Beta-caryophyllene for use in the treatment or prevention of pericarditis

Abstract

Beta-caryophyllene (BCP) is disclosed for use in the treatment or prevention of pericarditis. The effectiveness of BCP in reducing interleukin-1β (IL-1β) and IL-6 levels in an in vitro model is disclosed. Experiments are also disclosed showing that BCP is effective in reducing or preventing an increase in the pericardial cavity (an indicator of pericardial effusion) and pericardial thickness in an in vivo mouse model of pericarditis.

Description

[Technical Field]

[0001]

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 449,799, filed March 3, 2023, the entire contents of which are incorporated herein by reference.

[0002]

[0002] Field

[0003] The present invention relates generally to therapeutic agents and methods for treating or preventing cardiovascular diseases, disorders or conditions, and more particularly to therapeutic agents and methods for treating or preventing pericarditis. [Background technology]

[0003]

[0004] Background of the Invention

[0005] Pericarditis results from a strong pericardial inflammatory response, which involves the activation of a macromolecular intracellular complex called the NLRP3 (NACHT, leucine-rich repeat and pyrin domain-containing protein 3) inflammasome. Upon sensing injury and stress, the NLRP3 inflammasome triggers an inflammatory process accompanied by the release of proinflammatory cytokines, such as interleukin-1β, which act as downstream mediators.

[0004]

[0006] Pericarditis can be classified as acute, persistent, recurrent, and chronic. Acute pericarditis is a clinical syndrome characterized by transient incidents that resolve spontaneously or after treatment. Treatment primarily consists of nonsteroidal anti-inflammatory drugs (NSAIDs), aspirin, steroids, colchicine, and anakinra (4, 7, 8, 9). Episodes of acute pericarditis lasting more than 4–6 weeks but less than 3 months are called persistent pericarditis, while episodes lasting more than 3 months are known as chronic pericarditis. Recurrent pericarditis (RP) is defined as an episode of acute pericarditis occurring at least 4–6 weeks after resolution of the previous episode. RP has been reported to occur in 15–30% of patients with pericarditis, some of whom experience multiple recurrences in succession. In some cases, recurrence is due to inadequate treatment of the original episode, while in other cases, it is due to an inadequate response to current treatment.

[0005]

[0007] The pathogenesis of pericarditis is not fully understood (3). However, it is hypothesized that acute pericarditis represents a typical response to acute injury to pericardial mesothelial cells (4). Pericarditis is thought to be induced by an "irritant" such as a virus or cellular debris after viral infection (5, 6). Pericarditis can also be induced by cardiac procedures such as cardiac surgery (especially coronary artery bypass grafting), pacemaker insertion, radiofrequency ablation, transcatheter aortic valve implantation, and, rarely, percutaneous coronary intervention (7).

[0006]

[0008] According to Klein et al. (12), the entire contents of which are incorporated herein by reference, there is a high unmet need for a safe, convenient FDA-approved treatment that resolves recurrent events and reduces the risk of recurrence without imposing an undue treatment burden on patients. Following the publication of Klein et al., the FDA approved rilonacept (Arcalyst™), an interleukin-1 (IL-1) receptor blocker, as an orphan drug for the treatment of pericarditis and reducing the risk of recurrence in adult and pediatric patients aged 12 years and older. (11) Summary of the Invention [Problem to be solved by the invention]

[0007]

[0009] There is a continuing need for alternative therapeutic agents and methods for treating pericarditis, and the present invention aims to meet this need. [Means for solving the problem]

[0008]

[0010] Summary of the Invention

[0011] Through the following experiments, the present inventors found that β-caryophyllene (BCP) attenuates the increase in interleukin-1β (IL-1β) in an in vitro model of NLRP3 inflammasome activation. In another experiment, the present inventors found that BCP reduces the level of interleukin-6 (IL-6), which is secreted independently of the NLRP3 inflammasome pathway, indicating that the anti-inflammatory effect of BCP goes beyond inhibiting this pathway. Furthermore, after in vivo experiments using a mouse model of pericarditis, the present inventors found that BCP is effective in reducing pericardial effusion and pericardial thickness, two hallmarks of pericarditis.

[0009]

[0012] Thus, a first aspect of the present invention provides the use of BCP in the treatment or prevention of pericarditis.

[0010]

[0013] A second aspect of the invention provides the use of BCP in the preparation of a medicament for the treatment or prevention of pericarditis.

[0011]

[0014] According to a third aspect, the present invention provides a method of treating or preventing pericarditis in a subject in need thereof, the method comprising (optionally) identifying a subject suffering from or at risk of suffering from pericarditis, and administering to the subject an effective amount of BCP.

[0012]

[0015] A fourth aspect of the present invention provides BCP for use in the treatment or prevention of pericarditis.

[0013]

[0016] According to a fifth aspect, the present invention provides a composition comprising an effective amount of BCP and an effective amount of a pharmaceutically acceptable excipient, the composition being for use in the treatment or prevention of pericarditis.

[0014]

[0017] In some embodiments, the BCP is naturally occurring. In other embodiments, the BCP is synthetic. In yet other embodiments, the BCP is produced by a biotechnology process.

[0015]

[0018] Some embodiments of the uses and methods of the present invention involve the use of BCP to treat pericarditis, hi other embodiments, BCP is used to prevent recurrence of pericarditis.

[0016]

[0019] BCP can be used to reduce pericardial effusion and / or an increase in pericardial thickness in a subject (e.g., a subject suffering from or at risk of suffering from pericarditis) by, for example, at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, or up to about 100%, compared to the amount of pericardial effusion and / or pericardial thickness that would be present in the absence of administration of BCP. Furthermore, BCP can be used to reduce the release of IL-1β and IL-6 in a subject by, for example, at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

[0017]

[0020] Brief description of the diagram

[0021] The present invention may be better understood with reference to the following detailed description and drawings. [Brief explanation of the drawings]

[0018] [Figure 1A]

[0022] 1 is a bar graph showing the effect of a 10 μM dose of BCP on levels of IL-1β in an in vitro model of NLRP3 inflammasome activation. [Figure 1B]

[0023] 1 is a bar graph showing the effect of a 10 μM dose of BCP on the levels of IL-6 in another in vitro model. [Figure 2]

[0024] 1 is a bar graph showing the effect of BCP at a dose of 10 mg / kg on the pericardial cavity (an indicator of pericardial effusion) in an in vivo model of pericarditis. [Figure 3]

[0025] 1 is a bar graph showing the effect of BCP (10 mg / kg) on ​​pericarditis thickness in the same in vivo model of pericarditis. DETAILED DESCRIPTION OF THE INVENTION

[0019]

[0026] Detailed Description

[0027] definition

[0028] For clarity and to avoid ambiguity, certain terms are defined herein as follows.

[0020]

[0029] As used herein, the term "pharmaceutically active agent" may be used as disclosed herein and means a drug or agent intended for use in a subject to treat or prevent a disease, illness, physical injury, or pathological condition, or to affect the state, condition, or function of the body. Drugs used may be found in references such as, for example, the Rote Liste or the Merck Index. Examples that may be mentioned include, for example, BCP.

[0021]

[0030] When a composition of matter is described as having "at least X% purity," this means that one or more impurities may be present in an amount of up to 100-X% by weight, based on the total weight of the composition of matter. The purity of a component may be determined by high performance liquid chromatography (HPLC) or other suitable means.

[0022]

[0031] The term "subject" refers to members of the animal kingdom, including humans and other mammals. In some embodiments, the subject is a human.

[0023]

[0032] As used herein, the terms "treat," "treatment," and the like refer to halting or slowing the progression of a condition, disorder, or disease, or reducing the symptoms or the severity of its symptoms. The terms "prevent," "prevention," and the like refer to preventing, delaying, or reducing the risk of occurrence and / or development of a condition, disorder, or disease. The term is intended to encompass "improving the quality of life," "extending the lifespan," and "improving the clinical outcome" of a subject suffering from or at risk of suffering from a condition, disorder, or disease, and does not necessarily mean, but does not exclude, "curing" a condition, disorder, or disease.

[0024]

[0033] "Pharmaceutically acceptable excipient," as used herein, means any material that can be formulated with or present with a pharmaceutically active agent to achieve a desired function or functions, and that is not biologically or otherwise harmful when administered to a subject (e.g., a human). For example, the excipient should be non-toxic when administered and compatible with other ingredients in the formulation. One of ordinary skill in the art, given the teachings herein and information in the public domain, will know what compounds or ingredients would qualify as pharmaceutically acceptable excipients.

[0025]

[0034] The phrases "at least one," "one or more," and "and / or" are open-ended expressions that may be interpreted both conjunctively and disjunctively. For example, the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, respectively.

[0026]

[0035] The terms "a" or "an" mean "one or more." Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the term "or" is used in the sense of "and / or" unless the context clearly dictates otherwise. For the avoidance of doubt, reference herein to the use of BCP for treating or preventing pericarditis is a reference to the use of BCP for treating and / or preventing pericarditis.

[0027]

[0036] The term "comprise" means "including, but not limited to." Thus, for example, a composition that includes a set of compounds, components, or elements may include additional compounds, components, or elements not expressly listed. It should also be noted that the terms "comprise," "include," "contain," and "have" can be used interchangeably.

[0028]

[0037] The term "consisting of" means including the listed components and no additional components, except that, in the case of a compound or ingredient, it does not exclude other compounds or ingredients that may be present as natural or commercial impurities or additives. Natural and commercial impurities and additives will be apparent to those skilled in the art. For example, the beta-caryophyllene (BCP) used in the following experiments was plant-derived and supplied by Sigma-Aldrich. The Certificate of Analysis (C of A) states that the product contained 92.9% BCP and 5.9% other C15H24 terpene hydrocarbons as natural impurities. Additionally, other impurities were present in amounts of 1.2% (= 100 - 92.9 - 5.9%). Furthermore, the C of A specifies a target purity of 80%, but the actual purity was 92.9%. Thus, commercial sources of BCP may contain up to about 20, 17.5, 15, 12.5, 10, 7.5, or 5% impurities, including, in the case of plant-derived BCP, other plant constituents.

[0029]

[0038] The term "consisting essentially of" means "including the recited components (e.g., compounds, steps, ingredients, etc.) and any additional components that do not materially affect the basic and novel properties of the invention, which are the beneficial effects of BCP in the treatment and / or prevention of any form of pericarditis, i.e., acute pericarditis, persistent pericarditis, chronic pericarditis, and recurrent pericarditis."

[0030]

[0039] The terms "% w / w," "% wt.", "w / w%," "wt.%" and variations thereof refer to the amount of material calculated by dividing the weight of the material by the total weight of the formulation containing that material and multiplying by 100.

[0031]

[0040] The terms "% w / v," "w / v%," and variations thereof, refer to the mass of solute in grams divided by the volume in mL of solution in which the solute is dissolved, multiplied by 100. For example, a formulation containing 10 grams of solvent per 25 mL of solvent-containing solution would have 40 w / v% solvent, calculated as follows: (10 ÷ 25) × 100 = 40 w / v%.

[0032]

[0041] The term "about" refers to variations in the stated quantities that may occur, for example, through measuring procedures and liquid handling procedures used to make the pharmaceutical formulation, differences in the manufacture, source, or purity of the ingredients used to make the formulation, and / or differences due to different equilibrium conditions or different reaction levels of the ingredients in the formulation resulting from the initial mixture. For clarity, the term "about" includes variations in the stated value of up to ±5% or ±up to 10%. Whether or not a value is modified by the term "about," the claims include equivalents to the value.

[0033]

[0042] As used herein, the term "effective amount," in the context of the invention disclosed herein, means an amount that will produce a desired effect based on the purpose and function of the component. Implicit in "desired effect" is the absence of toxicity. For example, an effective amount of a pharmaceutically active agent is an amount that would be effective to provide a therapeutic effect while avoiding toxicity, such as may occur through long-term chronic administration. An effective amount of a solvent is an amount that would be effective, alone or together with other components, to solubilize other or remaining components of the formulation. What constitutes an effective amount can be determined by one of ordinary skill in the art through routine experimentation, given the teachings herein and / or publicly available information.

[0034]

[0043] As used herein, the phrase "Y-free" means that "Y" is not intentionally added, but may be present as an impurity or due to other factors. For clarity, a "Y-free" formulation either does not contain Y or contains only up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5% w / v Y based on the total formulation.

[0035]

[0044] Values ​​recited herein are intended to include all values ​​meeting the recited parameters, including those not explicitly recited. For example, a value less than 1.0 is less than 0.99, less than 0.98, less than 0.97, less than 0.90, less than 0.84, less than 0.56, less than 0.01, etc. Accordingly, all ranges disclosed herein should be understood to encompass all subranges subsumed therein. For example, a recited range of "1 to 10" should be considered to include all subranges between the minimum value of 1 and the maximum value of 10 (inclusive), such as 1 to 6.3, 5.5 to 10, 2.7 to 6.1, etc.

[0036]

[0045] This specification contemplates that any components may be omitted even if they are not expressly specified as being included or excluded herein.

[0037]

[0046] Beta-caryophyllene

[0047] The present invention contemplates the use of BCP and compositions (eg, pharmaceuticals) comprising same.

[0038]

[0048] BCP is a naturally occurring bicyclic sesquiterpene widely distributed throughout the plant kingdom (14). It is found in the essential oils of black pepper (Piper nigrum), melissa (lemon balm), guava leaf (Psidium guajava), hemp (Cannabis sativa), clove (Eugenia caryophyllata), ylang-ylang (Cananga odorata), and copaiba (Copaifera) species (17). BCP is a non-psychotropic, selective phytocannabinoid agonist of the CB2R and PPAR-γ receptors (15, 17, 18). BCP has been reported to have repellent, antibacterial or antibacterial, anticancer or antiproliferative, antifungal, AChE inhibitor properties, antioxidant, and anti-inflammatory properties. (18) Research suggests that BCP may be useful in treating anxiety, chronic pain, inflammation, cancer, depression, diabetes, neuroinflammatory conditions (e.g., Alzheimer's disease), endometriosis, interstitial cystitis, seizures, and nonalcoholic fatty liver disease (17, 18).

[0039]

[0049] The FDA has generally recognized as safe (GRAS) BCP and approved its use as a food additive and flavoring. However, BCP is classified as a Category 5 material (toxic at doses above 2000 mg / kg) according to OECD (Organization for Economic Cooperation and Development) Guideline 423 (15).

[0040]

[0050] The terms "beta-caryophyllene," "β-caryophyllene," and "BCP" are used interchangeably herein to refer to the compound (1S,4E,9R)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene.

[0041]

[0051] In practice, BCP occurs primarily as trans-caryophyllene ((E)-BCP) (1), with minor amounts of the isomers (Z)-caryophyllene ((Z)-BCP) (2) and α-humulene (α-caryophyllene) (3) and its oxidized derivative, α-caryophyllene oxide (BCPO) (4), as shown below (15, 17). [ka]

[0042]

[0052] The term BCP is intended to include all stereoisomers. The most commonly occurring form of BCP is one in which the stereocenter adjacent to the exocyclic double bond has the S configuration and the remaining stereocenters have the R configuration.

[0043]

[0053] Extraction of BCP from plant sources is accomplished by fractionation and other techniques known in the art (14). BCP can also be produced by synthetic and biotechnological processes. Chemical synthesis of BCP is described, for example, in Corey, EJ et al. J. Am. Chem. Soc. 1964 86 (3), 485-492 and also in Larionov et al., An unconventional approach to the enantioselective synthesis of caryophylloids. J. Am. Chem. Soc. 2008, 130, 2954-2955. Biotechnological techniques using microbial fermentation can also be used. However, due to the diversity of plants that produce BCP, obtaining BCP from natural sources is (currently) more convenient and less expensive than chemical synthesis and microbial fermentation (17).

[0044]

[0054] The BCP used in connection with the present invention has a purity of at least 80%, 85%, 90%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. Impurities that may be present in naturally occurring BCP are other C15H24 terpene hydrocarbons (e.g., isocaryophyllene and α-humulene, formerly known as α-caryophyllene). Synthetic BCP may contain impurities consisting of residual organic solvents and / or by-products or residues from production.

[0045]

[0055] The compositions may be in any dosage form known in the art, including, but not limited to, oral solutions, injectable or other parenteral formulations, tablets, gummies, gel capsules, chewing gum formulations, and the like.

[0046]

[0056] Oral solution and elixir formulations can be simple or highly complex, containing many types of excipients, including water-soluble organic solvents, water-insoluble organic solvents, surfactants, buffers, sugars, flavors, sweeteners, fragrances, dyes, antioxidants, and preservatives. Solutions may or may not contain water. Organic cosolvents, as known in the art, are typically used to solubilize poorly water-soluble drugs, including BCP, to the desired concentration in oral solutions. Oral solution embodiments include formulations containing BCP dissolved in water-insoluble solvents such as long-chain triglycerides, peanut oil, corn oil, soybean oil, sesame oil, olive oil, peppermint oil, hydrogenated vegetable oil, hydrogenated soybean oil, and medium-chain triglycerides. Medium-chain triglycerides can be synthetic or derived from coconut oil and palm kernel oil. Other water-insoluble solvents include beeswax, dl-α-tocopherol (vitamin E), and oleic acid.

[0047]

[0057] When in oral solution form, BCP may be present at a concentration of about 50, 75, 100, 150, or 200 mg / mL. Additionally or alternatively, the oral solution may contain BCP at a concentration of up to about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, or 250 mg / mL. In one embodiment, the oral solution contains BCP in an amount of about 100 mg / mL. In another embodiment, the oral solution contains BCP in an amount of about 250 mg / mL. In yet another embodiment, the oral solution contains BCP in an amount of about 500 mg / mL.

[0048]

[0058] When in the form of an injectable / parenteral solution, BCP may be present in concentrations as high as 500, 450, 400, 350, 300, or 250 mg / mL and / or as low as 200, 150, 100, or 50 mg / mL. In one embodiment, the injectable / parenteral solution contains BCP in an amount of about 100 mg / mL. In another embodiment, the injectable / parenteral solution contains BCP in an amount of about 250 mg / mL. In yet another embodiment, the injectable / parenteral solution contains BCP in an amount of about 500 mg / mL.

[0049]

[0059] As used herein, the term "subject" or "patient" refers to a mammal, including, but not limited to, humans and veterinary animals. In certain embodiments, the subject is a human.

[0050]

[0060] Additional Pharmaceutically Active Agents and Excipients

[0061] The mention of BCP herein does not exclude combination therapy with other pharmaceutically active agents. The mention of BCP herein does not exclude compositions containing additional pharmaceutically active agents. However, in some embodiments, only BCP is used to treat or prevent pericarditis. For example, other terpenes or terpenoids that may be present in essential oils containing BCP are explicitly excluded. In the same or other embodiments, the following pharmaceutically active agents are explicitly excluded from the uses, methods, and compositions of the present invention: α-caryophyllene (now called α-humulene), D-limonene, linalool, terpineol, terpinene, α-pinene, β-pinene, β-elemene, β-ocimene, camphene, nerolidol, euphor, citral, seratrol, falcarinol, salvinorin A, and pristimerin, alone or in any combination.

[0051]

[0062] In some embodiments, BCP is administered in combination with a therapeutic dose of another pharmaceutically active agent, such as a nonsteroidal anti-inflammatory drug, aspirin, a steroid, colchicine, and / or anakinra. Any desired combination of pharmaceutically active agents can be delivered simultaneously or sequentially (e.g., within 24 hours). The appropriate therapeutically effective dosage for each agent may be reduced due to additive or synergistic effects resulting from the combination therapy.

[0052]

[0063] Compositions according to the present invention may contain BCP together with at least one pharmaceutically acceptable excipient that does not impair the basic and novel properties of the present invention. For example, oral and injectable solutions according to the present invention may contain a solvent for BCP (e.g., a medium-chain triglyceride) and an effective amount of at least one lipophilic antioxidant to improve shelf life. Benzyl alcohol may be included in injectable solutions for its anesthetic properties. The at least one antioxidant may be selected from the group consisting of vitamin E (also known as α-tocopherol), carotenoids (xanthophylls and carotenes), propyl gallate, lecithin, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and monothioglycerol tert-butylhydroquinone (TBHQ). The term "effective amount" depends on the characteristics of the compound and refers to the amount of the compound or pharmaceutical composition that, when administered to a subject to treat (e.g., prevent or ameliorate) a condition, disorder, or state, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the compound being administered, as well as the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject being treated. Generally, the total amount of antioxidant will be greater than about 0.01% w / v and less than about 1.5% w / v. In some embodiments, the amount is greater than about 0.1% w / v and less than about 1% w / v.

[0053]

[0064] Additional excipients contemplated for use in practicing the present disclosure are those available to those skilled in the art, such as those found in the United States Pharmacopeia Vol. II and National Formulary Vol. XVII, US Pharmacopeia Convention, Inc., Rockville, Md. (1989).

[0054]

[0065] Administration method

[0066] BCP can be administered in a variety of ways known in the art, including orally and parenterally. The term "parenteral" is intended to include, but is not limited to, administration via intravenous (IV), subcutaneous (SC), intraperitoneal (IP), and intramuscular (IM) injection, and is used interchangeably herein with the term "injectable." Given that orally administered BCP is expected to have lower bioavailability, parenterally administered BCP doses will be lower than orally administered BCP doses.

[0055]

[0067] The compositions of the present invention can be administered at least once a week or at least once every 6, 5, 4, 3, or 2 days. The compositions can also be administered at least once a day or at least twice a day.

[0056]

[0068] For parenteral compositions, at least about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of BCP per kg of body weight can be administered per dose.

[0057]

[0069] For oral compositions, at least about 0.1, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 mg of BCP per kg of body weight can be administered per dose.

[0058]

[0070] The duration of treatment can be 6, 5, 4, 3, or 2 months, or 8, 7, 6, 5, 4, 3, 2, or 1 week. It is also envisioned that in the case of chronic conditions (e.g., chronic and / or recurrent pericarditis), the duration of treatment can be indefinite.

[0059]

[0071] In one embodiment, BCP is administered twice daily. In another embodiment, BCP is administered once daily. In yet another embodiment, BCP is administered twice weekly. In yet another embodiment, BCP is administered at least once weekly.

[0060]

[0072] An example dosing regimen involves administering up to 8 mg / kg body weight by subcutaneous injection daily for 5, 6, 7, 14, 21, or 28 days. Administration may involve splitting the dose and administering half the dose every 12 hours.

[0061]

[0073] Another example of a dosing regimen involves oral administration of up to 18 mg / kg body weight daily for 5, 6, 7, 14, 21, or 28 days. Administration may involve splitting the dose and administering half the dose every 12 hours. [Example]

[0062]

[0074] Example

[0075] The following experiments were performed using BCP (Cas number 87-44-5) with a purity of approximately 93%.

[0063]

[0076] In the following description of the experiments and / or figures, the following abbreviations are used:

[0064]

[0077] CNT = control

[0065]

[0078] LPS = lipopolysaccharide

[0066]

[0079] ATP = adenosine triphosphate

[0067]

[0080] BCP = beta-caryophyllene

[0068]

[0081] NaCl = normal saline or saline solution

[0069]

[0082] Veh = vehicle

[0070]

[0083] Zym = Zymosan A

[0071]

[0084] Example 1A - Effect of 10 μM BCP on IL-1β levels in an in vitro NLRP3 inflammasome activation model

[0085] Experiments were carried out to determine the effect of 10 μM BCP on NLRP3 inflammasome activation in an in vitro model as follows.

[0072]

[0086] J774A.1 murine macrophages were cultured in 96-well plates (15,000 cells / well) and divided into the following groups and subjected to various treatments described below over a 6-hour period: 1. Group 1 (control) - cells received no treatment during the 6 hour treatment period. 2. Group 2 (LPS+ATP) - Cells received LPS (1 μg / mL) for 5.5 hours and ATP (5 mM) for the last 30 minutes. 3. Group 3 (BCP) - cells were given BCP solubilized in methanol (10 μM) for 6 hours. 4. Group 4 (LPS+ATP+BCP) - Cells received BCP solubilized in methanol (10 μM) and LPS (1 μg / mL) for 5.5 hours and ATP (5 mM) for the last 30 minutes.

[0073]

[0087] After 6 hours, 100 µL samples were collected from each well of cells and loaded as a single measurement point in a specific ELISA assay used to measure the amount of IL-1β secreted by macrophages in each sample. IL-1β is an indicator of and positively correlates with NLRP3 inflammasome activation. The results are summarized in Figure 1A.

[0074]

[0088] Figure 1A shows that Group 2 cells (LPS + ATP) had significantly higher concentrations of IL-1β compared with Group 1 cells (control). Group 4 cells (LPS + ATP + BCP) had significantly lower concentrations of IL-1β than Group 2 cells (LPS + ATP). These results indicate that BCP was effective in attenuating the increase in IL-1β induced by administration of LPS and ATP in this in vitro model of NLRP3 inflammasome activation in J774A.1 murine macrophages.

[0075]

[0089] Example 1B - Effect of 10 μM BCP on IL-6 in an in vitro model of inflammation

[0090] Experiments were conducted to evaluate the anti-inflammatory activity and mechanism of action of BCP. IL-6 secretion is regulated by the NF-κβ pathway and activated by LPS.

[0076]

[0091] In the experiment, J774A.1 murine macrophages were cultured in 96-well plates (15,000 cells / well) and divided into the following groups and subjected to various treatments described below over a 6-hour period: 1. Group 1 (CNT) - Cells received no treatment during the 6-hour treatment period. 2. Group 2 (LPS) - Cells were treated with LPS (1 μg / ml) for 6 hours. 3. Group 3 (BCP) - cells were given BCP solubilized in methanol (MetOH) (10 μM) for 6 hours. 4. Group 4 (LPS+BCP) - Cells were treated with BCP solubilized in methanol (10 μM) and LPS (1 μg / mL) for 6 hours.

[0077]

[0092] After 6 hours, 100 μL samples were collected from each well of cells and loaded as a single measurement point in a specific ELISA assay used to measure the amount of IL-6 in each sample. The results are summarized in Figure 1B, which show that LPS significantly increased the levels of IL-6 secreted by macrophages, and BCP significantly attenuated this increase. Consequently, because IL-6 secretion is independent of the NLRP3 inflammasome pathway, the anti-inflammatory effect of BCP appears to extend beyond its inhibition of the NLRP3 inflammasome pathway.

[0078]

[0093] Example 2 - Testing of BCP in vivo

[0094] Experiments were performed using the mouse model of pericarditis described by Mauro et al. (10), which is incorporated herein by reference for its disclosure of the mouse model.

[0079]

[0095] Adult Institute of Cancer Research (IRC) mice (mean age 10 weeks) supplied by Harlan Laboratories (Harlan Sprague Dawley Inc.) were used and divided into the following treatment groups, each consisting of 4–5 mice: 1. Group 1 (Saline + Veh) - Mice received saline (0.9% w / v NaCl) during the surgical procedures described below, followed by treatment with a vehicle consisting of ethanol:Cremophor EL:water (1:1:18) for 7 days. 2. Group 2 (Saline + BCP) - Mice received saline (0.9% w / v NaCl) during the surgical procedure described below, followed by treatment with BCP at a dose of 10 mg / kg for 7 days. BCP was dissolved in the same vehicle used for Group 1. 3. Group 3 (Zym+Veh) - Mice received zymosan A during the surgical procedure described below, followed by treatment with the same vehicle used in Group 1 for 7 days. 4. Group 4 (Zym+BCP) - Mice received zymosan A during the surgical procedure described below, followed by treatment with BCP at a dose of 10 mg / kg for 7 days. BCP was dissolved in the same vehicle used for Group 1.

[0080]

[0096] Surgical preparation of mice

[0097] Mice in all four treatment groups were anesthetized, orally intubated, and placed in the right lateral position. A left thoracotomy was then performed. Under direct vision, a 30-gauge needle was used to deliver 1 mg of zymosan A (also referred to herein as "zymosan") dissolved in 50 μL of sterile saline (0.9% w / v NaCl) into the pericardial cavity of mice in Groups 3 and 4 by carefully lifting the pericardial sac with forceps until complete distribution of the solution within the pericardium was achieved. Mice in Groups 1 and 2 were sham-operated by injecting an equal volume of sterile saline instead of the zymosan A solution. All mice received perioperative analgesic treatment.

[0081]

[0098] All groups were treated intraperitoneally (IP) with BCP in vehicle or vehicle alone daily for 7 days, with the first treatment administered approximately 30 minutes after surgery.

[0082]

[0099] Transthoracic echocardiography [000100] On day 7, echocardiography was performed on all groups of mice under light anesthesia using a Prospect™ T1 ultrasound imaging system (Scintica, London, Canada). This procedure involved imaging the left ventricle in a two-dimensional (B-mode) parasternal short-axis view at the midventricular level. Images were optimized for the anterior wall and magnified to visualize anterior pericardial structures. After image optimization, a unidirectional (M-mode) image was acquired to optimize spatial and temporal resolution. An investigator blinded to group assignment measured the maximum pericardial cavity between the two layers of the pericardium in both M-mode and B-mode. The pericardial cavity is a one-dimensional measure of pericardial effusion.

[0083] [000101] The results are summarized in Figure 2, which shows that mice in Group 3 (Zym + Veh) exhibited significantly greater pericardial effusion compared with mice in Group 1 (Saline + Veh). Mice in Groups 2 (Saline + BCP) and 4 (Zym + BCP) did not exhibit greater pericardial effusion than mice in Group 1 (Saline + Veh), and the differences between these groups were not statistically significant. Thus, these results indicate that BCP was effective in attenuating the increase in pericardial effusion caused by zymosan administration in this mouse model of pericarditis.

[0084] [000102] Histochemical examination [000103] After echocardiography, mice were sacrificed on day 7, and their hearts were removed to culture medium and processed for pathological examination. Formalin-fixed, paraffin-embedded cross-sections of the hearts were stained with hematoxylin and eosin. An investigator blinded to group assignment measured pericardial thickness using computerized morphometry with Image-Pro™ Plus 6.0 software (Media Cybernetics, Silver Spring, MD).

[0085] [000104] The results are summarized in Figure 3, which shows that mice in Group 3 (Zym + Veh) exhibited significantly greater pericardial thickness compared to mice in Group 1 (Saline + Veh). Mice in Groups 2 (Saline + BCP) and 4 (Zym + BCP) did not exhibit pericardial thicknesses different from mice in Group 1 (Saline + Veh), and the differences between these groups were not statistically significant. These results indicate that BCP was effective in attenuating the increase in pericardial thickness caused by zymosan administration.

[0086] [000105] Human equivalent dose [000106] The human equivalent dose of 10 mg / kg used above in mice is 0.81 mg / kg. (13) Because BCP was administered parenterally in the in vivo mouse model described above, this dose would be applicable to parenteral formulations.

[0087] [000107] Embodiments [000108] Various embodiments ("items") of the present invention are contemplated as follows.

[0088] [000109] Item 1. Use of beta-caryophyllene (BCP) in the treatment of pericarditis.

[0089] [000110] Item 2. Use of BCP to prevent pericarditis.

[0090] [000111] Item 3. The use of Item 1 or 2, wherein the BCP is effective in reducing an increase in pericardial effusion in the subject.

[0091] [000112] Item 4. The use of Item 3, wherein the BCP is effective in reducing an increase in pericardial effusion by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the amount of pericardial effusion that would have been present if the BCP had not been administered.

[0092] [000113] Item 5. The use of Item 1 or 2, wherein the BCP is effective in preventing an increase in pericardial effusion in the subject.

[0093] [000114] Item 6. The use of any one of Items 1 to 5, wherein the BCP is effective in reducing an increase in pericardial thickness in a subject.

[0094] [000115] Item 7. The use of item 6, wherein the BCP is effective to reduce an increase in pericardial thickness by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the pericardial thickness that would have been present if the BCP had not been administered.

[0095] [000116] Item 8. The use of any one of Items 1 to 5, wherein the BCP is effective in preventing an increase in pericardial thickness in a subject.

[0096] [000117] Item 9. The use of any one of Items 1 to 8, wherein BCP is effective in attenuating increased levels of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a subject.

[0097] [000118] Item 10. The use of Item 9, wherein the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) is reduced by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

[0098] [000119] Item 11. The use according to any one of Items 1 to 8, wherein the BCP is effective in preventing an increase in the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a subject.

[0099] [000120] Item 12. Use of BCP in the preparation of a medicine for the treatment of pericarditis.

[0100] [000121] Item 13. Use of BCP in the preparation of a medicine for the prevention of pericarditis.

[0101] [000122] Item 14. A method for treating pericarditis in a subject in need thereof, comprising administering an effective amount of BCP to the subject.

[0102] [000123] Item 15. A method for preventing pericarditis in a subject in need thereof, comprising administering an effective amount of BCP to the subject.

[0103] [000124] Item 16. The method of Item 14 or 15, wherein the BCP is effective in reducing or preventing an increase in pericardial effusion in the subject.

[0104] [000125] Item 17. The method of Item 16, wherein the BCP is effective in reducing an increase in pericardial effusion by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the amount of pericardial effusion that would have been present in the absence of said administration.

[0105] [000126] Item 18. The method of any one of Items 14 to 17, wherein the BCP is effective in reducing or preventing an increase in pericardial thickness in the subject.

[0106] [000127] Item 19. The method of item 18, wherein the BCP is effective to reduce an increase in pericardial thickness by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the pericardial thickness that would have been present in the absence of said administration.

[0107] [000128] Item 20. The method of any one of Items 14 to 19, wherein BCP is effective in reducing or preventing an increase in the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the subject.

[0108] [000129] Item 21. The method of Item 20, wherein the level of at least one of interleukin-1β (IL-β) and interleukin-6 (IL-6) is reduced by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

[0109] [000130] Item 22. The method according to any one of Items 14 to 21, further comprising the step of identifying a subject suffering from or at risk of suffering from pericarditis prior to the administration.

[0110] [000131] Item 23. BCP for use in the treatment or prevention of pericarditis.

[0111] [000132] Item 24. BCP for use in the prevention of pericarditis.

[0112] [000133] Item 25. BCP for use in the treatment of pericarditis.

[0113] [000134] Item 26. A BCP for use in reducing an increase in pericardial effusion in a subject.

[0114] [000135] Item 27. BCP for use in preventing increased pericardial effusion in a subject.

[0115] [000136] Item 28. A BCP for use in reducing pericardial thickness increase in a subject.

[0116] [000137] Item 29. A BCP for use in preventing an increase in pericardial thickness in a subject.

[0117] [000138] Item 30. A composition comprising an effective amount of BCP and a pharmaceutically acceptable excipient, for use in treating pericarditis.

[0118] [000139] Item 31. A composition comprising an effective amount of BCP and a pharmaceutically acceptable excipient, for use in preventing pericarditis.

[0119] [000140] Item 32. The composition of Item 30 or 31, which is effective in reducing an increase in pericardial effusion in a subject.

[0120] [000141] Item 33. The composition of item 32, which is effective in reducing an increase in pericardial effusion by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the amount of pericardial effusion that would have been present if the composition had not been administered.

[0121] [000142] Item 34. The composition of Item 30 or 31, which is effective in preventing an increase in pericardial effusion in a subject.

[0122] [000143] Item 35. The composition of any one of Items 30 to 34, which is effective in reducing an increase in pericardial thickness in a subject.

[0123] [000144] Item 36. The composition of item 35, which is effective to reduce an increase in pericardial thickness by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the pericardial thickness that would have been present if the composition had not been administered.

[0124] [000145] Item 37. The composition of any one of Items 30 to 34, which is effective in preventing an increase in pericardial thickness in a subject.

[0125] [000146] Item 38. The composition of any one of Items 30 to 37, which is effective in reducing an increase in the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a subject.

[0126] [000147] Item 39. The composition of Item 38, wherein the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) is reduced by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the level that would be present if the composition were not administered.

[0127] [000148] Item 40. The composition of any one of Items 30 to 37, which is effective in preventing an increase in the level of at least one of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a subject.

[0128] [000149] Item 41. The use according to any one of Items 1 to 13, the method according to any one of Items 14 to 22, the BCP according to any one of Items 23 to 29, or the composition according to any one of Items 30 to 40, wherein the pericarditis is acute pericarditis.

[0129] [000150] Item 42. The use according to any one of Items 1 to 13, the method according to any one of Items 14 to 22, the BCP according to any one of Items 23 to 29, or the composition according to any one of Items 30 to 40, wherein the pericarditis is recurrent pericarditis.

[0130] [000151] Item 43. The use according to any one of Items 1 to 13, the method according to any one of Items 14 to 22, the BCP according to any one of Items 23 to 29, or the composition according to any one of Items 30 to 40, wherein the BCP does not contain impurities.

[0131] [000152] Item 44. The composition according to any one of Items 30 to 40, which does not contain any other pharmaceutically active agent.

[0132] [000153] Item 45. The composition according to any one of Items 30 to 40, which does not contain a compound selected from the group consisting of α-caryophyllene, α-humulene, D-limonene, linalool, terpineol, terpinene, α-pinene, β-pinene, β-elemene, β-ocimene, camphene, nerolidol, euphor, citral, serratrole, falcarinol, salvinorin A, and pristimerin.

[0133] [000154] Item 46. The composition of any one of Items 30 to 40, further comprising at least one additional pharmaceutically active agent.

[0134] [000155] Item 47. The composition of item 46, wherein the at least one additional pharmaceutically active agent is cannabidiol.

[0135] [000156] Statistical significance

[0136] [000157] In the experiments described above, data are expressed as continuous variables, such as the mean and standard error, and comparisons were made between three or more groups at each time point using one-way analysis of variance, followed by a Sidak test for multiple comparisons. p values ​​less than 0.05 are indicated using one asterisk (*). p values ​​less than 0.01 are indicated using two asterisks (**). p values ​​less than 0.001 are indicated using three asterisks (***). p values ​​less than 0.0001 are indicated using four asterisks (****).

[0137] [000158] The above experiments demonstrate that BCP may be a novel strategy for treating and preventing pericarditis, including acute and recurrent pericarditis. The above-described embodiments are merely examples and are not intended to limit the scope of the invention as described herein and defined by the following claims.

[0138] References 1.Adler Y, Charron P, Imazio M, et al., 2015 ESC Guidelines for the diagnosis and management of pericardial diseases: the Task Force for the Diagnosis and Management of Pericardial Diseases of the European Society of Cardiology (ESC) Endorsed by: The European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J 2015; 36: 2921-64. 2.Imazio M, Gaita F, LeWinter M., Evaluation and treatment of pericarditis: a systematic review. JAMA 2015; 314:1498-506. 3.Bonaventura A, Montecucco F., Inflammation and pericarditis: are neutrophils actors behind the scenes? J Cell Physiol 2019; 234:5390-8. 4.Buckley LF, Viscusi MM, Van Tassell BW, Abbate A., Interleukin-1 blockade for the treatment of pericarditis. Eur Heart J Cardiovasc Pharmacother 2018; 4:46-53. 5.Toldo S, Abbate A., The NLRP3 inflammasome in acute myocardial infarction. Nat Rev Cardiol 2018; 15:203-14. 6.Mauro AG, Bonaventura A, Mezzaroma E, Quader M, Toldo S., NLRP3 Inflammasome in Acute Myocardial Infarction. J Cardiovasc Pharmacol 2019; 74:175-87. 7.Chiabrando JG, Bonaventura A, Vecchie A, et al., Management of acute and recurrent pericarditis: JACC State-of-the-Art Review. J Am Coll Cardiol 2020; 75:76-92. 8.Bayes-Genis A, Adler Y, de Luna AB, Imazio M., Colchicine in pericarditis. Eur Heart J 2017;38: 1706-9. 9.Brucato A, Imazio M, Gattorno M, et al., Effect of anakinra on recurrent pericarditis among patients with colchicine resistance and corticosteroid dependence: the AIRTRIP randomized clinical trial. JAMA 2016; 316: 1906-12. 10.Mauro et al., The Role of NLRP3 Inflammasome in Pericarditis. JACC: BASIC TO TRANSLATIONAL SCIENCE Vol. 6, No. 2, 2021, pp. 137-150; doi.org / 10.1016 / j.jacbts.2020.11.016 11.“FDA Approves First Treatment for Disease That Causes Recurrent Inflammation in Sac Surrounding Heart”. U.S. Food and Drug Administration (FDA). 18 March 2021 (fda.gov / drugs / news-events-human-drugs / fda-approves-first-treatment-disease-causes-recurrent-inflammation-sac-surrounding-heart). 12.Klein et al., Clinical Burden and Unmet Need in Pericarditis: A Systematic Literature Review. Cardiology in Review; Vol. 30, No. 2, March / April 2022. 13.Nair Anroop B., Jacob Shery, A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31. doi: 10.4103 / 0976-0105.177703. 14.Scandiffio et al., Protective Effects of (E)-Caryophyllene (BCP) in Chronic Inflammation. Nutrients 2020, 12, 3273; doi:10.3390 / nu12113273. 15.Francomano et al. β-Caryophyllene: A Sesquiterpene with Countless Biological Properties. Appl. Sci. 2019, 9, 5420; doi:10.3390 / app9245420. 16.Sharma, C.; Al Kaabi, J.M.; Nurulain, S.N.; Goyal, S.; Amjad Kamal, M.; Ojha, S. Polypharmacological properties and therapeutic potential of β-caryophyllene: A dietary phytocannabinoid of pharmaceutical promise. Curr. Pharm. Des. 2016, 22, 3237-3264. 17.Johnson et al., A Systematic Review of Essential Oils and the Endocannabinoid System: A Connection Worthy of Further Exploration. Evidence-Based Complementary and Alternative Medicine Volume 2020, Article ID 8035301, 13 pages. doi.org / 10.1155 / 2020 / 8035301 18.Goncalves et al., Terpenoids, Cannabimimetic Ligands, beyond the Cannabis Plant. Molecules 2020, 25, 1567; doi:10.3390 / molecules25071567. 19.Al-Taee et al., β-caryophyllene, a dietary phytocannabinoid attenuates oxidative stress, inflammation, apoptosis and prevents structural alterations of the myocardium against doxorubicin induced acute cardiotoxicity in rats: an in vitro and in vivo study. European Journal of Pharmacology (2019), doi.org / 10.1016 / j.ejphar.2019.172467. 20.Meeran et al. β-Caryophyllene, a natural bicyclic sesquiterpene attenuates doxorubicininduced chronic cardiotoxicity via activation of myocardial cannabinoid type-2 (CB2) receptors in rats. Chemico-Biological Interactions 304 (2019) 158-167, doi.org / 10.1016 / j.cbi.2019.02.028. 21.Veleti et al., Protective Effect of Beta-Caryophyllene on Doxorubicin Induced Multiple Organ Toxicity in Rats. International Journal of Applied Pharmaceutical Sciences and Research Vol 5 Issue 2 April-June, 2020. 22.Yokubaitis et al., Effects of Cannabidiol and Beta-Caryophyllene Alone or in Combination in a Mouse Model of Permanent Ischemia. Int. J. Mol. Sci. 2021, 22, 2866. doi.org / 10.3390 / ijms22062866. 23.Younis et al., β-Caryophyllene as a Potential Protective Agent Against Myocardial Injury: The Role of Toll-Like Receptors. Molecules 2019, 24, 1929; doi:10.3390 / molecules24101929.

Claims

1. Use of beta-caryophyllene (BCP) in the treatment or prevention of pericarditis.

2. Use of BCP in the preparation of a medicament for the treatment or prevention of pericarditis.

3. BCP for use in the treatment or prevention of pericarditis.

4. A composition for use in the treatment or prevention of pericarditis, the composition comprising BCP and a pharmaceutically acceptable excipient.

5. A method for treating or preventing pericarditis in a subject in need thereof, comprising administering to said subject an effective amount of BCP.

6. 10. The use of claim 1 or 2, the BCP of claim 3, the composition of claim 4 or the method of claim 5, wherein the BCP is effective in reducing an increase in pericardial effusion in a subject.

7. 10. The use of claim 1 or 2, the BCP of claim 3, the composition of claim 4 or the method of claim 5, wherein the BCP is effective in preventing an increase in pericardial effusion in a subject.

8. 8. The use according to any one of claims 1, 2, 6 or 7, the BCP according to any one of claims 3, 6 or 7, the composition according to any one of claims 4, 6 or 7 or the method according to any one of claims 5 to 7, wherein the BCP is effective in reducing an increase in pericardial thickness in a subject.

9. 8. The use according to any one of claims 1, 2, 6 or 7, the BCP according to any one of claims 3, 6 or 7, the composition according to any one of claims 4, 6 or 7 or the method according to any one of claims 5 to 7, wherein the BCP is effective in preventing an increase in pericardial thickness in a subject.

10. The use of any one of claims 1, 2 or 6 to 9, the BCP of any one of claims 3 or 6 to 9, the composition of claims 4 or any one of claims 6 to 9 or the method of any one of claims 5 to 9, wherein the BCP is effective in attenuating increased levels of interleukin-1β (IL-1β) in a subject.

11. The use of any one of claims 1, 2 or 6 to 10, the BCP of any one of claims 3 or 6 to 10, the composition of claims 4 or any one of claims 6 to 10 or the method of any one of claims 5 to 10, wherein the BCP is effective in attenuating increased levels of interleukin-6 (IL-6) in a subject.

12. The use according to any one of claims 1, 2 or 6 to 11, the BCP according to any one of claims 1, 2 or 6 to 11, the composition according to any one of claims 4 or 6 to 11 or the method according to any one of claims 5 to 11, wherein the pericarditis is acute pericarditis.

13. The use according to any one of claims 1, 2 or 6 to 11, the BCP according to any one of claims 1, 2 or 6 to 11, the composition according to any one of claims 4 or 6 to 11 or the method according to any one of claims 5 to 11, wherein the pericarditis is recurrent pericarditis.

Images