected caspase-3/7 activity was very strong (acellular control).

TABLE 2Effect of 15(S)-HEPE EE on the viability offibroblasts after 48 hours of incubation15(S)-HE PE EE Stock solutionUnitprepared at 100 mg/ml in DM SOμg/mlControl0.0010.010.111030100300Visability (%)991009898989710196104971039999909996100103103989810097100969610298105105Mean1009896979710199104100sem113101203Morpholigical+++++++++.*observationsLegend+: normal population;+/−: growth reduction;−: toxicity;0: cell mortalityg: grains of compound;op: opacity of the compound;*morphological modification;ag: agglutinated cellssem: Standard error of the mean (standard deviation divided by sample size square root)

TABLE 3Effect of EPA EE on the viability of fibroblasts after 48 hours of incubationE PA EE Stock solution preparedUnitat 100 mg/ml in Ethanolμg/mlControl0.0010.010.111030100300Visability (%)10099979499100989598107101100104989895100931001061001009910199959499102105Mean1001009899979796100106sem022012211Morpholigical+++++++++observationsLegend+: normal population;+/−: growth reduction;−: toxicity;0: cell mortalityg: grains of compound;op: opacity of the compound;* morphological modification;ag: agglutinated cellssem: Standard error of the mean (standard deviation divided by sample size square root)

In control condition (Tables 4-5), the basal caspase-3/7 activity in non-stimulated fibroblasts was limited with a signal rather close to the background control. The apoptosis inducer staurosporine, tested at 100 nM, showed a strong stimulating effect on caspase-3/7 activity with a 10 fold increase between staurosporine-stimulated conditions and non-stimulated conditions (when considering the caspase-3/7 activity normalized versus protein quantity). Moreover, this activity was fully inhibited when adding in the reaction mix, the inhibitor Ac-DEVD-CHO at 0.1 μM, thus confirming the specificity of the detected caspase-3/7 activity. These results validated the assay.

In this assay, the 2 test compounds, i.e., 15(S)-HEPE EE and EPA EE were tested at the same range of 6 concentrations: 0.001, 0.01, 0.1, 1, 10 and 100 μg/ml.

Compound 15(S)-HEPE EE showed no effect on caspase-3/7 activity when tested from 0.001 to 10 μg/ml. However, at the highest concentration (100 μg/ml), this compound displayed opposite effects, with a very strong inhibitory effect for 15(S)-HEPE EE (83% of inhibition).

Compound EPA EE, showed no effect on caspase-3/7 activity when tested from 0.001 to 10 μg/ml. Compound EPA EE, at highest test concentration, displayed a strong inhibitory effect on caspase-3/7 activity (61% of inhibition).

At the concentration of 100 μg/ml, compounds 15(S)-HEPE EE and EPA EE both displayed a clear inhibitory effect on caspase-3/7 activity (with a stronger effect for compound 15(S)-HEPE EE).

As shown inFIG. 1, at the concentration of 100 μg/ml, compounds 15(S)-HEPE EE and EPA EE both displayed a clear inhibitory effect on caspase-3/7 activity (with a stronger effect for compound 15(S)-HEPE EE).