Patent Application
20240091169
Inflammatory diseases impact millions worldwide. However, if available, current therapeutics primarily treat disease symptoms and may have undesirable off-target effects. For instance, medicine for ulcerative colitis (UC), an idiopathic inflammatory disease of the large intestine, primarily exists to treat the symptoms of UC, these drugs (specifically antibiotics, corticosteroids, NSAIDs, and cytokine inhibitors). Said medicines can result in off-target effects and immunosuppression, further contributing to decreased quality of life for UC patients. Similarly, available colorectal cancer (CRC) therapies have limited efficacy and severe adverse effects. In fact, many CRC patients receiving chemotherapy experience issues of non-specific toxicity, pervasive off-target effects, and acquired drug resistance, resulting in reduced quality of life, treatment cessation, or need for alternative treatment regimens.
Ginseng is one of the most widely used herbal medicines and best-selling natural products worldwide. American Ginseng (AG) is a plant rich in bioactive phytochemicals with its active components (ginsenosides) documented to exert immunomodulatory, anti-inflammatory, anti-cancer, and chemo-protective effects. Isolated, active components of Ginseng exhibit more potent antioxidant and pro-apoptotic properties.
As such, what is needed in the art are clinically effective compositions and methods for treating and/or suppressing inflammatory diseases.
In general, disclosed herein are methods of treating an inflammatory disease or disorder by administering to the subject in need thereof a polyacetylene compound. The polyacetylene compound may be administered at a dose of from about 1 mg/kg body weight to about 10 mg/kg body weight.
Other features and aspects of the present disclosure are discussed in greater detail below.
A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures, in which:
Repeat use of reference characters in the present specification and drawings is intended to represent the same or analogous features or elements of the present invention.
Reference will now be made in detail to various embodiments of the presently disclosed subject matter, one or more examples of which are set forth below. Each embodiment is provided by way of explanation, not limitation, of the subject matter. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the disclosure. For instance, features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment. Thus, it is intended that the present disclosure cover such modifications and variations as come within the scope of the appended claims and their equivalents.
In general, disclosed herein are methods of treating an inflammatory disease or disorder by administering to the subject in need thereof a polyacetylene compound. The term “treating” as used herein refers to partially or completely alleviating, improving, relieving, inhibiting progression, and/or reducing incidence of one or more symptoms of an inflammatory disease or disorder.
In one embodiment, a subject in need thereof may be administered a polyacetylene compound. As used herein, a “polyacetylene compound” refers to compounds isolated from natural sources, such as fungi or plants. For instance, the polyacetylene compound may be an anti-tumor agent.
In one embodiment, the polyacetylene compound may be panaxynol (3(R)-(9Z)-heptadeca-1, 9-dien-4,6-diyn-3-ol. Panaxynol is an isolated, bioactive component in American Ginseng. Panaxynol has the following chemical formula: C17H24O. Panaxynol has the following chemical formula:
In one embodiment, the polyacetylene compound may be administered at a dose of from about 1 mg/kg body weight to about 10 mg/kg body weight, such as from about 1.5 mg/kg body weight to about 7.5 mg/kg body weight, such as from about 2 mg/kg body weight to about 5 mg/kg body weight, such as from about 2.5 mg/kg to about 4 mg/kg, or any range therebetween.
The duration of therapy will continue for as long as medically indicated or until a desired therapeutic effect (e.g., those described herein) is achieved. For instance, a subject can be treated until a complete response is acquired, such as until disease progression is delayed or inhibited. In one embodiment, the polyacetylene compound is administered daily for a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 6 weeks, about 8 weeks, or longer than 8 weeks following first administration. However, the course of treatment for any individual subject can be modified in clinical practice.
Methods disclosed herein are generally directed toward treating an inflammatory disease or disorder by administering to the subject in need thereof at least one panaxynol compound. The term “treating” may refer to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition, e.g., an inflammatory disease.
Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition (e.g., prior to an identifiable disease, disorder, and/or condition), and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
As used herein, an “inflammatory disease” refers to a disease caused by, resulting from, or resulting in inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. The inflammatory disease may include, but is not limited to, an inflammatory bowel disease, colon cancer, colitis-associated colon cancer, colorectal cancer, psoriasis, colorectal cancer, chronic inflammation, an autoimmune disease, Familial Adenomatous Polyposis (FAP), and Lynch Syndrome.
In one embodiment, the inflammatory disease may be an inflammatory bowel disease. For instance, the inflammatory bowel disease may be selected from the group consisting of Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, microscopic colitis, left-sided colitis, pancolitis, and ileocolitis. In one embodiment, the inflammatory disease may be colitis.
In one embodiment, the inflammatory disease may be colorectal cancer including, but is not limited to, colon cancer, rectal cancer, or colorectal adenocarcinoma.
The term “subject” refers to any organism to which aspects of the disclosure can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Subjects to which embodiments of the disclosure can be administered include mammals, such as primates, for example, humans. For veterinary applications, a wide variety of subjects are suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals, such as pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted above or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.
As used herein, the term “administering” refers to introducing a substance (e.g., a polyacetylene compound, an exogenous antigen, a cytotoxic agent, etc.) into a subject. The administration thereof can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. For instance, the polyacetylene compound may be administered orally, subcutaneously, intravenously, or intratumoral. In this regard, “oral” administration can refer to administration into a subject's mouth; “subcutaneous” administration can refer to administration just below the skin; “intravenous” administration can refer to administration into a vein of a subject; and “intratumoral” administration can refer to administration within a tumor.
Pharmaceutical compositions disclosed herein may be formulated to be compatible with its intended route of administration. As used herein, “routes of administration” may include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition can be sterile and should be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Compositions for parenteral delivery, e.g., via injection, can include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., corn oil) and injectable organic esters such as ethyl oleate. In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like that can enhance the effectiveness of the phenolic compound. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.
In one embodiment, a therapeutically effective amount of the polyacetylene compound may be administered to the subject. The term “therapeutically effective amount” refers to those amounts that, when administered to a subject in view of the nature and severity of that subject's disease or condition, will have a desired therapeutic effect, e.g., an amount which will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease or condition. A therapeutically effective dose further can refer to that amount of the therapeutic agent sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose can refer to that ingredient alone. When applied to a combination, a therapeutically effective dose can refer to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
A therapeutically effective dose can depend upon a number of factors known to those of ordinary skill in the art. The dosage can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; identity, size, condition, age, sex, health and weight of the subject or sample being treated; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion. These amounts can be readily determined by the skilled artisan.
Prior to or following administration of the polyacetylene compound, a biological sample may be obtained from a subject to measure the presence or level of one or more biomarkers. As used herein, “obtaining a biological sample” refers to a process for directly or indirectly acquiring a biological sample from a subject. For instance, a biological sample may be obtained (e.g., at a point-of-care facility, e.g., a physician's office, a hospital, laboratory facility) by procuring a tissue or fluid sample (e.g., blood draw, marrow sample, spinal tap) from a subject. Alternatively, a biological sample can be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject. The biological sample can be, for example, a “biopsy tissue” or a “tumor sample,” which refer to a sample of cells, tissues or fluids which is extracted from a subject, for example, in order to determine if the sample contains inflammation or to determine the gene expression profile or molecular profile of that tissue. The tissue or fluid may be examined to detect the presence or absence of one or more biomarkers of an autoimmune disease, including the presence of DNA and/or amino acid sequence mutations, molecular profile of the cells, and/or expression of the gene signature of cells.
The term “biomarker” may refer to mutations and/or molecules that can be evaluated in a biological sample (e.g., a biopsy tissue or a tumor sample) and are associated with a physical condition. For instance, biomarkers include expressed genes or their products (e.g., proteins) or antibodies to those proteins that can be detected from human samples, such as blood, serum, solid tissue, and the like, that is associated with a physical or disease condition. Such biomarkers include, but are not limited to, biomolecules comprising nucleotides, amino acids, sugars, fatty acids, steroids, metabolites, polypeptides, proteins (such as, but not limited to, antigens and antibodies), carbohydrates, lipids, hormones, antibodies, regions of interest which serve as surrogates for biological molecules, combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins), and any complexes involving any such biomolecules, such as, but not limited to, a complex formed between an antigen and an autoantibody that binds to an available epitope on said antigen.
In one embodiment, the biomarker may comprise a cytokine, a cell, a micro-RNA, or any combination thereof. As used herein, “cytokine” refers to small, secreted proteins that regulate the intensity and duration of the immune response by affecting the immune cells differentiation process involving changes in gene expression by which a precursor cell becomes a distinct specialized cell type. For instance, the biomarker may comprise interleukin 10 (IL-10), interleukin 17+(IL-17+), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 13 (IL-1β), transforming growth factor-beta (TGF-β), Foxp3, or any combination thereof.
In one embodiment, the biomarker may comprise a cell. For instance, the cell may be a regulatory T cell (e.g., CD8+ T cells) or T helper cell (e.g., CD4+ T cells). In one embodiment, the cell may comprise macrophages, including CD4+, CD3+, CD45+, CD8+, CD11b, or CD68.
As used herein, “macrophage” refers to a cell capable of phagocytosis. A macrophage is a relatively long-lived phagocytic cell of mammalian tissues, derived from blood monocytes, and may be static or mobile. Due to differences in receptor expression, cytokine production, and functions, a macrophage may be referred to as M1 or M2. In embodiments, M1 may refer to Type I macrophages; M2 may refer to Type II. M1 macrophages are cells capable of producing pro-inflammatory cytokines and are implicated in the killing of pathogens and tumor cells. M2 macrophages moderate the inflammatory response, eliminate cell wastes, and promote angiogenesis and tissue remodeling (e.g., repair). Non-limited examples of macrophages include adipose tissue macrophages, monocytes, Kupffer cells, sinus histiocytes, alveolar macrophages, tissue macrophages, Langerhans cells, microglia, Hofbauer cells, Intraglomerular mesangial cells, osteoclasts, epithelioid cells, red pulp macrophages, peritoneal macrophages, or Peryer's Patch macrophages. In embodiments the presence of M1 macrophages may be determined by measuring biomarkers for M1, for example CD38, G-protein coupled receptor 18 (Gpr18), Formyl peptide receptor 2 (Fpr2), or iNos. In embodiments the presence of M2 macrophages may be determined by measuring biomarkers for M2, for example Early growth response protein 2 (Egr2), c-Myc, or CD206.
In one embodiment, the biomarker may be pro-inflammatory markers associated with M1 macrophages including, but is not limited to, IL-1β and IL-6. In another embodiment, the biomarker may be pro-tumoral markers associated with M2 macrophages including, but are not limited to, IL-10, IL-13, and TGF-(β1.
Methods disclosed herein may be beneficial for treating an inflammatory disease. The expression level of a biomarker may be measured prior to and after administration of at least one polyacetylene compound disclosed herein. For instance, cytokine or regulatory T cell levels may be measured by any method well known in the art prior to and after administration of at least one compound disclosed herein.
In one embodiment, the expression level of a biomarker may be decreased by more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a controlled sample.
In one embodiment, the expression level of a biomarker may be increased by more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to a controlled sample.
The preceding description is exemplary in nature and is not intended to limit the scope, applicability or configuration of the disclosure in any way. Various changes to the described embodiments may be made in the function and arrangement of the elements described herein without departing from the scope of the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.
As used in this application and in the claims, the singular forms “a”, “an”, and “the” include the plural forms unless the context clearly dictates otherwise. Additionally, the term “includes” means “comprises”. The methods and compositions of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional ingredients, components or limitations described herein or otherwise useful in biocidal compositions.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percentages, and so forth, as used in the specification or claims are to be understood as being modified by the term “about”. Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximate unless the word “about” is recited.
As used herein, “optional” or “optionally” means that the subsequently described material, event or circumstance may or may not be present or occur, and that the description includes instances where the material, event or circumstance is present or occurs and instances in which it does not. As used herein, “w/w %” and “wt %” mean by weight as relative to another component or a percentage of the total weight in the composition.
The term “about” is intended to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Unless otherwise indicated, it should be understood that the numerical parameters set forth in the following specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, numerical parameters should be read in light of the number of reported significant digits and the application of ordinary rounding techniques.
The phrase “effective amount” means an amount of a compound that promotes, improves, stimulates, or encourages a response to the particular condition or disorder or the particular symptom of the condition or disorder.
Here and throughout the specification and claims, range limitations are combined and interchanged, such ranges are identified and include all the sub-ranges contained therein unless context or language indicates otherwise. For example, all ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other.
This written description uses examples to disclose the present disclosure, including the best mode, and also to enable any person skilled in the art to practice the disclosure, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the disclosure is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they include structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
Furthermore, certain aspects of the present disclosure may be better understood according to the following examples, which are intended to be non-limiting and exemplary in nature. Moreover, it will be understood that the compositions described in the examples may be substantially free of any substance not expressly described.
The present disclosure may be better understood with reference to the following examples.
Mice (C57BL/6) were housed 3-5/cage, maintained on a 12:12-h light-dark cycle in a low stress environment (22° C., 50% humidity, low noise), and given food and water ad libitum. All mice were fed an AIN-76A diet (Bioserv, Frenchtown, NJ, USA; catalog #:F1515), a purified, balanced diet that is phytoestrogen free. Dietary phytoestrogens have been shown to influence anxiety-related behaviors, fat deposition, blood insulin, leptin and thyroid levels as well as lipogenesis and lipolysis in adipocytes, all of which could nonspecifically impact tumorigenesis. Body weight, food, and water consumption were monitored on a weekly basis for the duration of the study.
Using the AOM/DSS protocol, CRC was chemically induced as illustrated in
To provide a more comprehensive study of colitis, two murine colitis models were utilized:
Experiment 1—Chronic Colitis: 14-week-old C57BL/6 female mice were given 3 cycles of DSS. Each cycle consisted of 7 days on DSS (cycle 1: 1%; cycle 2: 1.5%, cycle 3: 1%), immediately followed by 7 days of regular drinking water. Either vehicle or panaxynol was administered 3×/week beginning on the day prior to the first DSS cycle (day −1) until study completion (
Experiment 2—Acute Colitis: 14-week-old C57BL/6 female mice were given 1 cycle of 2% DSS in drinking water for 7 days, immediately followed by 7 days of regular drinking water. Either vehicle or panaxynol was administered 3×/week beginning on the day prior to the first DSS cycle (day −1) until study completion (
Panaxynol (PA; C17H24O) was purchased from Biopurify Pytochemicals Ltd in sealed 5 mg/mL vials in pure 95% ethanol and stored at −20° C. PA was prepared fresh on the day of treatment by diluting in H2O to 1 mg/mL for administration (p.o.) at a dosage of 2.5 mg/kg. This dosage was chosen following a pilot dose-response study to determine PA's efficacy. PA or VEH was administered 3×/week via oral gavage using flexible plastic tubing gavage needles (Instech, Cat #FTP-20) for a total of 12 weeks.
All mice underwent the same symptom monitoring and scoring. Body weights and symptom scores were determined 1×/week to quantify disease state, with a higher score indicative of a worsened disease state. Symptom scores were calculated by scoring percent body weight loss, stool consistency, and rectal bleeding as previously described. Briefly, fresh colonic evacuates were smeared onto “Hemoccult” tape (Beckman Coulter) to assess severity of diarrhea and were tested with developer (Beckman Coulter) to assess rectal bleeding. Stool consistency was scored as follows: solid cylinder (0), soft cylinder and easily spreadable (2), and noncylindrical or runny (4). Bleeding was scored as follows: no positive detection of blood (0), detection of blood but not grossly visible (2), and gross visibility of blood (4). Body weight was calculated as percent bodyweight loss: 0%-5% (0), 6%-10% (1), 11%-15% (2), 15%-20% (3), 20%-25% (4), and >25% (5). Scores of all three categories were summed to obtain an overall symptom score for each mouse.
Prior to euthanasia, colonoscopies were performed (n=3/group) to visualize the colonic tissue and quantify the severity of disease (colitis and colorectal tumors) using an established protocol. Briefly, mice were anesthetized with isoflurane to perform a detailed endoscopic examination in which the endoscope was gently inserted into the rectum and through the anus. Air was applied to slowly set apart the intestinal walls for examination. As the scope advanced, pictures were taken frequently and the following were meticulously assessed and scored by the blinded primary investigator using the well-established scoring system to integrate evaluation of intestinal inflammation (i.e., focal lesions (edematous areas of mucosa, erosions/reddened areas, ulcers), perianal region findings (diarrhea, bloody anal discharge, or prolapse), transparency of the colonic wall (visualization of the intramural blood vessels), and presence of intestinal bleeding) and tumors. Scoring was performed as previously described with no modifications. Upon completion of colonoscopies, mice were placed on a warm pad set at 37° C. for 5 min.
Following 12 weeks of PA treatment, mice were euthanized by isoflurane overdose following a 4 hr fast. Retro-orbital blood was collected for blood analysis. The colon was excised and carefully flushed with PBS to remove fecal material, cut longitudinally, and opened to be imaged on a dissection microscope in which all tumors were counted and measured based on size as follows: small (≤1 mm), medium (1-2 mm), and large (>2 mm). Following imaging, the colons in the first cohort of animals (n=5-6/group) were cut in half in which the proximal portion was snap frozen in liquid nitrogen for RT-qPCR gene expression analysis and the distal portion swiss rolled, fixed in 10% formalin, and paraffin embedded for histopathology, immunohistochemistry, and immunofluorescence. Colons from a separate cohort (n=5-8/group) were placed in ice-cold PBS until flow cytometry. In a third cohort, tumors were excised from the entire colon and snap frozen for RT-qPCR (n=4-5/group). The colon (without tumors; n=5-7/group) was then placed in ice-cold PBS until flow cytometry.
A blood panel analysis was performed (n=14-17/group for AOM/DSS groups and n=10 for CF group) using the VetScan HMT (Abaxis) for determination of total white blood cells (WBC), lymphocytes (LYM), monocytes (MON), and neutrophils (NEU). The neutrophil to lymphocyte ratio (NLR) was calculated from obtained values.
Lamina propria (LP) cells were isolated from colons to be used for flow cytometry to identify immune cell populations within the colonic LP. Colons were rinsed in sterile ice-cold PBS and minced with scissors. Tissues were subjected to a cell dissociation solution (HBSS without Ca2+Mg2+ containing 5 mM EDTA and 1 mM DTT) in a shaking incubator for 30 min at 37° C. Tubes containing dissociated tissues were vortexed and passed through a 100 μm cell strainer; flow through containing intestinal epithelial cells (IECs) was discarded. Remaining tissue was subjected to a cell digestion buffer (HBSS with Ca2+Mg2+ containing 5% FBS, 5 mg/mL Collagenase D, 50 μg/mL DNase 1, and 1 U/mL Dispase) in a shaking incubator for 30 min at 37° C. Tubes were vortexed and passed through a 40 μm cell strainer. Ice-cold PBS was added to filter to collect remaining cells. Tubes were spun at 2500 rpm for 15 min to pellet. Cells were passed through a Percoll gradient (40/80% (v/v) gradient) and spun at 2500 rpm for 20 min without acceleration and brakes. Cells at the 40/80 interface (leukocytes) were collected, spun at 2000 rpm at 4° C. for 10 min, washed with PBS, and prepared for flow cytometry analysis.
To identify macrophages, colonic LP cells were isolated as described above and first incubated with ZombieGreen (FITC; Bio-Rad) solution for 20 min at RT in dark to discriminate Live/Dead (n=5-8/group). Cells were then washed with flow buffer (HBSS without Ca2+Mg2+ containing 0.5% FBS, 10 mM HEPES (Gibco), and 2 mM EDTA (Gibco)), blocked for non-specific binding with FC block for 10 min at 4° C., and then incubated with fluorescently labelled antibodies (BioLegend) against macrophage cell-surface markers: CD45, CD11b, and CD68 for 20 min at 4° C., followed by 2 PBS washes, with the final resuspension in flow buffer. Colonic LP cells were gated for non-debris singlets and considered live immune cells with ZombieGreenNeg/Low and CD45+. From the Live CD45+ population, CD11b+CD68+ cells were identified as macrophages.
To identify neutrophils, colonic LP cells were isolated as described above and incubated with ZombieGreen (FITC; Bio-Rad) solution for 20 min at RT in dark to discriminate Live/Dead (n=2-3/group). Cells were then washed with flow buffer and blocked for non-specific binding with FC block for 10 min at 4° C., and then incubated with fluorescently labelled antibodies (BioLegend) against neutrophil cell-surface markers: CD45, CD11b, and Ly6g.
To identify T-cell phenotypes, colonic LP cells were isolated as described above and incubated in 0.02% Brefeldin A in complete media (cDMEM) for 4 hr (n=5-7/group). Cells were stained with fluorescently labelled antibodies against T-cell surface markers: CD45, CD4, and TCRβ. 1× fix was used to fix the cell surface stains, and 1× perm buffer was used to permeabilize cell membranes (BioLegend; Cat #424401). Cells were then stained with fluorescently labelled antibodies against intracellular marker FoxP3 to identify regulatory T-cells (T-regs). All cells were measured using a FACS Aria II (BD) and analyzed using FlowJo (BD Biosciences).
Proximal colons (n=5-7/group) and colonic tumors (n=4-5/group) were homogenized and RNA was extracted. RNA sample quality and quantities were verified using a Nanodrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) and determined to be of good quality based on A260/A280 and 260/230 values (>1.8) prior to cDNA synthesis using High-capacity Reverse Transcriptase kit (Applied Biosystems, Cat #4368814). Quantitative reverse transcriptase polymerase chain reaction (PCR) analysis was carried out as per the manufacturer's instructions and all primers used were TaqMan Gene Expression Assays (Applied Biosystems). TaqMan reverse transcription reagents were used to reverse transcribe and to analyze colon and tumor mRNA gene expression of the following markers: monocyte/macrophage (CD68, MRC1 (colon only), and MSR1 (colon only)), pro-inflammatory M1 macrophage markers (IL-1β (colon only) and IL-6), anti-inflammatory M2 macrophage markers (IL-10 (colon only), IL-13, and TGF-(β1), neutrophils (Ly6G), T-regs (Foxp3), pro-apoptotic Bc1-2-associated X-protein (Bax), genes associated with intestinal barrier integrity and function (occludin (OCLN), tight junction 3 (TJP3), mucin 1 (Muc1), and Muc2) was performed. Briefly, quantitative RT-PCR analysis was carried out as per the manufacturer's instructions (Applied Biosystems) using Taq-Man Gene Expression Assays on a Qiagen Rotor-Gene Q. Data were normalized to controls and compared to five reference targets (Hmbs, B2M, TBP, H2afv, and 18s), which were evaluated for expression stability using GeNorm.
To examine cell proliferation, immunohistochemical analysis of Ki67 (Abcam, #ab16667) was performed. Color was developed with DAB (Vector Laboratories, #SK-4100) for 6 min at RT and counterstained using CAT hematoxylin and Tacha's bluing for 30 secs. Representative images were taken at x20 using Keyence All-in-One fluorescence Microscope BZ-X800. Percentage of Ki67 cells (DAB-positive nuclei) over total cells, relative to area, were quantified using Material Image Processing and Automated Reconstruction (MIPAR) software. Five images were assessed for each mouse (n=3/group). TUNEL staining was carried out using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore, #S7100). Color was developed with DAB (Vector Laboratories, #SK-4100) for 6 min at RT and counterstained using 1% methyl green (R&D systems, #4800-30-18) for 10 min at RT. Representative images were taken at ×20 using Keyence All-in-One fluorescence Microscope BZ-X800. Percentage of apoptotic cells (TUNEL-positive nuclei) over total cells, relative to area, were quantified using Material Image Processing and Automated Reconstruction (MIPAR) software. Five images were assessed for each mouse (n=3/group).
Immunofluorescence of tumors within colon tissues (n=3/group) was performed. Briefly, sections were incubated with rodent decloaker (Biocare Medical, Cat #RD913M) for 30 min at 95° C. for antigen retrieval, 10% goat serum in 5% BSA/TB S for 1 hr at RT to block non-specific binding, and rat anti-mouse FitC-conjugated F4/80 antibody (BioRad; Cat #MCA497F) in 5% BSA/TBS overnight at 4° C. Sections were then washed with TBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml), washed with water, and mounted with a glycerol based mounting media. Representative fluorescent images were taken at χ20 using Echo Revolution microscope.
The distal portion of colon was swiss rolled and fixed overnight in 10% formalin, dehydrated with alcohol, and embedded in paraffin wax. Paraffin sections were stained with hematoxylin and eosin (H&E; Fisher HealthCare) and stained separately for goblet cells with Alcian Blue (AB; Newcomer Supply; Cat #9102A) (n=3/group).
Bone marrow-derived macrophages (BMDMs) were isolated from healthy 10-14-week-old C57BL/6 male mice. Briefly, mice were euthanized by isoflurane overdose. Femurs were cleared of muscle and debris. The epiphyses of the femurs were cut and placed knee-down and spun down in a 1.5 mL tube. Bone marrow was collected and resuspended in complete DMEM (cDMEM; 10% FBS, 1% penicillin/streptomycin) and passed through two 100 μm strainers. Cells were plated on an untreated 10 cm dish in cDMEM and allowed to incubate at 37° C. for 4 hr. Adherent/mature monocytes and dendritic cells were discarded, while floating/immature monocytes were collected. Cells were subjected to RBC lysis and resuspended in bone marrow maintenance media (BMMM; 20% L929-conditioned media in cDMEM). Cells were then plated on surface-treated 6-well plates at a 250 k cells/well seeding density. On day 3, fresh BMMM was added. On day 5, media was aspirated and fresh BMMM was added. For M2 macrophage polarization experiments, 6-day mature macrophages were given either cDMEM+VEH, M2 media (cDMEM with 10 ng/mL IL-4)+VEH, or M2 media+1 μM PA. Cells were incubated for 24 hr. For C26 colon tumor conditioned media (C26 CM) experiments, 6-day mature macrophages were given either serum-free media (SFM)+VEH, 33% C26-conditioned media (C26 CM)+VEH, or 33% C26 CM+1, 2.5, or 5 μM PA. Cells were incubated for 48 hr. After incubation with these different treatments, cells were washed twice with PBS and incubated with 5 mM EDTA PBS at 4° C. for 20 min. Cells were then gently scraped and transferred into flow tubes, spun at 1600 rpm at 4° C. for 5 min, and washed in PBS before antibody staining. Cells were stained and analyzed for macrophages via flow cytometry as described above with the addition of analyzing macrophage phenotypes. From the CD11b+CD68+ macrophage population, macrophage phenotypes were determined based on CD206 and CD11c expression. CD206−CD11c− cells were identified as M0 macrophages; CD206−CD11c+ as M1 macrophages; CD206+CD11c− cells as M2 macrophages; CD206+CD11c+ as M1-M2 transitional macrophages. All cells were measured using a FACS Aria II (BD) and analyzed using FlowJo (BD Biosciences). Three independent experiments were carried out for each in vitro assessment.
BMDMs were isolated as described above. On day 6 of maturation, BMDMs were subjected to 7 concentrations of PA to assess cell death: 0.001, 0.01, 0.1, 1.0, 5.0, 10.0, and 100.0 μM. On day 7 (i.e., 24 hr-post PA treatment), CCK-8 (Dojindo; Cat #CK04) assays were completed per manufacturer's instructions. C26 CRC cells were also subjected to these 7 concentrations of PA for 24 hr to assess cell death using CCK-8. Three independent experiments were carried out.
Statistical Analyses
All data was analyzed using commercial software (GraphPad Software, Prism 7, La Jolla, CA, USA). All in vivo outcomes were analyzed using a paired or unpaired Student's t-test, one-way ANOVA, or two-way ANOVA where appropriate. If a significant difference was observed for a one-way ANOVA, a Tukey's multiple comparisons test was performed to further define group differences. If a significant difference was observed for a two-way ANOVA, a Bonferroni's or Benjamini, Krieger, and Yekutieli multiple comparisons test was performed to further define group differences. Survival curve analysis was conducted by log-rank (Mantel-Cox) test. All gene expression analyses were analyzed using a Student's t-test. Statistical significance was set at p<0.05. Data are presented as mean±standard error of mean (SEM). All figures were generated using GraphPad Prism.
Results
The experimental design is presented in
After 12 weeks of treatment, colonoscopies were performed in a subset of mice (n=3/group) to visualize (
After colonoscopies, mice (n=14-17/group for AOM/DSS groups) were euthanized and colon was excised, imaged, and tumors counted. Representative images of tumors in the distal and proximal colon were taken and tumors were counted using a dissection microscope (
Given the reduction in tumor number and size with PA, its effects on apoptosis and proliferation in colonic tumors (n=3/group) was evaluated. Sections were stained with TUNEL for detection of apoptotic cells (
To investigate PA's effects on intestinal barrier function, colons were stained for H&E and AB (n=3/group), as well as processed for RT-qPCR gene expression analysis of vital markers of gut barrier functions (n=5-7/group). Representative images of H&α-stained distal colons were taken to examine any differences in histopathology and crypt architecture. Colons from CF mice exhibited normal crypt architecture with simple columnar epithelium (
To further examine PA's possible role in strengthening the gut barrier, the expression of colonic genes associated with intestinal barrier integrity including occludin (OCLN) and tight junction protein 3 (TJP3, also known as zonula-occuldin-3) was analyzed, which were both upregulated in PA-treated AOM/DSS mice (p≤0.05;
Previously, the role for macrophages contributing to tumorigenesis in the AOM/DSS CRC model was established. Given that PA reduced colitis through targeting macrophages, whether PA could modify macrophage behavior in CRC was examined. Flow cytometry was performed on colons (with tumors intact) and isolated colonic lamina propria (LP) cells which were gated for non-debris singlets and considered live immune cells with ZombieGreenNeg/Low and CD45+ (
To confirm the cellular findings of PA suppressing CD11b+CD68+ macrophages, RT-qPCR was performed on proximal colons (with tumors intact) of VEH- and PA-treated AOM/DSS mice (n=5-6/group). The expression of genes associated with pan-macrophages were examinedm which were all significantly suppressed in PA-treated AOM/DSS mice, including CD68, mannose receptor C type 1 (Mrc1), and mannose scavenger receptor 1 (MSR-1) compared to that of VEH (p≤0.05;
In addition to macrophages, neutrophils were assessed which are important inflammatory immune cells whose increased abundance is strongly associated with more severe tumorigenesis and worse prognosis in CRC. A small subset of mice (n=2-3/group) was used to grossly observe differences in neutrophils between groups. PA appeared to protect against the increased abundance of neutrophils in the colon (
To confirm that the suppression of macrophages by PA was not confounded by the fewer and smaller tumors in the colon of PA-treated mice, the assessment of macrophages in the colon following tumor removal (n=5-7/group) was repeated. Consistent with the aforementioned findings, the relative abundance of macrophages in colons were significantly increased in VEH-treated AOM/DSS mice compared to CF (p≤0.05;
In addition to macrophages, the effects of PA were assessed on regulatory-T cells (T-regs), important promoters of CRC development, inflammation, and intestinal damage (n=5-7/group). VEH but not PA-treated AOM/DSS mice exhibited significantly increased TCRβ+FoxP3+ T-regs in tumor-removed colons compared to CF (p≤0.05;
In vivo results of PA's suppressive effects on macrophages were confirmed in vitro using bone marrow derived macrophages (BMDMs). Three independent experiments were carried out for each assessment. First, CCK-8 assays were used to evaluate the cell viability of BMDMs after treatment with various concentrations of PA for 24 hr (
Next, in vitro experiments using C26 CRC cells were performed to confirm in vivo results of PA reducing CRC at least in part through its suppressive effects on macrophages. Three independent experiments were carried out for each assessment. CCK-8 assays were performed to evaluate PA's effects on cell viability of C26 CRC cells (
The experimental design for inducing chronic murine colitis is presented in
Since benefits of panaxynol with chronic colitis were observed, the effects of panaxynol on acute colitis and associated mechanisms were assessed. The experimental design for inducing acute colitis is presented in
H&E images revealed general colonic architecture as well as crypt architecture of the distal colon (
EM was leveraged to better visualize mucosal architecture. SEM 700× images showed Control mucosa had a plump, smooth, and velvety appearance documenting healthy in-tact epithelium and brush border (
AB images revealed positively-stained mucin protein (i.e., mucus) in the distal colon (
Macrophages play a key role in the regulation of colitis and intestinal homeostasis. To investigate the impact of panaxynol on macrophage phenotypes in the context of acute colitis, flow cytometry was performed in the colonic LP. DSS alone significantly increased relative LiveCD45+ immune cells (31.5±11.5% vs 78.0±3.3%, p=0.0004) compared to Controls. Controls were then excluded from macrophage and macrophage phenotype analysis, given the apparent changes in parent populations. Relative percentage of macrophage phenotypes in the colonic LP are shown in DSS-treated groups (
T-regs also play a vital role in combatting inflammation and consequently controlling colitis. To investigate whether panaxynol influenced T-regs in acute colitis, flow cytometry was performed in the colonic LP. Relative percentage of T-cell phenotypes in the colonic LP are shown in DSS-treated groups (
These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims.
This application claims the filing benefit of U.S. Provisional Patent Application Ser. No. 63/400,629, filed on Aug. 24, 2022, which is incorporated herein by reference.
This invention was made with government support under R01CA246809 awarded by National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63400629 | Aug 2022 | US |
