Patent Grant
11103628
This invention relates to detoxifying Gram negative bacterial lipopolysaccharide (LPS) and other inflammatory compounds in a person's blood. More particularly, an external biochemical reactor containing immobilized alkaline phosphatase enzymes for treating blood is described.
There are hundreds of different species of bacteria in the gastrointestinal (GI) tract including both beneficial bacteria (i.e., the commensal microbiome) and potentially pathogenic bacteria. Gram negative bacteria are one major group of potentially pathogenic bacteria in the GI tract. Gram negative bacteria have a cell wall surrounded by an outer membrane composed of lipopolysaccharides (LPS). Harmful endotoxins can include lipoglycans and LPS, which are large molecules consisting of a lipid and a polysaccharide composed of O-antigen, an outer and inner protein core joined by a covalent bond, and a lipid A moiety joined to the inner core by phosphate groups. The endotoxins produced by different Gram negative bacteria differ in their antigenicity due to differences in the O-antigen, but they all have the same biological effects which are mainly due to lipid A. Lipid A contains two phosphate groups that are believed to be essential for its toxicity
LPS in the bloodstream may be neutralized to some extent by many blood components including plasma lipids and proteins and LPS-binding protein (LPB). LPS binding to LPB elicits immune responses by presenting the LPS to cell surface pattern recognition receptors called CD14 and Toll-like receptors (e.g., TLR4) on macrophages, monocytes and endothelial cells. Interaction of LPS with these cellular receptors on monocytes and macrophages results in 1) production and release of cytokines including tumor necrosis factor alpha (TNFα), interleukins (e.g., IL-1, IL-6, IL-8) and platelet activating factor, resulting in activation of the arachidonic acid cascade to produce prostaglandins and leukotrienes, which are potent mediators of inflammation; 2) activation of the complement cascade C3 and C5a resulting in release of histamine which causes vasodilation, inflammation, and neutrophil chemotaxis; and 3) activation of the blood coagulation cascade that leads to acute disseminated intravascular coagulation, internal bleeding, hemorrhage and sepsis.
Sepsis is a life-threatening condition that develops when the body's response to infection causes injury to its own tissues and organs. Immediate, intensive treatment is crucial for surviving the condition and preventing septic shock because the risk of death from sepsis and septic shock increases with every passing hour. Toxic shock occurs when the body has an overwhelming response to infection—sometimes referred to as a “cytokine storm”—that causes the blood pressure to drop to dangerously low levels and triggers damaging changes to the organs causing them to become dysfunctional and stop working. Current treatment strategies include fluid replacement, antibiotics to control the infection, vasopressors to maintain adequate blood pressure, corticosteroids and anti-inflammatory drugs to lessen inflammation, and insulin to stabilize blood sugar levels. In some cases, a person might require surgery to remove abscesses and necrotic tissues that are a source of the microbial infection and toxins.
This disclosure describes a system, apparatus and method that can accomplish therapeutic removal of selected toxins within a biological system, including but not limited to those produced by Gram negative bacterial lipopolysaccharide (LPS) and other inflammatory compounds toxic to humans or animals.
In one embodiment, a detoxification method includes the steps of inducing flow of patient blood through an extracorporeal device inlet and outlet in fluid connection to a circulatory system of a patient. Biological agents contained within patient blood can be detoxified by passing patient blood over a biochemical reactor surface having attached or immobilized alkaline phosphatase enzyme, with the biochemical reactor being contained within the extracorporeal device.
In one embodiment, the alkaline phosphatase further comprises Saccharomyces boulardii alkaline phosphatase.
In one embodiment, the alkaline phosphatase detoxifies Gram negative bacterial lipopolysaccharide (LPS).
In one embodiment, the alkaline phosphatase detoxifies at least one of Gram negative or Gram positive bacterial extracellular lipoteichoic acid, ATP, DNA, RNA or flagellin, yeast and fungal extracellular ATP, DNA and RNA, viral extracellular DNA and RNA, and host extracellular ATP, DNA, or RNA.
In one embodiment, the alkaline phosphatase detoxifies biological agents contained within patient blood by dephosphorylation.
In one embodiment, detoxifying biological agents using S. boulardii alkaline phosphatase is used to therapeutically treat at least one of sepsis, septic shock, inflammation, bacteremia, yeast infections, fungal infections, viral infections, systemic inflammatory response syndrome (SIRS), Gram negative bacterial lipopolysaccharide (LPS)-exacerbated conditions, IBD, IBS, Crohn's disease, ulcerative colitis, enterocolitis, NEC, meningitis, meningococcemia, trauma or hemorrhagic shock, burns, liver disease, pancreatitis, periodontal disease, pneumonia, cystic fibrosis, asthma, A1AT deficiency, COPD, pulmonary fibrosis, tuberculosis, coronary heart disease, congestive heart failure, renal disease, hemolytic uremic syndrome, kidney disease, autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, cancer, Alzheimer's disease, diabetes, infections/abscess related diseases, and protein aggregation disorders including neurodegenerative diseases, Parkinson's disease, amyloidosis, and patients undergoing surgery, cardiovascular surgery, and transplants.
In one embodiment, alkaline phosphatase enzyme is immobilized by being covalently attached to the biochemical reactor surface.
In one embodiment, the biochemical reactor surface further comprises at least one of capillary tubing and microbeads.
In one embodiment, patient blood can be pumped through an extracorporeal device inlet and outlet in fluid connection to the circulatory system of a patient.
In one embodiment, the biochemical reactor surface is provided with a continuous blood flow from the patient that continues until the biological agent(s) being detoxified have been reduced to predetermined levels.
In one embodiment, a blood detoxification system includes an extracorporeal device having an inlet and outlet able to be placed in in fluid connection to the circulatory system of a patient. A biochemical reactor surface having attached alkaline phosphatase enzyme can act to detoxify biological agents contained within patient blood. The biochemical reactor can be contained within the extracorporeal device.
Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings.
This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary.
As used herein, the singular forms “a” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.
As used herein, the term “about”, when used in reference to numerical ranges, cutoffs, or specific values, is used to indicate that the recited values may vary by up to as much as 10% from the listed value. As many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can, and often times, will vary among different experiments. The values used herein should not be considered unduly limiting by virtue of this inherent variation. The term “about” is used to encompass variations of this sort up to, or equaling, 10%.
The term “attach,” “attached” or “attachment” as used herein, refers to connecting or uniting by a chemical bond, link, or force in order to keep two or more chemical compounds, polymers, proteins, polysaccharides, lipids, nucleic acids, or other biological or manufactured compositions together.
As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify a more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment.
Disclosed herein is an extracorporeal device, system or methods involving circulating, perfusing, or otherwise passing blood or other patient fluids through a system and device external to the body. One or more internal surfaces of the external or extracorporeal system include immobilized enzymatic agents to interact with one or more patient fluid borne biologic agents. The extracorporeal device, system or methods provide a platform that can be applied to numerous conditions and diseases involving circulating cells, compounds, or other biologic agents, such as those associated with bacterial, yeast, fungal, or viral infection, cell death, sepsis and many others.
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
Various types or isozymes of alkaline phosphatase can be used, including but not limited to human or animal derived intestinal AP (IAP), tissue-nonspecific AP (ALPL), placental AP (ALPP), germ cell AP (GCAP), or yeast derived alkaline phosphatase. Because of wide availability, ease of culture, and long duration of enzymatic activity, alkaline phosphatase produced by strains of the yeast Saccharomyces cerevisiae var. boulardii (S. boulardii) can be used in one embodiment. Advantageously, S. boulardii alkaline phosphatase has excellent stability and active lifetime, a pH activity profile suited for dephosphorylating toxins at normal pH blood, is less expensive to prepare than human or animal sources of alkaline phosphatase, and man-made alkaline phosphatase such as that made using recombinant DNA technology
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
In one embodiment of the device, system, or method of
1) Stand-alone treatment or in conjunction with other treatment strategies for bacteremia, sepsis and septic shock including oral dosing and IV injection of IAP or other AP isozymes;
2) Treatment to lessen morbidity due to LPS translocating from the gastrointestinal tract into the bloodstream when the intestinal permeability barrier becomes compromised due to dysbiosis caused by Gram negative bacterial infection and/or other acute or chronic metabolic disorders such as IBD, inflammatory bowel syndrome (IBS), Crohn's disease, cancer, liver disease, autoimmune diseases, diabetes, or aging;
3) Treatment to detoxify LPS exogenous ATP, DNA and flagellin in the bloodstream due to translocation across the intestinal barrier or due to microbial infections, bacteremia, abscesses or tissue necrosis at any site in the body;
4) Treatment to detoxify LPS exogenous ATP, DNA and flagellin in the bloodstream due to translocation following antibiotic treatment that alters the gut microbiome and disrupts homeostasis to allow increased contact of Gram negative bacteria in the intestinal lumen to gain access to Toll-like receptors on enterocytes, resulting in inflammation and decreased intestinal barrier function. In some situations, S. boulardii probiotics and/or oral IAP can additionally be used to accelerate return to normal homeostasis in the gastrointestinal tract;
5) Treatment to reduce the inflammatory response associated with necrotizing enterocolitis (NEC) and mitigate the septic response and end-organ injury;
6) Treatment of sepsis, septic shock, inflammation, bacteremia, yeast infections, fungal infections, viral infections, systemic inflammatory response syndrome (SIRS), Gram negative bacterial lipopolysaccharide (LPS)-exacerbated conditions, IBD, IBS, Crohn's disease, ulcerative colitis, enterocolitis, NEC, meningitis, meningococcemia, trauma or hemorrhagic shock, burns, liver disease, pancreatitis, periodontal disease, pneumonia, cystic fibrosis, asthma, AAT deficiency, COPD, pulmonary fibrosis, tuberculosis, coronary heart disease, congestive heart failure, renal disease, hemolytic uremic syndrome, kidney disease, autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, mast cell activation disorders, cancer, Alzheimer's disease, diabetes, infections/abscess related diseases, and protein aggregation disorders including neurodegenerative diseases, Parkinson's disease, amyloidosis, and patients undergoing surgery, cardiovascular surgery, and transplants;
7) Treatment of conditions associated with extracellular ATP, including hypoxia and ischemia that result in active release from cells and passive leakage from damaged/dying cells, and downregulation of ectonucleotidases. Examples include but are not limited to SIRS, AAT, COPD, IBD, IBS, diverticulosis, and diverticulitis;
8) Treatment of proinflammatory conditions derived from increased ATP, DNA and flagellin in patients with IBD, ulcerative colitis, or other disorder in which there is a decreased expression of IAP that results in excessive levels of proinflammatory compounds in the bloodstream;
9) Treatment to reduce levels of LPS, DNA, extracellular ATP and flagellin in the bloodstream that may be elevated following a course of systemic antibiotic treatment that altered the GI microbiome to favor non-commensal Gram negative bacteria including Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, and other bacteria, resulting in increased translocation of LPS, DNA, external ATP and flagellin into the bloodstream. Reduction of these toxins in the bloodstream can reduce immunological response and enable the body to re-establish the normal commensal microflora and homeostasis in the Gi tract; and
10) Treatment to reduce toxicity of LPS and microbial or host chemical compounds that may be proinflammatory including lipoteichoic acid, ATP, DNA and flagellin to reduce patient morbidity and result in fewer finger, hand, toe, foot, arm and leg amputations and surgery caused by sepsis and inflammation.
Various modifications to the foregoing described embodiments can be made. For example, multiple glass or plastic capillary tubing reaction chambers can be connected in series to act as biochemical reactors. In one embodiment, a first capillary tubing can contain immobilized SBAP to irreversibly dephosphorylate LPS, ATP and other proinflammatory compounds, and a second capillary tubing provided that contains immobilized Apyrase/CD73 to complete dephosphorylation of AMP to adenosine and inorganic phosphate if this reaction has not been completed in the first reaction chamber In other embodiments, a replaceable biochemical reactor can be used. The replaceable biochemical reactor can include immobilized SBAP or other alkaline phosphatase covalently attached to magnetic microbeads that are held by magnetic attraction to the inner surface of capillary tube. In still other embodiments magnetic microbeads with covalently attached enzymatic agents including SBAP and apyrase can be used in conjunction with a magnetic stirring system that is used to rotate magnetic cylinders within the biochemical reactor. Stirring cylinders can include surface attached and immobilized enzymatic agents to detoxify toxins or other undesired contaminants in the blood of the patient as the blood is pumped continuously through the biochemical reactor.
As will be appreciated, the described systems and methods of
In addition to alkaline phosphatase enzymes such as previously discussed, other phosphatase enzymes including any of human known alkaline phosphatase (AP) isozymes including intestinal AP (AP), tissue-nonspecific AP (TNAP), placental AP (PLAP), and germ cell AP (GCAP), any of other human phosphatase, AP, or nucleotidases including apyrase/CD39, CD73 (ecto-5′-nucleotidase), any of synthetic or man-made AP, such as AP made from recombinant DNA including E. coli, any of human or animal apyrase (cluster of differentiation 39=CD39) or AP and human nucleotidases human cluster of differentiation 73 (CD73)/ecto-5′-nucleotidase, bovine alkaline phosphatase (bAP), calf alkaline phosphatase (cIAP), potato apyrase, and AP from shrimp and recombinant DNA technology (E. coli alkaline phosphatase) can be used.
In some embodiments, an extracorporeal device with attached phosphatase biochemical reactor can be used as a stand-alone device or in conjunction with other treatment modalities. This can include but is not limited to devices or treatments including fluid replacement, corticosteroids, oral and IV administration of APs for treatment of sepsis, treatment of cytokine storm caused by serious acute respiratory virus SARS Covid-19 infection, injured/diseased organs and tissues, chronic inflammatory diseases such as diabetes and COPD, and following surgery, to reduce inflammation and speed recovery. As will be understood, treatment for Gram negative bacterial lipopolysaccharide (LPS) in patient blood, infection resulting in LPS or lipoteichoic acid in patient blood, abscesses resulting in LPS or lipoteichoic acid in patient blood, or other toxins in patient blood such as discussed herein are also contemplated.
In one example embodiment, a sterile hypodermic needle set can be used for accessing a patient's vein, (e.g., Blood Collection Set, Vaculet 21G×¾″ Winged, w/Multi-Sample Adapter, 12″ Tubing, or similar vein accessing device with a larger bore needle, if needed). A 36″ length of sterile plastic tubing can be used to pass through a peristaltic pump and connect the Vaculet with the biochemical reactor with a luer lock. The peristaltic pump or similar pumping device can be used for pumping blood from the patients arm to the biochemical reactor.
An external continuous-flow biochemical reactor is prepared by immobilizing S. boulardii alkaline phosphatase (SBAP) on the inner surface of polystyrene, polymethacrylate, or other plastic capillary tubing as described by Habja and Guttman “Continuous-flow biochemical reactors: Biocatalysis, bioconversion, and bioanalytical applications using immobilized microfluidic enzyme reactors”. J. Flow. Chem. 6(1):8-12, 2015, or Mohamad, et al. “An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotech. Biotechnol. Equip. 29(2):205-220, 2015. Enough capillary tubing (i.e., preferably up to 36 inches long) is used to allow immobilization of 50-1,500 IU AP, and preferably 200-500 IU SBAP in the tubing, which becomes the biochemical reactor. The plastic tubing with immobilized SBAP on the inner surface is sterilized and may be stored in the refrigerator at 4° C. for several months prior to use. When ready for use, the plastic tubing is placed into a 12″×12″×12″ chamber that has a lid that opens for placement of the tubing inside, a side opening with a luer lock for connection to the plastic tubing from the patient's arm (by way of the peristaltic pump), and a second side opening with a luer lock for connecting to the line that returns blood to the patient. Alternatively, the plastic tubing can be used without insertion into a chamber, with the pump and tubing together forming a portion of the extracorporeal device by themselves. The continuous-flow biochemical reactor can dephosphorylate approximately 50% of the LPS, external ATP, DNA and flagellin in blood per passage through it as the blood is pumped slowly (e.g., flow rate of 0.5-50 mL/min, and typically up to 10 mL/min) through the capillary tubing before returning it to the patient. Passage of the patient's blood through the biochemical reactor dephosphorylates and thereby detoxifies LPS, Gram negative or Gram positive bacterial extracellular ATP, DNA, RNA and flagellin, and host extracellular ATP, DNA, and RNA in the patient's blood before it is returned to the patient
A sterile hypodermic needle set can be used for accessing a patient's vein and a peristaltic pump can be used for pumping blood from the patients arm to a biochemical reactor. An external continuous-flow biochemical reactor is prepared by use of a sterile 250 mL closed container with a magnetic stirring bar that contains 10-150 g, and preferably 50 g of microbeads with immobilized SBAP, prepared by covalent bonding to have 50-1,500 IU SBAP, and preferably 200-500 IU SBAP prepared aseptically in the biochemical reactor. The external continuous-flow biochemical reactor has inlet and outlet connections for connecting with blood being pumped to and from the continuous-flow biochemical reactor. The container with SBAP covalently immobilized on the plastic beads is sterilized and may be stored in the refrigerator at 4° C. for several months prior to use. When ready for use, the container is placed onto a magnetic stirrer and stirring is started when blood begins to fill the container. A length of sterile plastic tubing is used to connect the biochemical reactor to the patient's arm vein for returning treated blood to the patient's arm or leg vein.
The peristaltic pump may be turned on after checking to ensure that all connections are tight so that they will not leak or allow the blood to become contaminated, and the pump is run continuously. Passage of the patient's blood through the biochemical reactor dephosphorylates and thereby detoxifies LPS, Gram negative or Gram positive bacterial extracellular lipoteichoic acid, ATP, DNA, RNA and flagellin, and host extracellular ATP, DNA, and RNA in the patient's blood before it is returned to the patient.
The biochemical reactor chamber is maintained at approximately body temperature (37° C.) by use of a thermostatically-controlled heating device. The patient's blood is pumped continuously through the biochemical reactor until analytical testing shows that levels of the LPS and inflammatory chemicals including, but not limited to ATP, DNA and flagellin, and/or selected markers (e.g., cytokines including TNFα, IL-6, or IL-8) have been reduced to undetectable or baseline levels and the patient's signs have returned to normal.
While specific embodiments have been illustrated and described above, it is to be understood that the disclosure provided is not limited to the precise configuration, steps, and components disclosed. Various modifications, changes, and variations apparent to those of skill in the art may be made in the arrangement, operation, and details of the methods and systems disclosed, with the aid of the present disclosure.
Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the present disclosure to its fullest extent. The examples and embodiments disclosed herein are to be construed as merely illustrative and exemplary and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having skill in the art that changes may be made to the details of the above-described embodiments without departing from the underlying principles of the disclosure herein.
This application is a continuation-in-part of U.S. patent application Ser. No. 16/862,378, filed on Apr. 29, 2020, the content of which being incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4246351 | Miyake | Jan 1981 | A |
| 4378435 | Takagi | Mar 1983 | A |
| 5395612 | Griffith | Mar 1995 | A |
| 5437861 | Okarma | Aug 1995 | A |
| 5635609 | Levy | Jun 1997 | A |
| 5656739 | Cubicciotti | Aug 1997 | A |
| 6193681 | Davidner | Feb 2001 | B1 |
| 6290952 | Poelstra | Sep 2001 | B1 |
| 7556768 | Brady | Jul 2009 | B2 |
| 8735087 | Brands | May 2014 | B2 |
| 10258734 | Federspiel | Apr 2019 | B2 |
| 10429385 | Winqvist | Oct 2019 | B2 |
| 10435457 | Watters | Oct 2019 | B2 |
| 10513546 | Watters | Dec 2019 | B2 |
| 10881781 | Orth | Jan 2021 | B1 |
| 20030120202 | Gordon | Jun 2003 | A1 |
| 20040014642 | Nicolau | Jan 2004 | A1 |
| 20040026322 | Nussbaumer | Feb 2004 | A1 |
| 20040092491 | Nieman | May 2004 | A1 |
| 20050249724 | Lihme | Nov 2005 | A1 |
| 20100092467 | Isenberg | Apr 2010 | A1 |
| 20110052560 | Brands | Mar 2011 | A1 |
| 20110212053 | Qian | Sep 2011 | A1 |
| 20130028880 | Chen | Jan 2013 | A1 |
| 20140008301 | Ostafin | Jan 2014 | A1 |
| 20140166578 | Ichim | Jun 2014 | A1 |
| 20150166978 | Cooney | Jun 2015 | A1 |
| 20150246170 | Miao | Sep 2015 | A1 |
| 20170002481 | Zussman | Jan 2017 | A1 |
| 20180169273 | Ferreira | Jun 2018 | A1 |
| 20190046717 | Laubrock | Feb 2019 | A1 |
| 20190054227 | Zheng | Feb 2019 | A1 |
| 20190060545 | Gruda | Feb 2019 | A1 |
| 20190247560 | Storr | Aug 2019 | A1 |
| 20200069790 | Bertin | Mar 2020 | A1 |
| Entry |
|---|
| Klaus Buttenschoen et al, “Endotoxin elimination in sepsis: physiology and therapeutic application”, Published in Langenbecks Arch Surgery, vol. 395, pp. 597-605, Jun. 27, 2010. (Year: 2010). |
| Margaret More et al, “Saccharomyces boulardii CNCM I-745 Improves Intestinal Enzyme Function: A Trophic Effects Review”, Published in Clinical Medicine Insights: Gastroenterology, vol. 11, pp. 1-14, Published Dec. 17, 2017. (Year: 2017). |
| Per H. Nilsson et al, “The creation of an antihrombotic surface by apyrase immobilization”, published online Mar. 7, 2010 in Biomaterials, vol. 21 of 2010, pp. 4484-449. (Year: 2010). |
| Per H. Nelson et al, “The creation of an antithrombotic surface by apyrase immobilization”, Published in Biomaterials, Jun. 2010, vol. 31 (16); pp. 4484-4491. (Year: 2010). |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16862378 | Apr 2020 | US |
| Child | 17109044 | US |
