Patent Application
20210177932
The embodiments described herein are generally directed to the prevention and treatment of immune deficiency and inflammation.
Diabetes refers to a group of metabolic diseases affecting various interrelated processes, including glucose uptake, energy utilization, immune function, weight control, and inflammation, and is used as a model of other immune-related and inflammation-related disorders.
Type 1 diabetes (T1D) is an autoimmune disease, affecting more than 1.25 million Americans, in which immune cells attack and destroy the insulin-producing β-cells of the pancreas, resulting in an inability to produce insulin and to regulate blood sugar levels.
Type 2 diabetes (T2D) is a metabolic disorder, defined by insulin resistance in peripheral tissues, such as liver, fat, and muscle, and ultimately leads to a loss of pancreatic β-cell function, resulting in severe diabetics and insufficient or no insulin secretion. Most obese subjects develop insulin resistance and some develop diabetes. From a global health and economic perspective (World Health Organization), the incidence of T2D is increasing with a reported 422 million people diagnosed in 2014 worldwide and about 30 million in the U.S. alone. If poorly controlled, T2D can lead to devastating health effects, including impaired wound healing, amputation, renal failure, cardiovascular disease, neuropathy, and retinopathy. Although T2D can be controlled through strict dietary regulation and routine intensive exercise, a large number of patients require clinical therapies.
Current treatments for diabetes primarily focus on lowering blood glucose levels. For example, current treatments for T2D include insulin, metformin, sulphonylureas, thiazolidinediones, α-glucosidase inhibitors, incretin hormone-based therapy, and sodium-dependent glucose co-transporter inhibitors. Each of these treatments can improve glycemia and, in some cases, even delay the onset of diabetes. However, none of these treatments have the disease-modifying characteristic of slowing the progressive decline in insulin sensitivity. The use of insulin as a treatment may also affect the risk of hypoglycemia.
Inflammation is a common mechanism in the pathophysiology of diabetes and obesity, since it increases insulin resistance and islet cell inflammation, leading to defects in β-cell secretion. Obesity-associated adipose tissue inflammation is a major cause of the decreased insulin sensitivity in T2D. Hyperglycemia increases glycation of hemoglobin, formation of reactive oxygen species, and dysregulation of nitric oxide synthase, resulting in increased activation of NF-κB and subsequent increases in pro-inflammatory cytokines (IL-1, IL-6, TNF-α), chemokines such as CCL-2, 58, 10, 12, and adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This leads to activation of endothelial cells, recruitment of inflammatory cells, increased levels of vascular endothelial growth factor (VEGF), and Angiopoietin 2. Targeting inflammation is a new strategy for the management of diabetes mellitus and its associated complications, which ideally will lead to better glycemic control and a decrease in micro- and macro-vascular complications, including cardiovascular disease. Early recognition of the inflammatory status of a predisposed individual may also lead to earlier initiation of therapy.
Recently, correlations between the immune system and diabetes have been established. High blood sugar levels can weaken the patient's immune system. T1D involves autoimmune destruction of the pancreatic β-cells, which produce insulin. Circulating immune cells from T1D subjects also displayed many aspects of a pro-inflammatory state by primed or activated CD11b+ monocytes. It has been proposed that problems with immune function, in response to bacterial and viral infections, may cause insulin deficiency in T1D. Patients with diabetes are known to have infections more often than those with normal glucose metabolism. Thus, diabetes causes a decrease in immune function and an increase in susceptibility to certain infections.
The main goal of therapy should be to stimulate the innate immune system to protect against cold, influenza, and many other infectious diseases and to suppress inflammatory diseases such as T1D. Optimal management should include the early initiation of combination therapy using multiple drugs with different mechanisms of action—particularly ones which target the pathophysiological defects present in T2D.
Accordingly, the inventors have developed an anti-metabolic drug, cyclic histidyl-proline (Cyclo [His-Pro]) plus zinc (Cyclo-Z), which modulates several components of inflammation and may be beneficial for the treatment of a host of other inflammatory/autoimmune diseases, including T1D and T2D.
In the studies described herein, Cyclo-Z treatment invariably increased immune cell levels and activities involved in normally presenting cells (dendritic cells, monocytes, natural killer cells), inducible immune cells (cytotoxic CD8+ and helper CD4+ T-cells, antigen presenting cells), and humoral cells (B-cells), when test subjects—in this case, rats—were young and the immune system still immature (three months old), in both diabetic and normal test subjects. Thus, Cyclo-Z is effective in the stimulation of cell growth and immune system components, which may be helpful in fighting infectious agents earlier than possible in young untreated target subjects (e.g., human patients). In older normal animals, Cyclo-Z treatment modulates levels of several components involved in inflammation (decreasing monocytes, CD8+ T-cells levels) and autoimmune diseases (increasing CD4+ helper T-cells levels), suggesting potential benefits in treatment of autoimmune diseases (such as T1D, allergies, psoriasis, Lupus, and arthritis) in target subjects.
Cyclo-Z treatment is a natural, revolutionary, anti-metabolic and anti-inflammatory therapeutic, which suppresses NF-κB signaling, effectively reversing diabetes, restoring β-cell activity, and improving body weight control without toxicity. It can be used to treat a wide variety of inflammatory and autoimmune-derangement syndromes, including T1D and T2D, and associated conditions.
There are two arms of the mammalian of immune system: innate and adaptive immunity. The innate immune system leukocytes include Natural killer (NK) cells, mast cells, eosinophils, basophils, and the phagocytic cells, including macrophages, neutrophils, and dendritic cells (DC), and function within the immune system by identifying and eliminating pathogens that might cause infection. Macrophages (CD11b+) are professional phagocytes and are highly specialized in the removal of dying or dead cells and cellular debris. NK cells are effector lymphocytes that control several types of tumors and microbial infections by limiting their spread and subsequent tissue damage. DCs are antigen-presenting cells that act as messengers between the innate and the adaptive immune systems. DCs' main function is to process antigen material and present it on the cell surface to the T-cells.
The disclosed study indicates that Cyclo-Z treatment modulated the population of several types of innate immune cells in both diseased and normal test subjects (rats). Cyclo-Z suppressed macrophage levels in STZ-induced diabetic test subjects, which exhibit high levels of macrophages in comparison to healthy subjects. However, Cyclo-Z treatment slightly increased macrophage levels in normal test subjects. DC levels were elevated in Cyclo-Z-treated diabetic test subjects, compared to control diabetic test subjects, and slightly elevated in normal test subjects. Cyclo-Z treatment did not affect CD161+ NK cell populations in mature normal test subjects. Although, at the initial stage of streptozotocin (STZ) injection, Cyclo-Z treatment increased levels of both CD161A+ NK and CD11b+ macrophage cells, at later stages of Cyclo-Z treatment, when invading antigen levels declined and inflammation is suppressed, NK and macrophage levels were reduced in both diseased and healthy test subjects. This role in immunity and chronic inflammation is important, as the early stages of inflammation are dominated by neutrophils, which are ingested by macrophages.
Adaptive immunity serves to protect the body from an infectious disease agent. It is mediated by B-lymphocytes and T-lymphocytes following exposure to a specific antigen, and characterized by immunological memory. There are two types of adaptive immune responses: humoral immunity, mediated by antibodies that are produced by B-lymphocytes; and cell-mediated immunity, facilitated by T-lymphocytes. CD4+ T-helper cells are white blood cells that are an essential part of the adaptive immune system. One of their main roles is to send signals to other types of immune cells, including CD8+ killer cells, which then destroy and inactivate the infectious particles.
Levels of both CD4+ and CD8+ T-cells did not change in young test subjects. However, when aged normal test subjects were treated with Cyclo-Z, CD4+ increased and CD8+ decreased. CD3+ T-cell co-receptor helps to activate both the cytotoxic T-Cell (CD8+ naive T-cells) and also T helper cells (CD4+ naive T-cells). Cyclo-Z treatment increased CD3+ cells in diabetic test subjects but decreased CD3+ cells in normal test subjects. CD86 and CD80 are proteins expressed on antigen-presenting (AP) cells that provide co-stimulatory signals necessary for T-cell activation and survival. However, CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting DCs and B-cells. Similarly to CD3, CD4, and CD8 T-cells, levels of CD86+ and CD80+ T-cells increased in Cyclo-Z-treated STZ-diabetic test subjects, while populations were suppressed in normal test subjects. The regulatory T-cell (Treg) is a subpopulation of T-cells which modulate the immune system, maintain tolerance to self-antigens, and preventing autoimmune reactions and disease. Tregs exhibit CD4+, FOXP3+, and CD25+ biomarkers and are thought to be derived from the same lineage as naïve CD4 cells. Because effector T-cells also express CD4 and CD25, Tregs are very difficult to effectively discern from effector CD4+, making them difficult to study.
The described data show a subset of cells express higher levels of CD25+ in STZ-induced diabetic test subjects than in normal test subjects, and Cyclo-Z treatment further increased this CD25+ population. The role of CD45RA on human B-cell function: anti-CD45RA antibody inhibits the activation of resting B-cells and antibody production of activated B-cells independently in humans. CD45RA+ cell levels are higher in diabetic test subjects than in normal test subjects. Cyclo-Z treatment further increased this CD45RA+ population in young STZ-induced diabetic test subjects, but lowered CD45RA+ levels in aged test subjects. Thus, Cyclo-Z treatment stimulates the adapted immune system CD4+ CD25+ T-cell (T-cell mediated) and the CD45RA+ (humoral) B-cell population of the adaptive immune system. The combined data demonstrate that Cyclo-Z treatment can regulate inflammation and different cell populations in both the innate and adaptive immune systems of diseased and healthy subjects depending on the age and level of the disease.
In an embodiment, a pharmaceutical composition to treat immune disorder and inflammation in mammals is disclosed. The composition comprises: zinc salt comprising zinc cation and anion; purified cyclic histidyl-proline (CHP); and a pharmaceutically-acceptable excipient. The zinc salt may comprise, for example, zinc chloride, zinc oxide, or zinc carbonate.
In another embodiment, the composition comprises a capsule that comprises: a zinc chelating agent; cyclic histidyl-proline (CHP); and one or more excipients. The capsule may be a hard or soft gel capsule. Each capsule may comprise 10-150 (e.g., 10-100 mg) of zinc and/or 3-50 mg (e.g., 3-30 mg) of CHP, depending on the age, gender, and/or physiological condition of the patient.
In another embodiment, a method of treating a disease of a patient is disclosed. The method comprises administering cyclic histidyl-proline plus zinc (Cyclo-Z) to the patient. The Cyclo-Z may be orally administered, for example, via a capsule taken 1-4 times per day between waking up and breakfast, between breakfast and lunch, between lunch and dinner, and/or between dinner and bedtime (e.g., 4 times per day consisting of once during each of these time periods, 2 times per day consisting of once between waking up and breakfast and once between breakfast and dinner or bedtime, 1 time per day at bedtime, etc.). The Cyclo-Z may comprise 5 mg per liter of cyclic histidyl-proline and 20 mg per liter of zinc. The disease(s), treated using the Cyclo-Z, may include an immune-disorder or inflammatory disease, including, for example, a cold, influenza, a bacterial infection, an allergy, type-one diabetes, and/or type-two diabetes.
The details of the present invention may be gleaned in part by study of the accompanying drawings, in which like reference numerals refer to like parts, and in which:
Embodiments of compositions and methods for treating and preventing immune deficiency and inflammation are described. After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, although various embodiments of the present invention will be described herein, it is understood that these embodiments are presented by way of example and illustration only, and not limitation. As such, this detailed description of various embodiments should not be construed to limit the scope or breadth of the present invention as set forth in the appended claims.
Diabetes refers to a group of metabolic diseases affecting various interrelated processes including glucose uptake, energy utilization, immune function, weight control, and inflammation, and is characterized by excessive amounts of glucose in the blood. This condition is triggered by the body's defect(s) in insulin production, insulin action, or both. Recently, correlations between the immune system and diabetes have been established. High blood sugar levels can weaken the patient's immune system. Viardot et al. reported that energy restriction after gastric banding attenuates activation of circulating immune cells of both the innate and adaptive immune system. Defects in the deletion of autoactive T-cells and in the function of T regulatory cells contribute to the autoimmune response characteristic of T1D. In T1D, autoimmune destruction of the pancreatic β-cells, which produce insulin, results in insulin deficiency directly inducing the diabetic condition. Circulating immune cells from T1D subjects also displayed many aspects of a pro-inflammatory state, as indicated by primed or activated monocyte CD11b. In T2D, it has been proposed that problems with immune function in response to bacterial and viral infections may cause insulin resistance and induce T2D. Patients with diabetes are known to have infections more often than those with normal glucose metabolism. Thus, it is not uncommon for inflammatory diseases, such as diabetes, to cause a decrease in immune function and an increase in susceptibility to certain infections.
Zinc plays an important role in the immune system—particularly in the development and normal functions of neutrophils and NK cells. Zinc levels should be taken into account whenever alterations of immune functions are observed in vitro or in vivo. Zinc-deficient patients have increased susceptibility to a variety of pathogens, and zinc deficiency may be responsible for decreased cell-mediated immune functions. The specific interaction of zinc with immunologically important proteins, signal transduction components, and membrane functions has been summarized in “The significance of zinc for leukocyte biology,” by Wellinghausen et al., J. Leukoc. Biol. 64:571-577 (1998), which is hereby incorporated herein by reference as if set forth in full. Zinc also has a profound effect on immunosuppression and inflammation, leading to the therapeutic use of it as a treatment for many immune system disorders. Zinc has a crucial effect for development and normal functions of neutrophils and NK cells, as discussed in “Zinc and the immune system,” by Shankar et al., Proc. Nutr. Soc. 59:541-552 (2000), which is hereby incorporated herein by reference as if set forth in full. In zinc-deficient patients, B-lymphocyte development and antibody production—particularly immunoglobulin G production—is compromised. Macrophage functions are also adversely affected by zinc deficiency, which can result in abnormal intracellular killing, cytokine production, and phagocytosis. In human studies in India, zinc supplementation has been shown to improve cellular immune status. Thus, improvement of zinc nutriture in children or zinc-deficient subjects may provide an important preventive intervention for high infectious disease morbidity and associated mortality in developing countries.
Cyclo (His-Pro) (CHP) is a naturally occurring cyclic dipeptide that is a metabolite of thyrotropin-releasing hormone (TRH) and functions as a transporter of zinc into cells. CHP is proven to have antibacterial and antifungal activities and can inhibit the growth of Saccharomyces cerevisae by inhibiting chitinase, as discussed in “Antifungal peptides in marine invertebrates,” by Fusetani, Invertebrate Survival J. 7:53-66 (2010), and “Structure-based exploration of cyclic dipeptide chitinase inhibitors,” by Houston et al., J. Med. Chem. 47:5713-5720 (2004), respectively, which are each hereby incorporated herein by reference as if set forth in full. It has a protective and therapeutic effect on STZ-induced cytotoxicity and apoptosis, and exerts anti-inflammatory effects by suppressing pro-inflammatory NF-κB signaling via Nrf2-mediated hemeoxygenase-1 activation, as discussed in “Cyclo (his-pro) exerts anti-inflammatory effects by modulating NF-κB and Nrf2 signaling,” by Minelli et al., Int. J. Biochem. Cell Biol. 44:525-535 (2012), which is hereby incorporated herein by reference as if set forth in full. CHP has also been shown to protect against glial inflammation and other neuro-inflammatory diseases, including lipopolysaccharide neurotoxicity, as discussed in “Neuroinflammation and endoplasmic reticulum stress are coregulated by Cyclo (His-Pro) to prevent LPS neurotoxicity,” by Bellezza et al., Int. J. Biochem. Cell Biol. 51:159-169 (2014), which is hereby incorporated herein by reference as if set forth in full. STZ-induced cytotoxicity and apoptosis was prevented in rat insulinoma cells (RINm5F), as discussed in “Protective effect of Cyclo (his-pro) on steptozotocin-induced cytotoxicity and apoptosis in vitro,” by Koo et al., J. Mol. Biotechnol. 21:218-227 (2011), which is hereby incorporated herein by reference as if set forth in full. CHP also exhibited anti-diabetic effects on STZ-induced diabetic mice, as discussed in “Synergistic anti-diabetic activities of zinc, cyclo (his-pro) and arachidonic acid,” by Song et al., Metabolism 50:53-59 (2001), which is hereby incorporated herein by reference as if set forth in full. Oral glucose tolerance test (OGTT) values significantly decreased, and plasma glucose levels dropped 60%, as compared to the STZ-induced diabetic control group without CHP treatment. Thus, CHP exhibits anti-diabetic and anti-toxicity effects on STZ-induced diabetic rats and cells.
Oral administration of Cyclo-Z (CHP+zinc) has been demonstrated to be effective in improving—and effectively reversing—diabetes in STZ-induced T1D rats, in T2D Goto-Kakizaki (G-K) rats, and in T2D ob/ob mice. In addition, Cyclo-Z improved body weight control in aged T2D G-K rats and obese and overweight Sprague-Dawley rats. All of these studies indicate that the combination of CHP+zinc in the Cyclo-Z formulation is much more effective than zinc or CHP alone.
Based on studies showing a link between zinc and immune functions, Cyclo-Z treatment may improve basic immune system parameters in both STZ-induced diabetic subjects and possibly in normal subjects. Thus, the inventors examined the effect of Cyclo-Z on the levels and activation of several major immune system cell components in STZ-induced diabetic (as a model of inflammatory and immune-deficient disease) and in normal control test subjects (in this case, rats).
A study was conducted on sixty Fisher 344 female and male rats, aged five to six weeks. Diabetes was induced by subcutaneous injection of streptozotocin (STZ, 30 mg/kg, Sigma-Aldrich, St. Louis, Mo.) in 0.1 M sodium citrate/citric acid buffer (pH 4.5). Rats were housed individually in separate cages and fed a low zinc (6 ppm) diet (Newco Distributors, Inc., Rancho Cucamonga, Calif.). Venous blood was collected from the tail for blood glucose determination starting one week after STZ injection at the initiation of CHP-plus-zinc treatment. Blood glucose levels were analyzed using an AlphaTRAK 2 portable glucose analyzer (Abbott Laboratories, Chicago, Ill.). Diabetes induction was considered to be successful when glucose levels were greater than 300 mg/dL. Rats were then divided into four groups of matched blood glucose levels: Group 1 diabetic rats receiving drinking water containing the Cyclo-Z formulation [CHP (5 mg/L) plus zinc (83 mg/L)]; Group 2 diabetic rats receiving normal drinking water; Group 3 normal rats receiving drinking water containing the Cyclo-Z formulation; and Group 4 normal rats receiving normal drinking water. Furthermore, urine glucose determinations were made by placing each test subject in a metabolic chamber, collecting their excreted urine, and measuring urine glucose levels (OneTouch Ultra 2 glucose meter, LifeScan, Inc. New Brunswick, N.J.). Weekly blood and urine glucose measurements, body weight, food, and water intake were measured throughout the timeframe of treatment. Once a month and under sterile conditions, peripheral blood was collected from the saphenous vein into heparin-rinsed tubes for FACS analysis. At the conclusion of the experiment (six-months old, four months of treatment), the animals were terminated with CO2 inhalation, and the spleen, and blood collected by cardiac puncture for analysis by FACS.
Two milliliters of red blood cell lysing solution was added to each tube containing up to 200 μl of whole blood and incubated at room temperature, protected from light, for fifteen minutes. Cell suspensions were washed, centrifuged, re-suspended, and incubated with specific antibodies. Samples were then analyzed on a Becton Dickinson (BD) FACSJazz™ flow cytometer with BD FACS™ sorter software. In addition, after four months of treatment, the spleens of each test subject were excised and processes by rubbing on 40 μm sterile filters to generate single cells, and then stained for FACS analysis. Antigen staining was carried out using conjugated primary antibodies at room temperature for twenty minutes. Fluorescently conjugated antibodies were purchased from eBiosciences (San Diego, Calif.) or BD Pharmingen (San Jose, Calif.) and are outlined in Table 1 below:
Cells were then washed in phosphate buffered saline (PBS) at 350×g for five minutes and re-suspended in 400 μL of PBS. For dendritic cell analysis only, the cell staining, washing, and suspension protocol was repeated with a secondary conjugated antibody, since no primary conjugated antibody was available. For each staining reaction, 20-50,000 FACS events were acquired. Unlabeled cells or cells labelled with isotype control antibodies were used as negative controls.
Animals were treated with Cyclo-Z beginning at the age of two months, and blood samples analyzed by FACS at one, two, and three months of treatment corresponding to three, four, and five months of age, respectively. Over a time course, blood was pooled from like-treated subjects and then analyzed as one sample for cell counts at each of these time points, (
5.1. T-Cells CD3
The Cluster of Differentiation (CD) 3 (CD3) T-cell co-receptor binds non-covalently to the T-cell receptor during early maturation of T-cells and results in activation of the T-lymphocytes, as discussed in “CD3 ITAMs count!,” by Malison, Nature Immunology 9:583-584 (2008), “Organization of proximal signal initiation at the TCR: CD3 complex,” by Guy et al., Immunology Reviews 232:7-21 (2009), and “T-Cell activation,” by Smith-Garvin et al., Annual Rev. Immunology 27:591-619 (2009), each of which is hereby incorporated herein by reference as if set forth in full. Defects in CD3 gene protein expression result in Severe Combined Immune Deficiency Disorder, characterized by severe defects in T-cell production or function. Thus, CD3 is an excellent marker for total T-cells.
As shown in
In normal test subjects, at time points other than the very young one-month-old test subjects, Cyclo-Z treatment significantly suppressed T-cell production. However, in STZ-treated test subjects, T-cells increased in the blood, but decreased in the spleen. These data indicate that Cyclo-Z treatment can modulate T-cell production depending on the physiological state to the subject.
Further analysis was conducted to evaluate the subset and activation state of the T-lymphocyte population.
5.2. T-Cells Subset CD4
CD4 is a glycoprotein found on the surface of T helper cells, monocytes, macrophages, and dendritic cells, as discussed in “CD4(+) T-cell subsets in intestinal inflammation,” by Shale et al., Immunol. Rev. 252:164-182 (2013), which is hereby incorporated herein by reference as if set forth in full. It is a co-receptor that assists the T-cell receptor (TCR) in communicating with an antigen-presenting cell. CD4 transfers the signal from TCR to the tyrosine kinase Lck, which initiates a signaling cascade, resulting in the production of various types of T-helper cells. To analyze the various T-lymphocyte sub-populations, FACS was used to first identify the CD3+ T-lymphocyte population. Then, the percentage of CD4+ and CD8+ T-cell subsets within that population were determined. The level of CD3+ CD4+ T-lymphocytes was approximately 60% over the entire time course (three- to five-months old), regardless of the health of the test subjects (
T helper cells (Th cells) are a type of T-cell that plays an important role in the immune system, and, particularly, in the adaptive immune system. Th cells help the activity of other immune cells by releasing T-cell cytokines, and help suppress or regulate immune responses. They are essential in B-cell antibody class switching, in the activation and growth of cytotoxic T-cells, and in maximizing bactericidal activity of phagocytes, such as macrophages. In general, Cyclo-Z treatment increased blood T-cells CD4 in normal test subjects. However, in STZ-treated test subjects, Cyclo-Z suppressed CD4 expression. These data also indicate that Cyclo-Z treatment modulates CD4 expression depending on the physiological conditions of the subject.
5.3. T-Cells Subset CD8
The CD8 antigen is a glycoprotein predominantly expressed on cytotoxic T-cells and can differentiate into two effector phenotypes, Tc1 and Tc2, secreting different cytokine patterns and having diverse functions including cytotoxicity for infected cells or an immune system suppressive effect as determined by the cytokine expression pattern, as discussed in “Functions of CD8 T-cell subsets secreting different cytokine patterns,” by Mosmann et al., Semin. Immunol. 9:87-92 (1997), which is hereby incorporated herein by reference as if set forth in full. Adoptive transfer of CD8+ T-cells with these phenotypes may have a role in the therapy of infectious disease and/or cancer, as discussed in “Tc17 CD8 T cells: functional plasticity and subset diversity,” by Yen et al., J. Immunol. 183:7161-7168 (2009), which is hereby incorporated herein by reference as if set forth in full.
Similarly to the CD4+ subset, the percentage of CD3+ CD8+ T-cells was relatively constant at 35-40% regardless of age or health. Over the time course, the tendency was for Cyclo-Z treatment to slightly increase levels of CD3+ CD8+ T-cells in diabetic test subjects and to decrease levels in non-diabetic test subjects (
CD8+ T-cells (or killer T-cells) are a subset of T lymphocytes (a type of white blood cell) that attack cancer cells, infected cells (particularly, cells infected with viruses), or cells that are damaged in other ways. Normal test subjects, treated with Cyclo-Z, have significantly reduced numbers of CD8 cells, whereas STZ-treated diabetic test subjects, treated with Cyclo-Z, have increased numbers of CD8 cells. These data indicate that CD8 cell levels are elevated with Cyclo-Z treatment in diseased subjects, but not in normal subjects.
Expression levels of RT1B and CD25 were also analyzed to look at activation states of the T-lymphocytes. To analyze activated populations, FACS was used to first identify the CD3+ T-lymphocyte population. Then, the percentage of RT1B+ or CD25+ cells within that population was determined.
5.4. T-Cells Activated RT1B
RT1B is a membrane glycoprotein that is used as a marker of thymocyte-activated immune stimulation. It is present on some T- and B-lymphocytes, dendritic cells, some macrophages, and certain epithelial cells, as discussed in “Identification of Ia glycoproteins in rat thymus and purification from rat spleen,” by McMaster et al., Eur. J. Immunol. 9:426-433 (1979), and “Two-domain MEW class II molecules from stable complexes with myelin basic protein 69-89 peptide that detect and inhibit rat encephalitogenic T-cells and treat experimental autoimmune encephalomyelitis,” by Burrows et al., J. Immunol. 161:5987-5996 (1998), each of which is hereby incorporated herein by reference as if set forth in full.
CD3+ RT1B+ T-cell activation increased with age (three to four months) over the time course in both normal non-diabetic and diabetic blood (
RT1B is a membrane glycoprotein that is used as a marker of thymocyte-activated immune stimulation. It is present on some T-lymphocytes and B-lymphocytes, dendritic cells, some macrophages, and certain epithelial cells. In normal cells from healthy subjects, RT1B-activated T-cell populations did not increase in the blood, but were significantly increased in the spleen. However, in the STZ-induced diabetic test subjects, RT1B-activated T-cell populations significantly increased in young (one- to two-months old) test subjects, but decreased in the blood and spleens of older (three- to four-months old) test subjects. These data indicate that, in healthy subjects, Cyclo-Z treatment increases the levels of activated immune cells in the spleen, but does not release the activated immune cells to the blood stream prior to injury (chemical or infectious organisms). In the diabetic test subjects, STZ-induced injury of the pancreas may have caused the release of immune cells from the pancreas into the blood, as demonstrated by the increased levels of RT1B+ T-cells in the blood and the decreased levels of RT1B+ in the spleens of the STZ-induced diabetic test subjects relative to the normal test subjects. Cyclo-Z treatment helps to normalize the levels of RT1B+ cell levels in diabetic subjects—increasing levels in the spleen and decreasing levels in the blood.
5.5. T-Cells Activated CD25
CD25 interacts with CD122 to form a heterodimer and serves as a high-affinity receptor of IL-2. It is a type I transmembrane protein present on activated T-cells, activated B-cells, some thymocytes, myeloid precursors, and oligodendrocytes. In mice, it has been used as a marker of CD4+ FoxP3+ helper T-cells (also known as regulatory Tregs) and has been identified on the majority of resting memory T-cells in humans, as discussed in “Defining a functionally distinct subset of human memory CD4+ T-cells that are CD25POS and FOXP3NEG,” by Triplett et al., Eur. K. Immunol. 42:1893-1905 (2012), which is hereby incorporated herein by reference as if set forth in full. Based on in vitro studies, IL-2 is considered a key growth and death factor for antigen-activated T-lymphocytes, as discussed in “The main function of IL-2 is to promote the development of T Regulatory cells,” by Malek, J. Leukoc. Biol. 74:961-965 (2003), which is hereby incorporated herein by reference as if set forth in full.
The results for relative CD3+ CD25+ T-lymphocytes levels were very similar to that of activated CD3+ RT1B+ T-cells. In STZ-diabetic rats, CD3+ CD25+ cell levels were increased at the age of three and four months, compared to controls, when treated with Cyclo-Z (one and two months of treatment, respectively), but decreased at the age of five months (three months of treatment) and were less than the non-treated control group (
Similarly to the CD3+ RT1B+ cell levels in the older (six-months old) diabetic test subjects' blood, STZ-untreated diabetic test subjects had significantly (p=0.0182) increased CD3+ CD25+ T-cell levels compared to untreated normal test subjects (
The results for the CD3+ RT1B+ and CD3+ CD25+ cell numbers indicate that both diabetic condition and age affect T-cell activation state in the blood and spleen. Cyclo-Z treatment is effective in stimulating T-cell activation in the blood of both younger diabetic and normal test subjects and in the blood and spleen of older normal test subjects.
Regulatory T-cells come in many forms. The most well-studied regulatory T-cells are those that express CD4, CD25, and FOXP3 (CD4+ CD25+ regulatory T-cells). Regulatory T-cells are involved in shutting down the immune responses after they have successfully eliminated invading organisms, and in preventing autoimmunity. In general, Cyclo-Z treatment in STZ-treated test subjects increased CD25+ cell levels in the blood, while Cyclo-Z treatment in normal test subjects decreased CD25+ cell levels in the blood. However, in the spleens of normal test subjects, Cyclo-Z significantly increased CD25+ cells, whereas, in the spleens of STZ-treated test subjects, Cyclo-Z decreased CD25+ T-cells. These data indicate that Cyclo-Z treatment may enhance regulatory T-cell activities in blood in cases of chemical injury or invasion of infectious organisms.
5.6. Antigen-Presenting Cells CD86
Cluster of Differentiation 86 (also known as CD86 and B7-2) is a protein expressed on AP cells that provides co-stimulatory signals necessary for T-cell activation and survival. The CD86 gene encodes a type I membrane protein that is a member of the immunoglobulin superfamily, as discussed in “Molecular cloning and expression of early T-cell costimulatory molecule-1 and its characteristic as B7-2 molecule,” by Chen et al., J. Immunol. 152:4929-4936 (1994), which is hereby incorporated herein by reference as if set forth in full. CD86 works in tandem with CD80 to prime T-cells.
CD86+ AP cell numbers were stimulated by Cyclo-Z treatment at all time points in diabetic test subjects (
CD86 is a protein expressed on AP cells that provides costimulatory signals necessary for T-cell activation and survival. In normal test subjects, Cyclo-Z treatment reduced CD86 expression, other than in the first month, both in the blood and spleen. In contrast, in STZ-treated diabetic test subjects, Cyclo-Z treatment increased CD86 expression in the blood, but depressed CD86 expression in the spleen. Thus, Cyclo-Z may stimulate the release of CD86+ AP cells from the spleen into the blood to protect against invasive foreign organisms.
5.7. Antigen-Presenting Cells CD80
Cluster of Differentiation 80 (CD80) is a protein found on activated B-cells and monocytes that provides a co-stimulatory signal necessary for T-cell activation and survival, as discussed in “Both extracellular immunogoblin-like domains of CD80 contain residues critical for binding T-cell surface receptors CTLA-4 and CD28,” by Peach et al., J. Biol. Chem. 270:21181-21187 (1995), which is hereby incorporated herein by reference as if set forth in full. CD80 works in tandem with CD86 to prime T-cells.
Similarly to CD86 data, CD80+ AP cell numbers were stimulated by Cyclo-Z treatment at all time points in diabetic test subjects and in the earliest time point (three-months old, one month of treatment) in normal test subjects, but decreased with Cyclo-Z treatment in normal test subjects at the latter two time points (
These data demonstrate that Cyclo-Z can regulate AP cell activity. These results, similarly to the CD3 data, suggest that when the immune system is not fully developed (young subjects), Cyclo-Z is effective in stimulating immunity in both diabetic and normal subjects. However, in fully developed subjects, Cyclo-Z may modulate and lead to lower normal adequate levels in healthy subjects while continuing to stimulate the immune system in fully developed diabetic subjects.
Although CD80 and CD86 are both expressed on monocytes, they seem to have different functions, since they are expressed differentially on distinct subsets of monocytes. Freshly isolated monocytes express CD86, but are CD80-negative. CD80 expression is weakly induced after six to eight hours of in vitro culture and is enhanced by stimulation with antigens. In studies, both CD86 and CD80 showed similar expression patterns and modulation, with Cyclo-Z treatment, in both normal and diabetic test subjects.
5.8. B-Cells CD45RA
CD45 antigen is involved in the activation, proliferation, and differentiation of human T- and B-lymphocytes, as discussed in “The role of CD45RA on human B-Cell function: anti-CD45RA antibody (anti-2H4) inhibits the activation of resting B cells and antibody production of activated B cells independently in human,” by Morikawa et al., Scand. J. Immunol. 34:273-283 (1991), which is hereby incorporated herein by reference as if set forth in full. It is a member of the Protein Tyrosine Phosphatase (PTP) family and found on all types of hematopoietic stem cells in various isoforms. These different CD45 isoforms can be used to distinguish between naïve and memory T-cells, as discussed in “Human B cells express a CD45 isoform that is similar to murine B220 and is down regulated with acquisition of the memory B cell marker CD27,” by Blessing et al., Cytometry B. Clin. Cytom. 51:1-8 (2003), and “Dual personality of memory T cells,” by Mackay, Nature 401-659-660 (1999), each of which is hereby incorporated herein by reference as if set forth in full. CD45RA is a high molecular weight form of CD45 found only on B-lymphocytes. It is present on the surface of more than 95% of B-lymphocytes in purified tonsillar B-cell preparations, as discussed in “The role of CD45 in the activation, proliferation and differentiation of human lymphocytes,” by Morikawa et al., Int. J. Hematol. 54:495-504 (1991), which is hereby incorporated herein by reference as if set forth in full. Anti-CD45RA antibody also had a suppressive effect on the early phase of B-cell differentiation and is directly involved in the regulatory function of B-cells, as discussed in “The role of CD45RA on human B-cell function: Anti-CD45RA antibody (anti-2H4) inhibits the activation of resting B-cells and antibody production of activated B-cells independently in humans,” by Morikawa et al., Scand. J. Immunol. 34:273-283 (1991), which is hereby incorporated herein by reference as if set forth in full. Two antibodies were used for FACS analysis of B-cell CD45RA protein expression, conjugated with either fluorescein isothiocyanate (FITC) or Phycoerythrin (PE). Similar results were obtained with both of the antibodies.
As shown in
CD45RA is expressed on B-cells, as well as the effector cells in both CD4 and CD8 T-cell subsets. In normal test subjects, Cyclo-Z treatment suppressed CD45RA+ B-cells, other than in the first month, but very significantly suppressed B-cell levels in the spleen. On the other hand, Cyclo-Z treatment generally increased CD45RA+ B-cells in STZ-induced diabetic test subjects. These results suggest that Cyclo-Z treatment stimulates B-cell activation, which would aid in the removal of foreign bodies.
5.9. Natural Killer CD
NK cells are large granular lymphocytes, producing inflammatory cytokines, which kill malignant, infected, or abnormal cells, as discussed in “Developmental pathways that generate natural killer cell diversity in mice and humans,” by Huntington et al., Nature Rev. Immunol. 7:703-714 (2007), which is hereby incorporated herein by reference as if set forth in full. NK activation is tightly controlled by cell surface receptors receiving both stimulatory and inhibitory signals. CD161A is a 60 kDa protein expressed on all NK cells and a small subset of T-lymphocytes. Two populations of CD161A+ cells were labelled with the CD161A antibody. The pattern of CD161A+ cell expression was very similar within both populations.
5.9.1. NK Cells Large Population
In STZ-induced diabetic test subjects, the CD161A+ population increased with Cyclo-Z treatment at the first two time points (three- and four-months old, one and two months of treatment), but was unchanged or decreased at the five-months-old time point (
Thus, these data suggest that Cyclo-Z treatment also has a modulatory effect on CD161A+ NK numbers by increasing the immune reaction in young diabetic or normal subjects and possibly acting to modulate inflammatory reactions in older non-diabetic subjects.
5.9.2. NK Cells Small Population
The second smaller CD161A+ population acted in a similar manner as the larger cell population in response to Cyclo-Z treatment in both diabetic and normal subjects (
The NK CD161A+ small population also showed similar effects as NK CD161A+ large population results shown in
CD161 (human NKR-P1A) is a major phenotypic marker in the NK gene complex and is thought to be involved in the regulation of NK and NKT cell function. Cyclo-Z treatment in normal subjects generally suppresses CD161A expression in both the blood and spleen NK cells. Mature STZ-treated test subjects expressed high levels of CD161, and Cyclo-Z treatment reduced CD161A expression in the NK cells. These data suggest that Cyclo-Z can down-regulate NK function of the immune system depending on the body need of immune stimulation.
5.10. Monocyte CD11b
CD11b is expressed on many leukocyte cell types, including macrophages, monocytes, granulocytes, and NK cells. It is involved in the innate immune system and mediates inflammation by controlling leukocyte cell adhesion and migration. Circulating immune cells from T1D subjects display many aspects of a pro-inflammatory state, indicated by activated monocytes, as discussed in “Increased expression of monocytes CD11b (Mac-1) in overweight recent-onset type 1 diabetic children,” by Cifarelli et al., Rev. Diabet. Stud. 4:113-120 (2007), which is hereby incorporated herein by reference as if set forth in full. It has also been observed that CD11b+ monocytes patrol the luminal surface of the endothelium within tissues, as if they are acting as guards for tissue damage or pathogen invasion, and has been suggested that, during infection, the ultimate tissue response is the result of an orchestrated interplay between the various monocytes subsets that are induced to leave the blood and the differentiative plasticity of macrophages in tissue, as discussed in “Monocyte subpopulation and their differentiation patterns during infection,” by Strauss-Ayali et al., J. Leukoc. Biol. 82:244-252 (2007), which is hereby incorporated herein by reference as if set forth in full.
Monocyte CD11b+ cell numbers increased after one month of Cyclo-Z treatment in both diabetic and normal subjects (
The CD11 protein is a heterodimer complex that consists of CD11b and CD18. CD11 is involved in numerous adhesion-related associations between immune cells (monocytes, macrophages, natural killer (NK) cells, and granulocytes). CD11 also regulates the uptake of complement-coated particles within cells. In normal test subjects, Cyclo-Z treatment exhibited no effects on blood CD11b+ monocyte cell levels. However, Cyclo-Z treatment in STZ-induced diabetic test subjects showed suppressed CD11b+ monocyte levels. This demonstrates that Cyclo-Z treatment is effective in suppressing inflammation in the mammalian immune system.
5.11. Dendritic Cells
Although B- and T-lymphocytes are the mediators of immunity, dendritic cells (DC) control their function, as discussed in “Dendritic cells and the control of immunity,” by Banchereau et al., Nature 392:245-262 (1998), which is hereby incorporated herein by reference as if set forth in full. DCs are antigen-presenting cells of the mammalian innate immune system, as discussed in “Dendritic cells and the immunity/tolerance decision,” by Usharauli, Med. Hypo. 64:112-113 (2005), which is hereby incorporated herein by reference as if set forth in full. DCs in the periphery capture and process antigens, express lymphocyte co-stimulator molecules, migrate to lymphoid organs, and secrete cytokines to initiate immune responses. Their main function is to act as messengers between the innate and adaptive immune system through interaction with T-cells. Thus, DCs can be a powerful tool for manipulation of the immune system.
Both STZ-induced T1D and normal subjects had increased levels of DCs at five months, in comparison to three months of age (
Diabetic subjects treated for four months (six-months old) had slightly increased (not significantly) levels of dendritic cells in the blood. Whereas, Cyclo-Z treatment did not affect levels in normal subjects (
DCs are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T-cells of the immune system. Thus, they act as messengers between the innate and the adaptive immune systems. In normal test subjects, DC levels are suppressed (or no change) in the blood, but increased in the spleen. However, in STZ-induced diabetic test subjects, DC levels increased with Cyclo-Z treatment compared to controls, but decreased in the spleen. These data suggest that Cyclo-Z treatment can modulate both the innate and adaptive immune system by regulating DC levels.
The immune system is a sophisticated collection of tissues that generally includes the blood and lymphatic system. The immune system is typically divided into two functional categories: innate and adaptive. Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of a foreign antigen's appearance in the body. Adaptive immunity is a specialized defense mechanism to respond to specific health threats (e.g., virus, cancer, etc.).
During infancy, there is a physiological delay in production of immune factors in mammals, as discussed in “Evolution of immunologic functions of the mammary gland and the postnatal development of immunity,” by Goldman et al., Pediatr. Res. 43:155-162 (1998), which is hereby incorporated herein by reference as if set forth in full. At birth, the mammalian immune system is immature, and immunity is conferred from the mother's immune system through breast milk. The infant immune system develops and matures after weaning from the mother. The results, disclosed herein, demonstrate that Cyclo-Z treatment stimulates production of several types of innate and adaptive immune system components at an early physiological stage where the subjects are normally still developing the immune system.
The innate immune response defense mechanisms include physical barriers, such as skin, chemicals in the blood, and immune system cells including macrophages, DCs, and neutrophils that attack foreign bodies that invade the body. Critical to the innate immune system is the DCs and NK cells. Cyclo-Z treatment invariably increased DC populations in the blood at the age of three months (one month of treatment), regardless of the overall health (disease or normal) of the subjects, before the subjects had a fully developed immune system (
This data supports the hypothesis that Cyclo-Z treatment modulates the innate immune system in mammals. NKs, a type of cytotoxic lymphocyte, respond and kill viruses and infected cells around three days after infection. They do not require activation in order to kill cells that are missing “self” markers of major histocompatibility complex (MHC) class 1(44). NKs are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus, where they then enter into the circulation.
Adaptive immunity, the second arm of the mammalian immune system, refers to antigen-specific immune responses. The adaptive immune response is more complex than the innate response. An antigen must first be recognized and processed. Once an antigen has been recognized, the adaptive immune system creates an army of immune cells (T- or B-cells) specifically designed to attack that antigen. T-cells develop in the thymus, and acquire specific receptors that can recognize a foreign virus or cell. There are two basic types of T-cells: T-helper cells that help regulate the immune system; and cytotoxic I-cells that destroy infected cells, cancer cells, or foreign cells. During this process, B-cells are also activated to produce antibodies to target abnormal or invading cells. Specific antibodies bind to an invading foreign cell, rendering it harmless. Then, the complex is delivered to the lymph system for destruction. A critical feature of adaptive immunity is the development of immunologic memory or the ability of the system to learn or record its experiences with various antigens or pathogens, leading to effective and rapid immune responses upon subsequent exposure to the same or similar pathogens.
T-cells are a major cell component of the adaptive immune system and can be identified with the presence of CD3+ protein marker. Cyclo-Z treatment enhanced levels of CD3+ T-cells in diabetic subjects at the age of three and four months, and in normal subjects only at three months of age. Older subjects (four- to six-months old) show a decline in CD3+ T-cell levels in normal subjects (
Levels of activated T-cells, as determined by the presence of RT1B or CD25 proteins on CD3+ T-cells, were increased in three- and four-month-old diabetic subjects with Cyclo-Z treatment (
CD3+ T-cells can also be subdivided into: 1) cytotoxic T-cells, expressing the CDS surface protein, which destroy pathogen infected cells, cancer cells, and foreign cells; or 2) helper T-cells, expressing the CD4 surface marker, which regulate both the cellular and humoral immune systems. CD4+ helper T-cells have been shown to reduce autoimmune reactions. Cyclo-Z significantly decreased levels of CD3+ CD8+ cytotoxic T-cells in the blood at six months of age (
CD86 and CD80 have opposing functions through CD28 and CTLA-4 that manipulate immune responses and tolerance in vivo, as discussed in “CD86 and CD80 differentially modulate the suppressive function in human regulatory T cells,” by Zheng et al., J. Immunol. 172:2778-2784 (2004), which is hereby incorporated herein by reference as if set forth in full. Cyclo-Z had similar effects on both CD86+ and CD80+ AP cell levels in blood (
CD45RA antigen is present on the surface of more than 95% of B-lymphocytes in the purified tonsilar B-cell preparation. All stages of pre-B and B-cells express CD45RA antigens, as discussed in “Selective expression of CD45 isoforms defines CALL+ monoclonal B-lineage cells in peripheral blood from myeloma patients as late stage B cells,” by Jensen et al., Blood 78:711-719 (1991), which is hereby incorporated herein by reference as if set forth in full. CD45RA+ B-cell levels were significantly increased with Cyclo-Z treatment at early stages of immune system development in diabetic (three- and four-months old) and normal (three-months old) subjects (
CD11b+ monocyte levels were also elevated in diabetic and normal subjects with Cyclo-Z treatment, compared to their respective controls at three months of age (
In conclusion, the clinical manifestation of inflammatory diseases (such as T1D and T2D) is expected to be associated with changes in activation-related biomarkers in circulating immune cells. Cyclo-Z treatment invariably increased immune cell levels and activities involved in the innate (dendritic cells, monocytes, natural killer cells), adaptive (cytotoxic CD8+ and helper CD4+ T-cells, antigen presenting cells), and humoral (B-cells) immune system when the subjects were young and the immune system still immature (three months old) in both diabetic and normal subjects. Thus, Cyclo-Z is effective in the stimulation of immune system components which may be helpful in fighting infectious agents earlier than possible in young naïve or immature subjects. In older normal subjects, Cyclo-Z treatment may modulate levels of several components involved in inflammation (decreasing monocytes, CD8+ T-cells levels) and autoimmune diseases (increasing CD4+ helper T-cells levels), suggesting potential benefits in the treatment of autoimmune disease.
This application claims priority to U.S. Provisional Patent App. No. 62/597,106, filed on Dec. 11, 2017, which is hereby incorporated herein by reference as if set forth in full.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/065012 | 11/11/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62597106 | Dec 2017 | US |
