# Patent

## Title
Adult and neonatal stem cell therapy to treat diabetes through the repair of the gastrointestinal tract

## Abstract

The anatomic and functional arrangement of the gastrointestinal tract suggests an important function of this organ is its ability to regulate the trafficking of metabolites as well as control the equilibrium between tolerance and immunity through gut-associated lymphoid tissue, the neuroendocrine network, and the intestinal epithelial barrier. Combining nucleated cells from various tissues and introducing them directly into the small intestine will have a positive effect on diabetes.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 12/697,537, filed Feb. 1, 2010, now U.S. Pat. No. 9,101,570, which claims the benefit of U.S. application Ser. No. 61/152,417, filed Feb. 13, 2009. The entire teachings of the above applications are incorporated herein by reference.


BACKGROUND OF THE INVENTION
Field of Invention
The invention relates to a method of treating diabetes. There are two types of diabetes, type 1 (T1D) and type 2 (T2D). About 10% of all Americans diagnosed with diabetes (more than 700,000) have T1D while the remaining other 90% of Americans that have diabetes, have type 2 diabetes. T2D (often referred to as adult onset diabetes) affects nearly 20 million people in the US. (CDC 2007) Diabetes, both T1D and T2D is the leading cause of blindness, renal failure and amputations, and patients have demonstrably shorter life spans. Increasingly diabetes is being understood as a metabolic disorder. This insight is leading to a new sort of pathology—the creation of inflammatory stress and an altered interaction between organ systems and between organ systems and the immune system resulting from the metabolism of certain substances. The evidence suggests that the organs themselves and the immune system are not impaired, but rather, they receive incorrect signals originating in the foregut due to the interaction of improper metabolites and the organ system. These signals then cause an improper cellular balance and function. While the dysfunction of the foregut in T1D and T2D is different, the original source of the incorrect signals leading to the pathology of the disease starts in the foregut.
Prior Art
T2D is a disease characterized by insulin insensitivity. Under normal circumstances, pancreatic islet cells release insulin in response to elevated levels of glucose in the bloodstream. Insulin drives glucose into peripheral tissues for use in building tissue and storing energy. However, in the setting of certain diets in susceptible individuals, glucose and insulin production is constantly switched on. As a protective mechanism, tissues subsequently become increasingly insensitive to insulin. Thus, T2D is a disease in which the environment (certain diets) damages the cellular niche of the foregut which triggers excess glucose production by the liver, which causes excess insulin production by the pancreas leading to insulin insensitivity in susceptible individuals.
T2D is caused by environmental insults due to the type of food consumed with the portion of the small intestine first exposed to food exiting the stomach most severely impacted. These environmental insults damage cellular niches in vulnerable individuals leading to the improper stimulation of the foregut during food digestion. Specifically, an imbalance of metabolites resulting from food digestion in the duodenum sends incorrect signals to the liver that over produces glucose that causes the pancreas to over produce insulin. Physiologic responses are eventually blunted as a protective mechanism in the face of constant signaling. Consequently, cells begin to look insulin resistant because there is too much glucose and insulin in the system. Overstressed pancreatic islets die because they cannot keep up with the required insulin production to overcome the excess amount of glucose being produced by the liver and the increasing insulin resistance of cells. In addition, some metabolites produced by the duodenum during food digestion need to communicate through nerve endings in the portal vein to signal hunger suppression to the brain. Advanced hyperglycemia damages these nerve endings exacerbating the problem. The following can be summarized as:
1) An improperly functioning duodenum that produces an imbalance of metabolites resulting in the excess production of glucose by the liver, insulin resistance, leading to a pancreatic burden that ends in islet necrosis; and
2) Improper hunger suppression due to damaged nerves primarily in the portal vein.
The combination of points 1 and 2 explains the high correlation between T2D and obesity.
Biliopancreatic diversion (BPD) and Roux-en-Y gastric bypass (RYGBP) effectively bypass the diseased portion of the intestinal tract so that the foregut (duodenum) is excluded and the distal small bowel becomes the new foregut. The distal small bowel is less impacted over time from the environmental insult that lead to the duodenum dysfunction because it is farther removed from the source of the insult which is certain partially undigested foods exiting the stomach. Consequently, immediately after the surgery, this undamaged portion of the small bowel becomes the new foregut, is attached to the stomach and remarkably, begins to produce proper metabolites in response to food intake. Thus, within days of surgery, blood sugar levels of patients return to a normal range and there is an increase in the release of certain hormones like GLP-1 that promote satiety. Interestingly, resolution of T2D following surgery is inversely proportional to duration of the disease; those who have had the disease for longer period of times prior to surgery experience lower rates of resolution. The longer a patient has lived with the disease, the greater the portion of the small bowel that is damaged, the greater the damage to the islets of the pancreas, and the greater the damage to nerve endings in the portal vein due to the prolonged impact of hyperglycemia.
With respect to type 1 diabetes (T1D), the goal of researchers is to 1) halt the progression of Type 1 diabetes (T1D) by re-introducing tolerance between the immune system and the pancreas and 2) regenerate the insulin producing capacity of the pancreas. Increasingly T1D is being understood as a metabolic disorder. This insight is leading to a new sort of Pathology—the creation of inflammatory stress and an altered interaction between the organ systems and the immune system resulting from the metabolism of certain substances. The evidence suggests that the immune system is itself not impaired, but rather, it receives incorrect signals due to the interaction of improper metabolites and the organ system. These signals then cause an improper cellular balance and function.
We believe that the cause of type 1 diabetes originates in the small intestine. The primary functions of the gastrointestinal tract have traditionally been perceived to be limited to the digestion and absorption of nutrients and electrolytes, and to water homeostasis. A more attentive analysis of the anatomic and functional arrangement of the gastrointestinal tract, however, suggests that another extremely important function of this organ is its ability to regulate the trafficking of macromolecules between the environment and the host through a barrier mechanism. Together with the gut-associated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions, controls the equilibrium between tolerance and immunity to nonself-antigens. When the finely tuned trafficking of macromolecules is deregulated due to certain environmental insults (most likely due to the ingestion of certain foods or pathogens) in genetically susceptible individuals, both intestinal and extra-intestinal autoimmune disorders can occur. Supporting this explanation are observations that diet modifies the incidence of diabetes and the phenotype of T-cells infiltrating the islets of Langerhans in animal models (NOD mice) of Type 1 diabetes. T-cells infiltrating the islets of Langerhans in Type 1 diabetics and in experimental models of autoimmune diabetes are intestinal in origin since they exhibit the b7-integrin receptor (gut associated homing receptor). Mesenteric lymphocytes from non-obese diabetic (NOD) mice can transfer diabetes to healthy recipients. We believe that concentrated nucleated cells injected into this environment can re-establishing proper intestinal function and arrest the autoimmune process by changing the interplay between altered epigenes and the environment.
We believe that the cause of type 1 diabetes originates in the small intestine. Specifically, T1D patients have an altered intestinal immune responsiveness. This altered mucosal immune system has been associated with the disease of T1D and is likely a major contributor to the failure to form tolerance, resulting in the autoimmunity that underlies type 1 diabetes. There are numerous cell types in the small intestine that play a role in proper immune system function, including the following:
Intraepithelial Lymphocytes.
Intraepithelial lymphocytes (IELs) are located at the basolateral side of the epithelial layer. Here they are exposed to a wide range of food and microbial antigens. One well-established function of IELs is their ability to protect the host from invasion by microorganisms that enter through the gastrointestinal tract. One subtype, the intestinal epithelial lymphocytes, are also at the mucosal interface and appear to play a key role in maintaining peripheral tolerance.
Intestinal Epithelial Cells.
Intestinal epithelial cells (IEGs) comprise the lining epithelium of the primitive intestine with the role of transduction of inflammatory signals from luminal microbes via toll-like receptors and other signaling mechanisms. These cells serve as the permeability barrier between the external and internal milieus of the body.
M-Cells.
M-cells do not have well-developed microvilli and allow macromolecular transport, are specialized for delivering foreign antigens and microorganisms to organized lymphoid tissues within the mucosa of the small and large intestines.
Goblet Cells.
Goblet cells are specialized mucus-secretory cells found throughout the intestine. Intestinal mucus is a complex gel that covers the surface of the villous epithelium and contributes significantly to cytoprotection, offering many ecological advantages for the microbiota.
Paneth Cells.
Paneth cells represent one of the four major epithelial cell lineages in the small intestine and are the only lineage that migrates downward into the crypt base after originating in the crypt stem cell region. The location of Paneth cells adjacent to crypt nucleated cells suggests that they play a critical role in defending epithelial cell renewal. In response to pathogen attack, the Paneth cells secrete a wide spectrum of antimicrobial peptides against gram-negative and gram-positive bacteria, fungi, protozoa, and viruses.
The focus of research and therapy for T1D has been on pancreatic islet regeneration through various methods to include allogeneic islet transplantation, venous or arterial infusion of concentrated marrow nucleated cells into the area of the pancreas, nonmyeloablative immune conditioning followed by a systematic autologous or allogeneic stem cell transplantation and immune suppression drugs. Unfortunately, there is no cure for T1D and patients diagnosed with this disease require exogenous insulin injections to live.
The focus of research and therapy for T2D has been on drugs to improve insulin sensitivity, weight loss through surgery or diet, and more recently, a Teflon sleeve that covers the duodenum. This sleeve allows the passage of food from the stomach to the distal portion of the small intestine without having the food contact the duodenum.
All of the major functions of the body are related. The source of diabetes is the foregut and when this organ is impaired, it results in incorrect protein signals being produced that cause problems in other organs. In the case of T1D, the incorrect signals cause the immune system to attack cells that make insulin. In the case of T2D, the incorrect signals cause the liver to overproduce glucose which then makes the pancreas have to over produce insulin and results in insulin insensitivity in peripheral tissues. The focus of prior art for both T1D and
T2D has been on organs and tissues which are damaged as a result of diabetes, with the primary focus being the pancreas. We believe in the case of both disease states, the focus needs to be on the organ (ie the gastrointestinal tract) the disease emanates from, and not the organs downstream (ie pancreas and other peripheral tissues) that are damaged as a result of the malfunctioning gastrointestinal tract.
U.S. Pat. No. 6,808,702 provides a method of implantation of stem cells into a gastrointestinal organ for purposes of repopulating various cellular components and I or providing a source of biological material for therapeutic intent. The source of these stem cells can be embryonic or adult neural and non-neural tissue, (e.g. bone marrow or fat tissue) U.S. Pat. No. 6,808,702 goes on to define a gastrointestinal organs to include hollow and solid organs. Hollow gastrointestinal organs include those that make up the alimentary tract, such as the mouth, esophagus, stomach, and bowels. Solid gastrointestinal organs include those that drain into the gastrointestinal alimentary tract such as the liver, gall bladder and pancreas. With respect to diabetes, U.S. Pat. No. 6,808,702 teaches that it is a disease of a solid organ as defined by the patent as a gastrointestinal organ and not a hollow organ as defined by the patent. Specifically, U.S. Pat. No. 6,808,702 teaches a method of producing enhanced levels of insulin in a patient by implanting stem cells and/or progeny thereof into the pancreas, which is considered a gastrointestinal organ as defined by the patent because it is a solid organ that drains into the gastrointestinal alimentary tract. For example U.S. Pat. No. 6,808,702 states“ Further, this invention can also be used to provide therapy for disorders that are not traditionally considered gastrointestinal disorders but are related to organs that are considered gastrointestinal organs (e.g. liver, gall bladder, and pancreas) in that the organs drain into the gastrointestinal alimentary canal. Such disorders include diabetes, which can be treated by means of implantation of stem cells into the pancreas of a patient to cause enhancement of insulin production.” It is important to note that U.S. Pat. No. 6,808,702 broadly defines gastrointestinal organs to include the pancreas; goes on to define diabetes as not being thought of as a gastrointestinal disorder; and instructs that stem cells be injected into the pancreas (a solid organ) as a possible therapy for diabetes. It is important to note that U.S. Pat. No. 6,808,702 states that diabetes is not traditionally thought of as a gastrointestinal disorder and specifically teaches away from the art disclosed here by instructing the injection of stem cells into the solid organ pancreas.
The art disclosed here is patently different from what is disclosed in U.S. Pat. No. 6,808,702. This art narrowly defines gastrointestinal tract to include hollow organs that make up the alimentary tract to include the stomach and bowels but does not include any solid organs. Opposed to the traditional view that diabetes is not a gastrointestinal disorder as taught by U.S. Pat. No. 6,808,702; the art disclosed herein specifically identifies diabetes as a disease that originates in the hollow organs of the gastrointestinal tract and not of the solid organ pancreas. This art then goes on to specifically teach that an effective therapy for diabetes is to implant nucleated cells from various tissues into the hollow organs of the gastrointestinal tract, specifically the duodenum. Conversely, U.S. Pat. No. 6,808,702 broadly defines gastrointestinal organs to include the pancreas, defines diabetes as being a disease of the solid organ pancreas, and in the art teaches to inject into the solid organ of the pancreas.
Some of the prior art referred to above use treating compositions that contain nucleated cells to include nucleated cells from bone marrow aspirates, fat aspirates, or mobilized peripheral blood. Specifically, the prior art teaches the delivery of the cells into the venous or arterial system. However, none of the prior art is delivering the nucleated cells directly into the appropriate tissue. This art teaches a method of injecting the treating composition directly into the tissue of gastrointestinal tract with a focus on the foregut. In view of the above, the present invention seeks to improve clinical outcomes by delivering a treating composition into the foregut which is the source of the disease. Compositions that contain nucleated cells are an obvious choice to include in the composition because nucleated cells have the demonstrated ability to regulate the cellular activity of surrounding cells and to repair the function of damaged tissue and immune systems. Tissues whose nucleated cell population are rich in cells that contain sub populations that are often referred to as stem cells include bone marrow aspirate, fat aspirate, cord blood, mobilized peripheral blood, Wharton's jelly, and other after birth tissue. Since diabetes ultimately damages other organ systems, it is reasonable to combine the treatment of the foregut with a method of treating other tissue affected by the disease. Thus combining a method of delivering cells into the foregut with a systemic delivery of nucleated cells into the venous or arterial system is appropriate.
SUMMARY OF THE INVENTION
Diabetes can be reversed through the repair the duodenum by implanting concentrated nucleated cells that include nucleated cells from various sources directly into the wall of the duodenum through an endoscopic procedure. The implantation can be carried out via local injection, as for example into a wall of the duodenum. This therapy can be augmented by combining direct injection into the gastrointestinal tract of the treating composition with arterial or venous delivery. Several different tissue sources rich in nucleated cell populations that contain nucleated cells can be used alone or in combination in preparing the treating composition.
DETAILED DESCRIPTION OF THE INVENTION
T2D can be reversed through the repair of the duodenum by injecting concentrated nucleated cells from various sources directly into the wall of the duodenum through an endoscopic procedure. This procedure can be combined with introducing a portion of the treating composition into the portal vein and or duodenal artery through a catheter based procedure. This procedure should 1) reset/repair the proper function of digestion and metabolite production in the duodenum and 2) repair of the nerve endings of the portal vein. The result will be an improvement in establishing normal glucose levels, support for the regeneration of the insulin producing capacity of the pancreas, improvement in insulin resistance, and the re-establishment of hunger suppression signals in response to food intake. Consequently, the therapy should be effective for both T2D and the co-morbidity of obesity associated with T2D.
The progression of T1D can be halted prior to the destruction of the pancreas by re-setting of the immune system. This can be accomplished through the repair of the small intestine by injecting a treating composition of nucleated cells into the wall of the duodenum through an endoscopic procedure to reset/repair the interaction between the immune system and the insulin producing islets of the pancreas.



DRAWINGS-FIGURES
FIG. 1 is a perspective view for explaining one embodiment of the present invention;
FIG. 2 is a perspective view for explaining an embodiment of the present invention whereby the cells are sourced from the patient being treated.



DETAILED DESCRIPTION—FIRST EMBODIMENT—FIGS 1
In the interest of clarity, numerous different surgical protocols and surgical devices currently available can be used to implant a treating composition into the wall of the gastrointestinal tract. Also, numerous different methodologies can be used for concentrating nucleated cells from the various tissues mentioned above in order to create the treating composition. The source of the cells can be the patient themselves or from other donors. Some of these tissues can be sourced, concentrated and delivered at point of care. Other sources of cells can be sourced, processed in a laboratory, and then delivered to the patient. It will, of course, be appreciated that in the development of any surgical protocol designed to implement the art described, numerous implementation-specific decisions must be made in order to fit a particular patient's profile and that these specific protocols will vary from one surgeon and patient to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure. For example, different injection patterns, needle gauges, and combinations of nucleated cells from various sources can be used to create and deliver the treating composition. The treating composition can be delivered through and endoscopic procedure or during an open procedure. In addition to cells, other types of treating compositions could be delivered in combination or individually as part of the treating composition that contain for example, platelets, growth factors or other proteins or therapeutic agents to include pharmaceuticals.
FIG. 1 is a perspective view of the stomach (102) which is connected to the foregut. (103) Several different endoscopic surgical tools exist to access the gastrointestinal tract and to inject cells into the wall of the duodenum. (100) Such tools are flexible in nature and are designed to move easily through the curved space within the gastrointestinal tract. These same tools can be used to implant a treating composition directly into the walls of the tissue comprising the gastrointestinal tract. Such direction injections can move in a spiral pattern along the walls of the tissue. (104,105,106)
FIG. 2 is a perspective of a surgical protocol of treating a patient (200) with tissue rich in stem cells sourced from their own body. (201) This tissue is then processed to concentrate the nucleated cells including the stem cells. (202) Such methods of concentration can be done point of care during same procedure as the sourcing of the cells and the delivery of the cells to the gastrointestinal tract. The treating composition is then delivered through and endoscopic tool (203) to the hollow organs of the gastrointestinal tract that include the stomach (206) and foregut (207.) Other organs such as the liver (204) and gallbladder (205) and pancreas (not shown) can also be treated by infusing a portion of the treating composition into the venous or arterial system.
In accordance with the present invention, the method of sourcing, concentrating, and delivering directly a treating composition rich in nucleated cells to include stem cells into the tissue of the foregut may be implemented using various types of laboratory and or surgical equipment. Thus the scope of the invention should be determined by the appended claims and their legal equivalents, and not by the examples given.
It will be appreciated that numerous different methods exist to concentrate the nucleated cells from various tissue to include bone marrow aspirate and fat aspirate. These methods can employ off site laboratory methods or devices designed to be used at the patients side (ie point of care) For example, their exists several centrifugation based protocols such as a ficoll separation method, cell washing technology, and apheresis technology for removing a large portion of the non-nucleated red blood cells and plasma from marrow aspirate and or cord blood. Such methods are well known in the art. For removing the extracellular matrix material from fat, whartons jelly, and other solid tissues, several well known protocols such as enzymatic digestion with collagenase are well known in the art. With all of these protocols, some material such as plasma and red cells remain in the concentrate.
Definitions: Gastrointestinal tract means hollow organs and include the alimentary tract to include the stomach and bowels.
T1D means type 1 diabetes.
T2D means type 2 diabetes.
Nucleated cell means a cell that contains a nucleus.

## Claims

1. A method to treat diabetes comprising:
implanting nucleated cells including stem cells from multiple source tissues directly into tissue of the duodenum to reset or repair the immune system or to reset or repair metabolite production in the duodenum, the nucleated cells being a heterogeneous population of cells and including cells from a bone marrow aspirate, a fat aspirate, cord blood, Wharton's Jelly, or afterbirth tissue.

2. The method according to claim 1 wherein the nucleated cells are implanted through an endoscopic procedure.

3. The method according to claim 1 wherein the nucleated cells are injected into the wall of the duodenum.

4. The method according to claim 1 wherein the nucleated cells include cells from a bone marrow aspirate.

5. The method according to claim 1 wherein the nucleated cells include cells from a fat aspirate.

6. The method according to claim 1 wherein the nucleated cells include cells from cord blood.

7. The method according to claim 1 wherein the nucleated cells include cells from Wharton's Jelly.

8. The method according to claim 1 wherein the nucleated cells are derived from autologous tissue.

9. The method according to claim 1 further comprising delivering a portion of the nucleated cells into the venous or arterial system.

10. The method according to claim 1 further comprising:
sourcing nucleated cells from a patient; and
preparing the nucleated cells using the nucleated cells sourced from the patient, wherein the sourcing, preparing and implanting are performed in one point-of-care procedure.

11. The method according to claim 1 wherein the nucleated cells are implanted into the tissue of the duodenum of a patient diagnosed with type-1 diabetes during the progression of the destruction of the islets of the patient's pancreas.

12. The method according to claim 1 wherein the nucleated cells include subpopulations of nucleated cells from respective source tissues.

13. The method according to claim 1 further comprising:
in one point-of-care procedure,
preparing a combination of nucleated cells from multiple source tissues, the nucleated cells being derived from autologous tissue; and
implanting the combination directly into tissue of the duodenum of a patient diagnosed with type-1 diabetes during the progression of the destruction of the islets of the patient's pancreas.

14. The method according to claim 1 where the nucleated cells are implanted through injections that move in a spiral pattern along the wall of the duodenum.