Resolvin e1 for mitigation of toxicity associated with cyclosporine a

Abstract

A method of reducing the toxicity of calcineurin inhibitors, such as cyclosporine A (CsA), by co-administering a therapeutic amount of an E-series resolvin, such as Resolvin E1. Toxicity is a common problem limiting drugs like CsA. Preventing liver and kidney complications due to CsA will provide immediate health benefits to many patients currently being treated with calcineurin inhibitors, such as CsA. Combination therapy using the CsA in conjunction with the lipid mediator Resolvin E1 was found to provide significant protection against organ toxicity and improved overall outcomes.

Description

TITLERESOLVIN El FOR MITIGATION OF TOXICITY ASSOCIATED WITH CYCLOSPORINE A STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under HT9425-23- 1-0435 awarded by the Department of Defense, and HL 159411 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION1. FIELD OF THE INVENTION

[0002] The present disclosure relates to the toxicity of calcineurin inhibitors and, more particularly, to the use of Resolvin El to reduce toxicity of calcineurin inhibitors such as cyclosporine A.2. DESCRIPTION OF THE RELATED ART

[0003] Cyclosporine A (CsA) is an eleven amino acid peptide antibiotic and calcineurin inhibitor having immunosuppressive properties that is used for organ transplantation and autoimmune diseases, such as severe aplastic anemia (SAA), where it has been shown to limit auto reactive T cells. It is the mainstay therapy for patients with SAA and is also used to treat a variety of autoimmune diseases. It is approved for the prophylactic use of organ rejection in allogeneic kidney, liver, and heart transplants, as well as to prevent bone marrow transplant rejection. It is also used for patients with severe and active rheumatoid arthritis when they become refractory to methotrexate. It is used for treating plaque psoriasis, eye conditions (keratoconjunctivitis sicca and vernal keratoconjunctivitis), and steroiddependent and steroid-resistant kidney disease. It is also used off-label to treat various autoimmune diseases such as atopic dermatitis, ulcerative colitis, connective tissue diseases, and idiopathic thrombocytopenic purpura. The main target in these diseases is an overactive immune response driven by T lymphocytes.

[0004] Toxicity is a common problem limiting use with calcineurin inhibitors like CsA and tacrolimus. Liver and kidney damage are the most reported adverse events with the use of CsA, and this is an important problem because evidence of organ damage limits use and dosage of CsA and, ultimately, reduces its efficacy. This is dangerous as it can halt necessary treatment. Tacrolimus is also associated with nephrotoxicity and can cause neurotoxicity. The precise mechanisms whereby calcineurin inhibitors drive organ damage and toxicity, which can limit their use, are complex and not well understood.123214059. V3-4 / 16 / 26

[0005] Preventing liver and kidney complications due to calcineurin inhibitor use will provide immediate health benefits to many patients currently being treated with calcineurin inhibitors, such as CsA. Accordingly, there is a need in the art for an approach that can provide protection against organ toxicity associated with calcineurin inhibitors, such as CsA. BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a combination therapy for use with calcineurin inhibitors such as CsA that involves the co-administration of the lipid mediator Resolvin El (RvEl) to provide significant protection against organ toxicity. Co-treatment with RvEl does not reduce the effectiveness of CsA and, in fact, improves the efficacy and durability of CsA. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0008] FIG. 1 is a series of graphs showing that RvEl therapy alleviates high-dose (10 mg / kg) CsA-mediated bone marrow inflammation and improves survival in mice with SAA. (A) SAA-induced mice were treated with 10 mg / kg CsA and vehicle or 0.0125 mg / kg RvEl. (B) CsA monotherapy yields ~75% while combinational therapy achieves 100% survival. (C) Absolute bone marrow cellularity and (D) representative hematoxylin and eosin (H&E) staining of the bone marrow. (E) Frequency of T cells, (F) quantity of T regulatory (Treg) and Thl7 populations, and (G) frequency of interleukin- 17A (IL-17A)+CD4+T cells. (H) Principal component analysis (PCA) plot and (I) heatmap of bone marrow cytokines, showing fold change relative to healthy mice and significance relative to SAA mice treated with CsA+RvEl.

[0009] FIG. 2 is a series of graphs showing that combinational therapy with CsA restores resolution of inflammation and hematopoiesis. (A) PCA plot of the polyunsaturated fatty acid metabolome in the bone marrow. (B) Total monohydroxy eicosatetraenoic acids (ETEs), (C) leukotriene B4 (LTB4), and (D) lipoxin A5 (LXA5). (E) Absolute quantity of phosphatidyl serine-coated microparticle (PS-MP)+phagocytes and (F) frequency of dead cells. (G) Frequency of bone marrow hematopoietic stem and progenitor cells (HSPCs), (H) myeloid cells in the blood, (I) kidney, and (J) liver.

[0010] FIG. 3 is a series of graphs showing that combinational therapy of CsA with RvEl limits toxicity and inflammation in the kidney and liver. (A) Heatmap of kidney cytokines, with significance relative to healthy controls. (B) Frequency of dead and apoptotic kidney cells. (C) Representative timelapse of (D) glomerular filtration rate (GFR) in live 223214059. V3-4 / 16 / 26mice. (E) Representative H&E staining of the kidney, demonstrating (1) fibrosis, (2) tubular atrophy, and (3) immune cell infiltration in CsA-treated kidneys only. (F) Heatmap of liver cytokines, with significance relative to healthy controls. (G) Frequency of dead liver cells and (H) serum alanine aminotransferase (ALT). (I) Representative H&E staining of the liver, showing (1) hepatocyte loss, (2) fibrosis, and (3) immune cell infiltration.

[0011] FIG. 4 is a series of graphs showing that RvEl therapy alleviates low-dose (3 mg / kg) CsA-mediated bone marrow inflammation, toxicity, and improves survival. (A) SAA-induced mice were treated with 3 mg / kg CsA and vehicle or 0.0125 mg / kg RvEl. (B) Low-dose CsA monotherapy yields ~35% while combinational therapy achieves ~75% survival. (C) Frequency of T cells and (D) quantity of bone marrow IL-17A. (E) Heatmap and (F) PC A plot of inflammatory bone marrow cytokines. (G) Absolute number of myeloid cells in the kidney and (H) liver.

[0012] FIG. 5 is a series of graphs showing that RvEl requires the receptor ChemR23 on immune cells, and particularly T cells, to alleviate bone marrow inflammation. (A) Schematic of bone marrow transplantation in which ChemR23 knockout (KO) host mice that received wildtype (WT) bone marrow have ChemR23 expression (dark gray) on immune cells only. WT mice that receive ChemR23 KO bone marrow have ChemR23 only on non-immune cells (dark gray). (B) Frequency of Sea- 1, which is unregulated during inflammation, on lineage (B220, Teri 19, CD1 lb, Gr-1, CD3) negative cells. (C) Frequency of interferon gamma (IFNy) and (D) IL-2 in CD4+T cells. (E) Frequency of IFNy+CD8+T cells. (F) Mice were induced with SAA using WT or ChemR23 KO splenocytes and then treated with CsA and vehicle or RvEl. Such experimental setup creates a condition in which T cells are selectively WT or KO. (G) Absolute number and frequency (H) of Thl7 / Treg subpopulations.

[0013] FIG. 6 is a series of graphs showing that dual-API (active pharmaceutical ingredient) formulation of CsA and RvEl alleviates CsA-mediated bone marrow inflammation, toxicity, and improves survival. (A) SAA-induced mice were treated with single- API (CsA; 10 mg / kg) or dual-API (CsA and RvEl; 10 mg / kg and 0.02 mg / kg, respectively) formulations. (B) Single-API CsA therapy yields 60% survival, while dual-API with CsA and RvEl yields 86% survival. (C) Absolute bone marrow cellularity. (D) Frequency of T cells and (E) Thl7 / Treg subpopulations. (F) Absolute number of phagocytes that have engulfed PS-MPs and (G) frequency of dead cells. (H) HSPCs per leg and (I) WBCs. (J) Myeloid cells in the kidney and (K) liver.323214059. V3-4 / 16 / 26DETAILED DESCRIPTION OF THE INVENTION

[0014] Referring to the figures, wherein like numerals refer to like parts throughout, the present invention comprises the co-treatment of a patient receiving cyclosporine A (CsA) with Resolvin El (RvEl) to protect against organ toxicity. CsA is used to suppress T Cells and is a first-line therapy for conditions such as severe aplastic anemia (SAA). CsA is the standard of care for SAA, but CsA therapy has high rates of refractoriness, failure, and often leads to secondary malignancies, such as myelodysplastic syndrome and acute myeloid leukemia. CsA also has a narrow therapeutic index, which requires close monitoring as the therapy also exhibits dose-associated toxicity. Patients undergoing CsA therapy require close monitoring, including daily blood tests, due to the drug’s known nephrotoxic and hepato toxic effects. Despite the widespread use of CsA, the mechanisms underlying its off-target effects and toxicity remain poorly understood.

[0015] CsA therapy was found to induce an influx of inflammatory immune cells and increased cell death in the kidney and liver, which is sustained 30 days post-treatment. CsA also caused prolonged reductions in platelets, alongside abnormal HSPC counts. The specialized proresolving mediator, Resolvin El (RvEl), enhances survival and the resolution of inflammation in SAA. In the present invention, it is further shown that RvEl mitigates chronic inflammation and toxicity associated with CsA and ultimately restores tissue homeostasis. RvEl normalizes the CsA-mediated influx of immune cells, restores HSPC balance, and alleviates toxicity.Remarkably, RvE 1 also increased the therapeutic efficacy of low-dose (3 mg / kg) CsA, improving survival rates from 35% to 75%, while simultaneously reducing toxicity. Mechanistically, RvEl directly acts on T cells through its receptor ChemR23.

[0016] These findings highlight the limitations of current immunosuppressants in restoring tissue homeostasis and demonstrate the therapeutic benefit of augmenting resolution pathways. While described herein in the context of CsA, the present invention is applicable to calcineurin inhibitors as a class, including tacrolimus and related agents that suppress T cell activation through inhibition of calcineurin-nuclear factor of activated T cells (NF AT) pathway. The NF AT family of transcription factors regulate T cell activation and differentiation.Calcineurin inhibition is associated with impaired resolution of inflammation, persistent immune activation, and toxicity. Accordingly, the administration of a specialized pro-resolving mediator (SPM), such as RvEl, restores resolution pathways, limits aberrant inflammation, and improves therapeutic outcomes.

[0017] In addition to RvEl, it should be recognized to those skilled in the art that other E-series resolvins may provide similar efficacy due to their shared ability to promote423214059. V3-4 / 16 / 26resolution of inflammation, enhance clearance of cellular debris, and restore tissue homeostasis, and thus also improve the efficacy of any adjunct calcineurin inhibitor treatment. These mediators may act through overlapping or distinct receptors, including ChemR23 and relegated pro-resolving pathways, to modulate immune cell function. The present invention may be administered orally or intravenously with an anhydrous water-miscible formula, with a bioavailability conversion based on the route of administration and the chosen oil or water base formula as an oil-based formula will have a lower bioavailability than a water-based formula. Orally dosed medicine will also have a lower bioavailability than injectable medicine. Any stability issues with RvEl can be overcome with adjustments to the formulation.

[0018] The present invention was demonstrated using an established animal model for immunosuppressant drug evaluation. More specifically, mice were induced to have severe aplastic anemia (SAA), a fatal disease driven by autoreactive T Cells. SAA-induced mice were treated with 3 mg / kg CsA or 10 mg / kg CsA starting day 7, when the bone marrow is already hypocellular. 3 mg / kg CsA increases survival to approximately 35%, while 10 mg / kg CsA enhances survival to ~75%. Tissue from surviving mice was harvested day 45.Surviving mice exhibited prolonged abnormalities in their blood and bone marrow, even 30 days after therapy has been completed. This included a stark expansion in the number of inflammatory Thl7 T cells, which are often implicated in autoimmune disease, and a marked decrease in Tregs, which are essential for restoring immune homeostasis. Inflammatory cytokines that are known drivers of disease, including TNFa, IFNy, and IL-2 are highly upregulated post-CsA therapy. Prolonged inflammation and immune dysregulation may contribute to relapse in SAA patients. Adult patients are typically prescribed doses of CsA in the 2-9 mg / kg per day range when administered intravenously. Oral doses range from 10-50 mg / kg and patients are instructed to take the medication two time per day, 12 hours apart. Monitoring of blood pressure, serum creatinine, and CsA levels is often required.

[0019] CsA-treated mice exhibited impairments in inflammation resolution. CsA binds cyclophilins, cytoplasmic proteins in many cells, and this complex binds to and inhibits calcineurin. This prevents translocation of specific transcription factors, like NF AT, that drive expression of genes that encode cytokines and growth factors that support T lymphocyte activation. Tacrolimus similarly inhibits calcineurin-NFAT. However, calcineurin-NFAT is required for various biological processes, including those essential for inflammation resolution and peripheral tissue function. Calcineurin-NFAT is essential for cyclooxygenase (COX-2), an enzyme necessary to produce lipid mediators that promote the 523214059. V3-4 / 16 / 26resolution of inflammation. COX-2 is also constitutively expressed in the kidney, where it regulates renal blood flow and tissue function. Low and high-dose CsA led to a substantial decrease in COX-2 expression. The clearance of dead and dying cells by phagocytes (i.e. efferocytosis) is an essential part of the resolution of inflammation, and calcineurin is used in various stages of phagocytosis. Phagocytes from CsA-treated mice exhibited a substantial decrease in their ability to clear PS-MPs, which mimic dead cells. This correlated with an increase in dead cells accumulating in the bone marrow, which can contribute to chronic inflammation and impaired resolution of inflammation. Calcineurin-NFAT activation in granulocyte-monocyte multipotent progenitors (MPPGM) normally restrains their proliferation. Inhibition of calcineurin, such as through CsA or tacrolimus, removes this proliferative checkpoint and enhances MPPGMproliferation. Our observation of a persistent expansion of MPPGMand expansion of myeloid populations in the periphery following CsA therapy aligns with this mechanism, demonstrating that systemic calcineurin inhibition can indirectly drive emergency myelopoiesis and peripheral toxicity.

[0020] CsA was also found to drive inflammation and toxicity in the kidney and liver. CsA-treated mice exhibited an increase in serum creatinine and ALT, common readouts of organ damage for the kidney and liver, respectively. CsA-treated mice also exhibited an influx of immune cells and increased cell death, suggesting increased inflammation and organ damage.

[0021] SAA patients who received 6 months of immunosuppressive therapy with CsA also exhibit skewed T cell responses, including enhanced Thl7 programs. CsA-treated patients also exhibit continuous impairments in inflammation resolution responses, including a decrease in enzymes essential for lipid mediator synthesis such as PTGS2 (encodes for COX-2). Furthermore, SAA patients exhibit an expansion of myeloid progenitors in the bone marrow. Such data demonstrates that the long-term dysregulation in immune homeostasis and impaired inflammation resolution post-CsA therapy observed in the preclinical murine model is also supported by SAA patient data.

[0022] Referring to FIG. 1, the co-administration of RvEl with CsA enhanced recovery, leading to an increase in survival and restoration of bone marrow cellularity.Furthermore, combinational therapy restored T cell homeostasis and reduced inflammatory marrow cytokines. These data strongly support the rationale of combining potent immunosuppression with pro-resolving therapy to effectively manage inflammation while simultaneously promoting resolution of inflammation and restoration of tissue homeostasis.623214059. V3-4 / 16 / 26

[0023] Referring to FIG. 2, combinational therapy also restored the polyunsaturated fatty acid metabolome by decreasing pro-inflammatory lipid precursors (monohydroxy ETEs) and pro-inflammatory LTB4, while increasing pro-resolving mediators like LXA5.Combinational therapy also improved the clearance of dead and dying cells, leading to a normalization in the frequency of dead cells in the bone marrow. Combinational therapy with RvEl also restored hematopoiesis, decreasing MPPGMand myeloid cells in peripheral tissues.

[0024] Referring to FIG. 3, combinational therapy with RvEl limited the toxicity and inflammation in the kidney and liver. Inflammatory cytokines and dead cells in the kidney and liver were both reduced by RvEl combinational therapy. Combinational therapy also alleviated kidney and liver damage, as evidenced by the improved GFR, ALT levels, and improved histological appearance of both tissues, relative to CsA alone. Such data illustrates the remarkable capability of RvEl to alleviate CsA-mediated toxicity.

[0025] Referring to FIG. 4, RvEl also enhanced the efficacy of low-dose (3 mg / kg) CsA therapy. Combinational therapy was capable of improving survival from 35% to 75%, decreasing T cell populations, reducing IL-17A and other inflammatory cytokines that drive disease (including CCL5 and IL-2). RvEl also alleviated low-dose CsA-mediated toxicity.

[0026] Referring to FIG. 5, mechanistically, RvEl required ChemR23 expression of immune cells, but not non-immune, to reduce inflammation and T cell activation. More specifically, ChemR23 on T cells was required for RvEl to restore Thl7 / Treg balance, demonstrating the direct, cell-specific effect of RvEl therapy.

[0027] Referring to FIG. 6, the present invention provides a pharmaceutical composition comprising 10 mg / kg CsA and 0.02 mg / kg RvEl formulated together as a dualAPI composition within a pharmaceutically acceptable carrier. The dual- API formulation improved survival over standard single- API (CsA) formulation. Dual- API formulation also restored bone marrow cellularity, T cell homeostasis, and hematopoiesis. In other embodiments, the calcineurin inhibitor and RvEl may be formulated separately and administered concurrently or sequentially. The formulation may comprise lipid-based carriers, nanoparticles, emulsions, or other delivery systems suitable for enhancing the stability, bioavailability, and therapeutic activity of RvEl. The relative amounts of each agent may be selected to optimize efficacy, including improved inflammation resolution, enhanced hematopoietic recovery, and reduced organ toxicity.

[0028] Thus, in an established model of SAA, a fatal form of fulminant bone marrow failure, administration of CsA can protect against mortality, but causes toxicity. Treatment with CsA in conjunction with RvEl provides protection against both liver and kidney damage 723214059. V3-4 / 16 / 26as demonstrated by GFR, ALT levels, and histological pathology. The concurrent therapy utilizing CsA and RvEl according to the present invention therefore demonstrates utility for both improving overall outcomes (survival) and protecting against toxicity.823214059. V3-4 / 16 / 26

Claims

CLAIMSWhat is claimed is:

1. A method of treating a patient, comprising administering a therapeutically effective amount of a calcineurin inhibitor and administering a therapeutically effective amount of an E-series resolvin.

2. The method of claim 1, wherein the therapeutically effective amount of an E-series resolvin is a therapeutically effective amount of Resolvin El.

3. The method of claim 1, wherein the therapeutically effective amount of the immunosuppressive agent is a therapeutically effective amount of a calcineurin inhibitor.

4. The method of claim 3, wherein the therapeutically effective amount of the calcineurin inhibitor is a therapeutically effective amount of cyclosporine A.

5. The method of claim 2, wherein the therapeutically effective amount of Resolvin El comprises a dosage of between 0.0125 mg / kg and 0.05 mg / kg.

6. The method of claim 4, wherein the therapeutically effective amount of cyclosporine A comprises a dosage of between 3 mg / kg and 50 mg / kg.

7. The method of claim 1, wherein the patient has a disease selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, psoriasis, scleroderma, systemic lupus erythematosus, Grave’s disease, Hashiomoto’s thyroiditis, multiple sclerosis, Guillan-Barre syndrome, Sjogren’s disease, hemolytic anemia, immune thrombocytopenia, idiopathic thrombocytopenic purpura, aplastic anemia, severe aplastic anemia, myelodysplastic disease, myelofibrosis, Fanconi anemia, Diamond-Blackfan anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, severe congenital neutropenia, and congenital amegakaryocytic thrombocytopenia.

8. The method of claim 1, wherein the patient has received an organ transplant.

9. The method of claim 1, wherein the step of administering a therapeutically effective amount of a calcineurin inhibitor and the step of administering a therapeutically effective amount of an E-series resolvin occur contemporaneously.

10. A pharmaceutical composition, comprising:a therapeutically effective amount of an immunosuppressive agent; anda therapeutically effective amount of an E-series resolvin.

11. The pharmaceutical composition of claim 10, wherein the therapeutically effective amount of E-series resolvin comprises a therapeutically effective amount of Resolvin El.923214059. V3-4 / 16 / 2612. The pharmaceutical composition of claim 10, wherein the therapeutic amount of the immunosuppressive agent comprises a therapeutically effective amount of a calcineurin inhibitor.

13. The pharmaceutical composition of claim 12, wherein the therapeutic amount of the calcineurin inhibitor comprises a therapeutically effective amount of cyclosporine A.

14. The pharmaceutical composition of claim 11, wherein the therapeutically effective amount of Resolvin El comprises a dosage of between 0.0125 mg / kg and 0.05 mg / kg.

15. The pharmaceutical composition of claim 13, wherein the therapeutically effective amount of cyclosporine A comprises a dosage of between 3 mg / kg and 50 mg / kg.1023214059. V3-4 / 16 / 26

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