Year of IP in review

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TimGDixon
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Year of IP in review

Post by TimGDixon »

The year isn't quite over but I thought I might share the new IP for 2021 in review. These are original works of Thomas and I along with some co-inventors like Dr. Veltmeyer, Dr. Lin, Ms. Ramos, Ms. O'Connor, Mr. Kaihani and a big thanks to all of them for their many contributions. This represents 20 new inventions between January and November that we all signed over to the Company so you can own it. This is for all of you who actually support us - let you have a little sneak peek into what is coming. But remember all of these things will take time - yeah I know Time.. old Mr. Time tick tick ticking away every day. Oh and please don't ask me to explain every graphic LOL. Just enjoy.



01-26-2021 Stimulation of Dendritic Cell Activity by Homotaurine and Analogues Thereof
Disclosed are means, methods, and compositions of matter useful for enhancement of dendritic cell activity. In one embodiment the invention provides the use of GABA agonists such as homotaurine for stimulation of dendritic cell activity. In one embodiment said dendritic cell activity is enhancement of natural killer cell activity and/or of T cell activity. In one embodiment NK cell activity is ability to induce cytotoxicity in neoplastically transformed cells, whereas T cell activity is either cytokine production for CD4 cells or cytotoxicity for CD8 cells.

Example 1 Homotaurine Increases Ability of StemVacs to Stimulate NK Activity
Peripheral blood mononuclear cells were obtained by ficoll centrifugation and plated with StemVacs umbilical cord derived dendritic cells. StemVacs was generated by culture of umbilical blood adherent cells with interleukin 4 and GM-CSF for 7 days with maturation step induced by 24 hour culture TLR agonist. Assessment of natural killer cell activity was performed subsequent to culture of cells with taurine (10 micrograms / ml) or homotaurine (10 micrograms /ml) for the indicated timepoints. Cytotoxicity against NK target cell line was performed using the flow cytometry Promega assay.
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Example 2 Homotaurine Increases Ability of StemVacs to Stimulate T cell Activity
Peripheral blood mononuclear cells were obtained by ficoll centrifugation and plated with StemVacs umbilical cord derived dendritic cells. StemVacs was generated by culture of umbilical blood adherent cells with interleukin 4 and GM-CSF for 7 days with maturation step induced by 24 hour culture TLR agonist. Assessment of T cell activity was performed subsequent to culture of cells with taurine (10 micrograms / ml) or homotaurine (10 micrograms /ml) for the indicated timepoints. T cell activity was assessed by ELISA quantification of interferon gamma production after stimulation with 5 micrograms per ml of phytohemagglutinin.
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02-08-2021 Stimulation of Natural Kill Cell Memory by Administration of Dendritic Cells
Disclosed are means, methods and compositions of matter useful for induction of natural killer cell memory by administration of dendritic cells and/or exosomes thereof. In one embodiment a mammal suffering from cancer is administered allogeneic cord blood derived dendritic cells that are not pulsed exogenously. In one embodiment the dendritic cells are stimulated to possess chemotactic activity towards the tumor by culture of dendritic cell progenitors in hypoxia. Natural killer cell memory is induced, in part, by triggering of upregulation of cytokines associated with homeostatic expansion such as interleukin 7 and interleukin 15.

Example
C57/BL6 mice were implanted with 500000 B16 melanoma cells and allowed to grow. StemVacs (human umbilical cord blood dendritic cells pulsed with poly IC (1 ug/ml for 4 hours) were administered to mice 5 days after tumor inoculation. The concentration of dendritic cells was 250,000 per mouse administrated intratumorally. Mice were observed until tumors regressed and given another 1 month after last tumor regression. Mice were then sacrificed and cells from transferred to naïve mice immunized with B16 melanoma. Cells were transferred 5 days after inoculation of B16. Control (diamond), CD8 (X) and CD4 (triangle) all had no protection. In contrast transfer of splenocytes (Square) or CD56 cells (X) resulted in suppression of tumor growth.

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02-22-2021 Ex Vivo Generation of Immunocytes Recognizing Brother of the Regulator of Imprinted Sites (BORIS) Expressing Cancer Stem Cells
Disclosed are means, methods and compositions of matter useful for induction of immunity towards cancer stem cells by providing a dendritic cell, wherein said dendritic cells express BORIS and/or peptides derived from BORIS, wherein said dendritic cell is cultured in the presence of one or more immunocytes. In one embodiment said dendritic cells are derived from umbilical cord blood sources and allogeneic to T cells, which are expanded ex vivo and used for the purposes of immunotherapy.

Example: Stimulation of Immunity to CD133 Positive PC-3 Cancer Stem Cells
Cord blood generated allogeneic dendritic cells were obtained by culture of adherent monocytes with GM-CSF (100 IU/ml) and IL-4 (100 IU/ml) for 7 days. Cells expressed CD11c and possessed typical dendritic morphology. Cells were incubated with full length BORIS protein and treated with 30 ng/ml Poly (IC) for 24 hours to induce dendritic cell maturation. Dendritic cells were cultured with allogeneic peripheral blood mononuclear cells for 7 days in the presence of IL-2 (10 IU/ml) and anti-CD3 and anti-CD28 beads (10(5) beads per ml. T cells were purified by Magnetic Activated Cell Sorting for CD3 (containing both CD8 and CD4 cells). PC-3 prostate cancer cells were cultured and separated into either control (no T cells added), unfractionated, CD133 negative and CD133 positive. Cells were co-cultured at a 1:1 ratio and cytotoxicity was determined by MTT assay.

As observed, T cells possessed a preferential ability to kill CD133 cells, which corresponds to cancer stem cell phenotype.

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Stimulation of Immunity to flk Positive PC-3 Cancer Stem Cells
Cord blood generated allogeneic dendritic cells were obtained by culture of adherent monocytes with GM-CSF (100 IU/ml) and IL-4 (100 IU/ml) for 7 days. Cells expressed CD11c and possessed typical dendritic morphology. Cells were incubated with full length BORIS protein and treated with 30 ng/ml Poly (IC) for 24 hours to induce dendritic cell maturation. Dendritic cells were cultured with allogeneic peripheral blood mononuclear cells for 7 days in the presence of IL-2 (10 IU/ml) and anti-CD3 and anti-CD28 beads (10(5) beads per ml. T cells were purified by Magnetic Activated Cell Sorting for CD3 (containing both CD8 and CD4 cells). PC-3 prostate cancer cells were cultured and separated into either control (no T cells added), unfractionated, flk negative and flk positive. Cells were co-cultured at a 1:1 ratio and cytotoxicity was determined by MTT assay.

As observed, T cells possessed a preferential ability to kill CD133 cells, which corresponds to cancer stem cell phenotype.
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Stimulation of Immunity to Rhodamine Positive PC-3 Cancer Stem Cells
Cord blood generated allogeneic dendritic cells were obtained by culture of adherent monocytes with GM-CSF (100 IU/ml) and IL-4 (100 IU/ml) for 7 days. Cells expressed CD11c and possessed typical dendritic morphology. Cells were incubated with full length BORIS protein and treated with 30 ng/ml Poly (IC) for 24 hours to induce dendritic cell maturation. Dendritic cells were cultured with allogeneic peripheral blood mononuclear cells for 7 days in the presence of IL-2 (10 IU/ml) and anti-CD3 and anti-CD28 beads (10(5) beads per ml. T cells were purified by Magnetic Activated Cell Sorting for CD3 (containing both CD8 and CD4 cells). PC-3 prostate cancer cells were cultured and separated into either control (no T cells added), unfractionated, Rhodamin negative and Rhodamin positive. Cells were co-cultured at a 1:1 ratio and cytotoxicity was determined by MTT assay.

As observed, T cells possessed a preferential ability to kill CD133 cells, which corresponds to cancer stem cell phenotype.

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03-04-2021 Therapeutic Monocytes for Prevention of Suicidal Ideation
The invention discloses compositions of matter, protocols, and therapeutic means for treatment of suicidal ideations and/or suppression of suicidal attempts. In one embodiment the invention provides the use of umbilical cord derived monocytes as a means of treatment. In another embodiment, monocytes are de-differentiated from adult monocytes using reprogramming means to create monocyte capable of producing anti-inflammatory as well as regenerative properties useful in reducing suicidal ideations and/or attempts.

Examples: Suppression of Inflammation Induced Memory Dysfunction by Cord Blood Derived Monocytes
The lipopolysaccharide administration model was used to replicate neuroinflammation associated with suicidal behavior. Female BALB/c mice were treated with control, daily lipopolysaccharide treatment (10 ng/mouse0, and some with cord blood derived monocytes. Cord blood derived monocytes were extracted by ficoll separation of cord blood mononuclear cells and 4-hour incubation on T175 plastic flasks. Non-adherent cells were washed off by rinsing with phosphate buffered saline and adherent cells were subsequently dissociated using trypsin and placed into another flask. Cord blood monocytes were cultured for 24 hours with 1 IU of oxytocin per million cells in complete DMEM media with 10% fetal calf serum. Cells were dissociated with trypsin, washed in phosphate buffered saline and administered intravenously vial tail vein at a concentration of 500,000 per mouse on days 0, 3, and 6.

To assess memory function, water filled basin which was 120 cm in diameter was broken into 4 quadrants. 10 cm diameter platform placed 1 cm below water. Mice were forced to swim to find the hidden platform, starting from all four different quadrants, each day for a total of 7 days. As seen below, mice receiving umbilical cord monocytes resisted the pathological effects of lipopolysaccharide treatment.

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Suppression of LPS Induced Plasma Interleukin-6 by Cord Blood Derived Monocytes
The lipopolysaccharide administration model was used to replicate neuroinflammation associated with suicidal behavior. Female BALB/c mice were treated with control, daily lipopolysaccharide treatment (10 ng/mouse0, and some with cord blood derived monocytes. Cord blood derived monocytes were extracted by ficoll separation of cord blood mononuclear cells and 4-hour incubation on T175 plastic flasks. Non-adherent cells were washed off by rinsing with phosphate buffered saline and adherent cells were subsequently dissociated using trypsin and placed into another flask. Cord blood monocytes were cultured for 24 hours with 1 IU of oxytocin per million cells in complete DMEM media with 10% fetal calf serum. Cells were dissociated with trypsin, washed in phosphate buffered saline and administered intravenously vial tail vein at a concentration of 500,000 per mouse on days 0, 3, and 6.

To assess interleukin 6 levels in plasma, mice were sacrificed at the indicated timepoints and cytokine was assayed by ELISA.

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03-16-2021 Pluripotent Stem Cell Derived Dendritic Cells and Engineered Dendritic Cells for Cancer Immunotherapy
Disclosed are populations of dendritic cells generated from stem cells capable of inducing immunity towards cancer. In one embodiment said dendritic cells are generated from allogeneic inducible pluripotent stem cells, for some uses, said pluripotent stem cells are genetically engineered/edited to induce cancer specific immunity and/or resist immunosuppressive effect of tumor derived microenvironment. In one embodiment pluripotent stem cells are transfected with cancer stem cell antigens such as BORIS and/or NR2F6.

Example 1: iPS Derived Dendritic Cells Induce Reduction of B16 Melanoma
iPSCs (ATCC-DYR0100 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1011™) were cultured on feeder layers of OP9 cells for 6 to 7 days in α-MEM supplemented with 20% FBS. The mesodermally differentiated cells were then harvested, reseeded onto fresh OP9 cell layers, and cultured in α-MEM supplemented with 20% FBS, 20 ng/mL GM-CSF, and 50 μmol/L 2-ME. On day 13 to 14, floating cells were recovered by pipetting. These cells were considered to be iPSC-derived myeloid cells (iPS-MCs). The cells were infected with lentivirus vectors expressing the c-Myc and the Brother of the Regulatory of Imprinted Sites (BORIS) gene, as well as shRNA encoding siRNA silencing VEGF-R in the presence of 8 ng/mL polybrene (Sigma-Aldrich), and were cultured in α-MEM supplemented with 20% FBS, 30 ng/mL GM-CSF, and 30 ng/mL M-CSF. After 5 to 6 days, proliferating cells appeared and were considered to be ESC- or iPSC-derived pMCs (ES-pMC or iPS-pMC, respectively). To induce the differentiation of these cells into DC-like cells (pMC-DC), they were cultured in RPMI-1640 supplemented with 20% FBS in the presence of 20 ng/mL IL4 plus 30 ng/mL GM-CSF for 3 days.

Mice were inoculated with 500,000 B16 melanoma. Mice were injected with saline (Control), unmodified stem cell derived DC (Unmodified), stem cell derived DC expressing BORIS (BORIS) and BORIS transfected DC with VEGF-R silenced (BORIS VEGF R Silenced). Tumor growth was assessed by calipers.

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03-23-2021 Chimeric Cells Comprising Dendritic Cells and Endothelial Cells Resembling Tumor Endothelium
Disclosed are means, methods and compositions of matter useful for induction of immunological responses towards tumor endothelial cells. In one embodiment the invention teaches fusion of dendritic cells and cells resembling tumor endothelial cells and administration of such chimeric cells as an immunotherapy for stimulation of tumor endothelial cell destruction. In other embodiments pluripotent stem cells are utilized to generate dendritic cells, wherein said dendritic cells are fused with pluripotent stem cell derived endothelial cells created in a manner to resemble tumor endothelial cells.

Example
iPSCs (ATCC-DYR0100 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1011™) were cultured on feeder layers of OP9 cells for 6 to 7 days in α-MEM supplemented with 20% FBS. The mesodermally differentiated cells were then harvested, reseeded onto fresh OP9 cell layers, and cultured in α-MEM supplemented with 20% FBS, 20 ng/mL GM-CSF, and 50 μmol/L 2-ME. On day 13 to 14, floating cells were recovered by pipetting. These cells were considered to be iPSC-derived myeloid cells (iPS-MCs). The cells were infected with lentivirus vectors expressing the c-Myc and the Brother of the Regulatory of Imprinted Sites (BORIS) gene, as well as shRNA encoding siRNA silencing VEGF-R in the presence of 8 ng/mL polybrene (Sigma-Aldrich), and were cultured in α-MEM supplemented with 20% FBS, 30 ng/mL GM-CSF, and 30 ng/mL M-CSF. After 5 to 6 days, proliferating cells appeared and were considered to be ESC- or iPSC-derived pMCs (ES-pMC or iPS-pMC, respectively). To induce the differentiation of these cells into DC-like cells (pMC-DC), they were cultured in RPMI-1640 supplemented with 20% FBS in the presence of 20 ng/mL IL4 plus 30 ng/mL GM-CSF for 3 days. These cells were fused with endothelial cells derived from iPSC cultured under conditions replicating tumor microenvironment, specifically, cells were grown in 10 ng/ml PGE2, 100 pg/ml TGF-beta, and 100 pg/ml VEGF. Cells were cultured for 7 days and subsequently sorted for expression of the cancer endothelial marker TEM-1. Fusion between the two cells was performed using the polyethelyne glycol method utilized to generate monoclonal antibodies.

Mice were inoculated with 500,000 lewis lung carcinoma cells. Mice were injected with saline (Control), BORIS expressing VEGF R silenced dendritic cells (StemVacs-V), iPSC derived endothelial cells (EC) and the hybrid (hybrid). Tumor growth was assessed by calipers.

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03-29-2021 Compositions Capable of Stimulating Immunity Towards Tumor Blood Vessels
Disclosed are novel means, protocols, and compositions of matter for eliciting an immune response against blood vessels supplying neoplastic tissue. In one embodiment pluripotent stem cells are transfected with one or more genes capable of eliciting immunity. In some embodiments such genes are engineered under control of specific promoters to allow for various specificities of activity. In one specific embodiment pluripotent stem cells engineered to endow properties capable of inducing expression of the α-Gal epitope (Galα1,3Galα1,4GlcNAc-R).

Example
iPSC expressing α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) were differentiated into tumor endothelial like cells by culture in VEGF (10 ng/ml), PDGF-BB (5 ng/ml), PGE-2 (100 pg/ml). Cells were incubated with human plasma and assessed for viability using MTT assay.

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04-13-2021 Amelioration and Treatment of Opioid Addiction
Disclosed are compositions of matter, protocols and treatment means for reducing and/or preventing opioid addiction. In one embodiment the invention teaches intranasal administration of umbilical cord blood plasma, or extracts thereof, together with pterostilbene or pterostilbene containing nanoparticles, and/or oxytocin, and/or human chorionic gonadotropin.

Example.
Neuroinflammation was induced by administration of LPS at 50 ng/mouse. Cord blood plasma and cord blood plasma with pterostilbene was administered intranasally. Assessment of TLR expression in brain homogenates were performed by flow cytometry.

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05-17-2021 Treatment of Major Depressive Disorder by Low Dose Interleukin-2
Disclosed are methods, compositions of matter, and protocols useful for treatment of major depressive disorder through administration of low dose interleukin-2 at a concentration and/or frequency sufficient to increase expansion of T regulatory cell numbers and/or enhancement of T regulatory cell activity. In some embodiments administration of interleukin-2 is provided as means of enhancing efficacy of standard antidepressant therapies. Furthermore, administration of interleukin-2 receptor agonists is also described in the current invention as a treatment of major depressive disorder.

Example
BALB/c female mice (10 per group) were administered lipopolysaccharides (LPS) (5 mg/kg) (Sigma-Aldrich) intravenously. Interleukin-2 was administered at 0.1 U per mouse for 3 days. Determination of depression was quantified by assessment of social interaction with uninjected mice. 10 mice per group were used for a total of 3 independent experiments.
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05-21-2021 Lithium as a Monotherapy and/or Stem Cell Adjuvant Therapy for Pulmonary Fibrosis
Disclosed are compositions of matter, therapeutics, and protocols useful for reduction and/or reversion of pulmonary fibrosis. In one specific embodiment lithium chloride is administered together with a regenerative cell in a patient suffering from, or at risk of pulmonary fibrosis. In one embodiment said lithium chloride is administered as an adjuvant to a regenerative therapy, wherein said regenerative therapy is a gene therapy, a protein therapy, a cell therapy, or a tissue transplant. In one embodiment lithium chloride, or a salt thereof is utilized alone, or with a regenerative means, to evoke preservation and/or elongation of telomere length in pulmonary tissue. In one embodiment the invention teaches administration of umbilical cord mesenchymal stem cells (MSC) and/or products derived from said cells in order to induce an inhibition of natural or pathological reduction of telomere length, to preserve telomere length or to enhance telomere length. In one embodiment the MSC described in the invention as useful are umbilical cord derived MSC.

Example: Lithium Enhances Ability of JadiCells to Increase T regulatory Cell Numbers in Mice.
10 week old Female BALB/c mice were administered 150 mg/kg of lithium chloride daily by the IP route. Additionally some animals received JadiCells at 500,000. Quantification of T regulatory cells was performed by intracellular staining for FoxP3 expression.

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Example 2: JadiCells are Superior to BM and Adipose MSC
• Bleomycin (BLM) administered intratracheally 3 mg/kg in female 10 week old C57/BL6 mice
• JadiCells™ or BM-MSC or adipose administered by tail vein at 5 days after BLM
• Animals sacrificed at day 12 and assessed for fibrosis (hydroxyproline content)
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Example 3: Lithium Chloride Enhances Antifibrotic Activities of JadiCells
• Bleomycin (BLM) administered intratracheally 3 mg/kg in female 10 week old C57/BL6 mice
• JadiCells™ was injected by tail vein at 5 days after BLM. Lithium was injected IP as described in example 1.
• Animals sacrificed at day 12 and assessed for fibrosis (hydroxyproline content)

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05-24-2021 Immunotherapies for Targeting of Tumor Vasculature
Disclosed are novel means, protocols, and compositions of matter for creating targeted immune responses and/or induction of immunological memory towards the tumor vasculature. In one embodiment pluripotent stem cells are transfected with one or more genes capable of eliciting immunity, induced to differentiate into endothelial-like cells which resemble the tumor endothelial cells, and utilized as a vaccine. In some embodiment’s genes are engineered under control of specific promoters to allow for various specificities of activity. In one specific embodiment pluripotent stem cells engineered to endow properties capable of inducing expression of the α-Gal epitope (Galα1,3Galα1,4GlcNAc-R). Addition of adjuvants to enhance antigen presentation of the vaccine composition, as well as means of stimulating systemic enhancement of circulating endothelial specific T cells are also disclosed.

Example
iPSC expressing α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) were differentiated into tumor endothelial like cells by culture in VEGF (10 ng/ml), PDGF-BB (5 ng/ml), PGE-2 (100 pg/ml). Cells were administered alone, 500,000 per mouse or together with Poly IC (5ng/mouse) into mice bearing B16 melanoma.

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07-06-2021 Treatment of Parkinson’s Disease by Immune Modulation and Regenerative Means
Disclosed are means, methods and compositions of matter for treatment Parkinson’s Disease through concurrent immune modulation and regenerative means. In one embodiment Parkinson’s Disease is treated by augmentation of T regulatory cell numbers and/or activity while concurrently providing regenerative cells such as mesenchymal stem cells, and/or dopamine secreting cells. In one embodiment administration of immunoglobulins such as IVIG together with low dose interleukin-2 and/or low dose naltrexone is disclosed as a preparatory means prior to administration of therapeutic cells such as stem cells. Other therapeutic means utilized in an adjuvant manner are also provided for hormonal rebalancing, transcranial magnetic stimulation, and deep brain stimulation.

Example: Decrease in Substantia Nigra Inflammation by Tol-DC and Preservation of Dopaminergic Neurons
Tol-DC (StemVacs) was generated by culture of umbilical cord adherent monocytes in GM-CSF 10 ng/ml and IL-4 (5 ng/ml) for 7 days. Cells were treated with 5 ng/ml IL-10 to generate (Tol-DC), whereas conventional DC were cells grown under identical conditions with no IL-10.

The cells were transferred i.v. at one and two weeks prior to intoxication with four 16 mg/kg doses of MPTP. Mice treated with PBS or MPTP alone served as controls. Two days after MPTP intoxication, mice were sacrificed, brains removed, frozen, and cryosectioned at 30 μm/section through the midbrain containing the substantia nigra. Sections were stained for Mac-1 expression by microglia.

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08-11-2021 Induction of Neurogenesis using Umbilical Cord Derived Mesenchymal Stem Cells and Derivatives Thereof
Disclosed are means, compositions of matter and protocols useful for treatment of neurological dysfunctions through stimulation of adult neurogenesis using administration of umbilical cord derived mesenchymal stem cells such as JadiCells. In one embodiment viral induced neuropathy is reduced by administration of JadiCells to stimulate neurogenesis. In another embodiment the neurogenic activity of selective serotonin reuptake inhibitors is enhanced by administration of JadiCells. In some embodiments administration of JadiCell exosomes, conditioned media, microvesicles and/or apoptotic bodies is utilized to stimulate neurogenesis.

Example: JadiCell Administration is Superior to other Stem Cells for Stimulating Dentate Gyrus Neurogenesis
BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations. After a period of 3 days animals were given BrdU and sacrificed the next day. Proliferation was assessed based on BrdU incorporation. 10 animals per group were used.

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08-18-2021 Enhancement of Umbilical Cord Mesenchymal Stem Cell Therapeutic Activity by Stimulators of T Regulatory Cells and/or Cells Expressing CD73
Disclosed are means, compositions of matter and protocols useful for treatment of COVID-19 and/or other inflammatory pathologies through stimulation of T regulatory cells and/or T cells expressing CD73 using administration of umbilical cord derived mesenchymal stem cells such as JadiCells. In one embodiment dosage of JadiCells needed to treat a patient is determined by the increase of T regulatory cells and/or CD73 expressing cells that are increased in number and/or activity subsequent to a test dose of JadiCells. In another embodiment stimulators of T regulatory cells and/or CD73 expressing T cells are utilized together with JadiCells in order to augment therapeutic activity. In some embodiments administration of JadiCell is performed with low dose interleukin-2 as a treatment for COVID-19 or other inflammatory related pathologies.

Example 1: JadiCells Increase the Number of T regulatory Cells
BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations. After a period of 3 days animals were sacrificed and T regulatory cells were measured by FoxP3 staining (Promega kit, following manufacturers instructions).

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Example 2: JadiCells Increase the Number of CD73 T Cells
BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations. After a period of 3 days animals were sacrificed and T regulatory cells were measured by CD73 staining (Promega kit, following manufacturers instructions).

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Example 3: Depletion of Treg Cells Decreases Therapeutic Potential of JadiCells
BALB/c mice were depleted of CD25 T regulatory cells by administration once every two days of anti-CD25 antibody one week before initiation of experiment. Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations. After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

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Example 4: Low Dose IL-2 Increases Therapeutic Potential of JadiCells
BALB/c mice were treated with IL-2 (10 IU/mouse) once every two days one week before initiation of experiment. Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations. After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

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08-23-2021 Umbilical Cord Mesenchymal Stem Cells for Treatment of Chronic Obstructive Pulmonary Disease and Lung Degeneration
The invention discloses means of treating lung degenerative diseases including chronic obstructive pulmonary disease (CODP) using umbilical cord mesenchymal stem cells such as JadiCells alone, and/or using said cells under conditions that are activated in order to endow enhanced regenerative activity. In one embodiment said activation of said mesenchymal stem cells is performed through stimulation with a toll like receptor agonist at a concentration and duration sufficient to induce a >50% increase in keratinocyte growth factor expression from said stem cells. In another embodiment the invention provides the use of JadiCells as a means of producing exosomes, wherein said exosomes possess therapeutic properties capable of reducing inflammation, fibrosis and degeneration associated with COPD, as well as stimulation of regenerative activity. In some JadiCells are activated by a treatment with Activated Protein C.

Examples
BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of elastase at the indicated concentrations. After a period of 3 days animals were sacrificed and concentration of neutrophils per viewing field was assessed. Animals receiving JadiCells had higher levels of MerTK and lower levels of TLR4.

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09-16-2021 Ivermectin Compositions for Treatment of COVID-19
Disclosed are novel mechanisms of action of ivermectin therapy as related to treatment of COVID-19 and means of augmenting therapeutic activities by co-administration with one or more of the following: pterostilbene, thymoquinone, epigallocatechin-3-gallate, and sulforaphane. In one embodiment the invention provides enhanced reduction of inflammation induced pulmonary leakage without augmenting immune suppressive mechanisms.

Example
BABL/c mice, 5-7 weeks of age, females, were intraperitoneally injected with 50 mg/kg pentobarbital. Lipopolysaccharides (LPS) (5 mg/kg) (Sigma-Aldrich) was delivered to the lungs through a tracheostomy. Group 1 received LPS alone, Group 2, ivermectin orally at 2 mg/kg for at time of challenge, Group 3 received QuadraMune, and Group 4 received the combination. Lung wait was compared to whole body weight.

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09-22-2021 Stimulation of Mesenchymal Stem Cell Therapeutic Activities by T Regulatory Cells
Disclosed are novel means of enhancing mesenchymal stem cell regenerative activities including, intra alia, production from pulmonary leakage and suppression of scar tissue formation by co-administration with T regulatory cells. In some embodiments the invention provides an interaction between T regulatory cells and mesenchymal stem cells in which T regulatory cells stimulate upregulation of mesenchymal stem cell activity in a GITR dependent manner.

Examples: Treg-JadiCell Synergy in LPS Model
BALB/c mice, 5-7 weeks of age, females, were intraperitoneally injected with 50 mg/kg pentobarbital. Lipopolysaccharides (LPS) (5 mg/kg) (Sigma-Aldrich) was delivered to the lungs through a tracheostomy. Group 1 received LPS alone, Group 2, Umbilical blood Treg cells at 500,000 per mouse, Group 3 received JadiCells 500,000 per mouse and Group 4 received the combination. Lung weight was compared to whole body weight.

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Treg-JadiCell Synergy in Poly IC Model
BALB/c mice, 5-7 weeks of age, females, were intraperitoneally injected with 50 mg/kg pentobarbital. Poly IC (1 mg/kg) (Sigma-Aldrich) was delivered to the lungs through a tracheostomy. Group 1 received Poly IC alone, Group 2, Umbilical blood Treg cells at 500,000 per mouse, Group 3 received JadiCells 500,000 per mouse and Group 4 received the combination. Lung weight was compared to whole body weight.

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Treg-JadiCell Synergy in LPS Model is Dependent on GITR
BALB/c mice, 5-7 weeks of age, females, were intraperitoneally injected with 50 mg/kg pentobarbital. Lipopolysaccharides (LPS) (5 mg/kg) (Sigma-Aldrich) was delivered to the lungs through a tracheostomy. Group 1 received LPS alone, Group 2, Umbilical blood Treg cells and JadiCells at 500,000 per mouse, Group 3 received both cells and control antibody and Group 4 received both cells and anti-GITR. Lung weight was compared to whole body weight.

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10-04-2021 Reduction of Neutrophil Extracellular Trap formation by Mesenchymal Stem Cells and their Exosomes
Disclosed are methods of reducing lung inflammation in acute respiratory distress syndrome elicited by various factors such as COVID-19 infection by reduction of neutrophil extracellular trap formation through administration of mesenchymal stem cells and/or exosomes thereof. The invention provides means of inhibiting neutrophil release of extracellular traps by mesenchymal stem cells and/or exosomes derived from said mesenchymal stem cells. Additionally, synergies are provided between mesenchymal stem cells and/or exosomes derived from mesenchymal stem cells and agents approaches which reduce neutrophil extracellular trap formation.

Example 1. Suppression of Neutrophil Extracellular Trap Formation by Exosomes from Poly IC activated Mesenchymal Stem Cells
Umbilical cord mesenchymal stem cells were cultured in DMEM media with 10% fetal calf serum. Cells were activated with 100 ng/ml Poly IC for 2 hours. Exosomes were purified using Exo-quick kit according to the manufacturer’s instructions. Exosomes were added at a concentration of 10 ng/ml based on protein content to neutrophils activated with PMA at 100 uM for the indicated timepoints. Neutrophil extracellular trap quantification was performed using the SYTOX method and quantified on flow cytometry as MFI.

Example 2: Exosomal Inhibition of Lung Pathology.
A COVID-like lung injury was induced by administration of LPS. Addition of exosomes collected from activated mesenchymal stem cells, as described in Example 1, resulted in suppression of fluid leakage in the treated animals.

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10-11-2021 Umbilical Cord Derived Regenerative and Immune Modulatory Stem Cell Populations
The present invention provides universal donor cellular populations derived from umbilical cords possessing ability to elicit immune modulation and evoke regeneration when administered into a mammalian host. Generation of cellular products for clinical use are provided including methodologies of expansion, characterization, and means of therapeutic implementation.

Example 1 Generation of Umbilical Cord Potentiated MSC
Umbilical cord tissue was obtained from healthy pregnancy and washed twice with phosphate buffered saline (PBS). The Wharton’s jelly portion of the cord was extracted. Upon extraction, mechanical mincing was performed utilizing sterile forceps and a scalpel. Minced pieces were cultivated in a cell culture dish with Dulbecco’s modified Eagle’s medium F12 (DMEM)-low glucose no L-Glutamine with 10% human AB serum, 1% 10.000 U/mL penicillin and 10.000 μg/mL streptomycin. Culture was performed at initial 12 hours at 2% oxygen, 24 hours at 5% oxygen, subsequent to which CD73 positive cells were selected using magnetic activated cell sorting and expanded. The culture-expanded cells were cryopreserved at P3 using standard cryopreservation protocols until their use in the following experiment. The cells were characterized at the time of cryopreservation with flow cytometric analysis to determine the expression of positive surface markers CD90, CD105, CD73, CD44, CD29, CD56 and negative for CD34, CD45 and CD14. Additionally, quality control analyses like mycoplasma analysis (using PCR), endotoxin analysis (using the LAL test and sterility analysis) were also completed. Cells were solubilized from cryopreservation before being made ready for injection. Average cell viability for each treatment was over 95%.

Example 2: Suppression of TNF-alpha Production from Activated Macrophages
Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers through the use of ficoll density gradient. A total of 5 million cells per ml were plated in DMEM complete media on T75 plates for a period of 6 hours in order to allow monocytes to adhere to the plates. Non-adherent cells were washed off with PBS with 3% serum albumin. Subsequently, adherent monocytes were extracted by trypsinization and plated at a one to one ratio of cells extracted from Example 1 (Invention MSC), as well as with conventional umbilical cord MSC (UC-MSC) which were not selected for CD73 expression or bone marrow MSC (BM-MSC). Cells received stimulation with the indicated amount of endotoxin for 48 hours and quantification of TNF-alpha was performed by ELISA.

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Example 3: Suppression of IL-6 Production from Activated Macrophages
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Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers through the use of ficoll density gradient. A total of 5 million cells per ml were plated in DMEM complete media on T75 plates for a period of 6 hours in order to allow monocytes to adhere to the plates. Non-adherent cells were washed off with PBS with 3% serum albumin. Subsequently,
adherent monocytes were extracted by trypsinization and plated at a one to one ratio of cells extracted from Example 1 (Invention MSC), as well as with conventional umbilical cord MSC (UC-MSC) which were not selected for CD73 expression or bone marrow MSC (BM-MSC). Cells received stimulation with the indicated amount of endotoxin for 48 hours and quantification of interleukin-6 was performed by ELISA.

Example 4: Suppression of IL-8 Production from Activated Macrophages
Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers through the use of ficoll density gradient. A total of 5 million cells per ml were plated in DMEM complete media on T75 plates for a period of 6 hours in order to allow monocytes to adhere to the plates. Non-adherent cells were washed off with PBS with 3% serum albumin. Subsequently, adherent monocytes were extracted by trypsinization and plated at a one to one ratio of cells extracted from Example 1 (Invention MSC), as well as with conventional umbilical cord MSC (UC-MSC) which were not selected for CD73 expression or bone marrow MSC (BM-MSC). Cells received stimulation with the indicated amount of endotoxin for 48 hours and quantification of interleukin-8 was performed by ELISA.

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Example 4: Suppression of Colitis by Administration of Umbilical Cord Cells of the Invention
Female BALB/c mice at eight weeks of age were treated for 7 days with dextran sodium sulfate (DSS) at 5% in their drinking water in order to induce a murine version of inflammatory bowel disease (IBD).
Mice were divided into groups of 10 mice per group.
• Group 1 saline;
• Group 2 DSS;
• Group 3 DSS and mesenchymal stem cells (500,000 intravenously days 1 and 3)
• Group 4 DSS and umbilical cord cells of example 1 (500,000 intravenously days 1 and 3);

Disease activity index (DAI) scores were calculated according to the Hamamoto et al. Clin Exp Immunol. 1999 Sep; 117(3):462-8.This is seen below

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Example 5: Production of Cytokines by Cells of the Invention Compared to Bone Marrow MSC and Adipose Tissue MSC
Cells (Invention cells or BM-MSC or Adipose-MSC) were utilized at passage 5. Cells were cultured in standard in fully humidified environment with 5% carbon dioxide. Cultured in 96 well plates for 48 hours, 50,000 cells per well. Unstimulated or stimulated with 50 or 100 ng/ml LPS, 10 or 20 ng/ml TNF, 10 or 20 ng/ml IL-1 beta. Cytokines assessed by ELISA

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11-01-2021 Induction of Concurrent Pulmonary Immune Modulation and Regeneration by Protein Mediated Conjugation of Immune Regulatory Cells with Endogenous Progenitor Cells
Disclosed are means, methods and compositions of matter useful for treatment of inflammatory pulmonary diseases such as COVID-19 through administration of agents that facilitate interaction between immune modulatory cells and endogenous pulmonary progenitor cells. In one embodiment a bispecific antibody capable of facilitating the interaction between CD25 on T regulatory cells and CD47 on pulmonary epithelial stem cells is described.

Examples:
One of the major pathologies of COVID-19 is the opening of the endothelial gap junctions allowing for fluid to enter the extracapillary spaces. This results in fluid accumulating in the alveoli, which makes breathing compromised, even in some cases during mechanical ventilation.

The TSOI-576 antibody was administered intravenously into BALB/c mice treated with 100 nanograms of endotoxin per mouse. Endotoxin functions by binding to toll like receptor 4 and causes a pathology that has been reported to be similar to COVID-19 induced ARDS. The lung weight of the animal was measured at time points of zero, 12 hours and 24 hours. Ten mice per group were assessed for the experiment. As can be seen, a significant dose-dependent suppression of pulmonary inflammation was seen as resulted of the antibody treatment. Additionally, examination of the lungs revealed TSOI-576 also resulted in dose-dependent suppression of neutrophil infiltration, interleukin-6 content in the lung and overall suppression of fibrosis in mice allowed to live 4 weeks and assessed for fibrotic tissue content by hydroxyproline staining.

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tempo
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Re: Year of IP in review

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Ah Tim, can you please explain all the graphics? :lol: :lol: :lol: :lol: :lol: :lol: :lol:
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Re: Year of IP in review

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Yes of course you would ask hahahahahahaha... By the way I am just giving you the date of filing, the abstract and then some examples of experiments we conducted prior to filing. These are not the actual patents.
tempo
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Re: Year of IP in review

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That’s incredible all of that accomplished in the year 2021! Thank you Tim and team! You guys definitely Rock!!
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Re: Year of IP in review

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Thank you Tempo. I don't think most people realize we have to run a lot of experiments to end up with something novel and some of those experiments can take weeks so work flow is critical - I think we have averaged something like 1.6 new patents a month since January. This is our life, this is who we are, this is how we think about these things...



tempo wrote: Thu Dec 09, 2021 4:19 pm That’s incredible all of that accomplished in the year 2021! Thank you Tim and team! You guys definitely Rock!!
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Re: Year of IP in review

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Although not in this list as it was filed on 06-15-2020 as "Nutraceuticals for Suppressing Indolamine 2,3 Deoxygenase" I thought I would share we got Notice of Allowance from the USPTO today for this patent meaning it will soon grant with the issued claims. Time for a clinical trial. For all the "science and patent lovers" out there.

https://therapeuticsolutionsint.com/the ... l-studies/


Nutraceuticals for Suppressing Indolamine 2,3 Deoxygenase
Disclosed are compositions of matter, treatments and protocols useful for reduction of expression and/or activity of indolamine 2,3 deoxygenase (IDO). In some embodiments the invention teaches the administration of a therapeutic combination of ingredients comprising of pterostilbene, nigella sativa, sulforaphane, and epigallocatechin-3-gallate (EGCG) to a mammal at possessing an increased expression and/or activity of said IDO in which reduction of number and/or activity is desired. In another embodiment, the invention teaches administration of said therapeutic combination to a mammal infected with viral and/or bacterial infections and/or neoplasia. In some embodiments dosage of said therapeutic combination is based on inflammatory and/or immunological parameters observed in patients.

Example 1: QuadraMune™ Preserves Memory in Inflammation Associated Memory Decline Model
Female BALB/c mice were treated with control, daily LPS treatment, and some with LPS and QuadraMune™. QuadraMune™ was administered daily by gavage at a concentration 1 (100 ug of broccoli sprout extract, nigella sativa, and green tea extract, and 50 ug of pterostilbene), and concentration 2 (200 ug of broccoli sprout extract, nigella sativa, and green tea extract, and 100 ug of pterostilbene). To assess memory function, water filled basin which was 120 cm in diameter was broken into 4 quadrants. 10 cm diameter platform placed 1 cm below water. Mice were forced to swim to find the hidden platform, starting from all four different quadrants, each day for 7 days. The time was recorded if they could find the hidden platform in 60 s…If not, mice are guided toward the platform and allowed to stand on it for 10 s.

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Example 2: QuadraMune™ Reduces Inflammation Associated Kynurenine Elevation
Blood samples were collected from mice in experiment 1 and assessed for Kynurenine content. Augmented levels of kynurenine were observed in LPS treated mice as compared to controls.

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tempo
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Re: Year of IP in review

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Congratulations Tim and team! This is awesome news!
Howzitgoing
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Re: Year of IP in review

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Tim, if QuadraMune can suppress IDO (Indolamine 2,3 Deoxygenase), is it also possible that it may suppress Tau, "a substance that builds up in Alzheimer's disease and damages brain cells essential for learning and memory. Tau buildup is caused by increased activity of enzymes that act on tau called tau kinases, which causes the tau protein to misfold and clump, forming neurofibrillary tangles."

Inching my way along here, but if QuadraMune can ameliorate, dare I say, prevent, cure Alzheimer's, well, you know the rest. The world will be grateful (understatement).
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Re: Year of IP in review

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You are thinking along the right lines there is supporting literature. We will certainly be exploring this.
Howzitgoing wrote: Fri Dec 10, 2021 6:07 pm Tim, if QuadraMune can suppress IDO (Indolamine 2,3 Deoxygenase), is it also possible that it may suppress Tau, "a substance that builds up in Alzheimer's disease and damages brain cells essential for learning and memory. Tau buildup is caused by increased activity of enzymes that act on tau called tau kinases, which causes the tau protein to misfold and clump, forming neurofibrillary tangles."

Inching my way along here, but if QuadraMune can ameliorate, dare I say, prevent, cure Alzheimer's, well, you know the rest. The world will be grateful (understatement).

Currently there are only 10 papers


1. PLoS One. 2013 Apr 22;8(4):e59749. doi: 10.1371/journal.pone.0059749. Print 2013.
Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain
.
Wu W(1), Nicolazzo JA, Wen L, Chung R, Stankovic R, Bao SS, Lim CK, Brew BJ, Cullen KM, Guillemin GJ.
Author information: (1)Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

DOI: 10.1371/journal.pone.0059749 PMCID: PMC3632609 PMID: 23630570 [Indexed for MEDLINE]

2. Neuropathol Appl Neurobiol. 2005 Aug;31(4):395-404. doi: 10.1111/j.1365-2990.2005.00655.x.
Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Alzheimer's disease hippocampus.

Guillemin GJ(1), Brew BJ, Noonan CE, Takikawa O, Cullen KM.
Author information: (1)Centre for Immunology, St Vincent's Hospital, Darlinghurst, NSW, Australia.

The present immunohistochemical study provides evidence that the kynurenine pathway is up-regulated in Alzheimer's disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO- and QUIN-immunoreactivity (-IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN-IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro-degeneration in AD. These results may open a new therapeutic door for AD patients.

DOI: 10.1111/j.1365-2990.2005.00655.x PMID: 16008823 [Indexed for MEDLINE]

3. J Alzheimers Dis. 2010;22(1):257-66. doi: 10.3233/JAD-2010-100684.
Oren-gedoku-to and its constituents with therapeutic potential in Alzheimer's disease inhibit indoleamine 2, 3-dioxygenase activity in vitro.

Yu CJ(1), Zheng MF, Kuang CX, Huang WD, Yang Q.
Author information: (1)Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China.

A well-known traditional Chinese medicinal prescription, Oren-gedoku-to (OGT), has been used in clinical therapies for many types of dementia in China and Japan. Additionally, it ameliorates the age-related deterioration of learning and memory in an Alzheimer's disease (AD) rat model. Indoleamine 2, 3-dioxygenase (IDO-1) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism, which ultimately leads to the production of the excitotoxin quinolinic acid (QUIN). IDO-1 has recently been established as one of the key players involved in the pathogenesis of AD. OGT is indicated to prevent cholinergic dysfunction and reduce oxidative stress; however, the exact mechanism underlying its ability to improve cognitive ability remains elusive. Here we present a novel mechanism of OGT's therapeutic potential in AD. We demonstrated that OGT significantly inhibited recombinant human IDO-1 (rhIDO-1) activity in vitro, and its four main constituents (i.e., berberine, palmatine, jatrorrhizine, and baicalein) were potent IDO-1 inhibitors. IC50 values, obtained from a cell-based assay, of HEK 293 cells and an enzymatic assay were much lower than the most commonly used IDO-1 inhibitor, 1-methyl tryptophan (1-MT). Berberine was the best inhibitor and had IC50 values of 7 μM (cell-based assay) and 9.3 μM (enzymatic assay). Jatrorrhizine and palmatine exhibited irreversible inhibition of rhIDO-1, whereas berberine and baicalein behaved as uncompetitive, reversible inhibitors with Ki values of 8 μM and 215 μM, respectively. In conclusion, constituents of OGT show strong IDO-1 inhibitory activity and may have significant therapeutic potential for AD.

DOI: 10.3233/JAD-2010-100684 PMID: 20847417 [Indexed for MEDLINE]

4. J Alzheimers Dis. 2018;62(2):523-547. doi: 10.3233/JAD-170929.
Alzheimer's Disease: Recent Concepts on the Relation of Mitochondrial Disturbances, Excitotoxicity, Neuroinflammation, and Kynurenines.

Zádori D(1), Veres G(1), Szalárdy L(1), Klivényi P(1), Vécsei L(1)(2).
Author information: (1)Department of Neurology, Faculty of Medicine, Albert Szent-Györgyi Clinical Center, University of Szeged, Szeged, Hungary. (2)MTA-SZTE Neuroscience Research Group, Szeged, Hungary.

The pathomechanism of Alzheimer's disease (AD) certainly involves mitochondrial disturbances, glutamate excitotoxicity, and neuroinflammation. The three main aspects of mitochondrial dysfunction in AD, i.e., the defects in dynamics, altered bioenergetics, and the deficient transport, act synergistically. In addition, glutamatergic neurotransmission is affected in several ways. The balance between synaptic and extrasynaptic glutamatergic transmission is shifted toward the extrasynaptic site contributing to glutamate excitotoxicity, a phenomenon augmented by increased glutamate release and decreased glutamate uptake. Neuroinflammation in AD is predominantly linked to central players of the innate immune system, with central nervous system (CNS)-resident microglia, astroglia, and perivascular macrophages having been implicated at the cellular level. Several abnormalities have been described regarding the activation of certain steps of the kynurenine (KYN) pathway of tryptophan metabolism in AD. First of all, the activation of indolamine 2,3-dioxygenase, the first and rate-limiting step of the pathway, is well-demonstrated. 3-Hydroxy-L-KYN and its metabolite, 3-hydroxy-anthranilic acid have pro-oxidant, antioxidant, and potent immunomodulatory features, giving relevance to their alterations in AD. Another metabolite, quinolinic acid, has been demonstrated to be neurotoxic, promoting glutamate excitotoxicity, reactive oxygen species production, lipid peroxidation, and microglial neuroinflammation, and its abundant presence in AD pathologies has been demonstrated. Finally, the neuroprotective metabolite, kynurenic acid, has been associated with antagonistic effects at glutamate receptors, free radical scavenging, and immunomodulation, giving rise to potential therapeutic implications. This review presents the multiple connections of KYN pathway-related alterations to three main domains of AD pathomechanism, such as mitochondrial dysfunction, excitotoxicity, and neuroinflammation, implicating possible therapeutic options.

DOI: 10.3233/JAD-170929 PMID: 29480191 [Indexed for MEDLINE]

5. Front Pharmacol. 2019 Sep 24;10:1044. doi: 10.3389/fphar.2019.01044. eCollection 2019.
Effects of the Novel IDO Inhibitor DWG-1036 on the Behavior of Male and Female 3xTg-AD Mice.

Fertan E(1), Stover KRJ(2), Brant MG(2), Stafford PM(2), Kelly B(2), Diez-Cecilia E(2), Wong AA(1), Weaver DF(2), Brown RE(1).
Author information: (1)Department of Psychology and Neuroscience, Dalhousie University, Halifax, NS, Canada. (2)Krembil Research Institute, University Health Network, Toronto, ON, Canada.

The kynurenine pathway metabolizes tryptophan into nicotinamide adenine dinucleotide, producing a number of intermediary metabolites, including 3-hydroxy kynurenine and quinolinic acid, which are involved in the neurodegenerative mechanisms that underlie Alzheimer's disease (AD). Indolamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of this pathway, is increased in AD, and it has been hypothesized that blocking this enzyme may slow the progression of AD. In this study, we treated male and female 3xTg-AD and wild-type mice with the novel IDO inhibitor DWG-1036 (80 mg/kg) or vehicle (distilled water) from 2 to 6 months of age and then tested them in a battery of behavioral tests that measured spatial learning and memory (Barnes maze), working memory (trace fear conditioning), motor coordination and learning (rotarod), anxiety (elevated plus maze), and depression (tail suspension test). The 3xTg-AD mice treated with DWG-1036 showed better memory in the trace fear conditioning task and significant improvements in learning but poorer spatial memory in the Barnes maze. DWG-1036 treatment also ameliorated the behaviors associated with increased anxiety in the elevated plus maze and depression-like behaviors in the tail suspension test in 3xTg-AD mice. However, the effects of DWG-1036 treatment on the behavioral tasks were variable, and sex differences were apparent. In addition, high doses of DWG-1036 resulted in reduced body weight, particularly in females. Taken together, our results suggest that the kynurenine pathway is a promising target for treating AD, but more work is needed to determine the effective compounds, examine sex differences, and understand the side effects of the compounds.

Copyright © 2019 Fertan, Stover, Brant, Stafford, Kelly, Diez-Cecilia, Wong, Weaver and Brown.

DOI: 10.3389/fphar.2019.01044 PMCID: PMC6773979 PMID: 31607909

6. J Neurol Sci. 2012 Dec 15;323(1-2):1-8. doi: 10.1016/j.jns.2012.08.005. Epub 2012 Aug 29.
The kynurenine pathway in neurodegenerative diseases: mechanistic and therapeutic considerations.

Tan L(1), Yu JT, Tan L.
Author information: (1)Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, China. dr.tanlan@163.com

The kynurenine pathway (KP), the primary route of tryptophan degradation in mammalian cells, consists of many metabolites including kynurenic acid (KYNA), quinolinic acid (QUIN), 3-hydroxykynurenine (3-HK) and picolinic acid (PIC). The former two are neuroactive, while the latter two are molecules with pro-oxidants and antioxidants properties. These agents are considered to be involved in aging and numerous neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). Several studies have demonstrated that altered kynurenine metabolism plays an important role in the pathogenesis of this group of diseases. The important metabolites and key enzymes show significant importance in those disorders. Both analogs of the neuroprotective metabolites and small molecule enzyme inhibitors preventing the formation of neurotoxic compounds may have potential therapeutic significance. In this review we discuss the mechanistic and therapeutic considerations of KP in aging and the main neurodegenerative diseases and review the updated knowledge in this therapeutic field.

Copyright © 2012 Elsevier B.V. All rights reserved.

DOI: 10.1016/j.jns.2012.08.005 PMID: 22939820 [Indexed for MEDLINE]

7. Brain Behav Immun. 2016 Aug;56:363-77. doi: 10.1016/j.bbi.2016.03.002. Epub 2016 Mar 7.
Indoleamine-2,3-dioxygenase mediates neurobehavioral alterations induced by an intracerebroventricular injection of amyloid-β1-42 peptide in mice.

Souza LC(1), Jesse CR(2), Antunes MS(1), Ruff JR(1), de Oliveira Espinosa D(1), Gomes NS(1), Donato F(1), Giacomeli R(1), Boeira SP(1).
Author information: (1)Laboratório de Avaliações Farmacológicas e Toxicológicas Aplicadas às Moléculas Bioativas, LaftamBio Pampa, Universidade Federal do Pampa, Itaqui, RS, Brazil. (2)Laboratório de Avaliações Farmacológicas e Toxicológicas Aplicadas às Moléculas Bioativas, LaftamBio Pampa, Universidade Federal do Pampa, Itaqui, RS, Brazil. Electronic address: cristianoricardojesse@yahoo.com.br.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by a progressive cognitive decline along with various neuropsychiatric symptoms, including depression and anxiety. Increasing evidence has been proposed the activation of the tryptophan-degrading indoleamine-2,3-dyoxigenase (IDO), the rate-limiting enzyme of kynurerine pathway (KP), as a pathogenic factor of amyloid-beta (Aβ)-related inflammation in AD. In the current study, the effects of an intracerebroventricular (i.c.v.) injection of Aβ1-42 peptide (400pmol/mice; 3μl/site) on the regulation of KP biomarkers (IDO activity, tryptophan and kynurerine levels) and the impact of Aβ1-42 on neurotrophic factors levels were investigated as potential mechanisms linking neuroinflammation to cognitive/emotional disturbances in mice. Our results demonstrated that Aβ1-42 induced memory impairment in the object recognition test. Aβ1-42 also induced emotional alterations, such as depressive and anxiety-like behaviors, as evaluated in the tail suspension and elevated-plus maze tests, respectively. We observed an increase in levels of proinflammatory cytokines in the Aβ1-42-treated mice, which led to an increase in IDO activity in the prefrontal cortex (PFC) and the hippocampus (HC). The IDO activation subsequently increased kynurerine production and the kynurenine/tryptophan ratio and decreased the levels of neurotrophic factors in the PFC and HC, which contributed to Aβ-associated behavioral disturbances. The inhibition of IDO activation by IDO inhibitor 1-methyltryptophan (1-MT), prevented the development of behavioral and neurochemical alterations. These data demonstrate that brain IDO activation plays a key role in mediating the memory and emotional disturbances in an experimental model based on Aβ-induced neuroinflammation.

Copyright © 2016 Elsevier Inc. All rights reserved.

DOI: 10.1016/j.bbi.2016.03.002 PMID: 26965653 [Indexed for MEDLINE]

8. Bioorg Med Chem Lett. 2021 Feb 15;34:127756. doi: 10.1016/j.bmcl.2020.127756. Epub 2020 Dec 24.
Novel BuChE-IDO1 inhibitors from sertaconazole: Virtual screening, chemical optimization and molecular modeling studies.

Zhou Y(1), Lu X(2), Du C(2), Liu Y(2), Wang Y(3), Hong KH(4), Chen Y(5), Sun H(6).
Author information: (1)College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China. Electronic address: zhouy701005@swu.edu.cn. (2)Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China. (3)College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China. (4)Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA. (5)School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China. (6)Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China. Electronic address: sunhaopeng@cpu.edu.cn.

In our effort towards the identification of novel BuChE-IDO1 dual-targeted inhibitor for the treatment of Alzheimer's disease (AD), sertaconazole was identified through a combination of structure-based virtual screening followed by MM-GBSA rescoring. Preliminary chemical optimization was performed to develop more potent and selective sertaconazole analogues. In consideration of the selectivity and the inhibitory activity against target proteins, compounds 5c and 5d were selected for the next study. Further modification of compound 5c led to the generation of compound 10g with notably improved selectivity towards BuChE versus AChE. The present study provided us with a good starting point to further design potent and selective BuChE-IDO1 inhibitors, which may benefit the treatment of late stage AD.

Copyright © 2020 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.bmcl.2020.127756 PMID: 33359445 [Indexed for MEDLINE]

9. J Alzheimers Dis. 2015;43(1):291-302. doi: 10.3233/JAD-140414.
The IDO inhibitor coptisine ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

Yu D(1), Tao BB(2), Yang YY(1), Du LS(1), Yang SS(1), He XJ(1), Zhu YW(1), Yan JK(1), Yang Q(1).
Author information: (1)State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China. (2)Department of Neurosurgery, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.

Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in the kynurenine pathway (KP) of tryptophan catabolism, was recently established as one of the potential players involved in the pathogenesis of Alzheimer's disease (AD). Coptisine is a main pharmacological active constituent of the traditional Chinese medicinal prescription Oren-gedoku-to (OGT) which has therapeutic potential for the treatment of AD. Our recent studies have demonstrated that OGT significantly inhibited recombinant human IDO activity, which shed light on the possible mechanism of OGT's action on AD. Here, we characterized the effects of coptisine in an AD mouse model on the basis of its IDO inhibitory ability. Coptisine was found to be an efficient uncompetitive IDO inhibitor with a Ki value of 5.8 μM and an IC50 value of 6.3 μM. In AβPP/PS1 transgenic mice, oral administration of coptisine inhibited IDO in the blood and decreased the activation of microglia and astrocytes, consequently prevented neuron loss, reduced amyloid plaque formation, and ameliorated impaired cognition. Neuronal pheochromocytoma (PC12) cells induced with amyloid-β peptide 1-42 and interferon-γ showed reduction of cell viability and enhancement of IDO activity, while coptisine treatment increased cell viability based on its reversal effect on the enhanced activity of IDO. In conclusion, our present findings provide further evidence supporting the critical links between IDO, KP, and AD, and demonstrate coptisine, a novel IDO inhibitor, as a potential new class of drugs for AD treatment.

DOI: 10.3233/JAD-140414 PMID: 25079795 [Indexed for MEDLINE]

10. Adv Exp Med Biol. 2003;527:167-76. doi: 10.1007/978-1-4615-0135-0_19.
Quinolinic acid in the pathogenesis of Alzheimer's disease.

Guillemin GJ(1), Williams KR, Smith DG, Smythe GA, Croitoru-Lamoury J, Brew BJ.
Author information: (1)Centre for Immunology, St Vincent's Hospital, Sydney, Australia. g.guillemin@cfi.unsw.edu.au

We propose that the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly quinolinic acid (QUIN), may play an important role in the pathogenesis of Alzheimer's disease (AD). In this study, we demonstrated that after 72 hours amyloid peptide (Abeta) 1-42 induced indoleamine 2,3-dioxygenase (IDO) expression and in a significant increase in production of QUIN by human macrophages and microglia. In contrast, Abeta11-40 and Prion peptide (PrP) 106-126 did not induce any significant increase in QUIN production. We also investigated the potential modulatory effect of QUIN and kynurenic acid (KYNA) on Abeta11-42 and Abeta1-40 aggregation. After 24 and 120 hours, we did not observe any significant difference in the level of aggregation compared to the control (Abeta alone). Abeta has been shown to induce IL1-beta mRNA expression by human foetal astrocytes and macrophages. We demonstrate that QUIN has the same effect. Interestingly, IL-1beta has been found in association with plaques in AD. All together these data imply that QUIN may be, locally, one of the factors involved in the pathogenesis of neuronal damage in AD.

DOI: 10.1007/978-1-4615-0135-0_19 PMID: 15206729 [Indexed for MEDLINE]
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