PALMITOYLETHANOLAMIDE TREATMENT FOR COVID-19-RELATED INFLAMMATION [0001] This application claims benefit of United States provisional patent application number 63/266,620, filed January 10, 2022, the entire contents of which are incorporated by reference into this application. REFERENCE TO A SEQUENCE LISTING [0002] The content of the XML file of the sequence listing named “GNCR001WOU1_Seq”, which is 8 kb in size, was created on January 9, 2023, and electronically submitted herewith the application. The sequence listing is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0003] SARS CoV-2 infection causes COVID-19, and COVID-19 cases continue to rise world- wide (total cases surpass 658 million). Globally, the number of deaths related to COVID-19 reached 6 million by March 7, 2022. To tackle this disaster, there are a number of parallel developments in public health policies, drug and therapeutic treatments and vaccines were presented at an unprecedented speed. However, along with the rising of new mutants and complications of the disease a number of publications have mathematically modeled that with the current effectiveness of policies, without more effective drug and/or vaccine treatment, COVID-19 is likely to linger for quite a while, even becoming seasonal like the flu (Gilbert et al., 2020; Wang et al., 2020). Already the waning immunity of vaccines is making the news. [0004] Beyond the renal and respiratory failure, COVID-19 associated coagulopathy is the leading cause of COVID-19 deaths (Levy et al., 2021). Coagulopathy broadly describes any imbalance in body’s natural hemostasis resulting in either excessive bleeding or clotting (Iba et al., 2020). This includes unnatural platelet activating and aggregation in the blood vessels leading to hypercoagulation or thrombosis. Hypercoagulability persists in the moderate and severe forms of COVID-19 and is strongly related to the signature systemic inflammation associated with the disease that trigger the blood clotting system (Litvinov et al., 2021). Indeed, many studies have demonstrated that the inflammatory “cytokine storm” created in COVID-19 correlates to the laboratory indicators of hypercoagulability in COVID-19 (Huang et al., 2020; Mangalmurti et al., 2020). This particular interplay represents a source of dire prognosis among the patients (Comer et al., 2021). The COVID-19 associated coagulopathy leads to venous thromboembolism which is prevalent in up to 69% in critically ill patients (Goshua et al., 2020). This may also be the reason why people with underlying comorbidities such as cardiovascular risk factors such as hypertension, diabetes are especially at risk of being the victim of COVID- 19 mortality via thrombotic events (Guzik et al., 2020; Khan et al., 2020; Schiffrin et al., 2020). [0005] There remains a need for effective treatment of inflammation related to coronavirus infections, and for the associated morbidities and post-acute sequalae.
SUMMARY [0006] These needs and more are met by the methods and compositions described herein, which provide an effective formulation of palmitoylethanolamide (PEA) for ameliorating the symptoms and inflammatory sequelae associated with coronavirus infection, including “long COVID”. The formulation of PEA described herein provides superior absorption and a surprising anti-inflammatory effect. [0007] In some embodiments, described herein is a method of reducing plasma levels of P- selectin in a subject, the method comprising administering to the subject an effective amount of palmitoylethanolamide (PEA). Also described is a method of reducing inflammation in a subject infected with SARS-CoV-2, the method comprising administering to the subject an effective amount of PEA. Additionally described herein is a method of ameliorating symptoms of SARS- CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of PEA. In some embodiments, the symptoms comprise immunothrombosis and/or post-acute sequelae of SARS-CoV-2 infection (PASC). [0008] In some embodiments, the PEA is formulated with a dispersing agent. In some embodiments, the dispersing agent comprises two or more components selected from the group consisting of a surfactant, a carrier oil, and a solvent. A representative example of a dispersing agent is marketed as Levagen+
TM (Gencor, Austin, Texas). Pharmacokinetic studies have demonstrated superiority of Levagen+
TM with an increased bioavailability of PEA of 1.75 times, compared to standard PEA. Due to its fatty nature, PEA has poor absorption in the body and has limited format options. This formulation uses the Lipisperse® dispersion technology, which reduces surface tension, thereby allowing particles to freely disperse in the watery gastrointestinal tract, and/or liquid formats (Pharmako Biotechnologies, New South Wales, Australia). This formulation results in superior absorption, and the dispersion technology is described in international patent publication number WO2018/187849 A1, published October 18, 2018. [0009] In some embodiments, the dispersing agent comprises an amphiphilic molecule and a carrier oil. In other embodiments, the dispersing agent comprises an amphiphilic molecule and solvent. In some embodiments, the dispersing agent comprises an amphiphilic molecule, a solvent and a carrier oil. In some embodiments, the dispersing agent comprises 10% (w/w) - 99% (w/w) total surfactant, 1 % (w/w) - 30% (w/w) total solvent and 5% (w/w) - 30% (w/w) total carrier oil. In some embodiments, the dispersing agent comprises 50% (w/w) - 75% (w/w) total surfactant, 2.5% (w/w) - 15% (w/w) total solvent and 1 % (w/w) - 10% (w/w) total carrier oil. [0010] For example, the dispersing agent may comprise 60% (w/w) - 75% (w/w) non-ionic surfactant, 0.2% (w/w) - 10% (w/w) phospholipid surfactant, 2.5% (w/w) - 15% (w/w) citrus oil, and 10% (w/w) - 25% (w/w) total carrier oil.
[0011] The dispersing agent may further comprise a preservative, such as an antimicrobial or an anti-oxidant. In an embodiment of the present invention, the preservative is an antioxidant is selected from the group consisting of ascorbyl palmitate, d alpha- tocopherol, dl-alpha- tocopherol, d-alpha-Tocopheryl acetate, dl-alpha-Tocopheryl acetate, d- alpha-Tocopheryl acid succinate, dl alpha-Tocopheryl acid succinate, Vitamin E and derivatives thereof, olive polyphenols and algal polyphenols. In some embodiments of the invention, the dispersing agent may comprise a preservative at a concentration of 0.1 % - 5% (w/w). [0012] The dispersing agent and the solid substance comprising the hydrophobic compound may be combined at any ratio that will facilitate the production of the liquid dispersible composition comprising the hydrophobic compound. In embodiments, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is from about 100:1 to about 1 : 1. Preferably, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is from about 20: 1 to about 5:1. In some embodiments, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is about 10:1 , about 9: 1 or about 8: 1. [0013] In some embodiments, the PEA is formulated for oral administration. In some embodiments, the PEA is formulated as a capsule. In some embodiments, the PEA is formulated for injection, or for transdermal delivery, or other modes. In some embodiments, the effective amount of PEA is 400-1,000 mg. In some embodiments, the effective amount of PEA is 600 mg. In some embodiments, the PEA is administered one to three times per day. In some embodiments, the PEA is administered twice per day. [0014] The subject is typically a mammal. In one embodiment, the mammal is human. In other embodiments, the mammal is a veterinary subject. Examples of veterinary subjects include, but are not limited to, equine, canine, bovine, porcine, ovine, and feline subjects. BRIEF DESCRIPTION OF THE DRAWINGS [0015] FIG.1. Schematic illustration of study participant flow. CON, placebo; LEV, 600 mg Levagen + twice daily. [0016] FIG.2. Supplemental Table 1. Nutrient intakes from 24-hour dietary recalls at baseline and post-trial for LEV and CON groups. [0017] FIG.3. Stacked bar charts representing types of symptoms reported by groups. A, CON; B, LEV.0, not reported; 1, reported.
DETAILED DESCRIPTION OF THE INVENTION [0018] The invention is based on the results of a randomized controlled trial demonstrating the effect of a novel food supplement, palmitoylethanolamide (PEA), on proinflammatory cytokines and biomarkers in an adult population recently (<10 days) diagnosed with COVID-19 who were asymptomatic or experiencing only mild symptoms. The data show an anti-inflammatory effect of PEA in seemingly healthy individuals with effective immune protection: those who recently tested positive for COVID-19 but were only mildly symptomatic. Since inflammation is likely to persist in many individuals infected with COVID (known as ‘long COVID), maintaining a strong protective immune profile following infection is important. [0019] Definitions [0020] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified. [0021] As used herein, an “effective amount”, or a “therapeutically effective” amount of a compound described herein is typically one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. It will be appreciated that different concentrations may be employed for prophylaxis than for treatment of an active disease, as well as for treatment of acute versus chronic symptoms. [0022] In some embodiments, a compound provided herein, or salt thereof, is substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art. [0023] As used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0024] As used herein, “contacting” means bringing at least two moieties together, whether in an in vitro system or an in vivo system. [0025] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.
[0026] As used herein, to “prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease. [0027] As used herein, “treating” or “treatment” includes prophylaxis and therapy, and results in amelioration of symptoms, delays progression, or otherwise improves the disease condition of the subject undergoing treatment. [0028] Compositions [0029] Described herein is an effective formulation of palmitoylethanolamide (PEA) for ameliorating the symptoms and inflammatory sequelae associated with coronavirus infection, including “long COVID”. PEA, a type of N-acylethanolamine, is an endogenous bioactive lipid that is produced naturally in the body in response to injury and stress. PEA is a naturally occurring fat-derived signaling compound in many animals and plant foods, as well as in animal cells and tissues. The main food sources of PEA are soy lecithin, soybean, peanut, and corn. Generally, PEAs are manufactured by reacting palmitic acid and ethanolamine. Ethanolamine is usually derived from fossil fuels, but can also be manufactured through the decarboxylation reaction of serine. The decarboxylation reaction can be performed using pyridoxal 5'-phosphate- dependent serine decarboxylase, and this enzyme can be isolated from various plants such as spinach. [0030] In some embodiments, the PEA is formulated with a dispersing agent. In some embodiments, the dispersing agent comprises two or more components selected from the group consisting of a surfactant, a carrier oil, and a solvent. A representative example of a dispersing agent is marketed as Levagen+
TM (Gencor, Austin, Texas). Pharmacokinetic studies have demonstrated superiority of Levagen+
TM with an increased bioavailability of PEA of 1.75 times, compared to standard PEA. Due to its fatty nature, PEA has poor absorption in the body and has limited format options. This formulation uses the Lipisperse® dispersion technology, which reduces surface tension, thereby allowing particles to freely disperse in the watery gastrointestinal tract, and/or liquid formats (Pharmako Biotechnologies, New South Wales, Australia). This formulation results in superior absorption, and the dispersion technology is described in international patent publication number WO2018/187849 A1, published October 18, 2018. [0031] In some embodiments, the dispersing agent comprises an amphiphilic molecule and a carrier oil. In other embodiments, the dispersing agent comprises an amphiphilic molecule and solvent. In some embodiments, the dispersing agent comprises an amphiphilic molecule, a solvent and a carrier oil. In some embodiments, the dispersing agent comprises 10% (w/w) - 99% (w/w) total surfactant, 1 % (w/w) - 30% (w/w) total solvent and 5% (w/w) - 30% (w/w) total carrier oil. In some embodiments, the dispersing agent comprises 50% (w/w) - 75% (w/w) total surfactant, 2.5% (w/w) - 15% (w/w) total solvent and 1 % (w/w) - 10% (w/w) total carrier oil.
[0032] For example, the dispersing agent may comprise 60% (w/w) - 75% (w/w) non-ionic surfactant, 0.2% (w/w) - 10% (w/w) phospholipid surfactant, 2.5% (w/w) - 15% (w/w) citrus oil, and 10% (w/w) - 25% (w/w) total carrier oil. [0033] The dispersing agent may further comprise a preservative, such as an antimicrobial or an anti-oxidant. In an embodiment of the present invention, the anti-oxidant preservative is selected from the group consisting of ascorbyl palmitate, d alpha- tocopherol, dl-alpha- tocopherol, d-alpha-Tocopheryl acetate, dl-alpha-Tocopheryl acetate, d- alpha-Tocopheryl acid succinate, dl alpha-Tocopheryl acid succinate, Vitamin E and derivatives thereof, olive polyphenols and algal polyphenols. In some embodiments of the invention, the dispersing agent may comprise a preservative at a concentration of 0.1 % - 5% (w/w). [0034] The dispersing agent and the solid substance comprising the hydrophobic compound may be combined at any ratio that will facilitate the production of the liquid dispersible composition comprising the hydrophobic compound. In some embodiments, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is from about 100:1 to about 1 : 1. Preferably, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is from about 20: 1 to about 5:1. In some embodiments, the ratio of the solid substance comprising the hydrophobic compound and the dispersing agent is about 10:1 , about 9: 1 or about 8: 1. [0035] In some embodiments, the PEA is formulated for oral administration. In some embodiments, the PEA is formulated as a capsule. In some embodiments, the PEA is formulated for injection, or for transdermal delivery, or other modes. In some embodiments, the effective amount of PEA is 400-1,000 mg. In some embodiments, the effective amount of PEA is 600 mg. In some embodiments, the PEA is administered one to three times per day. In some embodiments, the PEA is administered twice per day. [0036] Methods [0037] Described herein is a method of reducing plasma levels of P-selectin in a subject, the method comprising administering to the subject an effective amount of palmitoylethanolamide (PEA). Also described is a method of reducing inflammation in a subject infected with SARS- CoV-2, the method comprising administering to the subject an effective amount of PEA. Additionally described herein is a method of ameliorating symptoms of SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of PEA. In some embodiments, the symptoms comprise immunothrombosis and/or post-acute sequelae of SARS-CoV-2 infection (PASC). [0038] The subject is typically a mammal. In one embodiment, the mammal is human. In other embodiments, the mammal is a veterinary subject. Examples of veterinary subjects include, but are not limited to, equine, canine, bovine, porcine, ovine, and feline subjects.
[0039] Administration and Dosage [0040] In some embodiments, the composition is administered as a supplement to be ingested orally. In some embodiments, the composition is administered intragastrically. In some embodiments, subjects are administered 600 mg Levagen+ twice daily (LEV) for 2-24 weeks. In some embodiments, the administration is for 4-12 weeks. In some embodiments, the administration is for 4 weeks. [0041] Dosing is typically based on a subject’s body weight. In some embodiments, the PEA is administered at a dose of about 10–30ௗmg PEA/kg bodyweight. A 2013 review (16) reported on the role of PEA as an anti-inflammatory and therapeutic agent for influenza and the common cold. In six clinical trials in nearly 4000 volunteers, PEA demonstrated effectiveness and safety for the treatment of these indications. The effective dose range has consistently been 10–30ௗmg PEA/kg bodyweight. [0042] Levagen + also contains LipiSperse: a novel delivery system designed to increase the dispersion of lipophilic agents in aqueous environments and indicated to enhance PEA bioavailability by nearly 75% (24). As such, each pill contains 350 mg Levagen + with the label claim “Not less than 300 mg palmitoylethanolamide.” Ten percent of the 350 mg is LipiSperse; therefore, each pill contains 315 mg PEA. This is per USP regulations for finished products such that Levagen + contains 270–330 mg PEA (i.e., 90%–110% of the label's claimed ingredients). EXAMPLES [0043] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention. [0044] Example 1: Palmitoylethanolamide Reduces Proinflammatory Markers in Unvaccinated Adults Recently Diagnosed with COVID-19: A Randomized Controlled Trial [0045] Inflammation is at the core of many chronic and infectious conditions, including the severity of coronavirus disease 2019 (COVID-19) infection (1, 2). The SARS-CoV-2 virus, responsible for COVID-19, is classified in the ȕ genus of the Coronaviridae family (3). This family of enveloped viruses has a positive-strand RNA genome capable of encoding critical structural proteins, including the spike surface glycoprotein and the membrane, envelope, and nucleocapsid proteins, which play a pivotal role in the pathogenic outcomes of the virus (4). Upon exposure to SARS-CoV-2, largely through transmission by respiratory droplets, the viral spike protein interacts with angiotensin-converting enzyme 2 receptors on host cells, triggering subsequent changes in spike protein conformation and endosomal formation, events that facilitate viral entry into host cells (5). Once inside a cell, the virus releases its RNA and
replicates, and high numbers of formed virus leave the cell to invade other cells, furthering infection (6). [0046] Derangements in angiotensin-converting enzyme 2 activity caused by viral attachment in individuals infected with SARS-CoV-2 are supported by observed increases in plasma angiotensin II (7). Overactive immune responses may be tied to this loss in angiotensin- converting enzyme 2 activity and therefore inhibition of its protective mechanisms related to inflammation, such as its conversion of angiotensin II to angiotensin 1–7 and its inhibitory effects on macrophage cytokine expression (8). Furthermore, signaling mechanisms associated with the innate immune response against viral infection lead to the activation of transcription factors, such as NF-^B, which induce production of a diverse array of proinflammatory cytokines and chemokines (9). COVID-19 has also been reported to induce lymphopenia among dendritic cells, T cells, and NK cells and to upregulate other immune cells, such as neutrophils and monocytes (10, 11). This overproduction of cytokines leads to the enhanced expression of adhesion molecules on the surface of endothelial cells, further expanding the inflammatory sequelae. For example, elevated circulating P-selectin has been demonstrated to predict thrombosis in patients with COVID-19 (12). Excessive cytokine production, termed “cytokine storm,” can potentiate negative downstream consequences, including a variety of pulmonary and extrapulmonary complications, which are associated with severity of COVID-19 outcomes (13). Thus, investigations have utilized proinflammatory biomarkers to characterize disease severity and risk for death. Consequently, strategies to control inflammation are considered a key approach for slowing the progression of disease and tissue pathology. [0047] Whereas the heightened release of inflammatory mediators is typically observed in the acute stage of COVID-19 (14), some patients develop symptoms lasting beyond 12 wk, often occurring in those with mild-moderate infection severity. Recently, in a study of patients with mild-moderate SARS-CoV-2 infection, those experiencing persistent symptoms had increased immune cell activation and exhibited elevations in proinflammatory mediators 8 mo after infection (15). Hence, approaches to reduce inflammatory mediators in those with even mild- moderate symptoms could be of key importance to mitigating the acute and long-term impacts of COVID-19. [0048] Palmitoylethanolamide (PEA)—a fatty acid amide first isolated from lipid fractions of egg yolks, peanuts, and soybeans—has been documented as being beneficial for reducing inflammation (16). PEA is also endogenously produced in human tissues and functions as a lipid mediator targeting ion channels, nuclear hormone receptors, and G protein–coupled receptors, with wide-reaching effects on metabolism (17). One of the direct targets for PEA has been identified as nuclear receptor peroxisome proliferator–activated receptor Į (PPAR-Į), which is expressed in several cells, including immune cells, and supports PEA's ability for use in modulating inflammatory responses (18, 19). PEA's interaction with PPAR-Į has been shown to
mediate its anti-inflammatory effects, and PEA's binding to PPAR-Į on immune cells leads to reductions in proinflammatory and pain signals (20). Furthermore, PEA exerts its action on G protein–coupled receptor 55, a cannabinoid-like receptor, and indirectly influences activity of cannabinoid receptors 1 and 2 via inhibition of cannabinoid degradation, termed the “entourage effect” (21). Importantly, research has demonstrated that PEA inhibits the migration and degranulation of mast cells by direct and indirect mechanisms (17). Given the emerging evidence that COVID-19 infection activates mast cells via toll-like receptor 4, the ability of PEA to lessen immune cell–induced inflammation via toll-like receptor 4–dependent PPAR-Į activation may have important physiologic relevance (22). PEA has been utilized in several trials to treat influenza and the common cold and was shown to be effective in treating upper respiratory infections (16). [0049] Given the parallels between COVID-19 and the mechanisms by which PEA has been successful as an immunomodulator in conditions such as influenza, it may be a viable adjunctive treatment for COVID-19. Notably, PEA is endogenously produced and has not been associated with adverse side effects. Although PEA has been put forth as a possible adjunctive treatment strategy for COVID-19, there is a dearth of evidence on its use in patients with COVID-19 (23). In this context, the aim of this research is to expand this literature related to PEA in a novel manner and to evaluate the efficacy of 4-wk PEA supplementation to reduce the mediators of inflammation in adults who recently tested positive for COVID-19 but were not hospitalized, a group likely to experience elevations in inflammatory mediators following infection. We hypothesized that 4 wk of PEA supplementation would result in a favorable modulation in inflammatory mediators as compared with placebo supplementation. Given that inflammatory mediators would be expected to return toward homeostatic levels following infection, changes in inflammatory mediators are expected to improve to a larger degree in the treatment group as compared with the placebo group, all else being equal. [0050] Methods [0051] Participants [0052] Healthy adults between the ages of 18–65 years who recently tested positive for COVID- 19 and were not hospitalized for their illness were recruited via online advertisements, local news outlets, e-mail lists, and word of mouth to participate in this study. Inclusion in the study required a recent positive COVID-19 test result per PCR in asymptomatic/symptomatic individuals, although positive antigen test results were also accepted upon symptomatic infection consistent with COVID-19 per the symptoms outlined by the CDC. The exclusion criteria included the following: any unstable or serious illness; serious mood disorders; neurologic disorders, such as multiple sclerosis or cognitive damage; active smoking and/or nicotine or drug abuse; active regular marijuana or other cannabinoid use; other street/recreational drug use; chronic past and/or current alcohol use (>14 alcoholic drinks/wk);
allergies to any of the ingredients in the active or placebo formula, including peanuts and eggs; pregnancy or lactation; medical prescription of drugs that affect immune and/or inflammatory responses; malignancy treatment in the last 5 y; chronic use of steroids; and a BMI > 40 kg/m
2. Eligible respondents were sent an electronic consent form and scheduled to visit the test site based on CDC return-to-work guidance. All CDC guidance for workplace safety was followed at the test site, including hand hygiene practices, environmental infection control, and personal protective equipment. Study recruitment was conducted October 2020–March 2021, and all participants were unvaccinated for COVID-19. The Arizona State University Institutional Review Board approved this study (No.00,012,406), and all participants provided written consent. The study is registered at clinicaltrials.gov as NCT04912921. [0053] Study protocol [0054] The study followed a placebo-controlled randomized parallel-arm study design. Participants were stratified by age, sex, and BMI and randomly assigned by coin toss to receive the active ingredient or placebo treatment for 4 wk. Randomization was performed by a research team member not involved in data collection or analysis. Participants were screened for eligibility using an online questionnaire. Those who met the inclusion criteria were interviewed by phone for a secondary screening to confirm eligibility and to reduce physical contact with investigators during infectious periods. Eligible individuals were scheduled to visit the test site (Arizona Biomedical Collaborative Laboratory Building) based on CDC return-to- work guidance: 1) at least 10 d since symptoms first appeared or from positive test result in asymptomatic individuals, 2) at least 24 h since last fever without the use of fever-reducing medications, and 3) improvement of symptoms (e.g., cough, shortness of breath). [0055] Participants attended two in-person study visits and completed a health history questionnaire and 24-h dietary recall. Anthropometrics were collected during study visits, including height, weight, and BMI. At study visits, venous blood samples were collected by a certified staff phlebotomist into appropriate collection tubes for subsequent processing and analyses of inflammatory mediators. At study baseline, participants were provided with the assigned supplements; a calendar to track supplement intake; and directions on supplement ingestion, symptom reporting, and over-the-counter medication tracking. The final posttrial visit was scheduled after 4 wk, and the baseline assessments and blood draw protocol were repeated. Nutrient intakes obtained from 24-h dietary recalls at baseline and posttrial were assessed using Food Processor Nutrient Analysis Software (ESHA Research). [0056] Supplementation [0057] Participants in the active treatment group ingested 600 mg Levagen+ twice daily (LEV) for 4 wk. A 2013 review (16) reported on the role of PEA as an anti-inflammatory and therapeutic agent for influenza and the common cold. In six clinical trials in nearly 4000 volunteers, PEA demonstrated effectiveness and safety for the treatment of these indications.
The effective dose range has consistently been 10–30ௗmg PEA/kg bodyweight. Levagen + also contains LipiSperse: a novel delivery system designed to increase the dispersion of lipophilic agents in aqueous environments and indicated to enhance PEA bioavailability by nearly 75% (24). As such, each pill contains 350 mg Levagen + with the label claim “Not less than 300 mg palmitoylethanolamide.” Ten percent of the 350 mg is LipiSperse; therefore, each pill contains 315 mg PEA. This is per USP regulations for finished products such that Levagen + contains 270–330 mg PEA (i.e., 90%–110% of the label's claimed ingredients). Those in the placebo group (CON) ingested placebo capsules (maltodextrin) twice daily for 4 wk. LEV and CON supplements were identical in appearance, and supplement blinding was completed by an investigator not involved in data collection or analyses. For adherence checking, participants were asked to record pill ingestion daily and to return unemptied pill bottles. Participants recorded any physical symptoms and/or over-the-counter medication use daily during the trial. Participants also received weekly prompts via e-mail from investigators, which contained reminders related to the study protocol. [0058] Blood processing and analyses [0059] During baseline and posttrial visits, participant blood samples were collected from the antecubital vein by standard phlebotomy techniques, with blood collection sets in 4-mL dipotassium EDTA tubes and 8.5-mL serum separator tubes. After centrifugation of serum separator tubes, serum samples were treated with 1% Triton X-100 to inactivate SARS-CoV-2 virus (25). Whole blood collected in EDTA tubes was immediately lysed in Trizol RNA extraction buffer (TRIzol LS; Thermo Fisher Scientific) and prepared for qRT-PCR for gene expression analysis. Whole blood collected in EDTA tubes was transported and analyzed for complete blood count with differentials, specifically utilized for calculation of neutrophil/lymphocyte ratios by Sonora Quest Laboratories. Serum samples were analyzed for high-sensitivity C-reactive protein and ferritin by Sonora Quest Laboratories. Samples were stored at í80 °C until time of analyses. Serum cytokines and chemokines were analyzed by human focused 15-plex discovery assay (Eve Technologies Corporation): IL-6, IL-1ȕ, IL-1Ra, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, monocyte chemoattractant protein 1, IFN-Ȗ, granulocyte-macrophage colony-stimulating factor, and TNF-Į. Serum soluble P-selectin (sP-selectin) and intercellular adhesion molecule 1 were analyzed via commercially available ELISA methods (Invitrogen; Thermo Fisher Scientific). [0060] RNA isolation and qRT-PCR [0061] The expression of NF-^B, IL-6, and CD177 was quantified from whole blood samples. Trizol-treated whole blood was sonicated for 1 min and then centrifuged to remove cell debris. RNA was purified using the Direct-zol RNA MicroPrep Kit (R2062; Zymo Research), and cDNA was synthesized from total RNA using the cDNA Reverse Transcriptase Kit (4,374,966; Thermo Fisher Scientific) according to the manufacturers’ instructions. Amplification of NF-^B, IL-6,
CD177, and GAPDH genes was accomplished using SYBR Green Master Mix (A46012; Thermo Fisher Scientific) according to the following primers: NF-^B2 (F–
normalized with GAPDH and expressed as ǻCt. [0062] Statistical analysis [0063] The sample size (N = 60) was informed by a meta-analysis reporting 11 randomized controlled trials that investigated the impact of coenzyme Q10 on reducing IL-6 in chronic inflammatory diseases. In these trials, the sample size averaged 44 (range: 26–60), and power analyses based on several of these trials indicated that a sample size of 30–63 was adequate for 80% power at an alpha of 0.05 to detect a significant change in IL-6 (26). We examined histograms of input variables and performed log transformations as needed for analyses to reduce skewness. The primary outcomes included the change in circulating levels of IL-6, C- reactive protein, ferritin, intercellular adhesion molecule 1, sP-selectin, and neutrophil/lymphocyte ratio between groups. Secondary analyses were conducted on the changes in a panel of serum cytokines and chemokines and expression of NF-^B, IL-6, and CD177 in whole blood between groups. A series of linear regression models was built to test if the postchange as compared with the prechange in each inflammatory marker was associated with the treatment. In these models, the response variable was the postchange as compared with the prechange; the independent variable was the treatment group; and the covariates were age, sex, BMI, and existing conditions (binary). An overall P value <0.05 indicated statistical significance. The Benjamini-Hochberg procedure (27) was employed to account for multiple comparisons in analyses of primary outcomes with a false discovery rate of 10%. Data were analyzed using open source R software. [0064] Results [0065] From the 323 study respondents, 61 participants were enrolled in the trial. One LEV participant withdrew due to scheduling conflicts, and 60 participants (n = 30/group) completed the trial (Figure 1). Average compliance to supplement ingestion was high such that 87% and 91% of the tablets were taken during the study in the CON and LEV groups, respectively. Baseline characteristics of participants are shown in Table 1. Baseline characteristics of participants were comparable between study arms. Additionally, of the 60 participants who completed the trial, 2 in the CON group and 1 in the LEV group reported asymptomatic
infection. Types of COVID-19 symptoms reported by participants were also comparable between groups (Figure 3). [0066] Table 1: Baseline characteristics of study participants (N = 60) in the LEV and CON groups
1
1Data are presented as n (%) of participants or mean ± SD unless noted otherwise. CON, placebo; COVID-19, coronavirus disease 2019; LEV, 600 mg Levagen + twice daily [0067] Based on the 24-h dietary records obtained throughout the trial, no significant baseline or posttrial differences were seen between the LEV and CON groups in dietary intakes of energy, carbohydrates, proteins, fats, saturated fatty acids, sugar, total dietary fiber, vitamin C, vitamin D, calcium, iron, sodium, and zinc (Supplemental Table 1, presented in Figure 2).
[0068] There were no significant differences between groups in the reductions of IL-6, C- reactive protein, ferritin, intercellular adhesion molecule 1, or neutrophil/lymphocyte ratio (Table 2). However, the reduction in sP-selectin from baseline was significantly associated with LEV treatment compared with CON, which saw increases in these markers after adjustment for covariates and remained significant with a Benjamini-Hochberg false discovery rate ^ 0.10 (ȕ = í11.5; 95% CI: í19.8, í3.15; P = 0.0078). sP-selectin fell 8% in the experimental group (mean ± SD: –6.8 ± 17.3) and rose 5% in the control group (3.4 ± 15.3). [0069] Table 2: Status of primary outcome serum inflammatory biomarkers at baseline and after the 4-wk intervention and linear regression analyses on change from baseline scores in adults recently diagnosed with COVID-19 in the LEV and CON groups (n = 30/group)
1
1Data are presented as mean ± SD. Data were analyzed by multiple linear regression models with the change from baseline as the response variable regressed on the treatment group, adjusting for age, sex, BMI, and existing conditions. CON, placebo; COVID-19, coronavirus disease 2019; CRP, C-reactive protein; ICAM-1, intercellular adhesion molecule 1; LEV, 600 mg Levagen + twice daily; N/L, neutrophil/lymphocyte ratio; sP- selectin, soluble P-selectin. 2Data are presented as the regression estimate (95% CI) of the estimate for the group assignment variable from each model. *Significance is retained after correction for multiple comparisons: Benjamini-Hochberg false discovery rate ^ 0.10. [0070] The changes in circulating cytokines and chemokines and whole blood expression of inflammatory markers after 4 weeks of supplementation are presented in Table 3. The analyses did not reveal significant associations between the treatment group and the change from baseline in most serum inflammatory factors or whole blood expression of NF-^B, IL-6, or CD177. However, significant reductions from baseline in IL-1ȕ (ȕ = í22.9; 95% CI: í42.4, í3.40; P = 0.0222) and IL-2 (ȕ = í1.73; 95% CI: í3.45, –0.065; P = 0.0492) were associated with LEV treatment compared with CON (Table 3). [0071] Table 3: Status of inflammatory cytokines and chemokines in serum and RNA levels of inflammatory markers in whole blood determined by qRT-PCR and linear regression analyses on change from baseline scores in adults recently diagnosed with COVID-19 in the LEV and CON groups (n = 30/group)
1
1Data are presented as mean ± SD. Data were analyzed by multiple linear regression models with the change from baseline as the response variable regressed on the treatment group, adjusting for age, sex, BMI, and existing conditions. CON, placebo; COVID-19, coronavirus disease 2019; GM-CSF, granulocyte-macrophage colony-stimulating factor; LEV, 600 mg Levagen + twice daily; MCP-1, monocyte chemoattractant protein 1. 2Data are presented as regression estimate (95% CI) of the estimate for the group assignment variable from each model. Secondary outcome measures were not adjusted for multiplicity. [0072] Discussion [0073] These data show that daily PEA supplementation reduced sP-selectin concentrations in adults recently diagnosed with COVID-19. Specifically, sP-selectin fell 8% in the LEV experimental group and rose 5% in the CON group, a difference that was statistically significant after false discovery rate adjustment using the Benjamini-Hochberg procedure (P = 0.0078). Furthermore, LEV treatment was associated with significant reductions from baseline in IL-2 (P = 0.0492) and IL-1ȕ (P = 0.0222) compared with CON. P-selectin, a key thromboinflammatory marker, is stored in the Į-granules of platelets and Weibel-Palade bodies of endothelial cells and is translocated to the surface of the cell upon stimulation (28). This membrane glycoprotein is expressed on activated platelets and endothelial cells and contributes to the localized inflammation that ultimately eliminates pathogens and clears cell debris. P- selectin is involved in the initial attachment of platelets and endothelial cells to leukocytes and the rolling of immune cells to injured regions (29). In addition, P-selectin on activated platelets has been shown to stimulate exposure of tissue factor on monocytes, which may promote intravascular hemostasis and thrombosis (30). Although P-selectin is essential for optimal immune responses and the rapid elimination of infectious agents and foreign particles, under conditions of unresolved disease or chronic insult, P-selectin is related to the propagation and amplification of the inflammatory response. Importantly, P-selectin has also been linked to COVID-19 complications. [0074] There are reports of 6%–12% declines in sP-selectin following pharmaceutical interventions in patient populations. Nomura et al. (31) noted a 6% reduction in sP-selectin following administration of efonidipine to patients with hypertension and diabetes and suggested that this may prevent the development of cardiovascular complications caused by cell adhesion molecules or activated platelets and monocytes. Riondino et al. (32) noted a 12% reduction in sP-selectin following drug-induced normalization of blood pressure in older adults who were
hypertensive. Thus, the 8% reduction in sP-selectin noted herein is within the ranges cited in other intervention trials, suggesting its clinical relevance. [0075] Procoagulant responses and thrombosis are augmented in patients with COVID-19 (33). These derangements in hemostasis persist in moderate and severe COVID-19 (34) and are associated with the signature inflammatory imbalances of the cytokine storm and severity of disease outcomes (11, 35). P-selectin expression on the surface of platelets was recently shown to be significantly elevated in hospitalized patients with COVID-19, regardless of severity, compared with healthy donors (36). Furthermore, P-selectin is a key receptor for formation of platelet-leukocyte aggregation, which is increased in those infected with COVID-19 (36), and a marker of in vivo platelet activation during viral illness (37). Moreover, serum sP- selectin levels were found to be higher in moderate and severe cases of COVID-19 than in a healthy control group, suggesting the potential of sP-selectin as a prognostic marker for COVID- 19 disease (38). It has been noted that sP-selectin, the form in circulation, is a useful biomarker and potential contributor to vascular complications (39). Additionally, as patients with COVID-19 have highly stimulated circulating platelets, it has been suggested that P-selectin can be utilized as a marker of platelet activation in COVID-19 infection (40). Studies have further reported that crizanlizumab, a monoclonal antibody that targets sP-selectin, can decrease inflammation by binding to it and blocking leucocyte and platelet adherence to the vessel wall (41). Consistent with these findings, blocking of sP-selectin has been demonstrated to attenuate the hyperinflammatory and hyperthrombotic state characteristic of COVID infections (42). Given the prothrombotic consequences of increases in P-selectin on COVID-19 immunothrombosis, interventions targeting P-selectin may be favorable in dually targeting platelet and endothelial cell mechanisms of this disease. In the present study, ingestion of LEV for 4 wk induced a significant reduction in sP-selectin from baseline in adults recently diagnosed with COVID-19, as opposed to an increase in this marker in the CON group. [0076] Additionally, it has been noted that, whereas inflammatory mediators play a crucial role in viral defense, disproportionate immune responses are associated with the severity of COVID- 19. Several proinflammatory cytokines, chemokines, and markers of infection have been indicated to be upregulated in COVID-19: C-reactive protein, IL-6, IL-1ȕ, IL-2, TNF-Į, monocyte chemoattractant protein 1, granulocyte-macrophage colony-stimulating factor, and IFN-Ȗ (43). Of note, several of these factors are upregulated by increases in NF-^B expression. Therefore, it has been suggested that the NF-^B pathway could be a beneficial therapeutic target for mitigating COVID-19 severity (44). In the present study, we also noted significant mean decreases in IL-1ȕ (P = 0.0222) and IL-2 (P = 0.0492) associated with LEV treatment. [0077] Evidence from a 2016 study suggested that PEA administration reduces P-selectin, neutrophil infiltration, cytokine production (TNF-Į and IL-1ȕ), and NF-^B expression in rats following myocardial ischemia reperfusion injury (45). It also suggested, in support of the PEA
mechanisms previously reported, that the effect of NF-^B may be associated with PPAR-Į activation and expression. This finding agrees with a prior study demonstrating that the anti- inflammatory effects of PEA are in part mediated by PPAR-Į activation (46). Importantly, NF-^B and mast cell activation are linked to production of inflammatory cytokines, which can induce the expression of adhesion molecules on the endothelium (45). Regarding the results of the present study, although systemic reductions in sP-selectin, IL-2, and IL-1ȕ were observed in the experimental group (LEV), we did not observe reductions in expression of NF-^B, IL-6, CD177, and other circulating inflammatory factors. However, it is possible that these markers would be modestly upregulated in those with mild-moderate COVID-19 infections, as participants in this study were not hospitalized for COVID-19 illness. It is also plausible that whereas sP-selectin may be a simple diagnostic tool related to COVID-19 diagnosis and severity, alterations in markers associated with sP-selectin reduction at the level of specific target tissues (e.g., the lungs) may not be revealed by our analysis. [0078] A recent investigation suggested that murine alveolar macrophages challenged with the SARS-CoV-2 spike viral protein in vitro exhibited significant increases in proinflammatory markers, including NF-^B, IL-6, TNF-Į, and IL-1ȕ, and inflammasome expression, with this response being reduced significantly by treatment with PEA (47). It was additionally demonstrated that this response was mediated by PPAR-Į. Moreover, in a study of LPS- induced acute lung injury in rats, PEA administration reduced markers of neutrophil infiltration, immune cells quantity, and mast cell degranulation in the lungs and inhibited bronchoalveolar lavage fluid levels of the proinflammatory cytokines IL-6, TNF-Į, IL-1ȕ, and IL-18. Furthermore, PEA was suggested to block inflammatory cytokine production through inhibition of NF-^B activation in lung tissue (48). It is also possible that the reductions in sP-selectin and cytokines found in this study could be related to implications of PEA in the entourage effect. It has been postulated that PEA can inhibit degradation of endocannabinoids, thereby potentiating the effects at their targets. In line with this potential contributory mechanism, Zhao et al. (49) demonstrated that activation of CB2 receptors can inhibit the expression of P-selectin in an animal model of atherosclerosis, which was associated with reductions in macrophage infiltration. It has also been demonstrated that PEA may enhance the release of 2-arachidonoyl glycerol, an endocannabinoid that has been shown to reduce IL-2 secretion (50–52). [0079] The study participants were allowed to the test site following CDC return-to-work guidance; therefore, we were unable to capture responses immediately following viral infection. Yet, it is important to note that the LEV and CON groups were comparable in terms of the types of COVID-19 symptoms reported and the total number of symptoms reported. Furthermore, the CDC stated that inflammatory reactions and health problems related to COVID-19 can be experienced weeks after viral infection, even in those who experienced no or minimal symptoms during infection. With all of this noted, secondary outcome data were not adjusted for multiplicity, and caution should be exercised in interpreting findings of secondary outcome
analysis. In this trial, plasma concentrations of PEA were not measured; however, a previous report documented the pharmacokinetics and bioavailability of the Levagen + brand of PEA as well as the standard PEA formulation in humans (24). Elevations in plasma PEA were evident at 30 min postdose and remained elevated for at least 4 h postdose for both preparations, and bioavailability was elevated nearly 75% for the Levagen + PEA formulation compared with the standard PEA formulation (24). [0080] Though inflammatory mechanisms are crucial to an optimal immune response, unchecked secretion of cytokines and thromboinflammatory markers can promote the development of the inflammatory response in unresolved disease states and is implicated in COVID-19 complications. Therefore, the reduction in inflammatory markers noted herein shows that PEA exerts anti-inflammatory actions and can reduce the severity of COVID-19. P-selectin and inflammatory cytokines are also elevated in many chronic conditions linked to inflammation, including obesity, atherosclerosis, asthma, and cancer; hence, Levagen + administration may offer a degree of relief from inflammatory symptoms for many chronic conditions. [0081] References [0082] 1. Zhao Y, et al. MolBiosyst 2016;12(8):2318–41. [0083] 2. Amaral-Machado L, et al. Biomed Pharmacother 2021;134:111143. [0084] 3. Li X, et al. J Pharm Anal 2020;10(2)102–8. [0085] 4. Nilea SH, et al. Cytokine Growth Factor Rev 2020;54.66–70 [0086] 5. Zhang Q, et al. Signal Transduct Target Ther 2021;6(1):233. [0087] 6. Jackson CB, et al. Nat Rev Mol Cell Biol 2022;23(1):3–20. [0088] 7. Liu Y, et al. Sci China Life Sci 2020;63(3):364–74. [0089] 8. Banu N, et al. Life Sci 2020;256:117905. [0090] 9. Lee C, et al.Arch Pharmacal Res 2021;44(1):99–116. [0091] 10. Sallenave JM, Guillot L. Front Immunol 2020;11:1229. [0092] 11. Huang C, et al. Lancet 2020;395(10223):497–506. [0093] 12. Fenyves BG, et al. Am J Hematol 2021;96(12):E468–E471. [0094] 13. Gupta A, et al. Nat Med 2020;26(7):1017–32. [0095] 14. Carvalho T, et al. Nat Rev Immunol 2021;21(4):245–56. [0096] 15. Phetsouphanh C, et al. Nat Immunol 2022;23(2):210–6. [0097] 16. Keppel Hesselink JM, et al. Int J Inflam 2013;2013:1–8. [0098] 17. Petrosino S, et al. Br J Pharmacol 2017;174(11):1349–65.
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