Dicarboxylic acid esters for inducing analgesic effects

Abstract

The present disclosure provides compositions and methods for inducing an analgesic effect. In particular, the methods include administering to a subject in need thereof a pharmaceutical composition comprising a dicarboxylic acid ester.

Description

Technical Field

[0001]

[0002] The present disclosure relates to a method and a pharmaceutical composition containing a dicarboxylic acid ester for inducing an analgesic effect.

Summary of the Invention

[0002] [Introduction]

[0003] Anesthetics and analgesics are widely used in the treatment of pain.

[0003]

[0004] In recent years, Pavil et al. have described in detail the biochemical mechanism by which general anesthetics exert their pharmacological effects. Pavil has shown that inhaled anesthesia disrupts the order of lipid rafts and induces anesthesia by disrupting the order of lipid rafts. The most well-studied lipid rafts are membrane domains rich in sphingomyelin (e.g., monosialotetrahexosylganglioside 1 "GM1") that bind cholesterol and cholera toxin B (CT×B). GM1 lipid rafts contain phospholipase D2 (PLD2). Inhaled anesthesia increases the fluidity of GM1 lipid rafts, thereby disrupting GM1 lipid rafts, separating PLD2 from lipid rafts, and moving it proximal to TREK1. Then, PLD2 hydrolyzes phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. Next, PA binds to TREK1 and activates TREK1. When TREK1 is activated, the resulting potassium influx induces loss of consciousness and analgesia. Pavil et al. have also shown that the movement of PLD2 activates a second type of channel called TRAAK, a non-anesthetic-sensitive homolog of TREK1 [M.A. Pavel et al., PNAS, (2020) 117(24):13757~66].

[0004]

[0005] The inventors have found that, in one embodiment, diethyl azelaate (DEA) also enhances the fluidity of GM1 lipid rafts, and therefore inhibits the formation of membrane invasion complexes by both cholera toxin B subunit (CT×B) and anthrax toxin protective antigen (PA). This suggests that the dicarboxylic acid esters of the present disclosure are effective in relieving pain when applied topically. Therefore, the inventors assert that DEA modulates neurotransmission by means similar to inhalation anesthesia. Thus, dicarboxylic acid esters, represented by DEA, disrupt membrane fluidity and thus have an analgesic effect similar to inhalation anesthesia.

[0005]

[0006] At its most fundamental level, all types of pain other than psychogenic pain involve the transmission of harmful or noxious stimuli to the central nervous system via afferent nerves. The transmission of the resulting nerve impulses must cross the cell plasma membrane at multiple points in the signaling chain [A. Basbaum et al., Cell. (2009) 139(2):267~84]. The role of the plasma membrane in the pain mechanism has been demonstrated in vivo to reversibly reduce prostaglandin E2 (PGE2)-induced hyperalgesia by disrupting nerve signaling through cholesterol depletion by cyclodextrins, thereby increasing the fluidity of lipid rafts [Ferrari, L et al., J. Pain (2015) 16(1):60~6].

[0006]

[0007] Biochemical mediators of pain include cytokines, neuropeptides, lipids, growth factors, and neurotransmitters. Further cellular signals and sensors important for pain management include colony-stimulating factors (CSFs) and leptin.

[0007]

[0008] Some members of the CSF superfamily play beneficial roles in pain relief. Early systemic granulocyte colony-stimulating factor (G-CSF) therapy attenuates neuropathic pain after peripheral nerve injury [P. Chao et al., PLoS ONE (2012) 7(8):e43680]. G-CSF primes neutrophils, improving host defense and reducing the release of pro-inflammatory cytokines [T. Hartung et al., Blood. (1995) 85(9):2482~9]. In the central nervous system, GM-CSF reduces food intake and body weight [J. Reed et al., J. Clin. Invest. (2005) 115(11):3035~44]. However, in the above reports, CSF was administered exogenously as a biological agent. While increasing endogenous G-CSF production has been proposed as a new therapeutic strategy [S. Von Aulock et al., Curr. Opin. Investig. Drugs (2004) 5(11):1148~52], the only known method of inducing CSF to date is bacterial or viral antigen stimulation. Small molecule inducers of CSF, also known as secretion promoters, had not been identified until now.

[0008]

[0009] Leptin has been shown to reduce neuropathic pain induced by chronic nerve injury [X. Li et al., Mol. Pain (2013) 9:65].

[0009]

[0010] The inventors have demonstrated that dicarboxylic acid esters induce numerous changes in cellular and biological signaling pathways, and that these are therapeutically useful. In addition to other pharmacological activities of dicarboxylic acid esters, they induce the cellular release and / or production of signaling molecules with analgesic properties. Examples of responsive cellular molecules include leptin, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, the inventors discovered that treatment of mammalian cells and tissues with dicarboxylic acid esters can suppress the production of pain-related markers, including inducible nitric oxide synthase (iNOS) [Y. Kwok et al., PLoS One. 2012;7(8):e44232], S-nitrosylated cysteine ​​residues in cellular proteins [J. Zhang et al., Int. Anesthesiol. Clin. (2007) 45(2):27~37], prostaglandin E2 (PGE2) [A. Ahlawat et al., Eur. J. Pharmacol. (2018) 818:419~28], and adenosine triphosphate (ATP) [Mense, Dtsch Arztebl Int 2008;105(12):214~9].

[0010] [Overview of the prefecture]

[0011] Aspects of this disclosure relate to a method and formula I:R2OOC-(CH2) for inducing analgesic effects in a subject. n The present invention provides a pharmaceutical composition comprising a dicarboxylic acid ester of -COOR1 (wherein n is 4 to 10, and each R1 and R2 is independently a lower alkyl). Certain embodiments of this specification relate to inducing an analgesic effect in a subject, thereby inhibiting pain by activation of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), nucleotide oligomerized domain (NOD) receptors and / or dectin receptors.

[0011]

[0012] Certain embodiments of this specification relate to inducing analgesic effects and thereby inhibiting local pain in a subject by promoting the secretion of leptin, macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and / or granulocyte colony-stimulating factor (G-CSF).

[0012]

[0013] In some embodiments, the embodiments herein relate to methods and pharmaceutical compositions comprising the dicarboxylic acid ester of Formula I for inducing analgesic effects and thereby inhibiting local pain in a target by suppressing the expression and secretion of prostaglandin E2 (PGE2) and / or inducible nitric oxide synthase (iNOS) and / or adenosine triphosphate (ATP).

[0013]

[0014] In some embodiments, embodiments of this specification relate to a method for inducing an analgesic effect and thereby inhibiting local pain in a subject, the method comprising the step of administering a dicarboxylic acid ester of formula I to a subject, wherein the administration results in a reduction of symptoms associated with the pain state.

[0014]

[0015] In some embodiments, the embodiments herein relate to the use of dicarboxylic acid esters of formula I in the manufacture of pharmaceuticals for inducing an analgesic effect and thereby inhibiting local pain of a target.

[0015]

[0016] In some embodiments, the embodiments herein relate to the use of dicarboxylic acid esters of formula I for inducing an analgesic effect and thereby inhibiting local pain in a target.

[0016]

[0017] In some embodiments, the embodiments of this specification relate to the use of dicarboxylic acid esters of formula I in the manufacture of pharmaceuticals to induce an analgesic effect in a subject, thereby inhibiting local pain in the subject, such as acute pain, chronic pain, nociceptive pain, neuropathic pain, or dysalgesic pain.

[0017]

[0018] In some embodiments, the embodiments herein relate to the use of dicarboxylic acid esters of formula I in the manufacture of pharmaceuticals to induce analgesic effects in a subject, thereby inhibiting local pain in response to the modulation of one or more receptors of the subject as required, selected from iNOS, ATP, and pattern recognition receptors (PRRs) including Toll-like, NOD, and Dectin receptors.

[0018]

[0019] In some embodiments, the embodiments herein relate to the use of dicarboxylic acid esters of formula I in the manufacture of pharmaceuticals to induce analgesic effects and thereby inhibit local pain of a target by upregulating the activity and / or expression of one or more leptin, M-CSF, G-CSF, and GM-CSF, or by downregulating the activity and / or expression or release of at least one of ATP, iNOS, and PGE2. [Brief explanation of the drawing]

[0019]

[0020] The following drawings illustrate certain aspects of the present disclosure. The present disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Figure 1] This bar graph shows the regulation of ATP release in human plasmacytoid dendritic cells by a PRR agonist alone and in the presence of diethyl azelaate (DEA) according to certain embodiments of the present disclosure. [Figure 2] This graph shows the effect of local DEA on hindlimb response latency in an in vivo hot plate test according to a specific embodiment of the present disclosure. [Figure 3] These images show human peripheral blood mononuclear cells co-exposed to fluorescently labeled cholera toxin B subunit with and without DEA treatment (left column) and with DEA ​​treatment (right column), according to a specific embodiment of the present disclosure. [Modes for carrying out the invention]

[0020]

[0024] All applications, publications, patents, and other references cited in this specification are hereby incorporated by reference in their entirety.

[0021]

[0025] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "or" refers to a single element of the recited alternative elements or a combination of two or more such elements unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B" means "including A, B, or A and B" without excluding additional elements.

[0022]

[0026] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. As used herein, the term "about" is intended to qualify the numerical value that it modifies, and such a value is represented as a variable within the scope of error. When a particular range of error, e.g., standard deviation with respect to the mean value provided in a data chart or table, is not recited, the term "about" is understood to also mean a range that encompasses the recited value and ranges that are included by rounding or truncating that numerical value, taking into account significant figures.

[0023]

[0027] All numerical values or numerical ranges used in this specification, unless the context clearly indicates otherwise, include integers within such ranges, and values or fractions of integers within the ranges. Additionally, such ranges are also intended to include the numbers themselves and any sub-ranges between the numbers. This range may be an integral range between the two end values including both end values, or a continuous range. Thus, for example, a reference to a range of about 3 hours to about 10 hours includes 3 hours, 4 hours, 5 hours, etc., as well as 3 hours 1 minute, 3 hours 2 minutes, 3 hours 4 minutes, etc., 4 hours 1 minute, 4 hours 2 minutes, 4 hours 4 minutes, etc. and others. A reference to a range of 90 to 100% includes 92.2% to 97.5%, 91.5 to 94.5%, etc. A reference to a range of 1 to 25 days includes sub-ranges of 1 day to 5 days or 3 days to 7 days or 5 days to 25 days.

[0024]

[0028] The term "comprising" as used in this specification is intended to mean that a pharmaceutical composition (or composition) and method include the recited elements, but do not exclude others. The term "consisting essentially of" as applied to the compositions of this embodiment means that a composition can contain additional elements so long as the additional elements do not substantially change the composition. The term "substantially change" as applied to a composition refers to an increase or decrease in the therapeutic effectiveness of the composition compared to the effectiveness of the composition consisting of the recited elements. In other words, "consisting essentially of" means, when used to define a composition, excluding other components that give any essential significance to the composition. Thus, a composition consisting essentially of the components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers. "Consisting of" means excluding elements in excess of trace amounts of other components and substantial method steps for administering the compositions of the present invention. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0025]

[0029] As used herein, the terms “optional” or “optional” mean that the events or circumstances described thereafter may or may not occur, and that the descriptions include both instances in which such events or circumstances occur and instances in which they do not.

[0026]

[0030] As used herein, “subjects” refers to mammals, such as primates, and in one embodiment, humans. Examples of non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons. The term “subjects” also includes cats, dogs, ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, cattle, horses, pigs, sheep, goats, chickens, turkeys, ducks, pheasants, pigeons, doves, parrots, cockatoos, geese, and the like.

[0027]

[0031] As used herein, the “subject requiring” the method of this disclosure may be a subject suffering from a pain condition.

[0028]

[0032] As used herein, the term “therapeutic effective” refers to an amount of the active ingredient sufficient to induce a local analgesic effect in a subject. A therapeutic effective amount of the pharmaceutical composition of this disclosure may be effective in disrupting lipid rafts. A therapeutic effective amount of the pharmaceutical composition of this disclosure may be effective in upregulating (e.g., stimulating) the expression and / or secretion of leptin, M-CSF, G-CSF, GM-CSF, etc., from cells of the subject. A therapeutic effective amount of the pharmaceutical composition of this disclosure may also be effective in downregulating (e.g., inhibiting) the expression and / or secretion of ATP, iNOS, PGE2, etc., from cells of the subject. Alternatively, a “therapeutic effective” amount is an amount that provides some reduction, mitigation, decrease, or stabilization of at least one clinical symptom of the subject. Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some benefit is conferred to the subject. The effective amount may vary depending on factors, such as the wound or affected area, or the disease or condition being treated, the specific target construct administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular composition without requiring excessive experimentation.

[0029]

[0033] As used herein, the term "active ingredient" refers to a biologically active substance. In embodiments, the dicarboxylic acid esters of the Disclosure are the active ingredient of a pharmaceutical composition. In embodiments, the dicarboxylic acid esters of the Disclosure are the sole active ingredient of a pharmaceutical composition. In embodiments, the azelaic acid esters of the Disclosure are the active ingredient of a pharmaceutical composition. In embodiments, diethyl azelaic acid is the active ingredient of a pharmaceutical composition.

[0030]

[0034] As used herein, the term “pharmaceutically acceptable carrier” refers to any suitable adjuvant, carrier, excipient, or stabilizer, which may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions, and which does not cause significant irritation to the subject and does not inhibit the biological activity and properties of the administered active ingredient.

[0031]

[0035] As used herein, the term “disease” is intended to be generally synonymous with and interchangeable with the terms “disorder” and “condition” (as in a medical condition), all of which describe an abnormal condition of one or more parts of the human or animal body that impairs normal function, is typically manifested by prominent signs and symptoms, and causes a decline in lifespan or quality of life of the human or animal.

[0032]

[0036] As used herein, “treating” a disease or condition, or “treatment” a disease or condition, may mean preventing a disease or condition, slowing the onset or progression of a disease or condition, reducing the risk of progression of a disease or condition, preventing or slowing the progression of symptoms associated with a disease or condition, reducing or ending symptoms associated with a disease or condition, resulting in complete or partial regression of a disease or condition, or any combination thereof. Treatment may also mean preventive or prophylactic treatment of a disease or condition.

[0033]

[0037] As used herein, the term “inhibit” refers to a reduction, decrease, limit, and / or blockade of the occurrence of a particular effect, function, interaction, or symptom. The terms “inhibit,” “decreasing,” and “reducing” are interchangeable herein. In embodiments, the terms refer to reducing or suppressing in a subject a level of a particular effect, function, interaction, or the occurrence of a particular symptom (e.g., a level of a biomarker including, but not limited to, protein or non-protein molecules in tissues and body fluids) to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or less than the amount in the corresponding control. For example, pain is reduced, mitigated, or inhibited when a pain indicator, such as the blood concentration of prostaglandin PGE2, is reduced by at least about 10%, 20%, 30%, 50%, 80%, or 100%. In embodiments, pain is reduced, mitigated, or inhibited by at least about 1-fold, 2-fold, 3-fold, 4-fold, or more compared to pain before administration of the dicarboxylic acid ester of the Disclosure. Measurable changes may be objective (e.g., measurable by some test or marker, e.g., in vitro or in vivo assay or test or observation) or subjective (e.g., the subject shows signs of an effect or feels an effect).

[0034]

[0038] As used herein, the term “pain” refers to an unpleasant sensory and emotional experience associated with or described in relation to actual or potential tissue damage, including the nearly localized sensation of discomfort, pain, or suffering resulting from stimulation of differentiated nerve endings. There are various types of pain, whether acute or chronic, including, but not limited to, nociceptive pain, neuropathic pain, or dysalgesic pain. The purpose of inducing analgesic effects in a subject is to reduce the degree or severity of pain perceived by that subject.

[0035]

[0039] As used herein, the term "localized pain" refers to non-systemic pain experienced in a specific location or area of ​​a particular part of the body in question. In embodiments, the area of ​​pain is related to the primary lesion.

[0036]

[0040] In embodiments, the present disclosure relates to a method and a pharmaceutical composition for inducing an analgesic effect and thereby inhibiting pain of a target, wherein the formula is I:R2OOC-(CH2) n The present invention provides a method and a pharmaceutical composition comprising the step of administering a pharmaceutical composition containing a dicarboxylic acid ester having -COOR1 to a target. In embodiments, n is 4-10, 6-9, or 7-8. In embodiments, each R1 and R2 is independently a lower alkyl group. As used herein, the term "lower alkyl group" refers to a C1-C6 saturated linear or branched alkyl group. Examples of preferred lower alkyl groups (R1 and R2) of formula I include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, and similar groups. In embodiments, R1 and R2 are the same. In embodiments, R1 and R2 are the same. In embodiments, R1 and R2 are different.

[0037]

[0041] In the embodiment, the dicarboxylic acid ester is an azelaic acid ester. In the embodiment, the azelaic acid ester is diethyl azelaic acid, dimethyl azelaic acid, or a mixture thereof. In the embodiment, the azelaic acid ester is diethyl azelaic acid.

[0038]

[0042] In embodiments, the present disclosure provides a method for inducing an analgesic effect and thereby inhibiting pain in a target, comprising the step of administering a pharmaceutical composition comprising an azelaic acid ester to the target. In embodiments, the present disclosure provides a method for inducing an analgesic effect and thereby inhibiting pain in a target, comprising the step of administering a pharmaceutical composition comprising diethyl azelaic acid to the target.

[0039]

[0043] Local analgesia can be achieved by disrupting or increasing the fluidity of lipid rafts. Lipid rafts (also known as lipid microdomains) are individual lipid domains present in the outer leaflet of the plasma membrane. Lipid rafts are rich in cholesterol, sphingomyelin, gangliosides, and sphingolipids, such as monosialotetrahexosylganglioside 1 (GM1). Lipid rafts affect membrane fluidity and membrane protein transport, thereby regulating neurotransmission and receptor transport. Z. Korade et al., Neuropharmacology (2008) 55(8):1265~73.

[0040]

[0044] In embodiments, the analgesic effect is induced by the destruction of lipid rafts, which occurs when the dicarboxylic acid ester of formula I is brought into contact with the lipid rafts. When the dicarboxylic acid ester of formula I is applied topically to a target (e.g., an open wound, the area surrounding the wound, healthy skin, mucous membrane, or wound in the oral mucosa), the dicarboxylic acid ester comes into contact with lipid rafts located in the plasma membrane of the target and destroys the lipid rafts. In embodiments, the lipid rafts contain GM1.

[0041]

[0045] In the embodiment, the lipid raft contains phospholipase D2 (PLD2).

[0042]

[0046] In this embodiment, the dicarboxylic acid ester of formula I destroys the lipid raft containing GM1.

[0043]

[0047] In one embodiment, disruption of lipid rafts with the dicarboxylic acid ester of formula I initiated PLD2 from the lipid rafts, thereby activating TREK-1 and subsequently promoting the production of the signaling lipid phosphatidic acid (PA).

[0044]

[0048] In the embodiment, disruption of lipid rafts with the dicarboxylic acid ester of formula I regulates the membrane fluidity of the lipid rafts.

[0045]

[0049] In this embodiment, the disruption of lipid rafts by the dicarboxylic acid ester of formula I is due to an increase in the fluidity of the plasma membrane of the lipid raft.

[0046]

[0050] Pain can be subdivided into various types in different ways. Based on time, pain can be classified as chronic or acute. Chronic pain is recognized as pain that persists beyond the normal healing time, usually for more than three months. Chronic pain affects approximately 20% of people worldwide and accounts for approximately 20% of medical consultations. [R. Treede et al., Pain (2015) 156(6):1003~7]. Based on the mechanism, pain can be nociceptive, neuropathic, or dysalgesic. Nociceptive pain results from the stimulation of pain receptors (nociceptors) in response to tissue damage caused by mechanical, chemical, or thermal stimuli. Neuropathic pain results from damage to components of the nervous system. Dysalgesic pain arises from alterations in nociceptive perception.

[0047]

[0051] In the embodiments, the subject is suffering from pain. In the embodiments, the pain is acute or chronic. In the embodiments, the pain is nociceptive pain, neuropathic pain, or dysalgesic pain.

[0048]

[0052] In embodiments, the pain is acute. In embodiments, the subject may require treatment for acute pain. Examples of acute pain include, but are not limited to, traumatic pain, pain caused by procedures (surgery, dentistry, dermatology, etc.), wound pain, musculoskeletal pain (e.g., back pain, neck pain), toothache, infection (e.g., wound infection), and toxins (insects, animals, bacteria, fungi, etc.).

[0049]

[0053] In embodiments, the pain is chronic. In some embodiments, the subject may require treatment for chronic pain. Examples of chronic pain include, but are not limited to, fibromyalgia, arthralgia, pain due to iliotibial band syndrome, tennis elbow pain, cancer pain, musculoskeletal pain (e.g., back pain, neck pain), temporomandibular joint disorders, trigeminal neuralgia, chronic headache, pain associated with neurological diseases (MS, diabetic neuropathy, etc.), neuromas, pelvic inflammatory disease, endometriosis, herpes zoster and postherpetic neuralgia, and chronic neuropathy associated with infections (e.g., HIV), chemotherapy-induced neuropathic pain, surgery-induced neuropathic pain, trauma-induced neuropathic pain, vulvovaginal pain, atypical craniofacial pain, sciatica, phantom limb pain, toothache, and burning mouth syndrome.

[0050]

[0054] In embodiments, the pain is nociceptive pain. In embodiments, the subject may require treatment for nociceptive pain. Nociceptive pain is usually acute and progresses in response to a specific situation. Examples of nociceptive pain include, but are not limited to, pain from sprains, burns, contusions, surgical procedures, and fractures. Chronic nociceptive pain is caused by some condition if the pain lasts for more than six months. Examples of chronic nociceptive pain include pain from cancer, rheumatoid arthritis, osteoarthritis, and musculoskeletal conditions (e.g., back pain).

[0051]

[0055] In embodiments, the pain is neuropathic pain. In embodiments, the subject may require treatment for neuropathic pain. Neuropathic pain results from damage to components of the nervous system. Certain disease conditions may be the underlying cause of neuropathic pain. For example, the subject may suffer from metabolic diseases (e.g., diabetic neuropathy), autoimmune diseases (e.g., multiple sclerosis), viral infections (e.g., postherpetic neuralgia), vascular diseases (e.g., stroke), trauma and / or cancer. Campbell et al., Neuron (2006) 52(1):77~92; Dworkin et al., Arch Neurol (2003) 60;1524~34. In embodiments, the neuropathic pain is due to nerve damage resulting from metabolic diseases, trauma, ischemia or hemorrhage, inflammation, neurotoxicity, neurodegeneration, paraneoplastic conditions, vitamin deficiencies or cancer. Examples of neuropathic pain include, but are not limited to, postherpetic (or postherpetic) neuralgia, reflex sympathetic dystrophy / causalgia (neurotrauma), components of cancer pain, phantom limb pain, compressive neuropathy (e.g., carpal tunnel syndrome), and peripheral neuropathy (extensive nerve damage).

[0052]

[0056] In embodiments, the pain is dysalgesic pain. In embodiments, the subject may require treatment for dysalgesic pain. Dysalgesic pain is pain resulting from altered pain perception, despite the absence of clear evidence of actual or potentially triggering tissue injury causing activation of peripheral nociceptors, or evidence of a somatosensory disorder or lesion causing pain. Chimenti et al., Phys Ther. (2018) 98(5):302~314.

[0053]

[0057] In embodiments, pain is selected from acute pain, chronic pain, nociceptive pain, neuropathic pain, dysalgesic pain, traumatic pain, chemical pain, burn pain, ischemic pain, insect bite pain, stabbing pain, musculoskeletal pain (e.g., back pain, neck pain), rheumatoid arthritis pain, postoperative pain, bone pain (e.g., osteoarthritis), and pain due to various skin conditions (e.g., acne, psoriasis, psoriatic abscess, eczema, rosacea).

[0054]

[0058] In embodiments, the dicarboxylic acid ester of formula I is administered in an amount sufficient to induce an analgesic effect in a subject so as to reduce the pain response. In embodiments, the dicarboxylic acid ester of formula I is administered in an amount sufficient to reduce the pain response by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In the application, the dicarboxylic acid ester of formula I reduces the pain response to, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, The drug is administered in an amount sufficient to induce an analgesic effect in the subject, such that the analgesic effect is reduced by approximately 60% to 90%, 70% to 90%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, or 50% to 70%.

[0055]

[0059] In embodiments, the dicarboxylic acid ester of formula I is administered in an amount sufficient to induce an analgesic effect in a subject to reduce the pain response mediated by nociceptive pain, neuropathic pain, or dysalgesic pain. In embodiments, the dicarboxylic acid ester of formula I is administered in an amount sufficient to reduce the pain response mediated by nociceptive pain, neuropathic pain, or dysalgesic pain by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In this embodiment, the dicarboxylic acid ester of formula I reduces the pain response mediated by nociceptive pain, neuropathic pain, or dysalgesic pain by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, and about 40%. The drug is administered in an amount sufficient to induce an analgesic effect in the subject, reducing the pain level by approximately 90%, 50% to 90%, 60% to 90%, 70% to 90%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, or 60% to 80%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, or 50% to 70%.

[0056]

[0060] In embodiments, the present disclosure provides a method for modulating one or more pattern recognition receptor signaling pathways, including nucleotide oligomeric domain (NOD) receptors and dectin receptors, in a subject of interest, comprising the step of administering a therapeutically effective dose of the dicarboxylic acid ester of formula I to the subject. In embodiments, the reduction of pain (or reduction of the pain response) is achieved by modulating PRR-related pain activity.

[0057]

[0061] In some embodiments, pain reduction (or reduction of pain response) is achieved by regulating PRR activity.

[0058]

[0062] In one embodiment, the reduction of the pain response (i.e., the reduction of the pain response) is achieved by regulating NOD receptor activity.

[0059]

[0063] In some embodiments, the reduction of the pain response (i.e., the reduction of the pain response) is achieved by regulating dectin receptor activity.

[0060]

[0064] In embodiments, the present disclosure provides a method for inducing an analgesic effect and thereby modulating pain in a target by administering a pharmaceutical composition containing a therapeutically effective amount of the dicarboxylic acid ester of formula I to a target, thereby promoting the synthesis or release of one or more of leptin, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0061]

[0065] In embodiments, the present disclosure provides a method for inducing an analgesic effect and thereby modulating pain in a target by administering a pharmaceutical composition containing a therapeutically effective amount of the dicarboxylic acid ester of formula I to a target, thereby inhibiting the synthesis or release of at least one of inducible nitric oxide synthase (iNOS), adenosine triphosphate (ATP), and prostaglandin E2 (PGE2), or downregulating their activity and / or expression.

[0062]

[0066] In embodiments, the present disclosure provides a method for inducing an analgesic effect and thereby treating a subject suffering from pain by promoting a local increase in one or more of leptin, M-CSF, G-CSF, and GM-CSF at the administration site, or a decrease in iNOS, ATP, and PGE2, the method comprising the step of administering a pharmaceutical composition comprising a dicarboxylic acid ester of formula I to the subject.

[0063]

[0067] In the embodiment, the dicarboxylic acid ester of formula I is administered to a subject in an amount sufficient to induce the release of one or more of leptin, M-CSF, G-CSF, and GM-CSF, or to upregulate their activity and / or expression.

[0064]

[0068] In the embodiment, the dicarboxylic acid ester of formula I is administered to a subject in an amount sufficient to suppress the release of at least one of iNOS, ATP, and PGE2, or to downregulate their activity and / or expression.

[0065] Synthesis of dicarboxylic acid esters

[0069] Dicarboxylic acid esters of formula I may be commercially obtained or prepared by various methods known in the art. In embodiments, dicarboxylic acid esters can be prepared by the direct formation of esters from essential acids and alcohols. This condensation can be achieved by dehydration of the reaction mixture with a suitable agent or by heating of the acid and alcohol mixture. In embodiments, dicarboxylic acid esters can be prepared by reacting an alcohol with an activated acid. Examples of activated acids include acid halides, acid anhydrides including both homo and heteroanhydrides, reactions of the internal anhydride of the parent acid with an essential alcohol, and esters and anhydrides of both acids and alcohols, which are formed by the reaction of an essential acid or alcohol with p-toluenesulfonyl chloride to produce a tosyl anhydride or ester, which is then reacted with the alcohol or acid, respectively, to produce the desired final ester. Similarly, a simple organic acid anhydride, such as acetic anhydride, may be used instead of p-toluenesulfonyl chloride. In addition, the process may begin with one ester selected from the desired material composition, and the ester, which is the starting material for the acid, is converted into an ester that the alcohol will react with using a solution of the ester in the desired alcohol in the presence of a suitable acidic or basic catalyst. This method is also known in the art as transesterification.

[0066]

[0070] For example, one may start with a dimethyl ester of an acid, and a diethyl ester of an acid can be easily formed by an ester solution in ethanol in the presence of an acid or base. In addition, if a mixed ester of acids is desired, a suitable composite solution of two or more desired alcohols may be used by any of the methods described herein.

[0067]

[0071] The desired esters may be formed using halogenated intermediates or components. For example, thionyl chloride chlorinates both an acid and an alcohol, thereby yielding acyl and alkyl chlorides. These acyl and alkyl chlorides can then further react with the desired alcohol or acid to produce the desired ester product. Other common halogenating agents include, for example, oxalyl chloride and phosphorus chlorides and bromides, such as phosphorus pentachloride or trichloride and phosphorus pentabromide or tribromide or acid acid phosphorus.

[0068]

[0072] Ester formation by the action of a strong base on a mixture of acid and alcohol is a common practice. Examples of strong bases include lithium aluminum hydride and other metal hydrides, alkali metal alkoxides such as sodium ethoxide, and diisobutylaluminum hydride.

[0069] Method of administration

[0073] The pharmaceutical compositions of this disclosure can be administered to a subject in a variety of ways. For example, the pharmaceutical compositions can be administered topically, transdermally, intravenously, subcutaneously, intramuscularly, or orally. The pharmaceutical compositions can be applied topically to and / or around a target area or an area where treatment is desired.

[0070]

[0074] Treatment plans can vary depending on various factors typically considered by those skilled in the art. These factors include the route of administration, the nature of the formulation, the nature of the patient's disease, the size of the subject, weight, surface area, age, sex, other medications administered to the patient, and the judgment of the attending physician. Pharmaceutical compositions may be administered concurrently with or in addition to other treatments for reducing inflammatory conditions or pain.

[0071]

[0075] The pharmaceutical composition may be administered in combination with one or more additional therapeutic agents for the treatment of pain and / or inflammation. One or more additional therapeutic agents may be administered via the same or different routes of administration. Examples of one or more additional therapeutic agents include analgesics, anti-inflammatory agents, anesthetics, antibiotics, and antifungal agents.

[0072]

[0076] In embodiments, the pharmaceutical composition is administered topically to the target skin, for example, to an area where treatment is desired or at least to an area located near the area where treatment is desired. In embodiments, the pharmaceutical composition is administered to the target by topically rubbing it into the skin, thereby allowing the composition (or at least the dicarboxylic acid ester) to be absorbed into the skin.

[0073]

[0077] In embodiments, for preventative treatment, the pharmaceutical composition may be administered to a subject at intervals of 96 hours to immediately before the procedure (or surgery), 72 hours to immediately before the procedure (or surgery), 48 hours to immediately before the procedure (or surgery), 24 hours to immediately before the procedure (or surgery), 12 hours to immediately before the procedure (or surgery), 8 hours to immediately before the procedure (or surgery), 4 hours to immediately before the procedure (or surgery), 2 hours to immediately before the procedure (or surgery), 1 hour to immediately before the procedure (or surgery), or 0.5 hours to immediately before the procedure (or surgery), or within any range that can be delivered to the subject immediately before the procedure. The pharmaceutical composition may be applied topically to a healthy skin area at any time, for example, before hiking in one embodiment.

[0074]

[0078] In embodiments, for preventive treatment, the pharmaceutical composition is localized or administered topically to the subject before a procedure, such as venipuncture, injection, incision, depilation, or application and removal of a tattoo.

[0075]

[0079] In embodiments, the pharmaceutical composition is localized or administered topically to a subject during or after a procedure such as venipuncture, injection, incision, depilation, or application and removal of a tattoo.

[0076]

[0080] In the embodiment, the pharmaceutical composition is applied to the target once or more times. For example, the pharmaceutical composition can be administered at predetermined intervals. In the embodiment, for example, the pharmaceutical composition can be applied once a day, twice a day, three times a day, four times a day, or more than four times a day, or every other day, once every three days, once every four days, etc.

[0077]

[0081] In the embodiment, the pharmaceutical composition is administered to the subject in a therapeutically effective dose. When administered to the subject, the therapeutically effective dose depends on the specific condition being treated and the desired outcome.

[0078]

[0082] The pharmaceutical composition can be administered to the subject requiring it approximately every 1 to 24 hours, every 1 to 12 hours, every 2 to 8 hours, every 2 to 6 hours, every 4 to 6 hours, every 4 to 8 hours, every 12 hours, every 24 hours, every 48 hours, or more frequently. In embodiments, the pharmaceutical composition can be administered once, twice, three, four, five, six, seven, eight times a day, or more frequently, or in any combination thereof. In embodiments, the pharmaceutical composition can also be administered daily, every other day, every two days, every three days, every four days, or less frequently, or in any combination thereof. In embodiments, the pharmaceutical composition can be administered to the subject requiring it for a duration of 1 to 30 days, 1 to 25 days, 1 to 20 days, 1 to 15 days, or 1 to 10 days.

[0079] formulation

[0083] In embodiments, the pharmaceutical compositions of this disclosure may be formulated for delivery by any route of administration known in the art, including, but not limited to, topical, transdermal, intravenous, subcutaneous, intramuscular, or oral administration.

[0080]

[0084] The appropriate formulation depends on the chosen route of administration. As is understood in the art, any of the well-known techniques, carriers, and excipients, such as those disclosed in Remington's Pharmaceutical Sciences, Vol. 18 (Mack Publishing Company: Easton, Pa., 1990), which is incorporated herein by reference, may be used. The pharmaceutical compositions disclosed herein may be prepared by any means known in the art, such as conventional mixing, dissolution, granulation, elutriation, emulsification, encapsulation, encapsulation, or compression methods.

[0081]

[0085] The pharmaceutical compositions include those suitable for topical, transdermal, intravenous, subcutaneous, intramuscular, or oral administration, but the most suitable route may depend, for example, on the recipient's condition and disorder.

[0082]

[0086] In embodiments, the pharmaceutical compositions are suitable for topical and transdermal administration. Topical and transdermal administration of the azelaic acid esters of this disclosure may be in the form of treated gels, creams, lotions, solutions, ointments, suspensions, or emulsions. These pharmaceutical compositions may further comprise one or more suitable excipients disclosed herein.

[0083]

[0087] Parenteral administration of the dicarboxylic acid ester of formula I, for example, intravenous, subcutaneous, or intramuscular administration, may be in the form of a solution, suspension, or emulsion. In one embodiment, these formulations are prepared in physiological saline. These pharmaceutical compositions may further comprise one or more preferred excipients disclosed herein.

[0084]

[0088] In embodiments, the pharmaceutical composition is suitable for oral administration. The pharmaceutical composition can be conveniently presented in unit dosage forms and can be prepared by any method well known in the field of pharmacy. Typically, these methods involve the step of mixing a dicarboxylic acid ester of formula I and, optionally, any co-administered active ingredient with a carrier constituting one or more minor components. Generally, the pharmaceutical composition is prepared by homogeneously and completely mixing the active ingredient with a liquid carrier or a pulverized solid carrier or both, and then, if necessary, shaping the product into the desired composition. Pharmaceutical compositions suitable for oral administration and any optionally second active ingredient can be presented as individual units, e.g., capsules, cachets or tablets, each containing a predetermined amount of the active ingredient(s) as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient(s) can be presented as a bolus, lick or paste. These pharmaceutical compositions may further comprise one or more suitable excipients disclosed herein.

[0085] Excipients

[0089] In embodiments, the pharmaceutical compositions of the present disclosure may further comprise one or more excipients. The excipients may include carriers, such as insoluble polysaccharides or oligosaccharides. Examples of carriers include, but are not limited to, cellulose acetate, cellulose butyrate acetate, cellulose propionate acetate, cellulose phthalate acetate, chitosan, β-cyclodextrin, ethylcellulose, hydroxypropyl methylcellulose phthalate (HPMCP), crystalline cellulose, starch, and any combination thereof.

[0086]

[0090] Excipients may include thickeners, such as water-soluble polysaccharides. Examples of thickeners include, but are not limited to, hydroxypropyl methylcellulose (HPMC), acacia, alginic acid, colloidal silicon dioxide, carboxymethylcellulose calcium, gelatin, hydroxypropylcellulose, hydroxypropylcellulose (hypromellose), methylcellulose, sucrose, sodium alginate, sodium carboxymethylcellulose, and any combination thereof.

[0087]

[0091] In embodiments, the pharmaceutical compositions of the present disclosure may further include one or more pharmaceutical excipients, such as ascorbic acid, EDTA dihydrate, glycerin, citric acid monohydrate, sodium citrate dihydrate, sodium benzoate, sodium propionate, 70% sorbitol solution, sucralose, FD&C Yellow #6, artificial flavors (e.g., artificial peppermint flavor, artificial fruit flavor), purified water, or any combination thereof.

[0088]

[0092] In embodiments, the pharmaceutical compositions of the present disclosure may contain preservatives. Suitable preservatives include, but are not limited to, mercury-containing substances, such as phenylmercury salts (e.g., acetic acid, boric acid, and phenylmercury nitrate) and thimerosal; stabilized chlorine dioxide; quaternary ammonia compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride; imidazolidinyl urea; parabens, such as methylparaben, ethylparaben, propylparaben, and butylparaben, and their salts; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol; chlorobutanol; chlorocresol; phenylethyl alcohol; disodium EDTA; benzoic acid, benzyl alcohol, and sorbic acid, and their salts.

[0089]

[0093] In embodiments, the pharmaceutical compositions of the present disclosure may include one or more acceptable pH adjusters and / or buffering agents, such as acids, e.g., acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases, e.g., sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers, e.g., citric acid / glucose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within a pharmaceutically acceptable range. In embodiments, the pH of the pharmaceutical compositions of the present disclosure may be pH 4 to pH 7.5.

[0090]

[0094] In embodiments, the pharmaceutical compositions of the present disclosure may contain sugar alcohols. Examples of sugar alcohols include, but are not limited to, mannitol, glycerol, galactitol, fusitol, inositol, boremitol, maltotriitol, maltoethetreitol, polyglycitol, erythritol, treitol, ribitol, arabitol, xylitol, allitol, dulcitol, glucitol, sorbitol, althritol, iditol, maltitol, lactitol, isomalt, and any combination thereof.

[0091]

[0095] In embodiments, the pharmaceutical compositions of the present disclosure may include additives. Examples of additives include, but are not limited to, diluents, binders, surfactants, lubricants, coating materials, plasticizers, colorants, flavoring agents, or pharmaceutically inert materials. Examples of diluents include, for example, cellulose; cellulose derivatives, such as crystalline cellulose; starch; starch derivatives, such as corn starch and cyclodextrin; sugars; sugar alcohols, such as lactose and D-mannitol; and inorganic diluents, such as dried aluminum hydroxide gel, precipitated calcium carbonate, magnesium aluminometasilicate, and calcium hydrogen phosphate. Examples of binders include, for example, hydroxypropyl cellulose, methylcellulose, hydroxypropyl methylcellulose (hydroxypropyl methylcellulose), povidone, dextrin, pullulan, hydroxypropyl starch, polyvinyl alcohol, acacia, agar, gelatin, tragacanth, and macrogol. Examples of surfactants include fatty acid sucrose esters, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate, sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol, and quaternary ammonium salts (e.g., benzyldimethyltetradecylammonium chloride hydrate, benzethonium chloride, benzylcetyldimethylammonium chloride hydrate, benzyldimethylstearylammonium chloride hydrate, benzyldodecyldimethylammonium chloride hydrate, benzyldodecyldimethylammonium bromide). Examples of lubricants include stearic acid, calcium stearate, magnesium stearate, and talc. Examples of lubricants include dried aluminum hydroxide gel and magnesium silicate. Examples of coating materials include hydroxypropyl methylcellulose 2910, aminoalkyl methacrylate copolymer E, diethylaminoacetic acid polyvinyl acetal, macrogol 6000, and titanium dioxide.Examples of plasticizers include triethyl citrate, triacetin, and macrogol 6000.

[0092]

[0096] In embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of the dicarboxylic acid ester of formula I and a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the dicarboxylic acid ester of formula I in an amount of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total weight of the formulation. In the application, the pharmaceutical composition is contained in an amount of approximately 5% to 100% by weight, approximately 5% to 95% by weight, approximately 5% to 90% by weight, approximately 5% to 85% by weight, approximately 5% to 80% by weight, approximately 5% to 75% by weight, approximately 5% to 70% by weight, approximately 10% to 100% by weight, approximately 10% to 95% by weight, approximately 10% to 90% by weight, approximately 10% to 85% by weight, and approximately 10% by weight. ~80% by weight, approximately 10% by weight ~ approximately 75% by weight, approximately 10% by weight ~ approximately 70% by weight, approximately 15% by weight ~ approximately 100% by weight, approximately 15% by weight ~ approximately 95% by weight, approximately 15% by weight ~ approximately 90% by weight, approximately 15% by weight ~ approximately 85% by weight %, about 15% to about 80% by weight, about 15% to about 75% by weight, about 15% to about 70% by weight, about 20% to about 100% by weight, about 20% to about 95% by weight, about 20% to about 90% by weight, about 20% by weight Amount %~about 85 weight%, about 20 weight%~about 80 weight%, about 20 weight%~about 75 weight%, about 20 weight%~about 70 weight%, about 25 weight%~about 100 weight%, about 25 weight%~about 95 weight%, about 25 weight%~about 90 Weight%, about 25% to about 85% by weight, about 25% to about 80% by weight, about 25% to about 75% by weight, about 25% to about 70% by weight, about 30% to about 100% by weight, about 30% to about 95% by weight, about 3 0% to approximately 90% by weight, approximately 30% to approximately 85% by weight, approximately 30% to approximately 80% by weight, approximately 30% to approximately 75% by weight, approximately 30% to approximately 70% by weight, approximately 35% to approximately 100% by weight, approximately 35% to approximately 95% by weight, about 35% to about 90% by weight, about 35% to about 85% by weight, about 35% to about 80% by weight, about 35% to about 75% by weight, about 35% to about 70% by weight, about 40% to about 100% by weight,About 40% to about 95% by weight, about 40% to about 90% by weight, about 40% to about 85% by weight, about 40% to about 80% by weight, about 40% to about 75% by weight, about 40% to about 70% by weight, about 45% to about 100% by weight, about 45% to about 95% by weight, about 45% to about 90% by weight %, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 50% to about 100%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80% by weight Contains therapeutically effective amounts of the dicarboxylic acid ester of formula I in the following proportions: 1% by weight, approximately 50% to 75% by weight, approximately 50% to 70% by weight, approximately 55% to 100% by weight, approximately 55% to 95% by weight, approximately 55% to 90% by weight, approximately 55% to 85% by weight, approximately 55% to 80% by weight, approximately 55% to 75% by weight, approximately 55% to 70% by weight, approximately 60% to 100% by weight, approximately 60% to 95% by weight, approximately 60% to 90% by weight, approximately 60% to 85% by weight, approximately 60% to 80% by weight, approximately 60% to 75% by weight, and approximately 60% to 70% by weight.

[0093]

[0097] In embodiments, the pharmaceutical compositions of the present disclosure comprise a dicarboxylic acid ester of formula I, which can be formulated and blended into all pharmaceutical dosage forms known to those skilled in the art. Such pharmaceutical compositions can be used in cells, tissues, and organs of subjects requiring therapeutic and broad-dose administration. In embodiments, the dicarboxylic acid esters of the present disclosure can be administered orally, parenterally, topically, and locally to subjects at a daily dose of 0.001 mg / kg to 1000 mg / kg body weight per day.

[0094] kit

[0098] In embodiments, the Disclosure also provides a kit comprising, in particular, a pharmaceutical composition of the Disclosure for topical administration, a wipe for cleaning the site, a swab or brush for spreading the material to be applied, and an adhesive dressing to cover the site; optionally, the kit may include a waste disposal bag or container; optionally, the kit may include an absorbent element; optionally, the kit may include a pair of gloves (e.g., sterile nitrile or latex gloves). [Examples]

[0095] Materials and methods

[0099] Chemicals: Diethyl azelaate (purity >99%) was synthesized from azelaic acid and ethyl alcohol by acid-catalyzed esterification followed by fractional distillation, to a purity of 99% determined by gas chromatography-mass spectrometry (GC-MS). Unless otherwise specified, chemicals were sourced from Sigma Chemical (St. Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA).

[0096]

[0100] Cell proliferation and cytotoxicity assays: CellTiter 96 non-radioactive cell proliferation assay (MTT), CellTiter 96 aqueous mono-solution cell proliferation assay (MTS), and CellTiter-Glo bioluminescent cell viability (ATP) kits were purchased from Promega (Madison, WI). Protein concentrations were measured using the Micro BCA protein assay kit (Pierce Biotechnology, Rockford, IL).

[0097]

[0101] Isolation of Human Peripheral Blood Mononuclear Cells: Fresh human peripheral blood mononuclear cell (PBMC) samples were obtained from healthy volunteers. Blood was collected in BD Vacutainer CPT cell preparation tubes (Beckton Dickinson, Franklin Lakes, NJ), centrifuged at 1500 × g at room temperature, and PBMCs were isolated according to the supplier's protocol. Cell count and viability were assessed using the trypan blue exclusion method and measured with a hemocytometer.

[0098]

[0102] Membrane fluidity measurement: Membrane fluidity was measured using a membrane fluidity kit (PN M0271, Marker Gene Technologies, Eugene, OR). PBMCs were washed and 7 × 10⁶ cells were placed in phenol red-free RPMI cell culture medium containing 10% fetal bovine serum (FBS). 5 The solution was adjusted to cells / mL. 60,000 PBMCs per well were triple-sown in 96-well Acrowell filter plates (Pall Corporation, Port Washington, NY) and treated with 1 μM pyrendecanoic acid alone or in the presence of the test substance (DEA from cholesterol, 100 μM to 0.1 μM or 10 μM) while gently mixing in the dark for 20 minutes. Excimer fluorescence (excitation at 355 nm, emission at 460 nm) was corrected for monomer (excitation at 355 nm, emission at 400 nm).

[0099]

[0103] Multiplex immunoassay: Human and mouse cytokine, chemokine, and growth factor panels (including leptin and colony-stimulating factors G-CSF, GM-CSF, and M-CSF) were obtained from Affymetrix, Fremont, and CA. Samples were double or triple assayed as described for mouse assays [R. Streeper et al., Current Topics in Nutraceutical Research (2011) 9:1~12] and human assays [E. Izbicka et al., Cancer Genomics Proteomics (2012) 9(1):27~35]. Multiplex immunoassays were performed using the Luminex 100 IS system (Luminex Corporation, Austin, TX). Analyte concentrations were calculated from standard curves using Bio-Plex Manager 4.1.1 (Bio-Rad Laboratories, Hercules, CA).

[0100]

[0104] Pathogen-associated molecular pattern receptor agonist research: PRR agonist kit; human Toll-like receptors (TLR1-9; n=9 agonists), nucleotide oligomerized domains (NOD) NOD1 / NOD2 (n=10 agonists, represented as n1, n2, ..., n10), and Dectin-1 (single agonist) were purchased from InvivoGen, San Diego, CA. Human plasmacytoid dendritic cells (MatTek Corporation, Ashland, MA) were used in a 1.6 × 10⁶ study. 5 The analytes were diluted to cells / mL and incubated with an agonist for 24 hours in the absence of DEA or with 0.5% DEA at a moderate concentration recommended by the supplier. The measured levels of the analytes were normalized to the number of viable cells.

[0101] result Example 1

[0105] This experiment demonstrates that DEA modulates biomarkers of pain signaling in human epiderm (EpiDerm) tissue.

[0102]

[0106] Epiderm Bioassay: Epiderm® 3D skin models were purchased from MatTek Corporation (Ashland, MA). Test Substances: 25% DEA, 25% sodium azelaate, 1% sodium salicylate, 1% green tea extract, 1 μg / mL doxycycline high-crate (doxorubicin), 1% 4-ethoxybenzenealdehyde, and 0.025% total trans retinoic acid were tested in triplicate for 24 hours, either alone or in combination with 0.1% croton oil used as an irritant. The conditional medium was collected, and the remaining cells were homogenized on ice in the presence of protease and phosphatase inhibitors. The solubilized solution was purified by centrifugation. The medium and cell solubilized solution were rapidly frozen at -70°C. Proteins were degraded by gel electrophoresis and visualized by immunoblotting using human-specific antibodies against inducible nitric oxide synthase (iNOS), S-nitrosocysteine, and superoxide dismutase 2 (all from Abcam, Cambridge, and MA).

[0103]

[0107] Epiderm is a three-dimensional human tissue construct of untransformed human keratinocytes that exhibits in vivo-like morphological and growth characteristics, allowing for topical application to tissue surfaces that mimic human exposure pathways.

[0104]

[0108] When epiderm tissue was exposed to 0.1% croton oil used as a chemical irritant, treatment with 25% DEA neutralized the croton oil irritant toxicity in proliferation and viability assays and protected the tissue integrity as assessed by microscopy. Cell signaling experiments compared the effects of DEA with other compounds commonly used in topical formulations: azelaic acid, salicylic acid, green tea extract, doxorubicin, 4-ethoxybenzenealdehyde, and total trans retinoic acid. Among 57 test cytokines, chemokines, and growth factors, DEA downregulated most pro-inflammatory markers. Croton oil (chemical irritant) was used as a control. Results are presented as the percentage difference in marker levels between tissues treated with the test substance and tissues treated with croton oil alone (Table 1). As shown in Table 1, DEA simultaneously upregulated the anti-inflammatory cytokines IL-4, IL-10, IL-13, and IL-1ra. Furthermore, DEA also inhibited PGE2 and increased leptin, GM-CSF, and G-CSF levels. This pattern of tandem-up regulation of test markers in conjunction with PGE2 downregulation was unique to DEA and not observed with any other test substance.

[0105] Table 1: DEA simultaneously upregulates the anti-inflammatory markers leptin and colony-stimulating factor, while downregulating PGE2 in epidermal tissue (control) exposed to croton oil invasion. Results are expressed as a percentage of the control. [Table 1]

[0106] Example 2

[0109] This experiment demonstrates that DEA regulates cellular signaling from pattern recognition receptors.

[0107]

[0110] The inventors of this disclosure demonstrated changes in membrane protein function using a U-shaped dose-response pattern. At a concentration of 0.5%, DEA increased membrane fluidity by 18%, exceeding cholesterol control levels. The physiological consequences of DEA-induced membrane fluidity activity were further investigated in human plasmacytoid dendritic cells expressing multiple PRRs, including TLRs, NODs, and Dectin-1 receptors. Human plasmacytoid dendritic cells were stimulated with receptor agonists alone or in combination with 0.5% DEA.

[0108]

[0111] Human plasmacytoid dendritic cells (1.6 × 10⁻⁶) 5 Twenty different PRR agonists were exposed to 0.5% DEA ( / ml) for 24 hours either alone at concentrations within the physiological range of each agonist or in the presence of 0.5% DEA. Results are expressed as the ratio of ATP secreted from cells treated with PRR receptor agonists and DEA (treatment group) to the level of ATP secreted by cells treated with the agonist alone (control group). Agonists are positioned according to the localization of each agonist receptor on the cell surface or intracellularly. Extracellular levels were measured, and results are expressed as the ratio of ATP released from cells + receptor agonist + DEA to ATP from cells + receptor agonist alone. As shown in Figure 1, the ATP ratio was <1 in all cases except for two NOD receptor ligands (n9 and n10), where DEA did not show a clear effect on ATP signaling, but it effectively competed with the two NOD ligands based on different cytokine patterns in the presence and absence of DEA.

[0109]

[0112] Referring to Figure 1, DEA competed with all tested PRR agonists. The effect of DEA was greater against cell surface receptors than against intracellular receptors, and greater against multimeric receptors (TLRs and Dectin-1) compared to monomeric NOD receptors. It was further demonstrated that DEA downregulates cellular signaling from all tested PRRs in a pattern specific to and characteristic of each tested receptor agonist.

[0110]

[0113] As disclosed herein, the findings of DEA's ability to compete with all tested PRR agonists with its tremendous effect on cellular signaling are unexpected and unprecedented.

[0111] Example 3

[0114] This experiment demonstrates that localized DEA increases response latency to nociceptive thermal stimuli in vivo.

[0112]

[0115] In vivo study: Animal studies and all procedures were conducted at New Frontier Labs LLC, San Antonio, Texas, in accordance with the Animal Welfare Act Enforcement Regulations (9 CFR 3) and the NIH Guidelines on the Care and Use of Laboratory Animals. Both male and female Balb / c mice, 8-10 weeks old and weighing approximately 25g, were purchased from Taconic Farms (Hudson, NY). The animals were acclimatized to the environment for at least 72 hours after delivery. Before the experiment, the animals were randomized into treatment and control groups. Only male mice were used to evaluate the nociceptive effect of DEA (n=8 per group). The hot plate test was performed at a constant temperature of 42°C, as described in S. Hunskaar et al., Eur. J. Pharmacol. 1985;111(2):221-6, incorporated herein by reference. DEA was administered to the hind limbs either in pure form (100%) or diluted in ethanol (vehicle). The drug was administered 5 minutes before the heat test. The time from heat exposure to the animals first licking their hind limbs was measured using a stopwatch.

[0113]

[0116] Statistical Analysis: Statistical analysis was performed using Student's t-statistic; a p-value < 0.05 was considered significant. All samples / data were included in the analysis. Continuous distribution outcomes were summarized by mean and standard deviation. All statistical tests were two-sided at an experimentally wise 5% significance level using SAS version 9.2.

[0114]

[0117] Nociceptive thermal stimulation can induce pain, and a threshold of 40°C or higher is used to test the efficacy of therapeutic analgesics in rodents, as described by S. Hunskaar et al., Eur J Pharmacol. (1985) 111(2):221-6. Figure 2 summarizes the results of the effect of DEA on thermal sensitivity in mice. There was a clear dose-response in the protective effect of DEA, as demonstrated by the increase in response latency. At the highest concentration of 100%, DEA reduced thermal sensitivity by 33%, and the difference between DEA at 100% concentration and the untreated control was statistically significant (p=0.03). The degree of pain protection induced by topical DEA was comparable to that of acetylsalicylic acid and paracetamol applied intraperitoneally, as described by Hunskaar et al., Behav. Brain Res. (1986) 21(2):101-8.

[0115]

[0118] Transdermal adsorption of topical analgesics often limits their effectiveness, as reported by E. Beetge et al., Int J Pharm. (2000) 193(2):261~4. The physicochemical properties of DEAs, combined with their excellent safety and efficacy profile, are advantageous for their use in topical pain management.

[0116] Example 4

[0119] This experiment demonstrates that DEA disrupts the assembly of cholera toxin subunits in the plasma membrane of PBMCs.

[0117]

[0120] Visualization of lipid rafts: Fluorescent labeling of lipid rafts in human PBMCs was performed using the Vybrant Alexa Fluor 488 Lipid Raft Labeling Kit (PN V-34403, Life Technologies, Grand Island, NY). PBMCs (1 × 10⁶ lbs in 0.2 mL RPMI medium containing 10% FBS, in the absence or presence of 0.5% DEA) were used. 6Cells (quadruple repeats) were labeled with a fluorescent cholera toxin subunit B (CT-B) complex at 4°C for 10 minutes, transferred to polylysine-coated coverslips, washed with cold phosphate-buffered saline, and crosslinked with anti-CT-B antibody according to supplier instructions. The cells were fixed in 4% formaldehyde, washed with cold phosphate-buffered saline containing 0.1% bovine serum albumin, and placed on dry vector shields (Vectashield). Images were obtained by confocal microscopy, fluorescence imaging was performed using argon laser irradiation, and analysis was performed using ImageJ software version 1.53e (ImageJ.nih.gov).

[0118]

[0121] The regulation of lipid raft-related proteins by DEA was investigated using the multimeric AB5 toxin produced by the cholera toxin Vibrio cholerae. The B subunit of the toxin binds to the GM1 ganglioside receptor on the cell surface. The receptor-bound B subunit is localized to membrane microdomains called lipid rafts, where they form a pentameric membrane invasion complex. The A subunit of the toxin binds to the pentamer of the membrane receptor-bound B subunit, and the complex is internally translocated via endocytosis, releasing the toxin A subunit into the protoplasm. The integrity of the lipid raft is necessary for the cholera toxin to exert its toxic activity (S. Ray et al., J. Biol. Chem. 2012;287(36):30395~405).

[0119]

[0122] To investigate the effect of DEA on the formation of membrane invasion complexes, human PBMCs were exposed to fluorescently labeled cholera toxin B subunits (1) with DEA ​​treatment and (2) without DEA treatment. Individual cells were visualized using phase-contrast microscopy, and cell-bound cholera toxin B subunits were visualized using fluorescence microscopy. Figure 3 shows representative images of control cells without DEA treatment (left column) and cells with DEA ​​treatment (right column). Phase contrast and corresponding fluorescence images are shown in the upper and lower panels, respectively. Most control cells (77%) showed a fluorescence ring around the fluorescence focus, along with an enhanced fluorescence focus. In contrast, DEA-treated PBMCs showed faint diffuse fluorescence uniformly spread across the plasma membrane surface, and no enhanced fluorescence focus was detected.

[0120]

[0123] These results suggest that DEA disrupts B subunit pentamer formation and / or the binding of B subunits to the GM1 receptor, thereby interfering with the structuring of plasma membrane microdomains. The abbreviations used herein have the following meanings (unless otherwise specifically indicated, abbreviations not shown herein have their generally used meanings):

[0121] [Table 2]

[0122] [Related applications]

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 126,387, titled "DICARBOXYLIC ACID ESTERS FOR INDUCING AN ANALGESIC EFFECT," filed on 16 December 2020 in the names of inventors Robert T. STREEPER and Elzbieta IZBICKA. The entire contents of the aforementioned patent application are incorporated herein by reference.

Claims

1. A topical formulation for inducing a local analgesic effect in a subject suffering from pain, Diethyl azelaate in an amount of 20% to 100% by weight relative to the total weight of the aforementioned preparation, It contains a pharmaceutically acceptable carrier, The aforementioned subjects are patients suffering from pain selected from the group consisting of acute pain, chronic pain, nociceptive pain, neuropathic pain, dysalgesic pain, traumatic pain, chemical pain, burn pain, ischemic pain, pain from insect bites, stabbing pain, musculoskeletal pain, pain from rheumatoid arthritis or osteoarthritis, postoperative pain, bone pain, and skin-related pain, and the preparation is used for this purpose.

2. The formulation according to claim 1, characterized in that it is used to be administered every 4 to 8 hours.

3. The formulation according to claim 1 or 2, characterized in that it is used to be administered for a period of 1 to 30 days.

4. The formulation according to any one of claims 1 to 3, wherein the subject is a human.

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