Beta-caryophyllene for use in treating or preventing pericarditis

Abstract

Beta-caryophyllene (BCP) for use in treating or preventing pericarditis is disclosed. The effectiveness of BCP in attenuating the levels of interleukin-1β (IL-1β) and IL-6 in in vitro models is disclosed. Also disclosed are experiments showing BCP to be effective in attenuating or preventing an increase in the pericardial space (which is an indicator of pericardial effusion) and the pericardial thickness in an in vivo mouse model of pericarditis.

Description

BETA-CARYOPHYLLENE FOR USE IN TREATING OR PREVENTING PERICARDITIS

[0001] This application claims priority from U.S. provisional application 63 / 449,799 filed March 3, 2023, the entire content of which is incorporated herein by reference.

[0002] FIELD

[0003] The present invention relates generally to therapeutics and methods for treating or preventing cardiovascular diseases, disorders, or conditions and, more particularly, to therapeutics and methods for the treatment or prevention of pericarditis.

[0004] BACKGROUND TO THE INVENTION

[0005] Pericarditis results from an intense inflammatory reaction of the pericardium. This reaction involves the activation of a macromolecular intracellular complex called the NLRP3 (NACHT, leucine-rich repeat, and pyrin domain-containing protein 3) inflammasome. The NLRP3 inflammasome, when it senses injury and stress, triggers an inflammatory process that involves the release of proinflammatory cytokines, such as interleukin- ip, that act as downstream mediators.

[0006] Pericarditis can be categorized into acute, incessant, recurrent and chronic pericarditis. Acute pericarditis is a clinical syndrome characterized by a single incident that resolves on its own or following treatment. The treatments mostly consist of nonsteroidal anti-inflammatory drugs, aspirin, steroids, colchicine, and anakinra (4, 7, 8, 9). Episodes of acute pericarditis lasting more than 4 to 6 weeks but less than 3 months are called incessant pericarditis, while episodes lasting more than three months are known as chronic pericarditis. Recurrent pericarditis (RP) is defined as an episode of acute pericarditis that occurs at least 4-6 weeks after resolution of a prior episode. RP has been reported to occur in 15-30% of pericarditis patients some of whom go on to experience multiple recurrences. In some cases, recurrences are due to inadequate treatment of the original episode, but, in other cases, they are due to inadequate response to current treatments.

[0007] The pathogenesis of pericarditis is not completely understood (3); however, it has been hypothesized that acute pericarditis represents a stereotypical response to an acute injury of the mesothelial cells of the pericardium (4). It is believed to be triggered by an “irritant” such as a virus or cellular debris following a viral infection (5,6). It can also betriggered 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).

[0008] According to Klein et al. (12), the entire contents of which are incorporated herein by reference, there is a high unmet need for FDA-approved, safe, accessible treatments that resolve recurrent events and reduce recurrence risk without posing excessive treatment burden on patients. Since 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 reduction in risk of recurrence in adults and pediatric patients 12 years and older (11).

[0009] There is an ongoing need for alternative therapeutics and methods of treating pericarditis. The present invention is intended to meet this need.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have found, through experiments described below, that 0- caryophyllene (BCP) attenuated an increase in interleukin- 1 (IL-10) in an in vitro model of NLRP3 inflammasome activation. Through another experiment, the inventors found that the anti-inflammatory effect of BCP extends beyond inhibiting the NLRP3 inflammasome pathway as BCP was found to reduce the level of interleukin-6 (IL-6) which is secreted independently of this pathway. Furthermore, following in vivo experiments involving a mouse model of pericarditis, the inventors found BCP to be effective in reducing pericardial effusion and pericardial thickness, which are two characteristics of pericarditis.

[0012] Accordingly, a first aspect of the invention provides the use of BCP in treating or preventing pericarditis.

[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.

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

[0015] A fourth aspect of the invention provides BCP for use in treating or preventing pericarditis.

[0016] According to a fifth aspect, the invention provides a composition comprising an effective amount of BCP and an effective amount of a pharmaceutically acceptable excipient, wherein the composition is for use in treating or preventing pericarditis.

[0017] In some embodiments, the BCP is of natural origin. In other embodiments, the BCP is synthetic. In still other embodiments, the BCP is made by biotechnological processes.

[0018] Some embodiments of the present uses and methods entail using BCP to treat pericarditis. In other embodiments, BCP is used to prevent recurrence of pericarditis.

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

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] The invention may be better understood with reference to the following detailed description and figures, wherein,

[0022] Figure 1 A is a bar graph showing the effect of BCP at a dose of 10 pM on the level of IL- 10 in an in vitro model of NLRP3 inflammasome activation;

[0023] Figure IB is a bar graph showing the effect of BCP at a dose of 10 pM on the level of IL-6 in another in vitro model;

[0024] Figure 2 is a bar graph showing the effect of BCP at a dose of 10 mg / kg on the pericardial space (an indicator of pericardial effusion) in an in vivo model of pericarditis; and

[0025] Figure 3 is a bar graph showing the effect of BCP (10 mg / kg) on the pericardial thickness in the same in vivo model of pericarditis.

[0026] DETAILED DESCRIPTION

[0027] Definitions

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

[0029] As used herein the term “pharmaceutically active agent” means a drug or agent which can be employed as disclosed herein and is intended to be used in a subject to treat or prevent diseases, ailments, physical damage, or pathological symptoms; or to influence the state, the condition or the functions of the body. Drugs in use can be found in reference works such as, for example, the Rote Liste or the Merck Index. Examples which may be mentioned, include, for example, BCP.

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

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

[0032] When used herein, the terms “treat,” “treatment,” and the like expression mean stopping, delaying the progression of a condition, disorder, or disease, or reducing the symptoms or severity of the symptoms thereof. The terms “prevent,” “prevention,” and the like expression mean preventing, delaying, or reducing the incidence and / or risk of the onset of a condition, disorder, or disease. The terms are intended to encompass “improving quality of life,” “extending the life,” and “improving clinical outcomes” of a subject suffering from, or at risk of suffering from, the condition, disorder, or disease, and do not necessarily mean “curing” the condition, disorder, or disease, though such is not precluded.

[0033] “Pharmaceutically acceptable excipient” when used herein means any substance which can be formulated with or that is present alongside a pharmaceuticallyactive agent to achieve a desired function or functions and that is not biologically or otherwise undesirable when administered to a subject (e.g., human). For example, the excipient should be non-toxic when administered and compatible with the other ingredients in the formulation. The person skilled in the art will appreciate what compounds or ingredients would qualify as a pharmaceutically acceptable excipient given the teachings of the present specification and information in the public domain.

[0034] The phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of 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" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0035] The terms "a" or "an" means “one or more.” As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It should also be noted that the term "or" is employed in the sense of "and / or" unless the context clearly dictates otherwise. For an avoidance of doubt, references herein to the use of BCP to treat or prevent pericarditis are references to the use of BCP to treat and / or prevent pericarditis.

[0036] The term “comprising” means “including without limitation.” Thus, for example, a composition comprising a list of compounds, ingredients, or elements, may include additional compounds, ingredients, or elements not expressly recited. It is also to be noted that the terms "comprising," "including," “containing,” and "having" can be used interchangeably.

[0037] The term “consisting of’ means including the listed integers and no additional integers except that in the case of a compound or ingredient, other compounds or ingredients that may be present as natural or commercial impurities or additives are not excluded. Natural and commercial impurities and additives will be apparent to the person of ordinary skill in the art. For example, the beta-caryophyllene (BCP) used in the below experiments is of botanical origin and was 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. In addition, there are other impurities in amount of 1.2% (= 100 - 92.9 - 5.9%). Furthermore, the C of A specifies atarget purity of 80%, though the actual purity was 92.9%. Thus, commercial sources of BCP may containimpurities up to about 20, 17.5, 15, 12.5, 10, 7.5, or 5%, comprising other plant components, in the case of botanically sourced BCP.

[0038] The term "consisting essentially of means "including the listed integers (e.g., compounds, steps, ingredients, etc.) and any additional integers that do not materially affect the basic and novel properties of the invention. The basic and novel properties of the invention is the beneficial effect of BCP in treating and / or preventing pericarditis in any form, namely, acute pericarditis, incessant pericarditis, chronic pericarditis, and recurrent pericarditis.

[0039] The terms "% w / w," "% wt.," “w / w %,” “wt.%” and variations thereof, mean the amount of a substance in terms of the weight of that substance divided by the total weight of the formulation containing that substance, and multiplied by 100.

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

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

[0042] When used herein, the term “effective amount” means an amount that would bring about the desired effect, based on the purpose and function of the ingredient in the context of the invention disclosed herein. Implicit in “desired effect” is that toxicity does not arise. For example, an effective amount of a pharmaceutically active agent is that amount which would be effective to provide a therapeutic effect while avoiding toxicity, such as may occur via long-term chronic administration. An effective amount of a solvent is that amount which, alone or together with other ingredients, would be effective tosolubilize the other or remaining ingredients of the formulation. What constitutes an effective amount will be determinable by the person of ordinary skill in the art by routine experimentation, having regard to the teachings herein and / or information in the public domain.

[0043] When used herein, the expression “free of Y” means that “Y” is not deliberately added but may be present as an impurity or due to other factors. For the sake of clarity, a formulation that is “free ofY” will not contain Y or contain 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 ofY, based on the total formulation.

[0044] The values recited herein are intended to include all values that meet the stated parameters including those not expressly recited. For example, a value of less than 1.0 is intended to include 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. Thus, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10, e.g., 1 to 6.3, 5.5 to 10, 2.7 to 6.1, etc.

[0045] The present specification contemplates the possibility of omitting any ingredients even if they are not expressly named as included or excluded in the specification.

[0046] BETA-CARYOPHYLLENE

[0047] The present invention contemplates using BCP, as well as compositions (e.g., medicaments) comprising same.

[0048] BCP is a natural bicyclic sesquiterpene that is widely distributed in the plant kingdom (14). It can be found in the essential oils of black pepper (Piper nigrum), melissa (Melissa officinalis), guava leaf (Psidium guajava), hemp (Cannabis saliva), clove (Eugenia caryophyllata), ylang ylang (Cananga odor ata), and copaiba (Copaifera spp.) (17). BCP is a non-psychotropic, selective phytocannabinoid agonist of type 2 receptors (CB2-R) and of peroxisome proliferator-activated receptor gamma (PPAR-Y) receptors (15, 17, 18). It has been reported to have repellent, antimicrobial or antibacterial,anticancer or antiproliferative, antifungal, AChE inhibitor, antioxidant, and antiinflammatory properties (18). Studies suggest 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).

[0049] The FDA designates BCP as generally regarded as safe (GRAS) and have approved it for use as a food additive and flavoring. However, BCP is classified according to OECD (Organization for Economic Co-operation and Development) guideline 423 as a category five substance (toxic at doses greater than 2000 mg / kg) (15).

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

[0051] In nature, BCP mainly occurs as trans-caryophyllene ((E)-BCP) (1) mixed with small amounts of the isomers (Z) — caryophyllene ((Z)-BCP) (2) and a-humulene (a- caryophyllene) (3) and its oxidation derivative, -caryophyllene oxide (BCPO) (4) shown below (15, 17):

[0052] The term BCP is intended to include all stereoisomers. The most commonly occurring form of BCP is one wherein the stereocentre adjacent to the exocyclic double bond has the S configuration while the remaining stereocentre has the R configuration.

[0053] The extraction of BCP from plant sources is done by using fractionation and other techniques known in the art (14). BCP can also be made by synthetic and biotechnological processes. The chemical synthesis of BCP is described, for example, in Corey, E. J. 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 methods can also be used. However, due to the wide variety of plants that produce BCP, obtaining BCP from natural sources is (at present) more convenient and less expensive than by chemical synthesis and microbial fermentation (17).

[0054] The BCP used in the context of 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 BCP of natural origin are other C15H24 terpene hydrocarbons (e.g., isocaryophyllene and a-humulene, formerly called a- caryophyllene). Synthetic BCP may include impurities consisting of residual organic solvents and / or by-products or residues of manufacture.

[0055] The compositions can be in any dosage form known in the art, including, without limitation, oral solutions, injectable or other parenteral formulations, tablets, gummies, gel capsules, chewing gum formulations, and more.

[0056] Oral solutions and elixir formulations can be simple, or they can be quite complex, involving many types of excipients including water-soluble organic solvents, water-insoluble organic solvents, surfactants, buffers, sugars, flavors, sweeteners, aromatics, dyes, antioxidants, and preservatives. The solutions can include water or be free of water. Organic cosolvents are normally used to solubilize poorly water-soluble drugs, including BCP, to the desired concentration in oral solutions, as is known in the art. Embodiments of oral solutions include formulations comprising BCP dissolved in a water insoluble solvent, such as long-chain triglycerides, peanut oil, com oil, soybean oil, sesame oil, olive oil, peppermint oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides. Medium-chain triglycerides can be synthetic or derived from coconut oil and palm seed oil. Other water-insoluble solvents include beeswax, dl-a- tocopherol (Vitamin E), and oleic acid.

[0057] When in the form of an oral solution, BCP can be present in a concentration of from about 50, 75, 100, 150, or 200 mg / mL. Additionally, or alternatively, the oral solution can contain BCP in 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 oralsolution contains BCP in an amount of about 500 mg / mL.

[0058] When in the form of an injectable / parenteral solution, BCP can be present at a concentration 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.

[0059] As used herein, the term “subject” or “patient” refers to mammals and includes, without limitation, human and veterinary animals. In a preferred embodiment, the subject is human.

[0060] Additional Pharmaceutically Active Agents and Excipients

[0061] Recitation of BCP herein does not preclude combination therapies involving other pharmaceutically active agents. It also does not preclude compositions that comprise 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 expressly excluded. In the same or other embodiments, the following pharmaceutically active agents are expressly excluded from the present uses, methods, and compositions: a-caryophyllene (now called a-humulene), D-limonene, linalool, terpineol, terpinene, a-pinene, [3-pinene, [3-elemene, -ocimene, camphene, nerolidol, euphol, citral, celatrol, falcarinol, salvinorin A, and pristimerin, alone or in any combination.

[0062] In some embodiments, BCP is administered in combination with a therapeutic dose of another pharmaceutically active agent, e.g., nonsteroidal anti-inflammatory drugs, aspirin, steroids, colchicine, and / or anakinra. Any desired combination of pharmaceutically active agents can be delivered simultaneously or sequentially (e.g., within a 24 hour period). Suitable therapeutically effective dosages for each agent may be lowered due to additive or synergistic effects resulting from the combination therapy.

[0063] Compositions according to the present invention can contain BCP together with at least one pharmaceutically acceptable excipient that do not undermine the basic and novel properties of the invention. For example, oral and injectable solutions according tothe invention may contain a solvent for BCP (e.g., medium chain triglycerides) and an effective amount of at least one lipophilic antioxidant for improved shelflife. Benzyl alcohol may be included in injectable solutions for its anaesthetic properties. The at least one antioxidant can be selected from the group consisting of vitamin E (also referred to as a-tocopherol), carotenoids (xanthophylls and carotenes), propyl gallate, lecithin, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), and monothioglycerol tert-butylhydroquinone (TBHQ). What constitutes an “effective amount” depends on the identity of the compound and refers to that quantity of a compound or pharmaceutical composition that when administered to a subject for treating (e.g., preventing or ameliorating) a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending on the compound administered as well as the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. In general, the total amount of antioxidant(s) will be more than about 0.01 % w / v and less than about 1.5 % w / v. In some embodiments, the amount is more than about 0.1 % w / v and less than about 1 % w / v.

[0064] Additional excipients which are contemplated for use in the practice of the disclosure are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. II and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989).

[0065] Methods of Administration

[0066] BCP can be administered in a variety of ways known in the art, including orally and parenterally. The term “parenteral” is meant to include, without limitation, administration via intravenous (IV), subcutaneous (SC), intraperitoneal (IP), and intramuscular (IM) injection, and is used interchangeably with the term “injectable” herein. The dose of BCP when administered parenterally will be lower than the dose when administered orally, given that orally administered BCP is expected to have lower bioavailability.

[0067] The present compositions can be administered at least once weekly, or at least once every 6, 5, 4, 3, or 2 days. The composition can also be administered at least once daily, or at least twice daily.

[0068] For parenteral compositions, at least about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mg BCP per kg body weight can be administered per dose.

[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 BCP per kg body weight can be administered per dose.

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

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

[0072] One example dosage regime entails administering up to 8 mg / kg body weight, by subcutaneous injection, daily, for 5, 6, 7, 14, 21, or 28 days. The administration can entail dividing the dose and administering each half dose every 12 hours.

[0073] Another example dosage regime entails administering up to 18 mg / kg body weight, orally, daily, for 5, 6, 7, 14, 21, or 28 days. The administration can entail dividing the dose and administering each half dose every 12 hours.

[0074] EXAMPLES

[0075] The following experiments were performed using BCP (Cas. No. 87-44-5) having a purity of about 93%.

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

[0077] CNT = control

[0078] LPS = Lipopolysaccharide

[0079] ATP = Adenosine Triphosphate

[0080] BCP = beta-caryophyllene

[0081] NaCl = normal saline or saline

[0082] Veh = vehicle

[0083] Zym = Zymosan A

[0084] Example 1A - Effect of 10 uM BCP on level of IL-10 in in vitro NLRP3 Inflammasome Activation Model

[0085] An experiment was performed to determine the effect of 10 pM BCP on NLRP3 inflammasome activation in an in vitro model as follows.

[0086] J774A.1 mouse macrophages were cultured in 96-well plates (15,000 cells / well) and divided into the following groups to which various treatments described below were administered during a period of six hours:1. Group 1 (CONTROL) - cells received nothing during the 6-hour treatment period.2. Group 2 (LPS+ATP) - cells received LPS (1 pg / mL) for 5.5 hours and ATP (5 mM) for the last 30 minutes.3. Group 3 (BCP) - cells received BCP solubilized in methanol (10 pM) for 6 hours.4. Group 4 (LPS+ATP+BCP) - cells received BCP solubilized in methanol (10 pM) and LPS (1 pg / mL) for 5.5 hours and ATP (5 mM) for the last 30 minutes.

[0087] After 6 hours, 100 pL of a sample from each well of cells was collected and loaded as a single point in a specific ELISA assay that was used to measure the amount of IL-10 secreted by the macrophages in each sample. IL-1 is an indicator of and positively correlated with NLRP3 inflammasome activation. The results are summarized in Figure 1 A.

[0088] Figure 1A shows that the cells of Group 2 (LPS+ATP) had significantly higher concentrations of IL-10 compared to the cells of Group 1 (CONTROL). The cells of Group 4 (LPS+ATP+BCP) had significantly lower concentrations of IL- 10 than the cells of Group 2 (LPS+ATP). These results show that BCP was effective to attenuate the increase in IL-10 induced by administration of LPS and ATP in this in vitro model of NLRP3 inflammasome activation of J774A.1 mouse macrophages.

[0089] Example IB - Effect of 10 uM BCP on IL-6 in an In Vitro Model ofInflammation

[0090] An experiment was performed to evaluate BCP anti-inflammatory activity and mechanism of action. The secretion of IL-6 is regulated by the NF-K pathway and activated by LPS.

[0091] In the experiment, J774A.1 mouse macrophages were cultured in 96-well plates (15,000 cells / well) and divided into the following groups to which various treatments described below were administered during a period of six hours:1. Group 1 (CNT) - cells received nothing during the 6 hours treatment period.2. Group 2 (LPS) - cells received LPS (1 pg / ml) for 6 hours.3. Group 3 (BCP) - cells received BCP solubilized in methanol (MetOH) (10 pM) for 6 hours.4. Group 4 (LPS+BCP) - cells received BCP solubilized in methanol (10 pM) and LPS (1 pg / mL) for 6 hours.

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

[0093] Example 2 - Testing BCP In Vivo

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

[0095] Adult Institute of Cancer Research (IRC) mice (average age 10 weeks) supplied by Harlan Laboratories (Harlan Sprague Dawley Inc.) were used and divided into the following treatment groups, with each group consisting of 4-5 mice:1. Group 1 (Saline + Veh) - mice received normal saline (0.9 % w / v NaCl) in the surgical procedure described below and were subsequently treated for seven days with a vehicle consisting of ethanol :Cremophor EL:water (1:1:18).2. Group 2 (Saline + BCP) - mice received normal saline (0.9 % w / v NaCl) in the surgical procedure described below and were subsequently treated for seven days with BCP at a dose of 10 mg / kg. The BCP was dissolved in the same vehicle as the vehicle used for Group 1.3. Group 3 (Zym + Veh) - mice received Zymosan A in the surgical procedure described below and were subsequently treated for seven days with the same vehicle as the vehicle used for Group 1.4. Group 4 (Zym + BCP) - mice received Zymosan A in the surgical procedure described below and were subsequently treated for seven days with BCP at a dose of 10 mg / kg. The BCP was dissolved in the same vehicle as the vehicle used for Group 1.

[0096] Surgical Preparation of the Mice

[0097] The mice of all four treatment groups were anesthetized, intubated orotracheally, and placed in the right lateral decubitus position. A left thoracotomy was then performed. Under direct visualization and using a 30 Gauge needle, 1 mg of Zymosan A (referred to herein also as “zymosan”) dissolved in 50 pl of sterile normal saline (0.9 % w / v NaCl) was delivered into the pericardial space of the mice of Groups 3 and 4 by carefully lifting the pericardial sac with forceps until a complete distribution of the solution into the pericardium was achieved. Sham procedures were performed vis-a-vis the mice of Groups 1 and 2 by injecting an equal volume of sterile normal saline instead of the Zymosan A solution. All mice received analgesia for the peri-operative period.

[0098] For all groups, treatments with either BCP dissolved in vehicle, or vehicle alone, were administered intraperitoneally (IP) every day for seven days with the first treatment administered approximately 30 minutes after the surgery.

[0099] Transthoracic echocardiography[000100] On day seven, echocardiography was performed on the mice of all groups under light anesthesia using a Prospect™ T1 ultrasound imaging system (Scintica, London, Canada). In this procedure, the left ventricle in the parasternal short-axis view at the mid- ventricular level was visualized in the bi-dimensional mode (B-mode). The image was optimized for the anterior wall, and the image was zoomed in to visualize the anterior pericardial structures. After optimization of the image, a mono-dimensional mode (M-mode) was acquired for optimal spatial-temporal resolution. An investigator blinded to group allocation measured the maximal pericardial space between the 2 layers of the pericardium in both M- and B-mode. The pericardial space is a one-dimensional measure of pericardial effusion.[000101] The results are summarized in Figure 2 which shows that mice of Group 3 (Zym + Veh) developed a significantly larger pericardial effusion compared with the mice of Group 1 (Saline + Veh). The mice of Group 2 (Saline + BCP) and of Group 4 (Zym + BCP) did not develop a pericardial effusion greater than that of the mice of Group 1 (Saline + Veh) as the differences among these groups were not statistically significant. Thus, these results show that BCP was effective in attenuating the increase in pericardial effusion caused by zymosan administration in this mouse model of pericarditis.[000102] Histochemistry[000103] Following echocardiography, and on day seven, the mice were sacrificed, and their hearts were explanted and processed for pathology. Formalin -fixed paraffin- embedded transverse sections of the hearts were stained with hematoxylin and eosin. An investigator blinded to group allocation measured the pericardial thickness with the aid of computer morphometry using Image-Pro™ Plus 6.0 software (Media Cybernetics, Silver Spring, MD).[000104] The results are summarized in Figure 3 which shows that the mice of Group 3 (Zym + Veh) developed significantly larger pericardial thickness compared with the mice of Group 1 (Saline + Veh). The mice of Group 2 (Saline + BCP) and of Group 4 (Zym + BCP) did not develop a pericardial thickness different from that of the mice of Group 1 (Saline + Veh) as the differences among these groups were not statistically significant. Theseresults show that BCP was effective in attenuating the increase in pericardial thickness caused by zymosan administration.[000105] Human Equivalent Dose[000106] The human equivalent dose of 10 mg / kg used above in mice is 0.81 mg / kg (13). This dose would be applicable to parenteral formulations since BCP was administered parenterally in the above in vivo mouse model.[000107] Embodiments[000108] Various embodiments of the invention (“items”) are contemplated as follows.[000109] Item 1. Use of beta-caryophyllene (BCP) in treating pericarditis.[000110] Item 2. Use of BCP to prevent pericarditis.[000111] Item 3. The use of item 1 or 2, wherein the BCP is effective to attenuate an increase in pericardial effusion in a subject.[000112] Item 4. The use of item 3, wherein the BCP is effective to attenuate 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 be present if the BCP was not administered.[000113] Item 5. The use of item 1 or 2, wherein the BCP is effective to prevent an increase in pericardial effusion in a subject.[000114] Item 6. The use of any one of items 1 to 5, wherein the BCP is effective to attenuate an increase in pericardial thickness in a subject.[000115] Item 7. The use of item 6, wherein the BCP is effective to attenuate 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 be present if the BCP was not administered.[000116] Item 8. The use of any one of items 1 to 5, wherein the BCP is effective to prevent an increase in pericardial thickness in a subject.[000117] Item 9. The use of any one of items 1 to 8, wherein the BCP is effective to attenuate an increase in the level of at least one of interleukin- 1 (IL- 10) and interleukin-6 (IL-6) in a subject.[000118] Item 10. The use of item 9, wherein the level of at least one of interleukin- 10 (IL-10) and interleukin-6 (IL-6) is attenuated by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.[000119] Item 11. The use of any one of items 1 to 8, wherein the BCP is effective to prevent an increase in the level of at least one of interleukin- 10 (IL- 10) and interleukin-6 (IL-6) in a subject.[000120] Item 12. Use of BCP in the preparation of a medicament for the treatment of pericarditis.[000121] Item 13. Use of BCP in the preparation of a medicament for the prevention of pericarditis.[000122] Item 14. A method of treating pericarditis in a subject in need thereof, comprising administering an effective amount of BCP to the subject.[000123] Item 15. A method of preventing pericarditis in a subject in need thereof, comprising administering an effective amount of BCP to the subject.[000124] Item 16. The method of items 14 or 15, wherein the BCP is effective to attenuate or prevent an increase in pericardial effusion in the subject.[000125] Item 17. The method of item 16, wherein the BCP is effective to attenuate 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 be present in the absence of said administration.[000126] Item 18. The method of any one of items 14 to 17, wherein the BCP is effective to attenuate or prevent an increase in pericardial thickness in the subject.[000127] Item 19. The method of item 18, wherein the BCP is effective to attenuate an increase in pericardial thickness by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or95%, compared to the pericardial thickness that would be present in the absence of said administration.[000128] Item 20. The method of any one of items 14 to 19, wherein the BCP is effective to attenuate or prevent an increase in the level of at least one of interleukin- 1 [3 (IL-ip) and interleukin-6 (IL-6) in the subject.[000129] Item 21. The method of item 20, wherein the level of at least one of interleukin- ip (IL-0) and interleukin-6 (IL-6) is attenuated by at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.[000130] Item 22. The method of 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 said administration.[000131] Item 23. BCP for use in the treatment or prevention of pericarditis.[000132] Item 24. BCP for use in preventing pericarditis.[000133] Item 25. BCP for use in treating pericarditis.[000134] Item 26. BCP for use in attenuating an increase in pericardial effusion in a subject.[000135] Item 27. BCP for use in preventing an increase in pericardial effusion in a subject.[000136] Item 28. BCP for use in attenuating an increase in pericardial thickness in a subject.[000137] Item 29. BCP for use in preventing an increase in pericardial thickness in a subject.[000138] Item 30. A composition comprising an effective amount of BCP and a pharmaceutically acceptable excipient, wherein the composition is for use in treating pericarditis.[000139] Item 31. A composition comprising an effective amount of BCP and a pharmaceutically acceptable excipient, wherein the composition is for use in preventing pericarditis.[000140] Item 32. The composition of item 30 or 31, wherein the composition is effective to attenuate an increase in pericardial effusion in a subject.[000141] Item 33. The composition of item 32, wherein the composition is effective to attenuate 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 be present if the composition was not administered.[000142] Item 34. The composition of item 30 or 31, wherein the composition is effective to prevent an increase in pericardial effusion in a subject.[000143] Item 35. The composition of any one of items 30 to 34, wherein the composition is effective to attenuate an increase in pericardial thickness in a subject.[000144] Item 36. The composition of item 35, wherein the composition is effective to attenuate 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 be present if the composition was not administered.[000145] Item 37. The composition of any one of items 30 to 34, wherein the composition is effective to prevent an increase in pericardial thickness in a subject.[000146] Item 38. The composition of any one of items 30 to 37, wherein the composition is effective to attenuate an increase in the level of at least one of interleukin- ip (IL-1 ) and interleukin-6 (IL-6) in a subject.[000147] Item 39. The composition item 38, wherein the level of at least one of interleukin- ip (IL-1 P) and interleukin-6 (IL-6) is attenuated 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 was not administered.[000148] Item 40. The composition of any one of items 30 to 37, wherein the composition is effective to prevent an increase in the level of at least one of interleukin- 1 [3 (IL-ip) and interleukin-6 (IL-6) in a subject.[000149] Item 41. The use of any one of items 1-13, the method of any one of items 14-22, the BCP of any one of items 23-29, or the composition of any one of items 30-40, wherein the pericarditis is acute pericarditis.[000150] Item 42. The use of any one of items 1-13, the method of any one of items 14-22, the BCP of any one of items 23-29, or the composition of any one of items 30-40, wherein the pericarditis is recurrent pericarditis.[000151] Item 43. The use of any one of items 1-13, the method of any one of items 14-22, the BCP of any one of items 23-29, or the composition of any one of items 30-40, wherein the BCP does not contain impurities.[000152] Item 44. The composition of any one of items 30-40, wherein the composition is free of other pharmaceutically active agents.[000153] Item 45. The composition of any one of items 30-40, wherein the composition is free of compounds selected from the group comprising a-caryophyllene, a- humulene, D-limonene, linalool, terpineol, terpinene, a-pinene, P-pinene, P-elemene, P- ocimene, camphene, nerolidol, euphol, citral, celatrol, falcarinol, salvinorin A, and pristimerin.[000154] Item 46. The composition of any one of items 30-40, wherein the composition further comprises at least one additional pharmaceutically active agent.[000155] Item 47. The composition of item 46, wherein the at least one additional pharmaceutically active agent is cannabidiol.[000156] Statistical Significance[000157] In the experiments described above, data were expressed as continuous variables as mean and standard error, and one-way ANOVA was used to compare between 3 or more groups at each time point, followed by Sidak test for multiple comparisons. P values less than 0.05 are shown using one asterisk (*). P values less than 0.01 are shownusing two asterisks (**). P values less than 0.001 are shown using three asterisks (***). p values less than 0.0001 are shown using four asterisks (****).[000158] The above experiments show that BCP may represent a novel strategy for treating and preventing pericarditis, including acute and recurrent pericarditis. The embodiments described above are by way of example only and are not intended to limit the scope of the invention as described herein and defined by the following claims.REFERENCES1. 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, Gattomo 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.2020.11.01611. "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- recurr ent-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 / nul2113273.15. Francomano et al. P-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 P-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 / 803530118. Goncalves et al., Terpenoids, Cannabimimetic Ligands, beyond the Cannabis Plant. Molecules 2020, 25, 1567; doi:10.3390 / molecules25071567.19. Al-Taee et al., P-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. P-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., P-Caryophyllene as a Potential Protective Agent Against Myocardial Injury: The Role of Toll-Like Receptors. Molecules 2019, 24, 1929; doi:10.3390 / molecules24101929.

Claims

CLAIMS1. Use of beta-caryophyllene (BCP) in treating or preventing pericarditis.

2. Use of BCP in the preparation of a medicament for the treatment or prevention of pericarditis3. BCP for use in treating or preventing pericarditis.

4. A composition for use in treating or preventing pericarditis, wherein the composition comprises BCP and a pharmaceutically acceptable excipient.

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

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

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

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

9. The use of claim 1, 2, 6, or 7, BCP of claim 3, 6, or 7, composition of claim 4, 6, or 7, or method of claim 5, 6, or 7, wherein the BCP is effective to prevent an increase in pericardial thickness in a subject.

10. The use of any one of claims 1, 2, and 6-9, BCP of any one of claims 3, and 6-9, composition of any one of claims 4, and 6-9, or method of any one of claims 5-9, wherein the BCP is effective to attenuate an increase in the level of interleukin- 1 (IL-10) in a subject.

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

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

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

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