Formulation and its use in blood storage and transfusion

Abstract

To provide methods for prolonging viability of blood and enhancing efficacy of transfusion.SOLUTION: Provided is a method for preparing a blood sample in which the onset of storage lesion of red blood cells ex vivo in the blood sample during storage is delayed or the progression thereof is retarded, the method comprising a step of mixing the sample with a solution comprising an effective amount of a curcuminoid, wherein the concentration of the curcuminoid in the sample ranges from about 0.1 to about 1 mM.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application is a divisional application of U.S. Patent No. 11,484,493, a continuation - in - part application of U.S. Application No. 18 / 051,266 filed on October 31, 2022, which is a continuation of International Application No. PCT / US2021 / 058611 filed on November 9, 2021, which claims the benefit of priority of Provisional Application No. 63 / 111,160 filed on November 9, 2020, Provisional Application No. 63 / 161,696 filed on March 16, 2021, and Provisional Application No. 63 / 235,880 filed on August 23, 2021. The entire disclosures of all of these are hereby incorporated by reference in their entirety.

[0002] Technical Field Disclosed herein are formulations containing curcuminoids for blood storage and transfusion. Methods for extending the viability of blood and methods for enhancing the effectiveness of transfusion are also provided.

Background Art

[0003] The delivery of many therapeutic agents remains problematic due to bioavailability issues that prevent achieving therapeutic levels systemically or locally at the target site. Insufficient bioavailability results from a variety of factors, including low solubility, low uptake from the intestine, and rapid elimination from the circulation (first - pass limitation) due to rapid degradation by the liver. As a result, many promising therapeutic agents with appropriate activity cannot be delivered directly in an effective manner to affected areas such as the lungs, arthritic joints, inner ear, paranasal sinuses, etc. On the other hand, oral or IV administration routes often do not provide appropriate therapeutic levels at the target local site.

[0004] Nitric oxide (NO) has known systemic benefits, including the ability to reverse inflammation, prevent and reverse endothelial dysfunction, repolarize activated macrophages, inactivate activated platelets, protect / restore the endothelial glycocalyx of blood vessels, and possess antibacterial / antiviral activity. Inflammatory injury enhances the production of reactive oxygen species (ROS) that drive the onset and persistence of endothelial dysfunction. Enhanced levels of ROS initiate multiple events that cause dysregulation of the vascular system. ROS degrades the glycocalyx, which results in loss of vascular integrity, enhanced access and adhesion of circulating cells (monocytes, erythrocytes, neutrophils, and platelets) to the endothelial surface, loss of shear stress-mediated NO production, and loss of superoxide dismutase that limits ROS. When ROS is enhanced, endothelial nitric oxide synthase (eNOS) ceases NO production in the endothelium and more ROS actually begins to be generated. Increasing NO levels either by direct supplementation or enhanced production can dramatically reverse this cycle and restore vascular homeostasis. However, systemic delivery of NO is challenging because the NO molecule has a short lifespan and limited access to the circulation.

[0005] CD38 plays a central role in numerous organ systems and tissues. CD38 is a major enzyme for the degradation of nicotinamide adenine dinucleotide (NAD) in mammalian cells. Reduced NAD levels are closely related to metabolic syndrome and age-related diseases. In the vascular system, in terms of the treatment of endothelial dysfunction, reducing CD38 levels significantly alleviated angiotensin II (Ang II)-induced vascular remodeling in mice, as shown by reduced blood pressure; it decreased vascular medial thickness, the media-to-lumen ratio, and collagen deposition; and restored elastin expression. Reducing CD38 levels significantly alleviated Ang II-induced vascular aging by suppressing the biosynthesis, secretion, and internalization of senescence-associated small extracellular vesicles (SA-sEVs), which promote the aging of adjacent non-damaged VSMCs. Furthermore, the protective effect of CD38 deficiency against VSMC aging was related to the restoration of lysosomal dysfunction, particularly regarding the maintenance of sirtuin-mediated mitochondrial homeostasis and the activation of the mitochondria-lysosome axis in VSMCs. Therefore, CD38 activity and the resulting related intracellular NAD reduction are essential for Ang II-induced VSMC (vascular smooth muscle cell) aging and vascular remodeling, and thus, CD38 and its related intracellular NAD reduction are essential for Ang II-induced VSMC aging and vascular remodeling that can cause cardiovascular disease and renal failure. Similar results have been reported regarding the centrality of CD38 / NAD+ activity in other disease states, including osteoarthritis, lupus, neuropathy, and age-related physical and cognitive decline. The central role of CD38 results, at least in part, from any of a number of inflammatory triggers, including the accumulation of senescent cells, which causes its overexpression in activated immune cells such as macrophages and microglial cells and on the activated intima of all blood vessels.

Prior Art Documents

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Summary of the Invention

[0008] This patent document provides a transdermal formulation for the delivery of one or more active agents to a subject in the treatment of a disease or condition associated with elevated CD38 levels that in turn cause a decrease in NAD+ levels. Low NAD+ promotes mitochondrial dysfunction and limits the activity of sirtuins. Sirtuin activity is necessary to maintain NO production in the endothelium and to shut down oxidative and nitrosative stress imposed by activated immune cells such as the M1 population of macrophages and microglial cells. CD38 is expressed on the surface of macrophages, microglial cells and other immune-activated leukocytes as well as on the intima of blood vessels. Increased levels of CD38 are caused by diseases or conditions such as trauma, inflammation, infection, radiation, chemotherapy, and an excess of senescent cells. Enhanced levels of CD38, in turn, promote further cellular senescence, inflammation and oxidative stress, which creates a cycle of persistent inflammation that is difficult to break and further stimulates more CD38 production.

[0009] The transdermal formulations disclosed herein result in a decrease in CD38 ectoenzyme activity, such as NADase activity; thereby enhancing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. By doing so, this approach provides both new methods of preventing and treating a variety of diseases, including those resulting from endothelial dysfunction following acute or chronic inflammatory injury, age-related decline in physical and cognitive skills, age-related cardiac tissue changes, vascular hypertrophy, osteoarthritis, peripheral neuropathy and long COVID. Furthermore, this approach addresses the negative consequences of CD38 overproduction with respect to the ability of stem cells to differentiate into mature cells, which is at least partially responsible for chronic inflammatory anemia and loss of effectiveness of native stem cells and stem cell therapies to repair damaged tissue.

[0010] The formulations disclosed in this specification for local delivery result in direct delivery to both plasma and blood cells and thus avoid first-pass elimination by the gastrointestinal tract and liver. Furthermore, red blood cells loaded with the active agent can function as a stealth delivery vehicle that is not noticed by the immune system and the liver. Additionally, uptake of the delivery by activated CD38-loaded (M1 population) circulating macrophages and other immune-active white blood cells can result in rapid re-polarization (return to the M2 population), which rapidly decreases the levels of CD38 and the production of inflammatory cytokines. This strategy should also reduce the senescent cell burden. By combining a potent anti-inflammatory agent (curcuminoid) that re-polarizes M1 macrophages (and thus reduces CD38 levels) and promotes sirtuin activity, with senolytic agents (e.g., quercetin, apigening, fisetin, luteolin), the prospect of disrupting the inflammatory cycle in chronic diseases and promoting tissue repair by enabling stem cell differentiation and proliferation is significantly maximized.

[0011] One aspect of this patent document is a transdermal formulation for enhancing systemic nitric oxide (NO) via the CD38 / NAD+ pathway, comprising: (a) an effective amount of one or more curcuminoids and an effective amount of at least one senolytic flavonoid, wherein the curcuminoid comprises one or more of, for example, curcumin, demethoxycurcumin, and bisdemethoxycurcumin, and the flavonoid from the senolytic agent comprises one or more of quercetin, fisetin, apigenin, luteolin, and rapamycin; (b) a sufficient amount of a polyol to dissolve the effective amount of curcuminoid and flavonoid; and optionally (c) a fatty acid, wherein the polyol and the fatty acid are in a ratio in the range of about 10:1 to about 50:1 by weight, comprising Disclosed is a transdermal formulation in which the amounts of polyol and fatty acid are selected such that an effective amount of the active agent(s) is delivered transdermally or transmucosally after topical application.

[0012] In some embodiments, the curcuminoid is curcumin. In some embodiments, the other active agent is at least any one of the following flavonoids: quercetin, apigenin, fisetin, luteolin, or another small bio - compatible senolytic drug such as rapamycin. In some embodiments, the formulation comprises a combination of a curcuminoid and a senolytic drug such as quercetin and apigenin.

[0013] The formulations and kits disclosed herein can be applied to the treatment of various conditions or diseases. Examples include transdermal treatment of acute and chronic inflammatory conditions, percutaneous prevention and reversal of endothelial dysfunction, reduction of the risk of cytokine storm or cytokine storm phenomenon (release of abnormal or higher - than - normal levels of inflammatory cytokines) in patients having conditions that cause underlying endothelial dysfunction (such as COVID - 19 or SARS - CoV - 2 infection), topical treatment of dermatological conditions, anti - aging skin treatment, ophthalmological conditions, aerosol - based treatment of lung conditions, topical treatment of infections, treatment of red blood cells to improve storage characteristics and reverse storage damage, thus improving the safety and efficacy of stored red blood cells, loading therapeutic agents onto red blood cells prior to transfusion, stabilizing red blood cells via intravenous (IV) delivery of an RBC stabilizer, IV intervention for treating cytokine storm phenomenon and related acute inflammatory crises, aerosol treatment and prevention of acute respiratory distress syndrome (ARDS) and other conditions that destroy lung tissue by excessive oxidative damage and subsequent inflammation, and loading therapeutic agents onto medical sponges for use, for example, in the ear, nose, mouth, rectum, and vagina. Sustained transdermal systemic delivery can also be achieved to utilize the therapeutic potential of NO for various chronic diseases and conditions.

[0014] The formulation further includes polyphenols, flavonoids, stilbenoids, seco-steroids, and other phytochemicals and natural products that promote the formation of NO. In some embodiments, the NO booster includes at least one of the agents selected from the group consisting of polyphenols, flavonoids, stilbenoids, seco-steroids, and natural products. In some embodiments, the curcuminoid includes at least one of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, and vitamin D.

[0015] In some embodiments, the polyol is polyethylene glycol having a molecular weight in the range of 100 to about 1000. In some embodiments, the fatty acid is myristic acid.

[0016] In some embodiments, the formulation provides a sustained release of an NO releasing agent from an NO booster or an NO precursor (e.g., an S-nitrosothiol-containing molecule) over a period of approximately about 8, about 10, about 15, about 20, about 24, or about 48 hours.

[0017] In some embodiments, the formulation further includes a thickening agent that maintains the transdermal formulation in a semi-solid or solid form. In some embodiments, the thickening agent is petrolatum, cocoa butter, or a polyalkylene glycol with an MW exceeding 2 kDa.

[0018] Another aspect of the present invention provides a kit or a transdermal delivery system incorporating the transdermal formulation disclosed herein. In some embodiments, the transdermal delivery system is a kit or a nebulizer.

[0019] Another aspect of the present invention provides a method for preparing a blood sample to slow the occurrence or progression of ex vivo red blood cell storage damage in a blood sample during storage as compared to an untreated reference. The method includes the step of mixing the red blood cells with a solution containing an effective amount of curcuminoid and optionally a flavonoid. In some embodiments, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the red blood cells remain viable after 10, 15, 20, 30, 40, 50, 60, 80 days or longer.

[0020] In some embodiments, the effective amount is selected such that inflammation, hemolysis, or microparticle formation is reduced by at least 20% as compared to an untreated reference over the same period. In some embodiments, the effective amount is selected such that the adenosine triphosphate (ATP) level and / or the 2,3-diphosphoglycerate (2,3-DPG) level is reduced by less than 5%, less than 10%, less than 20%, less than 30%, or less than 40% over a specified period. In some embodiments, the effective amount is selected such that the adenosine triphosphate (ATP) level and / or the 2,3-diphosphoglycerate (2,3-DPG) level is more than 5%, more than 10%, more than 20%, more than 30%, or more than 40% higher over the same specified period as compared to an untreated reference or control over the same period. In some embodiments, the specified period is 10, 15, 20, 30, 40, 50, 60, 80 days, or longer.

[0021] In some embodiments, the sample is maintained at a temperature in the range of about -30°C to about 37°C. The sample is collected from a healthy individual.

[0022] In some embodiments, the concentration of the curcuminoid in the solution ranges from about 1 mM to about 10 M, about 10 mM to about 10 M, about 100 mM to about 10 M, about 1 M to about 10 M, or about 5 M to about 10 M before mixing with red blood cells. In some embodiments, the solution has a pH in the range of about 5.5 to about 7.0, about 6 to about 7.0, or about 6.5 to about 7.0. In some embodiments, the solution further contains a fatty acid. In some embodiments, the fatty acid is myristic acid.

[0023] In some embodiments, the concentration of the curcuminoid in the sample ranges from about 0.2 to about 20, about 0.5 to about 10, about 1 to about 10, or about 1 to about 5 mM in the sample.

[0024] In some embodiments, the method includes a step of depleting oxygen and / or carbon dioxide in the sample.

[0025] In some embodiments, the solution contains a solvent selected from the group consisting of polyethylene glycol, ethanol, acetone, ethyl acetate, acetonitrile, DMF, THF, DMSO, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, and any combination thereof. In some embodiments, the solvent contains polyethylene glycol. In some embodiments, the solvent consists essentially of PEG. In some embodiments, the solvent and the curcuminoid are in a ratio in the range of about 5:1 to about 40:1, about 5:1 to about 30:1, about 5:1 to about 20:1, or about 5:1 to about 10:1 by weight. In some embodiments, the curcuminoid is curcumin. In some embodiments, the solution is substantially free of water.

[0026] Another aspect of the invention is an ex vivo blood sample comprising red blood cells and an agent comprising a curcuminoid and optionally a flavonoid in a solvent, wherein the agent is in an amount effective to extend the viability of the red blood cells in the sample or slow the occurrence or progression of storage damage of the red blood cells ex vivo, and the solvent is sufficient to maintain the agent in a dissolved state in the sample. The ex vivo blood sample can be provided by the methods described herein. In some embodiments, the concentration of the agent in the sample ranges from about 0.2 to about 20, about 0.5 to about 10, about 1 to about 10, or about 1 to about 5 mM. In some embodiments, the agent comprises curcumin. In some embodiments, the agent comprises curcumin and at least a flavonoid.

[0027] A related aspect provides a method of transfusing blood to a subject in need thereof. The method comprises the step of transfusing the blood sample disclosed herein to the subject.

[0028] Another aspect is a method of enhancing the effectiveness of a blood transfusion comprising the step of administering a transdermal formulation to a subject in need thereof, wherein the transdermal formulation (a) an effective amount of a curcuminoid selected from the group consisting of curcumin, demethoxycurcumin, and bisdemethoxycurcumin; (b) an amount of a polyol sufficient to dissolve the effective amount of the curcuminoid; and optionally (c) a fatty acid, wherein the polyol and the fatty acid are in a ratio in the range of about 10:1 to about 50:1 by weight and the amounts of the polyol and the fatty acid are selected such that an effective amount of an NO booster is transdermally delivered upon administration.

[0029] In some embodiments, the curcuminoid is curcumin. In some embodiments, the transdermal formulation further comprises at least a flavonoid selected from quercetin, apigenin, fisetin, luteolin, and rapamycin. In some embodiments, the transdermal formulation comprises curcumin and further comprises at least one of quercetin and apigenin. In some embodiments, the transdermal formulation comprises from about 3 wt% to about 10 wt% of a curcuminoid.

[0030] In some embodiments, the transdermal formulation further comprises at least one flavonoid, and the curcuminoid and the at least one flavonoid are in a ratio in the range of about 1:1 to about 10:1. In some embodiments, the transdermal formulation comprises quercetin and apigenin in a ratio in the range of about 1:1 to about 10:1.

[0031] In some embodiments, the transdermal formulation comprises myristic acid. In some embodiments, the polyol is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, and glycerol. In some embodiments, the polyol is polyethylene glycol having a molecular weight in the range of 200 to about 600. In some embodiments, the polyol and the curcuminoid are in a ratio in the range of about 8:1 to about 12:1.

[0032] In some embodiments, the blood is an ex vivo blood sample disclosed herein and shows an extension of the viability of red blood cells or a delay in the occurrence or progression of storage damage. In some embodiments, the transdermal formulation is administered prior to blood transfusion.

[0033] Aspect [1] A method for preparing a blood sample in which the occurrence of ex vivo storage damage of red blood cells during storage is delayed or the progression thereof is retarded, the method comprising the step of mixing the sample with a solution containing an effective amount of a curcuminoid. The method of [1], wherein the effective amount is selected such that red blood cells remain substantially viable for at least about 60 days. [3] The method of [1] or [2], wherein the effective amount is selected such that inflammation, hemolysis, or particulate formation is reduced by at least 20% compared to an untreated reference over the same period. [4] The method of any one of [1] to [3], wherein the concentration of curcuminoid in the sample ranges from about 0.1 to about 1 mM. [5] The method of any one of [1] to [4], wherein the solution further contains myristic acid. [6] The method of any one of [1] to [5], wherein the adenosine triphosphate (ATP) level and / or 2,3-diphosphoglycerate (2,3-DPG) level in the sample decreases by less than 20% over a 50-day period. [7] The method of any one of [1] to [6], wherein the solution contains a solvent selected from the group consisting of polyethylene glycol, ethanol, acetone, ethyl acetate, acetonitrile, DMF, THF, DMSO, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, and any combination thereof. [8] The method of any one of [1] to [7], wherein the solvent contains polyethylene glycol (PEG). [9] The method of any one of [1] to [7], wherein the solvent consists essentially of PEG.

[10] The method of any one of [1] to [9], wherein the solvent and the curcuminoid are in a ratio in the range of about 5:1 to about 20:1 by weight.

[11] The method of [9], wherein the PEG has a molecular weight in the range of 200 to about 600.

[12] The method of [9], wherein the PEG and the curcuminoid are in a ratio in the range of about 8:1 to about 30:1.

[13] The method of any one of [1] to

[12] , wherein the sample and the solution are in a ratio in the range of about 8000:1 to about 3000:1 by weight.

[14] The method of any one of [1] to

[13] , wherein the curcuminoid is curcumin.

[15] The method according to any one of [1] to

[14] , wherein the curcuminoid is in the range of about 3% to about 15% by weight in the solution.

[16] The method according to any one of [1] to

[15] , wherein the solution is substantially free of water.

[17] The method according to any one of [1] to

[16] , wherein the solution further comprises at least a flavonoid.

[18] The method according to

[17] , wherein the flavonoid is selected from quercetin, apigenin, fisetin, luteolin, and rapamycin.

[19] An ex vivo blood sample prepared by the method according to any one of [1] to

[18] .

[20] A method of transfusing blood to a subject, comprising the step of transfusing the blood sample of

[19] to the subject in need thereof.

[21] A method for preparing a blood sample in which the occurrence of ex vivo storage damage of red blood cells during storage is delayed or its progression is retarded, comprising the step of mixing a solution containing an effective amount of curcuminoid with the blood sample, wherein the concentration of curcuminoid in the sample is in the range of about 0.1 to about 1 mM.

[22] A method for preparing a blood sample in which the occurrence of ex vivo storage damage of red blood cells during storage is delayed or its progression is retarded, comprising the step of mixing a solution containing an effective amount of curcuminoid with the sample, wherein the effective amount is selected such that the red blood cells remain substantially viable for at least about 60 days.

[23] An ex vivo blood sample comprising blood mixed with a solution, wherein the solution contains an effective amount of curcuminoid for delaying the occurrence of storage damage of red blood cells or retarding its progression, and the concentration of curcuminoid in the blood sample is in the range of about 0.1 to about 1 mM.

[24] Use of the ex vivo blood sample according to claim 15 for treating a disease or condition, wherein the ex vivo blood sample comprises blood mixed with a solution, and the solution contains an effective amount of curcuminoid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] DETAILED DESCRIPTION OF THE INVENTION Aspects of the present disclosure provide transdermal formulations and methods for enhancing systemic NO levels. Formulations and methods are also provided for enhancing ex vivo erythrocyte viability and improving the effectiveness of transfusions. Since NO has known systemic benefits, including the ability to reverse inflammation, prevent and reverse endothelial dysfunction, reprogram activated macrophages, inactivate activated platelets, protect the endothelial lining of blood vessels, and possess antibacterial / antiviral activity, the formulations of the present disclosure can be applied to the treatment of various diseases and conditions. In some aspects, the treatment is based on the transdermal delivery of an agent that can improve nitric oxide production in the endothelium or directly release NO from a suitable S-nitrosothiol-containing molecule. The formulations disclosed herein can promote the production of NO in the endothelium and reduce ROS levels through a decrease in CD38 levels.

[0035] The transdermal formulations described herein have the advantage of reduced side effects compared to conventional oral administration of active agents. For example, oral curcumin can cause GI disturbances (hypermotility, increased acid production in the stomach), especially when used chronically. Delivery of the agent on the skin or mucosa bypasses the GI tract and achieves the desired therapeutic effect while minimizing side effects.

[0036] The following text may refer to or exemplify specific aspects of the formulations, kits, or methods for the treatment or prevention of diseases, but is not intended to limit the scope of the formulations, kits, or methods to such specific references or examples. Various modifications can be made by those skilled in the art taking into account practical and economic considerations such as the specific form of the formulation and the amount or frequency of administration of the formulation for treating or preventing a disease or condition.

[0037] As used herein, the articles "a" and "an" refer to "one or more" or "at least one" unless otherwise indicated. That is, any reference to an element or component of an embodiment by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements or components may be present.

[0038] As used herein, the term "about" refers to plus or minus 10% of the numerical indication being referenced. In some embodiments, "about" refers to plus or minus 5% of the numerical indication being referenced.

[0039] The term "agent" or "active agent" refers to a molecule or compound that prevents, alleviates, or improves the symptoms of a disease, extends the lifespan of a subject being treated, or achieves a desirable / acceptable medical or hygienic state. The agent in the NO booster increases the systemic production of NO in the subject. The agent in the NO precursor releases NO or undergoes a reaction to produce another agent that releases NO after transdermal delivery into the bloodstream.

[0040] The term "body cavity" includes any opening on the body of a subject and / or the surface area within the opening. Non-limiting examples of body cavities include the nose, sinuses, mouth, ear, rectum, vagina, open wound, blister, buccal cavity, and mucosal surfaces (e.g., gums).

[0041] The term C 1~30 Alkyl includes alkyl groups that are branched or unbranched and have any number of carbons in the range of 1 to 30. Non-limiting examples include methyl, ethyl, propyl, and butyl.

[0042] The term "cytokine storm" refers to an abnormal systemic release of dysregulated inflammatory cytokines leading to disease, and is also referred to as "cytokine release syndrome" or "inflammatory cascade". In many cases, a cytokine storm or cascade is part of a sequence where one cytokine typically leads to the production of multiple other cytokines that can enhance and amplify the immune response. Generally, these inflammation-inducing mediators are divided into two subgroups: early mediators and late mediators. For example, early mediators such as tumor necrosis factor, interleukin-1, and interleukin-6 are not sufficient therapeutic targets for re-establishing homeostasis because they dissipate within the time frame when a patient visits a clinic for medical treatment. In contrast, so-called "late mediators" have been targeted because it is during this later "inflammatory cascade" that a patient becomes aware of being ill.

[0043] The term "ex vivo" refers to biological material outside of a living body in the context of biological materials (e.g., blood, tissue, or organ).

[0044] The term "inflammatory disease or disorder" can refer to any disease, disorder, or syndrome in which an excessive or unregulated inflammatory response results in a transient inflammatory state, damage to host tissue, or loss of tissue function. "Inflammatory disease" also refers to pathological conditions mediated by granulocyte influx and / or neutrophil chemotaxis, as well as transient inflammatory states including leaky gut syndrome and "brain fog" resulting from chemotherapy.

[0045] The term "long COVID" refers to the adverse effects or symptoms resulting from COVID that become apparent after a person appears to have recovered from the initial infection. Non-limiting examples of long COVID symptoms include brain fog, fatigue, pain, clotting problems, myocarditis, and edema.

[0046] The term "NO booster" refers to an agent or mixture of agents that increases the systemic production of NO in a subject. An NO booster itself does not release NO.

[0047] The term "NO precursor" refers to an agent or mixture that releases NO either directly or indirectly via a derivative. The NO precursor can be or include an agent containing an NO-releasing moiety, which is transdermally delivered into the bloodstream of a subject and then releases NO. Non-limiting examples of such NO-releasing agents include S-nitroso-glutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Alternatively, the NO precursor may include an agent that yields a derivative containing an NO-releasing moiety, and the derivative releases NO after being transdermally delivered into the bloodstream. Non-limiting examples of agents that yield NO-releasing derivatives include glutathione, N-acetylcysteine (NAC), N-acetylpenicillamine, and cysteine, which can be nitrosated at the thiol group to produce S-nitrosothiol-containing derivatives.

[0048] The term "semi-solid" refers to a flexible and deformable solid form. Free-flowing liquids and hard solid forms are excluded from semi-solids. Non-limiting examples include gels, ointments, creams, emulsions, microemulsions, nanoemulsions, pastes, balms, lotions, and mousses.

[0049] The term "subject" includes any animal, but preferably includes mammals such as humans, non-human primates, dogs, cats, horses, cows, or rodents. More preferably, the subject is a human.

[0050] The term "pharmaceutically acceptable salt" means a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity. Non-limiting examples of such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfates, aromatic sulfates, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic proton present can react with an inorganic or organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that the particular anion or cation that forms part of any salt of the present invention is not critical as long as the salt as a whole is pharmaceutically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0051] The term "therapeutically effective amount" or "effective amount" refers to an amount of an active agent that is effective to prevent, alleviate, or improve the symptoms of a disease, extend the survival period of a subject being treated, or achieve a desirable / acceptable medical or hygienic state. Determination of a therapeutically effective amount or effective amount is well within the ability of one of ordinary skill in the art, particularly in light of the detailed disclosure provided herein. In the context of blood storage or transfusion, the term "effective amount" as used herein also refers to an amount of an agent that maintains the viability or function of a biological sample (e.g., cells, tissues, or organs).

[0052] The term "treating" or "treatment" of any disease or condition, in some embodiments, refers to ameliorating the disease or disorder (i.e., preventing or reducing the onset of at least one of the disease or its clinical symptoms). In some embodiments, "treating" or "treatment" refers to improving at least one physical parameter that may not be discernible by the subject. In some embodiments, "treating" or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In some embodiments, "treating" or "treatment" refers to delaying or even preventing the onset of the disease or disorder. For example, treatment can be "preventive treatment" and should be construed as any mode of treatment used to prevent the progression of a disease or for prophylactic purposes in a person at risk of developing the condition.

[0053] The term "transdermal" or "transdermally" refers to the delivery, administration or application of a formulation containing an active agent by direct contact with the skin or mucosa followed by transport of the agent through the skin or mucosa into the subject's blood circulation. Such delivery, transport, administration or application is also known to include the dermal, transdermal, transmucosal and buccal routes. As used herein, "dermal" includes skin and mucosa, which includes oral, buccal, nasal, rectal and vaginal mucosa. In some embodiments, the term also refers to the delivery and transport of an agent into a cell through a cell wall (e.g., a red blood cell wall).

[0054] The term "transdermal formulation" refers to a composition or formulation of an agent that delivers an agent or a derivative of an agent (e.g., an S-nitrosothiol-containing molecule derived from a thiol-containing molecule) across the skin or mucosa (or any other surface described above) upon application to the skin or mucosa. Transdermal formulations can be in the form of a solution, suspension, gel, ointment, cream, emulsion, microemulsion, nanoemulsion, paste, balm, magma, lotion, mousse, wax, or liposome. A kit or transdermal delivery system incorporating a transdermal formulation can be in the form of, for example, a patch, swab, nebulizer, sprayer, sponge or pouch.

[0055] The terms "viable cells", "viable tissue", and "viable organ" each refer to one or more cells, tissues, and / or organs that at least include a first population of living cells that are capable of surviving and substantially maintaining their native biological functions under the essential biological conditions (e.g., nutrients, incubation temperature, etc.) that are effective to maintain the viability of such cells, tissues, or organs sufficient for transplantation into a suitable recipient host, and are collected, stored, maintained, cultured, transported, and / or transplanted under such conditions. If x% (e.g., 60% or 80%) of the blood cells in a processed blood sample remain viable after a certain period, the value of that percentage refers to the blood cells that maintain their viability ex vivo among the initial total population of blood cells. Alternatively, if x% (e.g., 60% or 80%) of the blood cells remain in the processed blood sample after a specified period, the percentage refers to the cells that remain in circulation over the specified period. Transfused refers to cells.

[0056] Transdermal preparation Aspects of the present disclosure provide a transdermal formulation for transdermally delivering a therapeutically effective amount of a drug. The formulation generally comprises the following: (a) An effective amount of an active drug, such as a CD38 inhibitor, a sirtuin-1 (SIRT1) activator, a NO booster and / or a NO precursor, wherein the NO booster increases the systemic production of NO, and the NO precursor comprises or induces a NO releasing agent, an effective amount of an active drug; (b) A sufficient amount of a solvent to dissolve an effective amount of the NO booster or NO precursor; and optionally (c) A fatty acid and Upon administration, an effective amount of an active agent (e.g., a sirtuin-1 (SIRT1) activator, a NO booster and / or a NO-releasing agent) is delivered transdermally. In some embodiments, the active agent is delivered into a blood vessel for the active agent to enter the systemic circulation from an artery or a vein. In some embodiments, the active agent is delivered into a deep layer of the skin (e.g., the upper epidermis layer or the lower epidermis layer under the stratum corneum).

[0057] In some embodiments, the agent in the transdermal formulation is a flavonoid or a curcuminoid, or a combination thereof. One or more flavonoids and / or one or more curcuminoids can be included in the formulation. For example, the formulation may contain one or two flavonoids such as quercetin and apigenin, optionally in combination with curcumin, and each of the components or constituents is in a therapeutically effective amount.

[0058] In some embodiments, the transdermal formulation comprises one or more flavonoids or curcuminoids. Non-limiting examples include combinations of quercetin and apigenin, combinations of quercetin and curcumin, combinations of curcumin and apigenin, and combinations of quercetin, apigenin, and curcumin. Whether the combination contains one or more flavonoids or one or more curcuminoids, the weight ratio of each flavonoid to each curcuminoid is independently in the range of about 1:100 to about 100:1, about 5:100 to about 100:5, 1:10 to about 10:1, 2:10 to about 10:2, 3:10 to about 10:3, 4:10 to about 10:4, 5:10 to about 10:5, 6:10 to about 10:6, or 8:10 to about 10:8. Non-limiting examples of the ratios include about 100:1, about 80:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 8:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:8, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:80, about 1:100, and any range between any two of the foregoing values. In some embodiments, the combination contains curcumin and one or both of quercetin and apigenin, and the mass ratio of curcumin to each of one or both of quercetin and apigenin is independently in the range of about 1:5 to about 10:1, about 1:5 to about 3:1, about 1:2 to about 3:1, or about 1:2 to about 2:1.

[0059] When the formulation contains two or more flavonoids (e.g., quercetin, apigenin, fisetin, luteolin, and rapamycin), the weight ratio of the two flavonoids can independently be in the range of about 1:100 to about 100:1, about 5:100 to about 100:5, 1:10 to about 10:1, 2:10 to about 10:2, 3:10 to about 10:3, 4:10 to about 10:4, 5:10 to about 10:5, 6:10 to about 10:6, or 8:10 to about 10:8. Non-limiting examples of the ratios include about 100:1, about 80:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 8:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:8, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:80, about 1:100, and any range between any two of the foregoing values. In some embodiments, the formulation comprises quercetin and apigenin in a ratio in the range of about 1:1 to about 10:1, about 2:1 to about 10:1, about 5:1 to about 10:1, about 1:1 to about 1:10, about 1:1 to about 1:5, or about 1:1 to about 1:2.

[0060] The level of NO in a subject can be measured using known techniques such as those disclosed in, for example, U.S. Patent Nos. 9,044,182 and 8,425,428. In some embodiments, an effective amount is an amount sufficient to have a measurable effect against, for example, hypertension, inflammation, osteoarthritis, endothelial dysfunction, dermatological conditions, ophthalmological conditions, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomena, cerebral malaria, Chagas disease, and abnormal hemoglobinopathies such as sickle cell disease and HbE / β thalassemia, type 2 diabetes, and diseases including lupus. In some embodiments, an effective amount is an amount sufficient to have a measurable positive effect on blood flow and / or vasodilation and / or a measurable negative effect on blood pressure. In some embodiments, the effect on blood flow and / or vasodilation is observed locally at the site of topical application. In some embodiments, an effective amount is an amount sufficient to have a measurable effect against inflammatory diseases such as inflammatory skin diseases, inflammatory bowel diseases, and inflammation of the systemic vasculature including the blood-brain barrier resulting from chemotherapy, as evidenced by improvement in appropriate clinical parameters such as Physician's Global Assessment, after treatment with the formulation. In some embodiments, an effective amount is an amount sufficient to obtain a systemic or local level of nitric oxide to have a measurable positive effect on, for example, blood flow and / or vasodilation, a measurable negative effect on blood pressure, and / or a measurable effect against inflammatory skin diseases such as inflammatory skin diseases, as evidenced by appropriate clinical parameters, for the desired effect.

[0061] Non-limiting examples of SIRT1 activators, active agents, include polyphenols, flavonoids, stilbenoids, seco-steroids, and other phytochemicals or natural products that promote the formation of NO.

[0062] After a NO booster or NO precursor has been delivered transdermally, it results in the production of NO within the body of the subject. The NO booster or NO precursor is in an amount effective to increase NO systemically or locally to a level high enough to achieve the purpose of treating a disease or condition. In some embodiments, the NO booster includes polyphenols, flavonoids, stilbenoids, seco-steroids, or natural products that promote NO production. In some embodiments, the NO precursor includes S-nitrosothiol-containing molecules, or thiol-containing molecules and a source of nitrous acid. In some embodiments, the NO booster contains one or more of curcuminoids, flavonoids, berberine, resveratrol, a source of vitamin D, and pharmaceutically acceptable salts and derivatives thereof. Curcuminoids are linear diarylheptanoids and include, for example, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Flavonoids have a 3-hydroxyflavone backbone and include, for example, 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, kaempferol, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnazin, and rhamnetin. Non-limiting examples of sources of vitamin D include vitamin D2 and vitamin D3, as well as any precursor of vitamin D. Non-limiting examples of polyphenols include plant extracts, brazilein, and theaflavins (e.g., theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3)).

[0063] In some embodiments, the NO booster consists essentially of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, a source of vitamin D, and any combination thereof.

[0064] In some embodiments, the formulation contains both a NO booster and a NO precursor. For example, a combination of curcumin and a NO releasing agent (e.g., an S-nitrosothiol-containing molecule or a thiol-containing agent) in a polyol / fatty acid system is a powerful formulation for treating localized inflammation and infections, while at the same time being able to provide the systemic benefits of curcumin for controlling systemic inflammation.

[0065] Additional examples of curcuminoids include methylcurcumin, demethoxycurcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloylmethane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin 1), 1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonylcurcumin), 1,7-bis(2-hydroxynaphthyl)-1,6-heptadiene-2,5-dione (2-hydroxynaphthylcurcumin), and 1,1-bis(phenyl)-1,3,8,10-undecatetraene-5,7-dione. In some embodiments, the NO booster is curcumin, or a synthetic curcumin that is 80%, 85%, 90% or 98% pure difeuroylmethane.

[0066] In some embodiments, the transdermal formulation contains one or more curcuminoids, and optionally one or more of polyphenols, flavonoids, stilbenoids, seco-steroids, or natural products that promote NO production, as active ingredients for treating a disease or condition. In some embodiments, the transdermal formulation contains one or both of curcumin and quercetin, and optionally one or more of polyphenols, flavonoids, stilbenoids, and seco-steroids.

[0067] The NO precursor is either an S-nitrosothiol-containing molecule or a mixture containing a thiol-containing molecule and a nitrite source. When in contact with an acid source, the nitrite source generates nitrous acid, which can then nitrosate the reactive thiol of the thiol-containing molecule. The S-nitrosothiol-containing molecule releases NO to the subject in need thereof.

[0068] A variety of thiol-containing molecules can be used as precursors. Examples include glutathione, N-acetylcysteine (NAC), N-acetylpenicillamine, cysteine, and their derivatives. Either the amino group of cysteine or NAC can be acetylated with acetyl or another carbonyl of different carbon chain lengths (e.g., COC 2~30 alkyl). By adjusting the length of the carbon chain, the solubility and lipophilicity of the molecule can be modified. Similarly, the carboxy group of cysteine can be converted to an ester (e.g., ethyl ester, or other substituted or unsubstituted C 3~30 alkyl ester) or an amide having an NR2 moiety (wherein each R is independently H or another substituted or unsubstituted C 3~30 alkyl). Changes in the carbon chain enable the regulation of the properties of the molecule.

[0069] A variety of inorganic compounds can serve as nitrite sources. Non-limiting examples of nitrite sources include alkali metal nitrates, alkaline earth metal nitrates, transition metal nitrates, and ammonium nitrite. In some embodiments, the nitrite source is potassium nitrite, sodium nitrite, rubidium nitrite, strontium nitrite, barium nitrite, calcium nitrite, copper nitrite, zinc nitrite, or a mixture thereof. Nitrites can also include natural sources such as extracts of lettuce and spinach. In some embodiments, the nitrite source is saturated in a polyol solvent. The nitrite source can also be composed of nitrite-loaded nanoparticles.

[0070] Nitrite-loaded nanoparticles can be prepared by techniques known in the art, including, for example, the procedure reported in U.S. Patent No. 8,333,997, the entire disclosure of which is incorporated herein by reference. To limit NO release from the nanoparticles during manufacture, the medium should maintain a pH greater than about 7.5 throughout the preparation. The nitrite-loaded nanoparticles can then be mixed with a solvent system of polyol and fatty acid (e.g., PEG400 and myristic acid) and remain stable until exposed to an aqueous environment. In the presence of thiol-containing molecules, the nitrite-loaded nanoparticles will allow for the formation of S-nitrosothiols when the mixture is exposed to an acid source or a slightly acidic aqueous environment on the skin.

[0071] The use of nitrite-loaded nanoparticles allows for the use of high concentrations of nitrite under hydrophobic conditions. The combination of nitrite-loaded nanoparticles and a solvent system of polyol and fatty acid (e.g., PEG400 and myristic acid) allows for a stable mixture that releases NO and S-nitrosates thiols when exposed to an aqueous environment, regardless of other included deliverables. There is no release of nitrite or production of NO in the viscous solvent until water or an acid source is introduced.

[0072] The acid source can be packaged separately from the mixture containing the thiol-containing molecule and the nitrite source and mixed with the nitrite prior to administration. For example, the nitrite source and the acid source can be separately encapsulated in a permeable or frangible pouch. The amount and concentration of the acid can be adjusted according to the amount of other agents and the nature of the acid. Non-limiting examples of acids include acetic acid, oxalic acid, and citric acid.

[0073] Non-limiting examples of S-nitrosothiol-containing molecules include S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Similar to thiol-containing molecules, these S-nitrosothiol-containing molecules can be modified by varying the carbon chain in their respective ester, amide, or N-acyl moieties to fine-tune their properties.

[0074] Solvents such as polyols suitable for delivery systems enable high concentrations of poorly soluble drugs. In addition, it should have a profile as being biocompatible and safe for biomedical applications. Further, it should ideally promote skin and mucosal permeation to enable transdermal delivery. Non-limiting examples of polyols include polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, and glycerol. In some embodiments, the polyol is polyethylene glycol (PEG). In some embodiments, the PEG has a molecular weight in the range of about 100 to about 2000, about 100 to about 1000, about 100 to about 800, about 100 to about 600, about 200 to about 600, or about 200 to about 400 daltons. In some embodiments, the formulation is substantially free of water.

[0075] Other examples of solvents for the formulations disclosed herein may include glycols, ethanol, acetone, ethyl acetate, acetonitrile, DMF, THF, DMSO, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, PEG, and any combination thereof. For example, the solvent may include PEG together with one or more of acetone, tetrahydrofuran, and n-hexane. In a further example, the solvent may be a combination of acetone and tetrahydrofuran, a combination of acetonitrile and tetrahydrofuran, or a combination of n-hexane and tetrahydrofuran.

[0076] In some embodiments, the solvent of the formulation consists essentially of a polyol. In some embodiments, the formulation may include one or more additional solvents. Non-limiting examples include essential oils such as mineral oil, petrolatum, castor oil, eugenol, menthol, cineole, or rose oil, n-methylpyrrolidone, vegetable oil, oleyl alcohol, dipropylene glycol, polyoxyethylene derivatives of sorbitan esters, saturated polyglycolized C 8~10 glycerides, polyoxyethylated fatty acid glycerides, oleic acid, dimethyl sulfoxide (DMSO), fatty alcohols, isopropyl myristate (IPM), triacetin, ethyl oleate, isostearic acid, medium-chain fatty acids and other fats, and mixtures thereof. In addition to dissolving the drug, these solvents can also function as plasticizers, and thus the formulation can be flexible, stretchable, moldable and / or otherwise skin-friendly.

[0077] In some embodiments, the polyol solvent is a low molecular weight polyethylene glycol (PEG) having a molecular weight in the range of about 50 to about 2000, about 50 to about 1000, about 100 to about 1000, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 200 to about 800, about 200 to about 600, or about 200 to about 400 Daltons. Non-limiting examples of the molecular weight of the polyol solvent include about 100, about 200, about 300, about 400, about 500, about 600, about 800, about 1000. Short-chain PEG molecules such as PEG200 and PEG400, which are liquid at ambient temperature, are particularly useful.

[0078] Other solvents that can be used alone or in combination with a polyol to dissolve curcuminoids or flavonoids include deep eutectic solvents (DES) and natural deep eutectic solvents (NADES). Non-limiting examples of NADES include sugars (e.g., glucose, sucrose, fructose), organic acids (e.g., lactic acid, malic acid, citric acid), urea, and choline chloride. NADES can also be used in combinations including derivatives of organic acids (e.g., maleic acid (emalic acid), proline, betaine), choline chloride (e.g., choline chloride, d(−)fructose; choline chloride, A-l rhamnose; choline chloride, lactic acid), various sugars (fructose, sucrose; glucose, sucrose, fructose), and other combinations (betaine, sucrose; betaine, d-(+)glucose, proline).

[0079] The solvent may be based on a colloidal delivery system, and these include, for example, micelles (e.g., micelles made from surfactants having a hydrophobic core for encapsulating curcumin), microemulsions (e.g., microemulsions made from an aqueous phase and an organic phase stabilized by a surfactant suitable for curcumin incorporation into the hydrophobic core), conventional emulsions and nanoemulsions (an organic phase (oil) surrounded by a hydrophilic emulsifier; encapsulation of curcumin within the core oil phase), Pickering emulsions (water-in-oil emulsions stabilized by nanoparticles suitable for encapsulating curcumin within the core oil), multiple emulsions (e.g., multiple emulsions prepared by layer-by-layer deposition (lbl) of charged emulsifier layers surrounding an internal oil phase incorporating curcumin), solid lipid nano / micro particles (SLNs / SLMs) (e.g., particles made by cooling a water-in-oil emulsion to form crystallized lipid particles capable of incorporating lipophilic curcumin), liposomes and nanoliposomes (e.g., particles having a hydrophobic shell prepared from phospholipids capable of encapsulating a hydrophilic core and curcumin), hydrogels (micro / nanogels) (e.g., a porous network of a biopolymer network suitable for encapsulating curcumin).

[0080] Other solvents include dimethyl sulfoxide (DMSO), ethanol (EtOH), polyethylene glycol 400 (PEG 400), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), dimethyl isosorbide (DMI), propylene glycol, glycerin, propylene carbonate, ethanol, acetone, ethyl acetate, acetonitrile, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, and any combination thereof. The aforementioned solvents can be used alone or in combination, and the amount can be easily adjusted using conventional techniques without undue experimentation.

[0081] Fatty acids serve as permeation enhancers. Non-limiting examples of fatty acids include myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristic acid, and any combination thereof. In some embodiments, the fatty acid is myristic acid. Medium-chain fatty acids such as myristic acid and / or other fatty acids of equivalent size / molecular weight are particularly useful. In some embodiments, the formulation does not contain fatty acids or contains only trace or small amounts of fatty acids.

[0082] In some embodiments, the permeation enhancer consists essentially of a fatty acid or an ester thereof. In some embodiments, the formulation may contain one or more additional permeation enhancers. Non-limiting examples include surfactants, alcohols, fatty alcohols and glycols, esters, fatty acid esters and fatty alcohol esters, esters of long-chain fatty acids with methyl, ethyl, isopropyl alcohol, esters of fatty alcohols with acetic acid, lactic acid and oleic acid, diethanolamine, essential oils, terpenes and terpenoids, amides, urea, polyoxyethylene fatty alcohol ethers, polyoxyethylene fatty acid esters, sulfoxides, ether alcohols, pyrrolidone, transcutol, capsaicin derivatives, dimethylamino acid esters, peptides, iminosulfurane, dicarboxylic acid esters, nanocarriers, triglycerides, hydrocarbons, phospholipids, alone or in combination thereof.

[0083] By adjusting the amounts and ratios of the polyol, fatty acid, and NO booster or NO precursor, the solubility of the fatty acid and the agent in the NO booster or NO precursor, as well as the physical state of the formulation (e.g., liquid or gel or semi-solid) and the release profile of the active agent can be controlled. The ratio of the polyol to the fatty acid affects the form of the solution and generally ranges from about 5:1 to about 500:1, about 5:1 to about 100:1, about 1:1 to about 100:1, about 20:1 to about 100:1, about 30:1 to about 100:1, about 20:1 to about 80:1, about 20:1 to about 60:1, about 50:1 to about 10:1, or about 30:1 to about 50:1, about 40:1 to about 10:1, about 40:1 to about 10:1, about 20:1 to about 60:1, about 20:1 to about 15:1, about 18:1 to about 12:1 by weight. In some embodiments, the concentration of the fatty acid in the polyol ranges from about 0.01 M to about 1 M, about 0.01 M to about 0.8 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.2 M, about 0.01 M to about 0.15 M, about 0.01 M to about 0.1 M, about 0.02 M to about 0.2 M, about 0.02 M to about 0.1 M, about 0.04 M to about 0.08 M, or about 0.06 M to about 0.1 M. In further exemplary embodiments, the concentration of the fatty acid in the polyol is about 0.01 M, about 0.02 M, about 0.03 M, about 0.04 M, about 0.06 M, about 0.08 M, about 0.1 M or about 0.12 M. In some embodiments, the fatty acid is saturated in the polyol. In some embodiments, the polyol is PEG. In some embodiments, the fatty acid is myristic acid.

[0084] The ranges and amounts of the polyol and fatty acid are as described above and can be modified by those skilled in the art in view of practical needs without undue experimentation. In some embodiments, the fatty acid is selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and any combination thereof. In some embodiments, the polyol is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, glycerol, and combinations thereof. In some embodiments, the polyol is polyethylene glycol having a molecular weight in the range of 200 to about 600. In some embodiments, the polyol and fatty acid are in a ratio in the range of about 5:1 to about 100:1 by weight.

[0085] The fatty acid can be in the range of approximately about 0.1% to about 30% (from about from about 0.1% to about 30%), about 0.5% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 2% to about 8%, or about 4% to about 8% of the total weight of the NO booster or NO precursor, polyol and fatty acid (if present) or the total weight of the formulation. Non-limiting examples of the amount of fatty acid include about 1 wt%, about 3 wt%, about 5 wt%, about 7 wt%, about 8 wt%, or about 10 wt%.

[0086] Water can cause aggregation and particle formation. In some embodiments, the transdermal formulation is anhydrous or substantially free of water. Minimizing or removing water from the formulation helps maintain a uniform distribution of fatty acids and / or active ingredients (such as NO boosters or NO precursors) in the polyol (e.g., PEG). In some embodiments, the water in the transdermal formulation is less than 5 wt%, less than 2 wt%, less than 1 wt%, less than 0.5 wt%, less than 0.1 wt%, or less than 0.01 wt%.

[0087] In some embodiments, the transdermal formulation includes at least one water repelling agent, also referred to as a water repellant. Examples of water repelling agents include silicones such as cyclomethicone, dimethicone, simethicone, C 26~28 alkyldimethicone, C 26~28 alkylmethicone, polyphenylsisquioxane, trimethylsiloxysilicate, and a copolymer of cyclopentasiloxane and dimethicone / vinyltrimethylsiloxysilicate, and blends thereof. The water repelling agent can be particularly useful in embodiments where the topical vehicle is used with a water-reactive agent, such as a nitric oxide releasing agent (e.g., diazeniumdialate or sodium nitrite) that releases nitric oxide in the presence of water. In other cases, such as when the active agent is not water-sensitive, the water repelling agent may or may not be included.

[0088] Depending on the treatment goal of the formulation, the active agent in the formulation (e.g., a CD38 inhibitor, an NO booster or NO precursor, or a mixture thereof) is in the range of about 0.05% to about 80%, about 0.05% to about 50%, about 0.05% to about 35%, about 0.05% to about 30%, about 0.05% to about 20%, about 0.05% to about 10%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% to about 5%, about 1% to about 20%, about 1% to about 10%, or about 1% to about 5% of the total weight of the NO booster or NO precursor, polyol, and fatty acid (if present) or the total weight of the formulation. Non-limiting examples of the amount of active agent in the formulation include about 1 wt%, about 3 wt%, about 5 wt%, about 7 wt%, about 8 wt%, about 10 wt%, about 12 wt%, and about 15 wt% in the formulation. In some embodiments, the amount of active agent in the formulation or in a dosage unit of the formulation is, independently, in the range of about 0.001 mg to about 20 g, about 0.002 mg to about 20 g, about 0.004 mg to about 20 g, about 0.006 mg to about 20 g, about 0.008 mg to about 20 g, about 0.01 mg to about 20 g, about 0.05 mg to about 20 g, about 0.1 mg to about 20 g, about 0.1 mg to about 5 g, about 0.1 mg to about 2 g, about 0.1 mg to about 1 g, about 1 mg to about 5 g, about 1 mg to about 1 g, about 10 mg to about 100 mg, about 5 mg to about 50 mg, or about 10 mg to about 30 mg in a dosage unit of the formulation. The dosage unit can be in physically distinct package forms (e.g., capsules, patches, vials). The dosage unit can also be a predetermined portion of the formulation for each individual administration. For example, a suitable amount as a dosage unit of the formulation can be taken out of the container for direct topical application, for loading onto a patch or any suitable carrier, before being topically applied. The amount or size of the dosage unit can be easily adjusted according to the purpose of use and the area of application.Non-limiting examples of the amount of active agent in a dosage unit include about 0.001 mg, about 0.002 mg, about 0.004 mg, about 0.006 mg, about 0.008 mg, about 0.01 mg, about 0.02 mg, about 0.04 mg, about 0.06 mg, about 0.08 mg, about 0.1 mg, about 0.2 mg, about 0.4 mg, about 0.06 mg, about 0.08 mg, about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 80 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 800 mg, about 1 g, about 2 g, about 5 g, about 10 g, about 15 g, about 20 g, about 25 g, about 30 g, about 35 g, about 40 g, about 50 g, about 60 g, about 80 g, about 100 g, and any range between any two of the foregoing values. In some embodiments, each active agent in the dosage unit is, independently, in the range of about 0.01 to about 1 mg, about 0.01 to about 0.5 mg, or about 0.1 to about 0.5 mg. In some embodiments, the active agent is a curcuminoid, which is selected from one, two, or three of curcumin, demethoxycurcumin, and bisdemethoxycurcumin, and optionally, is a combination with one or more of the foregoing flavonoids. The dosage unit can be administered once, twice, three times, or as needed per day. In some embodiments, the dosage unit is administered once daily, every two days, every three days, every four days, every five days, every six days, every seven days, or every ten days.

[0089] In some embodiments, the ratio of each active agent to the polyol is independently in the range of about 1:5 to about 1:100, about 1:5 to about 1:50, about 1:5 to about 1:30, about 1:5 to about 1:20, about 1:8 to about 1:15, or about 1:10 to about 1:15 by weight. In some embodiments, the polyol is PEG. In some embodiments, the fatty acid is myristic acid. In some embodiments, the formulation is substantially free of piperine. Depending on the disease or condition being treated and the site of administration, the ratios and amounts of PEG, fatty acid, and active agent can be selected such that the resulting formulation is in a liquid, gel, or other suitable form. Additional agents may be added to control the physical state of the formulation.

[0090] In some embodiments, the transdermal formulation contains PEG, myristic acid and / or other fatty acids of equivalent size / molecular weight, and one or more active agents. In some embodiments, the transdermal formulation contains PEG, myristic acid and / or other fatty acids of equivalent size / molecular weight, an active agent, and a second agent. In some embodiments, the transdermal formulation contains PEG, myristic acid, and an active agent selected from at least one of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, and vitamin D. In some embodiments, the transdermal formulation contains PEG, myristic acid, and one or more active agents selected from at least one of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, and vitamin D, and a NO precursor or a second agent. In some embodiments, the transdermal formulation contains PEG having a molecular weight in the range of about 200 to about 500 (e.g., PEG200, PEG300, PEG400, or PEG500). The ratio of PEG to myristic acid (and / or other fatty acids of equivalent size / molecular weight) is in the range of about 5:1 to about 100:1 by weight (e.g., 6:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, or 80:1). The ratio of PEG to a NO booster (e.g., curcumin) is in the range of about 5:1 to about 100:1 by weight. Non-limiting examples of the ratio of PEG to a NO booster include, for example, 6:1, 8:1, 10:1, 12:1, 15:1, 18:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 80:1, and any range between any two of the foregoing values. In some embodiments, the formulation contains curcumin. In some embodiments, the formulation contains curcumin, demethoxycurcumin, bisdemethoxycurcumin, or any combination thereof. In some embodiments, the formulation contains vitamin D.In some embodiments, the concentration of the individual active ingredient in the formulation ranges from about 0.01 M to about 1 M, from about 0.05 M to about 0.5 M, from about 0.05 M to about 0.3 M, or from about 0.1 M to about 0.2 M. Non-limiting examples of the concentration of the active ingredient (e.g., curcumin) in the polyol (e.g., PEG) include about 0.06 M, about 0.08 M, about 0.1 M, about 0.12 M, about 0.14 M, about 0.16 M, about 0.18 M, about 0.20 M, about 0.25 M, about 0.30 M, about 0.40 M, about 0.60 M, and about 0.80 M.

[0091] The transdermal formulation has an extended shelf life with minimal degradation of the active ingredient(s). In some embodiments, the active ingredient(s) of the formulation remain stable at greater than 95% or greater than 99% for a period of at least 1 month, at least 3 months, at least 6 months, or at least 12 months. In some embodiments, the transdermal formulation comprises one or more curcuminoids, myristic acid, and PEG. The one or more curcuminoids range from about 2% to about 10%, from about 3% to about 8%, or from about 4% to about 6% by weight in the formulation. Myristic acid ranges from about 1% to about 10%, from about 1% to about 8%, from about 2% to about 8%, or from about 4% to about 6% by weight in the formulation. PEG ranges from about 60% to about 95%, from about 70% to about 90%, from about 80% to about 90%, or from about 95% to about 90% by weight in the formulation. In some embodiments, the PEG is PEG400.

[0092] In some embodiments, the formulation further comprises a gelling agent or thickening agent that maintains it in a semi-solid or solid form. Non-limiting examples of gelling agents or thickening agents include carbomers, methylcellulose, hydroxypropylmethylcellulose, poloxamers, polyacrylic acid, alginates, chitosan, xanthan gum, gellan gum, xyloglucan, paraffin, silicone, petrolatum, cocoa butter, and high molecular weight polyalkylene glycols.As additional examples, polyethylene oxide, ammonium methacrylate, carrageenan, aqueous solutions of cellulose acetate phthalate such as CAPNF manufactured by Eastman, sodium carboxymethyl cellulose, carboxypolymethylene, cellulose, cellulose acetate (microcrystalline), cellulose polymers, divinylbenzene styrene, ethyl cellulose, ethylene vinyl acetate, silicone, polyisobutylene, shellac (FMC BioPolymer), guar gum, guar rosin, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, and cellulose derivatives such as methyl cellulose, hypromellose phthalate (hydroxypropyl methyl cellulose phthalate), methyl acrylate, microcrystalline wax, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetate phthalate such as Suretic manufactured by Colorcon, PVP ethyl cellulose, polyvinyl pyrrolidone (PVP), acrylate, PEG / PVP, trimethylsiloxysilicate, maleic acid / anhydride copolymer, polyacrylic, poloxamer, polyglactic acid / poly l-lactic acid, terpene resin, locust bean gum, prolamin (Zein), acrylic copolymer, polyurethane dispersion, gelatin (both type A and type B from various sources such as pig, cow, and fish), dextrin, starch, polyvinyl alcohol-polyethylene glycol copolymer, methacrylic acid-ethyl acrylate copolymer such as BASF's Kollicoat polymer, methacrylic acid and methacrylate-based polymers such as poly(methacrylic acid) copolymer and methyl methacrylate copolymer, esters of polyvinyl methyl ether / maleic anhydride copolymer such as Gantrez ES-425, Gantrez ES-225 manufactured by ISP, and mixtures thereof. Non-limiting examples of high molecular weight polyalkylene glycols include PEG and polypropylene glycol (PPG).The polyalkylene glycol may have a molecular weight greater than 1 kDa, greater than 2 kDa, greater than 3 kDa, greater than 4 kDa, greater than 6 kDa, greater than 8 kDa, greater than 10 kDa, greater than 15 kDa, greater than 20 kDa, greater than 25 kDa, or greater than 30 kDa. Without limiting the scope, the semi-solid formulation can be in the dosage form of an ointment, gel, cream, emulsion, paste, lotion or liposome.

[0093] In some embodiments, the formulation comprises a combination of small and large polyalkylene glycols having an MW difference in the range of 500 - 5000, 1000 - 3000, 1000 - 2000, or 1500 - 2000 daltons. By adjusting the ratio between two or more polyalkylene glycols, the viscosity and both the rate / extent of skin penetration and uptake by circulation can be controlled. For example, the combination can include one or both of PEG and PPG having an MW in the range of 100 - 2000, 200 - 2000, 400 - 1000, or 500 - 800 daltons, respectively. The combination can also include one or both of PEG and PPG having a higher MW in the range of 800 - 5000, 1000 - 3000, or 1000 - 2000 daltons, respectively. In a further exemplary embodiment, one of the polyalkylene glycols has an MW of 100, 200, 400, 600, or 800, and another polyalkylene glycol has an MW of 1000, 1500, 2000, 2500, or 3000. In some embodiments, the combination includes PEG of 400 daltons and PEG of 2000 daltons. In some embodiments, the ratio of the low MW polyalkylene glycol to the high MW polyalkylene glycol is in the range of about 10:1 to about 1:10, about 5:1 to about 1:5, about 2:1 to about 1:2 by weight. Further exemplary ratios of low MW polyalkylene glycol (e.g., PEG and / or PPG) to high MW polyalkylene glycol (e.g., PEG and / or PPG) include 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8 and 1:10.

[0094] In some embodiments, the formulation does not contain additional therapeutic agents other than the NO booster or NO precursor. In some embodiments, the active agent in the formulation consists essentially of the NO boosters and / or NO precursors described herein. In some embodiments, the formulation may contain additional therapeutic agents, such as, for example, antioxidants, antibiotics, antiviral agents, and / or antifungal agents.

[0095] The formulation may contain other components, such as, for example, solubilizers, skin penetration enhancers, surfactants, co-solvents, thickeners or viscosifying agents, preservatives, isotonic agents, isosmotic agents, absorption promoters for the drug, mucoadhesive polymers, non-mucoadhesive polymers, chelating agents, stabilizers, antioxidants, and mixtures thereof.

[0096] In some embodiments, the thickener is selected from one or more of carbomer, methylcellulose, hydroxypropylmethylcellulose, poloxamer, polyacrylic acid, alginate, chitosan, xanthan gum, gellan gum, xyloglucan, paraffin, silicone, petrolatum, and cocoa butter.

[0097] Non-limiting examples of solubilizing agents include diethylene glycol monoethyl ether (ethoxydiglycol, commercially available as Transcutol®) and diethylene glycol monoethyl ether oleate (Soficutol®), commercially available under the trade name Poly(TM); polyethylene castor oil derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil; polyethylene glycol, especially low molecular weight polyethylene glycol; polyethylene glycol derivatives such as glyceryl caprylate / caprate (commercially available as Labrasol®); alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DMA, but are not limited thereto. Many solubilizing agents can also function as absorption enhancers. A single solubilizing agent can be incorporated into the formulation or a mixture of solubilizing agents can be incorporated into the formulation.

[0098] Non-limiting examples of skin penetration enhancers assist in promoting the passage of therapeutic levels of active agents through a moderately sized area of intact skin. Suitable enhancers are well known in the art and include, for example, lower alcohols such as methanol, ethanol and 2-propanol; alkyl methyls such as dimethyl sulfoxide (DMSO), decyl methyl sulfoxide (C10MSO) and tetradecyl methyl sulfoxide, sulfoxides; urea; 2-pyrrolidone, N-methyl-2-pyrrolidone and N-pyrrolidone such as N-(hydroxyethyl)pyrrolidone N,N-diethyl-m-toluamide; C2-C6 alkanediols; dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and various miscellaneous solvents such as tetrahydrofurfuryl alcohol; and 1-substituted azacycloheptan-2-ones, particularly 1-N-dodecylazacycloheptan-2-one (laurocapram, available from Whitby Research Incorporated, Richmond, Va. under the trade name Azone®).

[0099] Among surfactants, for example, polyethoxylated glycerides, polysorbates, poloxamers, sodium lauryl sulfate, phospholipids such as phosphatidylcholine or phosphatidylglycerol and their derivatives, polyoxyethylated hydrogenated castor oil, polyoxyethylated fatty acids, optionally a mixture of mono-, di-, and triglycerides of polyoxyethylated fatty acids, and mixtures thereof can be mentioned.

[0100] Among preservatives, for example, benzalkonium chloride, boric acid, benzoic acid, C1-4 alkyl esters of p-hydroxybenzoic acid, chlorobutanol, benzyl alcohol, phenylethyl alcohol, organometallic derivatives of mercury, polyquaternium such as polyquaternium 1, and mixtures thereof can be mentioned.

[0101] Among tonicity agents and osmotic pressure agents, for example, inorganic salts such as sodium chloride, glucose, trehalose, mannitol, amino acids, and mixtures thereof can be mentioned.

[0102] Among mucoadhesive polymers, for example, hyaluronic acid, polygalacturonic acid, polyacrylic acid, carboxymethyl amylose, carboxymethyl chitin, chondroitin sulfate, methylcellulose, gelatin, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, xanthan gum, chitosan, carbopol, polycarbophil, gellan gum, carrageenan, alginate, pectin, poloxamer, and mixtures thereof can be mentioned. Among non-mucoadhesive polymers, for example, polyvinyl alcohol can be mentioned. Among chelating agents, for example, disodium edetate and sodium cromoglycate can be mentioned. Among antioxidants, for example, sodium metabisulfite, sodium bisulfite, acetylcysteine, ascorbic acid, and mixtures thereof can be mentioned.

[0103] By adjusting the amounts and ratios of the polyol, fatty acid, and one or more active agents, the solubility of the fatty acid and active agent, the physical state of the formulation, and the release profile of the active agent can be controlled. In some embodiments, the polyol, fatty acid, active ingredient, and other necessary components are formulated in ratios such that the formulation provides a rapid onset of action within about 5 minutes, about 10 minutes, about 15 minutes, or about 30 minutes.

[0104] The transdermal formulations disclosed herein can provide sustained or continuous release of a drug (e.g., a CD38 inhibitor, an NO booster, or an S-nitrosothiol-containing molecule). In some embodiments, the formulation provides sustained release (transdermal delivery into the bloodstream) of the drug for a period of 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, about 2 days, about 3 days, about 5 days, or about 7 days. The release rate can also be controlled by selecting the polyol solvent and fatty acid in suitable ratios. In some embodiments, one, two, or three of the following parameters can be achieved for the formulation: (a) Less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% of the drug is delivered into the bloodstream within about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours; (b) About 25% to about 90%, about 30% to about 85%, about 35% to about 70%, about 40% to about 70%, about 50% to about 60%, about 35% to about 50%, about 40% to about 60%, or about 35% to about 80% of the drug is delivered into the bloodstream within about 6 hours, about 8 hours, about 10 hours, about 12 hours, or about 14 hours; and (c) More than 60%, more than 70%, or more than 80% of the drug is delivered into the bloodstream at about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, or about 48 hours.

[0105] In some embodiments, the active agent and carrier (e.g., polyol or fatty acid) in the formulation and their amounts are selected such that the therapeutic effect window is maintained for about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 24 hours, about 2 days, about 3 days, about 5 days, or about 7 days, and the plasma concentration of the active agent varies by less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30% or less than 40% during such a window. In some embodiments, the window begins within about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, or about 2 hours after the formulation is administered.

[0106] The formulation may include a second agent. Examples of the second agent include antihypertensive agents, antibacterial agents, anti-inflammatory agents, analgesics, anesthetics, antihistamines, preservatives, immunosuppressive agents, antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilm agents, and mixtures thereof. Alternatively, the second agent may be in a separate formulation and / or administered separately from the transdermal formulation described herein.

[0107] Examples of anti-inflammatory agents include non-steroidal anti-inflammatory agents (NSAIDs); propionic acid derivatives such as ibuprofen and naproxen; acetic acid derivatives such as indomethacin; enolic acid derivatives such as meloxicam and acetaminophen; methyl salicylate; monoglycol salicylate; aspirin; mefenamic acid; flufenamic acid; indomethacin; diclofenac; alclofenac; diclofenac sodium; ibuprofen; ketoprofen; naproxen; pranoprofen; fenoprofen; sulindac; fenclofenac; clidanac; flurbiprofen; fentiazac; bufexamac; piroxicam; phenylbutazone; oxyphenbutazone; clofezone; pentazocine; mepirizole; tiaramide hydrochloride;Steroids, such as clobetasol propionate, betamethasone dipropionate, halbetasol proprionate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, desoxymethasone, triamcinolone acetonide, mometasone furoate, fluticasone propionate, betamethasone dipropionate, triamcinolone acetonide, fluticasone propionate, desonide, fluocinolone acetonide, hydrocortisone valerate, prednicarbate, triamcinolone acetonide, fluocinolone acetonide, hydrocortisone and others known in the art, prednisone, dexamethasone, fluocinolone acetonide, hydrocortisone acetate, prednisone acetate, methylprednisolone, dexamethasone acetate, betamethasone, betamethasone valerate, flumethasone, fluorometholone, beclomethasone dipropionate, fluocinonide, topical corticosteroids, including hydrocortisone, hydrocortisone-21-monoesters (e.g., hydrocortisone-21-acetate, hydrocortisone-21-butyrate, hydrocortisone-21-propionate, hydrocortisone-21-valerate, etc.), hydrocortisone-17,21-diesters (e.g., hydrocortisone-17,21-diacetate, hydrocortisone-17-acetate-21-butyrate, hydrocortisone-17,21-dibutyrate, etc.), alclometasone, dexamethasone, flumethasone, prednisone, or methylprednisolone, etc., may be one of the lower potency corticosteroids, or may be a higher potency corticosteroid such as clobetasol propionate, betamethasone benzoate, betamethasone dipropionate, diflorasone diacetate, fluocinonide, mometasone furoate, triamcinolone acetonide, etc.;

[0108] In some embodiments, the formulation contains an antiviral agent, such as acyclovir, trifluridine, idoxuridine, penciclovir, famciclovir, cidofovir, ganciclovir, valaciclovir, podophyllox, podophyllotoxin, ribavirin, abacavir, delavirdine, didanosine, efavirenz, lamivudine, nevirapine, stavudine, zalcitabine, zidovudine, amprenavir, indinavir, nelfinavir, ritonavir, saquinavir, amantadine, interferon, oseltamivir, ribavirin, rimantadine, zanamivir, and combinations thereof. The antiviral treatment can be used for the treatment of both localized and systemic viral infections such as Covid-19, oral herpes or genital herpes.

[0109] Examples of antibacterial agents include penicillin and related drugs, carbapenems, cephalosporins and related drugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, sodium fusidate, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines, vanomycin, teicoplanin, streptogramins, folic acid antagonists including sulfonamides, trimethoprim and its combinations as well as pyrimethamine, nitrofurans, synthetic antibacterial agents including mandelic acid methenamine and hippuric acid methenamine, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, viomycin, everninomicin, glycopeptides, glycylcyclines, ketolides, oxazolidinones; imipenem, amikacin, netilmicin, fosfomycin, gentamicin, ceftriaxone, dirithromycin, linezolid, synercid, aztreonam, and metronidazole, epiroprim, sanfetrinem sodium, biapenem, dynemicin, cefluprenam, cefoselis, sanfetrinem cilexetil, cefpirome, mersacidin, rifalazil, cothiamycin, lenapenem, beneplim, slopenem, lipipenem acoxil, cyclothialidine, mikacocidin A, carmonam, cefozopran and cefetamet pivoxil.

[0110] Examples of antihistamines include diphenhydramine hydrochloride, diphenhydramine salicylate, diphenhydramine, chlorpheniramine hydrochloride, chlorpheniramine maleate, isothipendyl hydrochloride, tripelennamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride, and the like. Examples of local anesthetics include dibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, 2-(diethylamino)ethyl p-butylaminobenzoate hydrochloride, procaine hydrochloride, tetracaine, tetracaine hydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonine, and dyclonine hydrochloride.

[0111] Examples of preservatives include alcohol, quaternary ammonium compounds, boric acid, chlorhexidine and chlorhexidine derivatives, iodine, phenol, terpene, bactericides, thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, chlorhexidine, povidone iodine, cetylpyridinium chloride, eugenol, and disinfectants containing trimethylammonium bromide.

[0112] Examples of analgesics include alfentanil, benzocaine, buprenorphine, butorphanol, butabarbene, capsaicin, clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone, indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine, pramoxine, propalacaine, propoxyphene, proxymetacaine, sufentanil, tetracaine, and tramadol.

[0113] Examples of anesthetics include alcohols such as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol; pramoxine; resorcinol; triclosan; procaine drugs such as benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine; dexibupivacaine; diamocaine; dibucaine; etidocaine; hexylcaine; levobupivacaine; lidocaine; mepivacaine; oxethazaine;prilocaine; procaine; propanocaine; propoxycaine; pyrocaine; lysocaine; rodocaine; ropivacaine; tetracaine; and derivatives such as pharmaceutically acceptable salts and esters including bupivacaine HCl, chloroprocaine HCl, diamocaine cyclamate, dibucaine HCl, dyclonine HCl, etidocaine HCl, levobupivacaine HCl, lidocaine HCl, mepivacaine HCl, pramoxine HCl, prilocaine HCl, procaine HCl, propanocaine HCl, propoxycaine HCl, ropivacaine HCl, and tetracaine HCl.

[0114] Examples of hemostatic agents include thrombin, phytomenadione, protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome, carbaxochrome sodium sulfanat, rutin, and hesperidin. Further examples of a second or additional agent include chemotherapeutic agents (such as tyrosine kinase inhibitors, immune checkpoint inhibitors, VEGF inhibitors, etc.) and glucose-lowering agents (such as metformin).

[0115] As further examples of the second agent, there are daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis(chloroethyl)nitrosourea, busulfan, mitomycin C, actinomycin D, mitramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimethoprim, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol. In some embodiments, second agents that can be used in combination with the formulations disclosed herein or incorporated into the same transdermal formulation include anti-inflammatory agents, analgesics, antibacterial agents, antifungal agents, antibiotics, vitamins, and antioxidants. In some embodiments, the second agent is selected from piperine, anthranilic acid, benzophenone, camphor derivatives, cinnamic acid esters (e.g., octyl methoxycinnamate), dibenzoylmethane (e.g., butyl methoxydibenzoylmethane), p-aminobenzoic acid (PABA) and its derivatives, salicylic acid esters, and PDE5 inhibitors (e.g., sildenafil (Viagra), tadalafil (Cialis), vardenafil (Levitra), and avanafil (Stendra)).

[0116] Any of the second agents described herein can be incorporated into the same transdermal formulation. Alternatively, in some aspects of any of the methods disclosed herein, the second agent can be administered separately from the transdermal formulation via any suitable route, including oral, transdermal, and parenteral routes.

[0117] In some aspects, the combination of active agents in the transdermal formulation produces a synergistic therapeutic effect. For example, curcumin and a PDE5 inhibitor, when incorporated into the same transdermal formulation or administered sequentially in combination, can result in earlier symptom recovery (fever, cough, sore throat, and shortness of breath), reduced exacerbation, and reduced red flag signs in patients with viral infections (e.g., COVID-19).

[0118] Transdermal delivery system The kit or transdermal delivery system may contain any combination of the components described herein in an amount sufficient for at least one agent, and may further include instructions recorded in tangible form for the use of the components. Depending on the use, one or more components may be provided in pre-measured single-use amounts in individual, typically disposable patches, tubes, or equivalent containers.

[0119] The formulations disclosed herein can be incorporated into a transdermal delivery system or kit and used as a patch, swab, aerosol, cream, sponge, nebulizer, or via other suitable means. The transdermal delivery system may also include instructions for the administration of the formulations disclosed in this patent document and one or more treatment methods. Liquid or semi-solid formulations can be applied directly to the skin using, for example, a swab or sponge. Alternatively, the transdermal delivery system can include a layer coated or impregnated with a liquid, semi-solid, or solid transdermal formulation. For example, the patch can have a layer impregnated with a liquid formulation or coated with a semi-solid or solid formulation. The transdermal delivery system may also include an adhesive member for attaching it to the skin.

[0120] The transdermal delivery system or kit can be of any suitable shape for application to the subject in need thereof. For example, a sponge loaded with the formulations disclosed herein can be shaped as circular, cylindrical, conical, planar, tubular, and other symmetric or asymmetric shapes for insertion into a body cavity or attachment or application to a target location, and can include an applicator or an applicator portion. The sponge can be made of a material that absorbs liquid by capillary action. Alternatively, the material can be hydrophilic or hygroscopic, or can be coated with a hydrophilic or hygroscopic layer that exhibits an affinity for an aqueous solution, particularly moisture from a site where a reservoir is disposed or from a body cavity. A sponge with absorption properties can be made from natural or synthetic materials including, for example, polyester, polyurethane, and vegetable cellulose.

[0121] In some embodiments, the formulation is incorporated into a liquid reservoir. The reservoir can be used independently, attached to a sponge, or partially or fully encapsulated within a sponge. Alternatively, the contents of the liquid reservoir can be loaded onto a sponge for application after being mixed with the necessary treatment or additional agents. In formulations containing a source of nitrous acid that requires an acid to generate nitrous acid and NO, the acid can be added to the reservoir containing nitrite prior to administration. Alternatively, a dual liquid reservoir system can be employed. For example, one pouch contains a thiol-containing molecule and nitrite in a polyol solvent system, and another pouch contains a source of acid. Optionally, additional pouches can be used to separately encapsulate the thiol-containing molecule or nitrite or fatty acid. Prior to administration or upon contact with the skin, the contents of the pouches are mixed to initiate the reaction of the acid with the nitrite and subsequent nitration of the thiol-containing molecule. Pouches for encapsulating the NO precursor or acid source are generally fragile or permeable containers that do not contact each other or are separated by a non-permeable removable barrier prior to administration of the formulation. Upon administration, the acid and NO precursor can permeate from their respective pouches and mix with each other under pressure from the user after removal of the barrier. The acid and NO precursor can also be mixed by simply breaking the pouch during or prior to administration. In a further exemplary embodiment, the acid and NO precursor are mixed in a container prior to administration. The resulting mixture is taken up by a cotton swab, sponge, or absorbent patch and then applied to the skin.

[0122] In some embodiments, the pouch has a permeable or semi-permeable membrane surface optionally coated with an adhesive for attaching the membrane to the skin. Instead of an adhesive coating, the pouch can be attached to the skin by holding the pouch against the skin and then covering the pouch with an adhesive patch or encapsulating sheet. Commercially available pouch stock materials such as DuPont's SURLYN® can also be used for the liquid reservoir. Additional examples include coextruded ethylene acrylic acid / low density polyethylene (EAA / LDPE) materials, or BAREX® (acrylonitrile-methyl acrylate) made by INEOS.

[0123] In some embodiments where the NO precursor is a mixture of a thio-containing molecule and nitrite, the formulation can be incorporated into a patch. The layers of the patch are impregnated with the NO precursor in a polyol and fatty acid solvent system, while the acid source is placed in a separate layer. The two layers do not contact each other until the patch is attached to the skin or prior to administration. By applying pressure to the patch, the contents of the different layers can be mixed. Alternatively, the patch can include an impermeable barrier between the two layers, and the nitrite and acid can be mixed by removing the barrier prior to administration.

[0124] In some embodiments, the formulation is a solid containing a thickening or solidifying material such as cocoa butter. In some embodiments, the formulation is a solid or semi-solid containing petrolatum. The solid or semi-solid formulation can be applied to the skin or can melt when rubbed in while applying pressure to the skin.

[0125] In some embodiments, the formulation is filled into a nebulizer or sprayer, which delivers the drug in aerosol form to the nose, mouth, or lungs of the subject in need. Carbon dioxide or other suitable gas can be used as the propellant.

[0126] The system or kit can include any number of additional reagents or substances useful for practicing the methods of the invention. The kits or systems of the invention can be provided at any temperature. For example, for storage of kits containing certain S-nitrosothiol-containing molecules in a liquid or gel, they can be provided and maintained at a suitable temperature or about 0°C.

[0127] The kit or system can also include instructions for use and packaging materials for holding the containers or combinations of containers. Instructions such as written instructions or video-recorded demonstrations detailing the use of the transdermal formulation for treating target diseases and conditions can be included in the kit or system. Typical packaging materials for such kits and systems include solid matrices (such as glass, plastic, paper, foil, etc.) that hold the components in any of a variety of configurations (such as pouches, tubes, etc.).

[0128] Such kits or systems can also include information such as scientific literature references, package insert materials, clinical trial results, and / or summaries thereof that indicate or establish the activity and / or advantages of the composition and / or describe dosing, administration, side effects, drug interactions, or other information useful to a healthcare provider. Such information can be based on the results of various studies, such as studies using experimental animals including in vivo models and studies based on human clinical trials. The kits or systems described herein can be provided, sold, and / or promoted to healthcare providers including physicians, nurses, pharmacists, formulary bureaus, etc. The kits can also, in some aspects, be sold directly to consumers.

[0129] In connection with the formulations disclosed herein and their effects on blood, aspects of this patent document provide biological material samples (e.g., cell samples, tissue samples, organ samples, and blood samples) containing biological materials that are treated with the formulations disclosed herein. The samples can be prepared for testing, for storage, for transfusion, or for any other suitable purpose. Kits containing biological materials stored in containers are also provided herein. The containers can be formed for storage, transportation, transfusion, or any other use of the sample.

[0130] Biological samples can be prepared, for example, by washing, rinsing, or mixing the biological samples with the formulations disclosed herein. In some aspects, the biological samples include the formulations and biological materials ex vivo. In some aspects, the biological material is blood.

[0131] Biological materials can be recovered from one animal and transplanted into an animal of the same species (allotransplantation) or an animal of a different species (xenotransplantation). Tissues can be derived from the whole or part of an organ such as a heart valve or aorta, or a specific site of an animal, e.g., the cartilage or tendon of the knee joint.

[0132] Exemplary types of mammalian cells that can be recovered, stored, and / or transported using one or more of the methods and formulations described herein include, but are not limited to, chondral cells, cartilagenous cells, osteochondral cells, pancreatic islet cells, osteoprogenitor cells, nerve cells, bone cells, bone marrow cells, adipocytes, fibroblasts, muscle cells, blood, blood components, stem cells, and embryonic stem cells. In some aspects, the biological material is blood.

[0133] Exemplary types of mammalian tissue that can be recovered, stored, and / or transported in accordance with the present invention include, but are not limited to, skin, cartilage, tendon, ligament; fascia, tibia, patella, and other bones, heart valves, semi-tendinous tissue, blood vessels, vertebral discs, corneas, lenses, menisci, hair, adipose tissue, fibrous tissue, nerve tissue, connective tissue, and striated, smooth, or cardiac muscle tissue. Cells or tissues can be recovered from a human or animal subject and then processed and / or cryopreserved (frozen) for subsequent transplantation. Allografts, including heart valves and portions of heart valves, aortic roots, aortic walls, connective tissue including fascia and dura mater, vascular grafts (including arteries, veins, and biological tubes), and orthopedic soft tissues, e.g., tendons or ligaments with or without bone, are often subjected to cryopreservation. In this way, a rapid supply of these valuable tissues becomes available for subsequent transplantation into mammals, particularly humans. In addition, viable xenograft tissues from transgenic animals or tissues derived from human or non-human cells that may contain differentiated cell types, stem cells, or genetically modified cells of various origins can also be appropriately processed, cryopreserved, and stored for subsequent transplantation. Further examples include engineered cells of tissue or constructs made by tissue engineering.

[0134] Any explanted animal tissue, cell population, and recovered mammalian organ stored or maintained by any one of the methods or processes disclosed herein, or any explanted mammalian cell, tissue, or organ stored in one or more of the disclosed compositions, is preferably suitable for transplantation into a selected recipient animal, particularly a selected recipient mammal. Examples of mammalian species into which the explanted tissue can be transplanted include, but are not limited to, humans, cows, horses, sheep, pigs, goats, rabbits, dogs, cats, and non-human primates.

[0135] In some aspects of any of the methods disclosed herein, cell types can include chondral cells, cartilagenous cells, osteochondral cells, pancreatic islet cells, osteoprogenitor cells, nerve cells, bone cells, bone marrow cells, adipocytes, fibroblasts, muscle cells, blood cells, and stem cells; animal tissues can include skin, bone, cartilage, tendon, ligament, intervertebral disc, cornea, lens, meniscus, hair, skeletal muscle, smooth muscle, cardiac muscle, adipose tissue, fibrous tissue, nerve tissue, and connective tissue; or mammalian organs can include cochlea, testis, ovary, stomach, lung, heart, liver, pancreas, kidney, intestine, and eye.

[0136] Cell populations, tissues, and organs prepared by the processes provided herein can be of any origin, but those of animal origin, particularly mammalian origin, are preferred. Exemplary biological materials to be explanted can be obtained from one or more animals including, but not limited to, cows, dogs, goats, horses, cats, chickens, humans, rabbits (lapine), wild rabbits (leporine), wolves (lupine), mice, sheep, pigs, foxes, or non-human primates.

[0137] Due to their high affinity for red blood cells, curcuminoids (e.g., curcumin) and / or flavonoids are expected to further act as therapeutic agents through enhancement of the production and release of either nitric oxide or small molecules that generate / release nitric oxide (nitrosothiols and iron-NO releasing hemes) when loaded onto red blood cells. On the other hand, curcuminoids (as well as other phytochemicals) loaded onto red blood cells are not readily induced by the immune system or liver for excretion, and thus are expected to show improved pharmacokinetics.

[0138] Since the formulation is directly mixed with blood cells in a blood sample, the amount of solvent is therefore increased such that it retains the curcuminoid and / or flavonoid dissolved in the mixture or prevents precipitation of the drug. As described above, the solvent may contain one or more components. The ratio of the solvent to one or more drugs can range from about 5:1 to about 500:1, about 10:1 to about 200:1, about 20:1 to about 200:1, about 20:1 to about 100:1, or about 50:1 to about 100:1 by weight. Non-limiting examples of the ratio include about 5:1, about 8:1, about 10:1, about 15:1, about 20:1, about 30:1, about 50:1, about 80:1, about 100:1, about 150:1, about 200:1, about 400:1, and any range between any two of the foregoing ratios. In some embodiments, each drug in the mixture or sample independently has a concentration in the range of about 0.001 to about 100, about 0.01 to about 50, about 0.01 to about 10, about 0.05 to about 10, about 0.05 to about 5, or about 0.1 to about 1 μg / mL. Non-limiting examples of the concentration include about 0.001 μg / mL, about 0.005 μg / mL, about 0.01 μg / mL, about 0.05 μg / mL, about 0.1 μg / mL, about 0.2 μg / mL, about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 5 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 30 μg / mL, about 50 μg / mL, about 100 μg / mL, about 200 μg / mL, or any range between any two of the foregoing concentrations. In some embodiments, the formulation disclosed herein has a concentration of curcuminoid in a solution in the range of about 1 mM to about 10 M, about 10 mM to about 10 M, about 100 mM to about 10 M, about 1 M to about 10 M, or about 5 M to about 10 M before mixing with the blood sample.

[0139] The agent, solvent, and their amounts are selected such that more than 40% of the red blood cells remain substantially viable over a period of at least about 24 hours. In some embodiments, at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, or at least 99% of the red blood cells remain viable for at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 45 days, at least 2 months, at least 3 months, at least 4 months, or at least 6 months. In some embodiments, the agent is curcumin or consists essentially of curcumin.

[0140] In some embodiments, the agent, solvent, and their amounts are selected such that the blood sample exhibits an enhanced increase in circulation time after being re-infused into the subject as compared to a reference sample not treated with the formulation under the same test conditions. The increase can be in the range of about 2% to about 30%, about 5% to about 20%, or about 8% to about 15%. Non-limiting examples of increases in circulation time include about 3%, about 5%, about 8%, about 10%, about 12%, about 15%, about 20%, and any range between any two of the foregoing values. Thus, the blood sample can function as both a physiological agent (oxygen delivery) and a therapeutic agent (curcumin delivery) when transfused into a patient having an inflammation-related co-morbidity including, for example, hemorrhagic shock.

[0141] In animal studies, histopathological examination shows a significant decrease in the % area involved in vascular congestion and iron deposition in male HbSS in the spleen and liver infarction and iron deposition in the liver as compared to the vehicle. Collectively, the decrease in hemolysis, and the increase in Hb, hematocrit, and ATP suggest erythrocyte stabilization, improvement in mitochondrial metabolism, and reduction of oxidative stress and organ damage resulting from the action of the formulations disclosed herein.

[0142] Other biomarkers affected by the formulation include enhanced ATP levels, reduced protein carbonylation, reduced vascular congestion and / or iron deposition, reduced levels of the general inflammatory marker serum amyloid-P, reduced inflammatory cytokines in the skin secretome with or without concomitant reduction of interleukins, reduced monocyte chemoattractant protein 1 (MCP-1), reduced interferon gamma (IFN-γ), reduced granulocyte macrophage colony-stimulating factor (GM-CSF), and regulated on activation normal T cell expressed and secreted protein (RANTES). Each biomarker independently increases or decreases in the range of about 2% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 20%, or about 10% to about 15% compared to a reference blood sample having the same components except that it has not been mixed with or treated by the formulation disclosed herein. Non-limiting examples of increases or decreases for each biomarker independently include about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, or any range between any two of the foregoing values.

[0143] These biomarkers are associated with various diseases or conditions. For example, MCP-1 contributes to neuropathic pain and inflammation; GM-CSF stimulates the differentiation and growth of granulocytes. Both RANTES and MCP-1 can activate mast cells. Thus, the formulation disclosed herein results in a significant reduction in degranulation of skin mast cells in sickle subjects compared to a vehicle or reference sample. The formulation disclosed herein provides an anti-inflammatory effect by targeting cytokine release and inhibiting granulocyte activity, either by transdermal administration or by mixing with blood ex vivo.

[0144] The above-described effects on various biomarkers by ex vivo mixing of the formulation and blood can also be achieved via administration to a subject with the transdermal formulations disclosed herein. Both administration of the transdermal formulations disclosed herein and transfusion of the processed ex vivo blood samples can significantly improve hyperalgesia (chronic pain), inflammation, hemolysis, oxidative stress, and organ damage, improve mitochondrial function and hematological parameters of SCD pathophysiology, and provide other disease-modifying and anti-nociceptive effects.

[0145] The mixture or blood sample may contain additional components such as stabilizers, antibacterial agents, and anti-inflammatory agents. For example, myristic acid and other medium-chain fatty acids that may be beneficial to health can be included as additives. Saturated fatty acids such as lauric acid, which has been shown to possess antibacterial and anti-inflammatory properties against Propionibacterium acnes (P. acnes), can also be included in the blood sample.

[0146] Method of use The transdermal formulations provide rapid intervention against various diseases and conditions by locally and / or systemically introducing high levels of curcuminoids and / or other potent anti-inflammatory agents and / or antioxidants.

[0147] A physiologically natural way to enhance the endothelial production of NO and reduce ROS levels is by decreasing CD38 levels. High levels of CD38 create low levels of NAD+, which in turn leads to: i) mitochondrial dysfunction that results in the overproduction of ROS; and ii) a decrease in the activity of sirtuin proteins. Collectively, these CD38-induced effects result in a decreased production of NO from endothelial cells, as well as an overproduction of ROS and peroxynitrite from activated macrophages and microglia, which promotes endothelial dysfunction. Furthermore, there is an interaction between senescent cells and CD38, with each promoting the accumulation of the other. The accumulation of senescent cells in the endothelium not only impedes the repair of the vascular intima but also promotes persistent endothelial dysfunction, including pathological vascular remodeling and a continuous decrease in NO production.

[0148] Formulations for the sustained transdermal delivery of active agents result in a decrease in CD38 ectoenzyme activity, such as NADase activity; thereby enhancing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. By doing so, this approach provides both new preventive and treatment methods for a variety of diseases, including those resulting from endothelial dysfunction following acute or chronic inflammatory injury, age-related decline in physical and cognitive skills, age-related cardiac tissue changes, vascular hypertrophy, osteoarthritis, peripheral neuropathy, and long COVID. Furthermore, this approach addresses the negative consequences of CD38 overproduction with regard to the ability of stem cells to differentiate into mature cells, which is at least partially responsible for chronic inflammatory anemia and the loss of effectiveness of native stem cells and stem cell therapies in repairing damaged tissues.

[0149] The transdermal formulation can be administered via any suitable route for delivering the active ingredient across the skin, mucosa, or membrane of the subject's body. Non-limiting examples of suitable routes include, for example, topical (e.g., drops and mucosal routes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols including those by nebulizer), intratracheal, intranasal, and epithelial routes. In some embodiments, the transdermal formulation comprises one or both of curcumin and quercetin, and optionally one or more of polyphenols, flavonoids, stilbenoids, and seco-steroids.

[0150] Without being limited to any particular theory, the transdermal formulations disclosed herein are hypothesized to deliver the active agent transdermally to the subject and enhance the systemic or local NO level in the subject. In some embodiments, the formulation contains an effective amount of an NO booster to increase the systemic or local NO level in the subject. In some embodiments, the formulation contains an effective amount of an NO precursor, and the method converts the NO precursor to, for example, an S-nitrosothiol-containing molecule, which releases NO transdermally to the subject. In some embodiments, the formulation may contain both an NO booster and an NO precursor. In some embodiments, the formulation may also contain a second agent as defined above.

[0151] The transdermal formulation achieves NO enhancement through a pathway that includes upregulation of endothelial nitric oxide synthase (eNOS), enhancement of eNOS activity, and reduction of ROS levels. For example, ROS scavenge NO, cause eNOS decoupling, lead to cessation of eNOS-related NO synthase, and instead result in further production of ROS by eNOS. On the other hand, endothelial loss results in i) loss of the fluid-mediated mechanotransduction mechanism for controlling NO production from eNOS; and also loss of the acellular region adjacent to the endothelium that prevents NO scavenging by hemoglobin in red blood cells. The transdermal formulation of this patent document provides an effective amount of an active agent capable of enhancing NO levels in the inner endothelial layer of blood vessels by inhibiting ROS from scavenging NO and limiting degradation of the inner glycocalyx layer of the endothelium.

[0152] The transdermal formulation can be applied to the body surface or body cavity of a subject. For example, a method of enhancing systemic or local NO levels or treating a disease or condition can include inserting a sponge loaded with the formulation disclosed herein between the cheek and the gum.

[0153] In diseases or conditions associated with elevated CD38 levels, the transdermal formulations disclosed herein can reduce CD38 levels / activity, restore nicotinamide adenine dinucleotide (NAD), and thus prevent and reverse many of the consequences of CD38 enhancement. NAD+ (NAD) is an important coenzyme found in all cells in the body that is involved in hundreds of metabolic processes such as cellular energy and mitochondrial health. It is required for the activity of SIRT1 and SIRT3, which are essential for the control of inflammation, oxidative stress, and cell repair. CD38 is an enzyme found primarily but not exclusively on the surface of macrophages and microglial cells. CD38 has NADase activity (breaks down NAD). The amount of CD38 on macrophages and microglial cells increases with aging and the onset of acute and chronic inflammation, and as a result, NAD levels decrease, leading to decreased energy, fatigue, increased inflammatory processes, and an inability to repair damage to cellular components including DNA. The transdermal formulations disclosed herein can effectively reduce CD38 levels with little or no side effects, and thus it can prevent and limit severe endothelial dysfunction following acute inflammatory injury, limit age-related physical and cognitive skill decline, reverse age-related heart tissue changes, and help stabilize red blood cells.

[0154] Certain flavonoids, such as apeginin and quercetin, have additional advantages compared to curcumin due to their senolytic activity, in addition to many anti-inflammatory and antioxidant stress properties similar to curcumin. Senescent cells induce an increase in CD38 levels. By removing senescent cells, CD38 levels can be decreased and thus NAD levels can be maintained or restored. Furthermore, CD38 can be decreased when activated M1 macrophages are re-polarized to M2 macrophages. Curcumin and flavonoids have the ability to re-polarize macrophages. Uptake of these active substances by circulating M1 macrophages via the transdermal / transmucosal route is proposed to be a mechanism for the rapid decrease in CD38 activity. A CD38 inhibitor can be a drug that re-polarizes macrophages and then results in a decrease in CD38 activity.

[0155] A disease or condition associated with elevated CD38 levels refers to a disease or condition in which a subject is detected to have a level of CD38 higher than normal levels. Normal levels of CD38 can be readily obtained from healthy individuals using well-known procedures and statistically acceptable analyses.

[0156] Various diseases or conditions can be treated with the transdermal formulations disclosed herein. Non-limiting examples of diseases and conditions include age-related physical and / or cognitive decline, lupus, rheumatoid arthritis, multiple sclerosis, leukemia and multiple myeloma, cardiovascular diseases, neurodegenerative diseases, COVID-19 symptoms, severe and persistent ones (long COVID), diabetes, hypertension, neuropathic pain, osteoarthritis, anemia in chronic diseases, ALS, Parkinson's disease, ischemia-reperfusion injury, hypoxia-reoxygenation injury, transfusion-induced injury, radiation-induced injury including dermatitis, and neuroinflammation.

[0157] Other diseases or conditions treatable with the formulations disclosed herein include muscle structure disorders, neuronal activation disorders, muscle fatigue disorders, muscle mass disorders, metabolic disorders, vascular diseases, ocular vascular diseases, myogenic eye diseases, kidney diseases, hypertension, inflammation, endothelial dysfunction, dermatological conditions, ophthalmological conditions, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomena, sickle cell disease, the inflammatory consequences of acute sickle cell crisis and other abnormal hemoglobinemias (including HbE / β-thalassemia), Chagas disease, type 2 diabetes, lupus, and transient inflammatory conditions including leaky gut syndrome and "brain fog" resulting from chemotherapy.

[0158] In some embodiments, the disease or condition is one of the following: a muscle structure disorder selected from Bethlem myopathy, central core disease, congenital fiber type disproportion, distal muscular dystrophy (MD), Duchenne and Becker MD, Emery-Dreifuss MD, facioscapulohumeral MD, hyaline body myopathy, limb-girdle MD, muscle sodium channelopathy, myotonic chondrodystrophy, myotonic dystrophy, myotubular myopathy, nemaline body disease, oculopharyngeal MD, or stress urinary incontinence; a nerve cell activation disorder is selected from amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Guillain-Barré syndrome, Lambert-Eaton syndrome, multiple sclerosis, myasthenia gravis, neuropathy, peripheral neuropathy, spinal muscular atrophy, tardy ulnar palsy, and toxic myoneuropathy; a muscle fatigue disorder is selected from chronic fatigue syndrome, diabetes (type I or II), glycogen storage disorder, fibromyalgia, Friedreich's ataxia, intermittent claudication, lipid storage myopathy, MELAS, mucopolysaccharidosis, Pompe disease, or thyrotoxic myopathy; a muscle mass disorder is cachexia, cartilage degeneration, cerebral palsy, muscle compartment syndrome, critical illness myopathy, inclusion body myositis, polymyositis, muscular atrophy (inactive), sarcopenia, steroid myopathy, and systemic lupus erythematosus; and a β-oxidation disorder is selected from systemic carnitine transporter, carnitine palmitoyltransferase (CPT) II deficiency, very long chain acyl-CoA dehydrogenase (LCHAD or VLCAD) deficiency, trifunctional enzyme deficiency, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, short chain acyl-CoA dehydrogenase (SCAD) deficiency, and riboflavin-responsive disorder of β-oxidation (RR-MADD);Metabolic disorders are selected from hyperlipidemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, low HDL cholesterolemia, high LDL cholesterolemia and / or HLD non-cholesterolemia, high VLDL proteinemia, abnormal lipoproteinemia, apolipoprotein A-I hypoproteinemia, atherosclerosis, arteriosclerosis, cardiovascular diseases, cerebrovascular diseases, peripheral circulatory diseases, metabolic syndrome, syndrome X, obesity, diabetes (type I or II), hyperglycemia, insulin resistance, impaired glucose tolerance, hyperinsulinemia, diabetic complications, heart failure, myocardial infarction, cardiomyopathy, hypertension, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), thrombosis, Alzheimer's disease, neurodegenerative diseases, demyelinating diseases, multiple sclerosis, adrenoleukodystrophy, dermatitis, psoriasis, acne, skin aging, hirsutism, inflammation, arthritis, asthma, irritable bowel syndrome, ulcerative colitis, Crohn's disease, and pancreatitis; cancers are selected from colon cancer, colorectal cancer, skin cancer, breast cancer, prostate cancer, ovarian cancer, and lung cancer; vascular diseases are selected from peripheral vascular insufficiency, peripheral vascular diseases, intermittent claudication, peripheral vascular disease (PVD), peripheral arterial disease (PAD), peripheral arterial occlusive disease (PAOD), and peripheral occlusive arterial disease; ophthalmic vascular diseases are selected from age-related macular degeneration (AMD), Stargardt disease, hypertensive retinopathy, diabetic retinopathy, retinopathy, macular degeneration, retinal hemorrhage, and glaucoma; muscular ophthalmic diseases are selected from strabismus, progressive external ophthalmoplegia, esotropia, exotropia, refractive and accommodative disorders, hyperopia, myopia, astigmatism, anisometropia, presbyopia, accommodative disorders, and internal ophthalmoplegia; renal diseases are selected from glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, acute nephritis, recurrent hematuria, persistent hematuria, chronic nephritis, rapidly progressive nephritis, acute renal failure, chronic renal failure, diabetic nephropathy, and Bartter syndrome.;

[0159] In some embodiments, the disease or condition is selected from hereditary lipodystrophy, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal ischemia / reperfusion injury (IRI), cardiac ischemia / reperfusion injury, Duchenne and Becker muscular dystrophy, diabetes (type I or II), obesity, and sarcopenia. The disease or condition is selected from hereditary lipodystrophy, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), renal ischemia / reperfusion injury (IRI), cardiac ischemia / reperfusion injury, Duchenne and Becker muscular dystrophy, diabetes (type I or II), obesity, and sarcopenia.

[0160] In some embodiments, the disease or condition is selected from Alzheimer's disease, CPEO - chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome (KSS), Leber hereditary optic neuropathy (LHON), MELAS - mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes, MERRF - myoclonic epilepsy and ragged-red fiber disease, NARP - neurogenic muscle weakness, ataxia, and retinitis pigmentosa, Pearson syndrome, platinum-based chemotherapy-induced ototoxicity, cocaine syndrome, xeroderma pigmentosum A, Wallerian degeneration, and HIV-induced lipodystrophy.

[0161] In some embodiments, the disease or condition is a neurodegenerative disease including dementia, Alzheimer's disease (AD), Parkinson's disease.

[0162] In some embodiments, the disease or condition is a tumor selected from glioblastoma, lung cancer, colon cancer, liver cancer, breast cancer, gastric cancer, bladder cancer, and melanoma.

[0163] In some embodiments, the disease or condition is an autoimmune disease selected from diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE).

[0164] In some embodiments, the disease or condition is an inflammatory disease selected from asthma, chronic obstructive pulmonary disease (COPD), pneumonia, and non-alcoholic steatohepatitis (NASH).

[0165] In further exemplary embodiments, the transdermal formulations and systems prevent, manage, and reverse the clinical outcomes of inflammatory diseases including diabetes, COVID-19 infection, and sickle cell disease, provide topical treatment for hypertension or topical treatment for osteoarthritis, reverse the acute inflammatory cascade (cytokine storm), improve the safety and efficacy of stored red blood cells to be transfused, treat cerebral malaria or Chagas disease, or can be used as a transdermal therapy for treating other early-stage acute inflammatory diseases.

[0166] Phytochemicals (e.g., curcuminoids) have been shown to have antiviral activity. For example, the glycocalyx has recently been shown to prevent viral access to the ACE2 binding receptor on endothelial cells and thus can limit uptake and replication. When the glycocalyx is degraded by endothelial dysfunction, viral access to the ACE2 binding site increases. Curcumin and many of these other phytochemicals protect and preserve the glycocalyx by reducing ROS production and enhancing endothelial NO production (see below). These phytochemicals also reduce diet- and obesity-induced inflammatory injury by normalizing lipid and glucose metabolism, including insulin production and utilization. To treat inflammation caused by toxic chemicals and metals, curcumin and other phytochemicals can chelate and remove toxins from the blood. They also limit the inflammatory response to inhaled toxicants and thus reduce the tendency to progress to ARDS. Phytochemicals stabilize red blood cells and thus minimize toxin-induced hemolysis, a potent trigger for inflammation.

[0167] The formulations disclosed herein can address inflammatory injuries, including acute inflammatory injuries caused by specific viral infections (e.g., SARS CoV2, dengue fever, and influenza), obesity and glucose-induced inflammatory triggers, as well as inflammation caused by exposure to toxic metals and chemicals. For example, in patients with long COVID, the adverse effects resulting from COVID, which appear after seemingly recovering from the primary infection, include brain fog, fatigue, pain, coagulation problems, myocarditis, edema, etc. Most of these long COVID symptoms may be due to a continuous imbalance between pro-inflammatory and anti-inflammatory factors that promote the onset and persistence of endothelial dysfunction. The formulations and methods disclosed herein can be applied to the treatment of these clinical symptoms of long COVID.

[0168] In some aspects of the treatment method, the transdermal formulations disclosed herein are applied to the treatment of diseases or conditions generally associated with a "cytokine storm" including, but not limited to: COVID-19 infection, sepsis, systemic inflammatory response syndrome (SIRS), cachexia, septic shock syndrome, traumatic brain injury (e.g., brain cytokine storm), graft-versus-host disease (GVHD), or as a result of treatment with activated immune cells, e.g., IL-2 activated T cells, T cells activated by anti-CD19 chimeric antigen receptor (CAR) T cells. In addition to the effect on endothelial function, an active agent at a sufficient concentration, such as curcumin, acts to efficiently block the binding of the spike protein on SARS CoV 2 to the ACE2 binding site on endothelial cells and lung epithelial cells, thus inhibiting viral replication in vulnerable subjects.

[0169] In some aspects of the treatment methods disclosed herein, the transdermal formulation is administered to treat vascular leakage caused by a disease or condition. Non-limiting exemplary diseases or conditions include vascular leak syndrome, infectious diseases, inflammatory diseases, particularly sepsis, lupus, irritable bowel disease, inflammatory bowel disease, and inflammation of the systemic vasculature including the blood-brain barrier resulting from chemotherapy. Vascular leakage is characterized by hypotension, peripheral edema, and hypoalbuminemia. Vascular leakage may also be associated with diseases caused by pathogens, particularly viruses and bacteria.

[0170] In some aspects of the treatment methods disclosed herein, the transdermal formulation is administered to treat or reduce the risk of cardiovascular diseases associated with endothelial dysfunction. Endothelial cells are an important component of blood vessels and play an important role in cardiovascular homeostasis by regulating blood fluidity and fibrinolysis, vasotension, angiogenesis, monocyte / leukocyte adhesion, and platelet aggregation. The occurrence of endothelial dysfunction disrupts endothelial barrier permeability, which is part of the inflammatory response in the development of cardiovascular diseases. Non-limiting examples of cardiovascular diseases include coronary artery disease (CAD), such as angina pectoris and myocardial infarction (commonly known as a heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, and venous thrombosis.

[0171] In some embodiments, the amount / dosage of the active agent(s) is selected and / or the dosing schedule is configured such that the level of a biomarker associated with cardiovascular disease in a subject is increased or decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% as compared to a control (no treatment with the transdermal formulation) or a level prior to treatment with the transdermal formulation disclosed herein. Non-limiting examples of biomarkers associated with cardiovascular disease include white blood cell count (WBC), erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), cardiac troponin, creatine kinase (CK), CK-MB, and myoglobin. In some embodiments, the subject prior to treatment has an abnormal level of one or more biomarkers associated with cardiovascular disease, and the abnormal level of the one or more biomarkers is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% higher or lower than a normal level or the level of a healthy subject.

[0172] Additional examples of "cytokine storms" or infectious diseases commonly associated with vascular leakage include, but are not limited to, coronavirus (including COVID-19 / (SARS-CoV-2) coronavirus infection), malaria, avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), and severe acute respiratory syndrome (SARS). Certain infectious agents include, but are not limited to, Ebola, Marburg, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Fini, Machupo, Sabia, Guanarito, Garrissa, Ireshia, or Lassa fever virus. In some embodiments, the infectious disease is caused by a virus, bacterium, fungus, worm, protozoan, or hemorrhagic infectious agent. In some embodiments, the infectious disease is caused by a coronavirus (including cov including covid-19), Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, or Rhabdoviridae virus. In some embodiments, the transdermal formulations and methods described herein can be applied to the treatment of septic shock syndrome, chronic inflammatory responses to infectious diseases.

[0173] The methods disclosed herein can also be applied to the treatment of various types of pain, including, for example, neuropathic pain, surgery-related pain, trauma, periodontal or other dental procedure-related pain, and orthopedic or arthritis pain. For example, pain associated with periodontal or other dental procedures can be treated with a sponge inserted into the subject's mouth at a suitable location, such as between the cheek and the gum. The transdermal formulation can be administered before or after the onset of pain. For example, the formulation can be administered to the subject before a surgical procedure as a prophylactic method of reducing pain.

[0174] Conventional drugs for neuropathic pain have various levels of side effects. The transdermal formulations disclosed herein can be used alone or in combination with conventional drugs including, for example, gabapentinoids as first-line drugs, tricyclic antidepressants, and / or selective serotonin-norepinephrine reuptake inhibitors, lidocaine, capsaicin, and / or tramadol as second-line drugs, and morphine, oxycodone, botulinum toxin A, and other opioids as third-line treatments. As a result, the transdermal formulations offer the advantage of reducing dependence on conventional analgesics and minimizing side effects.

[0175] The transdermal formulations can also be applied to postoperative pain management. For example, for individuals undergoing surgeries including bypass, and chest surgery, coronary artery, inguinal hernia repair, and lower limb amputation, the transdermal formulations can serve as alternative therapeutic agents for treating these aforementioned pain conditions.

[0176] As described above, the formulations can be administered in any suitable form for the methods disclosed herein. In some embodiments of any of the formulations or methods disclosed herein, the formulations are in semi-solid or solid form and are rubbed in or spread on the skin or mucosal surface of the subject. In some embodiments, patches are coated or impregnated with formulations in liquid, semi-solid or solid form. In some embodiments, one or more of the NO booster, NO precursor and acid source are in a liquid reservoir prior to administration. In some embodiments, the formulations are administered via a nebulizer or a sprayer. In some embodiments, the subject is a human. In some embodiments, the presence of symptoms, signs, and / or risk factors of the disease or condition being treated is determined before initiating administration of the formulation.

[0177] The transdermal preparation of this patent document can be administered to activate nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin-1 (SIRT1) in a subject. Therefore, various diseases or conditions associated with dysfunctional SIRT1 can be treated. Sirtuins are a type of NAD+-dependent protein deacetylase enzyme that regulate a wide variety of cellular activities that promote cell survival and extend lifespan in response to environmental stress. Sirtuins exert their effects by removing acetyl groups from specific target proteins in the presence of oxidized nicotinamide adenine dinucleotide (NAD+). For example, the yeast sirtuin enzyme Sir2 (silent information regulator 2), originally identified for its role in the silencing of DNA transcription, has also been shown to promote cell survival in response to calorie restriction. Similarly, in C. elegans, the sirtuin enzyme SIR-2.1 has been shown to extend lifespan. In mammalian cells, the sirtuin enzyme SIRT1 (a homolog of the yeast Sir2 and C. elegans SIR-2.1 enzymes) deacetylates the tumor suppressor p53 to promote cell survival. SIRT1 has been reported to regulate various pathways, including restoration of angiogenesis function and secretion of angiogenesis-promoting factors in endothelial progenitor cells. Influential papers have demonstrated that SIRT1 is involved in protection against excessive inflammation and oxidative stress by deacetylating NFκB and forkhead box O transcription factors. Furthermore, SIRT1 inhibits cellular senescence, promotes keratinocyte differentiation, and protects against UV-induced DNA damage. Some studies have also demonstrated that downregulated or dysfunctional SIRT1 is associated with various diseases such as the diabetic environment, and that overexpression of SIRT1 improves glucose intolerance and insulin sensitivity and protects against diabetes. Therefore, sirtuins appear to be activated as part of a beneficial cellular response to stress, leading to cell survival and lifespan extension.

[0178] Therefore, activators of sirtuins can be beneficial in bringing about fundamental cellular processes that protect cells from stress, prevent or treat various diseases or conditions, and extend healthspan.

[0179] Transdermal delivery of active agents (e.g., NO enhancing and SIRT1 activating therapeutics) enables easy combination with oral treatments that target other related disease pathways not effectively addressed via transdermal agents. The method includes administering to a subject in need thereof a transdermal formulation disclosed herein. In some embodiments, the formulation comprises (a) a therapeutically effective amount of a SIRT1 activator; (b) an amount of a polyol solvent sufficient to dissolve the SIRT1 activator; and (c) a fatty acid. The SIRT1 activator can be one or more of the NO boosters described above. In some embodiments, the SIRT1 activator comprises one or more of curcuminoid, berberine, quercetin, resveratrol, and fisetin. The amount of the activator can be adjusted according to the nature of the agent and the disease or condition being treated. In some embodiments, the activator ranges from about 0.05 wt% to about 40 wt% in the formulation. In some embodiments, the formulation provides sustained release of the activator over a period of about 15 hours.

[0180] Treatment of acute and chronic diseases or other conditions can benefit from enhanced systemic nitric oxide levels in the endothelium and / or activation of the SIRT1 and NRF2 signaling pathways. Non-limiting diseases or conditions include sickle cell disease, HbE / β thalassemia and other thalassemias, diabetic retinopathy, glaucoma, dry eye syndrome, and surgery-induced inflammation.

[0181] In some embodiments, the method enhances SIRT1 activity in a subject. The scope and composition of the formulation are as described above. In some embodiments, the formulation comprises (a) a therapeutically effective amount of a SIRT1 activator; (b) an amount of polyol solvent sufficient to dissolve the SIRT1 activator; and optionally (c) a fatty acid. The SIRT1 activator can be one or more of the NO boosters described above. In some embodiments, the SIRT1 activator comprises one or more of curcuminoid, berberine, quercetin, resveratrol, and phycetin. The amount of the activator can be adjusted according to the nature of the drug and the disease or condition being treated. In some embodiments, the activator ranges from about 0.05 wt% to about 40 wt% in the formulation. In some embodiments, the formulation provides sustained release of the activator over a period of about 1, about 2, about 4, about 8, about 10, about 15, or about 24 hours. In some embodiments, the disease or condition is selected from aging, chronic and acute inflammatory conditions, chemically induced vascular inflammation, viral infections, bacterial infections, and fungal infections. In some embodiments, diagnosing a subject as having a transdermal formulation, endothelial dysfunction, or a disease or condition associated with endothelial dysfunction. In some embodiments, the subject is diagnosed as having a disease or condition selected from the group consisting of neurodegenerative diseases, diabetic nephropathy, diabetes, cardiovascular diseases, endothelial dysfunction, muscular dystrophy, pain, neuropathic conditions, abnormal vascular homeostasis, and lupus.

[0182] The transdermal formulation of this patent document can be administered to promote a therapeutic effect or reduce the adverse events of another therapy. In some aspects of any of the methods disclosed herein, the transdermal formulation of this patent document can be administered before, simultaneously with, or after another treatment, including, for example, a drug therapy administered orally, an intravenous infusion, an intramuscular injection, a topical medical procedure, and / or surgery. In some aspects, the transdermal formulation is administered before an additional therapy for a disease or condition. For example, a topical pretreatment with the formulation disclosed herein before a blood transfusion can maximize tissue perfusion and minimize transfusion-related inflammation. The topical pretreatment with the formulation or the combined administration with another therapy can also reduce adverse events related to the treatment (e.g., side effects related to glucose-lowering drugs such as metformin, skin rashes, oral mucositis / stomatitis related to chemotherapy).

[0183] The transdermal formulation can also enhance endothelial function in a subject. Thus, various diseases or conditions associated with dysfunctional or imbalanced endothelial function can be treated. The endothelium has two major interrelated elements essential for vascular homeostasis: the glycocalyx and endothelial nitric oxide synthase (eNOS). The hair-like projections from the endothelium called the glycocalyx are involved in the following: i) Maintaining vascular integrity and thus restricting vascular leakage and access of macrophages and lipids to deeper layers of the vessel wall (trigger for plaque formation); ii) Controlling the overproduction of reactive oxygen species (ROS) by acting as a depot for superoxide dismutase (SOD), a powerful antioxidant; iii) Regulation of blood flow in response to physiological demands by shear stress-controlled production of nitric oxide by eNOS; iv) Restricting access and binding of blood-derived cells (RBCs, monocytes, leukocytes), platelets, and infectious agents to the endothelium; v) Restricting platelet activation; vi) Preventing blood flow stasis; vii) Ensuring continuous NO production by eNOS by preventing eNOS decoupling as a result of excessive ROS. In the decoupled state, eNOS no longer generates NO but instead generates ROS, leading to more inflammation; viii) Maintaining a cell-free area along the endothelium and thus preventing scavenging of endothelium-generated NO by RBCs in close proximity to the endothelium. On the other hand, nitric oxide generated by endothelial nitric oxide synthase (eNOS) is essential for vascular homeostasis. Important functions of eNO include the following: i) Maintaining tissue perfusion / oxygenation; ii) Preventing blood flow stasis; iii) Preventing a procoagulant environment; iv) Repolarizing activated macrophages, thus promoting tissue repair and limiting tissue damage; v) Regulating the pro-inflammatory and anti-inflammatory processes (the balance between pro-inflammatory iNOS activity that generates harmful peroxynitrite, anti-inflammatory eNOS activity that generates eNO, and activation of SIRT1); vi) Preventing inflammatory damage resulting from ischemia-reperfusion and hypoxic-reoxygenation; vii) Preventing ROS-induced damage, including lipid peroxidation and glycocalyx degradation; viii) Creating a depot of nitrosothiols stored within the endothelium and surrounding vascular layers that can rapidly supply NO under conditions where it is necessary to increase NO levels, such as in cases of extreme muscle activity.

[0184] Endothelial dysfunction is a physiological impairment of normal biochemical processes carried out by the endothelium, the cells that line the inner surface of blood vessels. The hallmark of endothelial dysfunction is the impairment of endothelium-dependent vasodilation mediated by nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS), a constitutive form of NOS mainly expressed in endothelial cells. In a healthy vascular system, NO produced by the endothelium diffuses into vascular smooth muscle cells (VSMCs), which activates guanylate cyclase, stimulates the production of cyclic guanosine monophosphate (cGMP), thereby promoting relaxation of VSMCs and, as a result, promoting vasodilation. Other functions of the endothelium, such as inhibition of platelet aggregation, inhibition of leukocyte adhesion, and inhibition of VSMC proliferation, are also mediated by NO. In dysfunctional endothelium, NO production is impaired. Endothelial dysfunction can be clinically detected, for example, by an increase in the number of circulating endothelial cells (CECs).

[0185] Endothelial dysfunction is associated with various diseases, such as hypertension, coronary artery disease, heart failure, stroke, peripheral artery disease, diabetes, chronic kidney disease, abnormal vascular smooth muscle cell proliferation and other cardiovascular diseases, type 2 diabetes, insulin resistance and other metabolic syndromes, lupus, HIV, radiation and drug therapy (e.g., chemotherapy)-induced inflammation, abnormal hemoglobinopathies (sickle cell disease, HbE / β-thalassemia), cytokine storm-related conditions induced by viral diseases (e.g., SARS CoV 2, dengue fever, influenza, hemorrhagic shock, hemorrhagic fever), erectile dysfunction following surgery-induced inflammation, and inflammation associated with an increase in the population of senescent cells typically associated with aging. Furthermore, endothelial dysfunction is considered an important event in the development of atherosclerosis and has been present for years before clinically evident vascular pathology. Endothelial dysfunction has also been shown to be prognostically important in the prediction of vascular events, including stroke and myocardial infarction. In addition, endothelial dysfunction has been shown to be involved in inflammation, infectious diseases, immune system dysfunction, sleep apnea, sepsis, chronic obstructive pulmonary disease, and exposure to inflammatory agents.

[0186] The methods disclosed herein are applicable to the treatment of acute and chronic consequences of endothelial dysfunction. Examples of chronic consequences of endothelial dysfunction include the diseases and conditions described above. In some embodiments, the methods are applicable to the treatment of acute consequences, such as, for example, cytokine storms, and associated hypoxic / ischemic organ damage caused by physical activity or diet (e.g., heart attacks due to inadequate tissue perfusion / oxygenation), stroke, microemboli and macroemboli, pulmonary embolism, ischemia-reperfusion injury, hypoxia-reoxygenation injury, and long COVID, which is a consequence of ongoing chronic inflammation / endothelial dysfunction. In some embodiments, the methods are applicable to the treatment of chronic consequences, such as, for example, cardiovascular disease (CVD), coronary artery disease (CAD), renal insufficiency, enhanced predisposition to cognitive decline and dementia, hypertension, sexual dysfunction, slow-healing wounds, accelerated stent failure / closure, coronary artery bypass failure, slow-healing wounds, reduced tolerance to physical activity due to mitochondrial dysfunction, accelerated age-related conditions, osteoarthritis, transient ischemic events, diabetic retinopathy, reduced insulin production due to initiation of inflammation damage to pancreatic β-cells, HIV-induced CVD, CVS, and CAD secondary to any ongoing regular episodes of sleep apnea and / or blood flow stasis (e.g., sickle cell disease). Other uses of the methods include transfusions, including either RBC or hemoglobin-based oxygen carriers (HBOCs), and kidney dialysis.

[0187] Transdermal formulations can be administered to treat various local conditions associated with endothelial dysfunction. For example, local conditions such as slow-healing lower limb ulcers and erectile dysfunction are related to a fundamental and often severe endothelial dysfunction that restricts blood flow to damaged tissues. Methods for treating slow-healing lower limb ulcers include the elimination of biofilms and infections that impede the therapeutic effect of agents designed to accelerate wound closure and the sustained local delivery of nitric oxide. In parallel, the transcellular delivery of active agents such as curcumin normalizes the systemic vasculature and thus promotes tissue oxygenation and enables the migration and development of stem cells. Transdermal formulations can be administered in combination with an antibiotic agent or any suitable wound healing agent. In the case of erectile dysfunction, the transdermal delivery of agents such as curcumin can be used alone or in combination with local nitric oxide and / or oral PD5 inhibitors to restore systemic vascular health and reduce systemic inflammation. Considering that systemic NO boosters (such as curcumin) enhance NO production in the endothelium and oral supplementation with PD5 inhibitors prolongs the duration of action of NO, the combination of transdermally delivered active agents and oral PD5 inhibitors will likely accelerate endothelial recovery in patients with endothelial dysfunction, including long COVID and cytokine storm.

[0188] The transdermal formulations disclosed herein can also be administered to a subject in need thereof to reduce ROS production, peroxynitrite production (through inactivation of iNOS activity), and / or increase eNO production in the endothelium. Without being bound by any particular theory, the formulations are hypothesized to provide a multifaceted effect on a plurality of anti-inflammatory and antioxidant enzymes, including sirtuin 1 (SIRT1) and other inflammatory regulatory sirtuins, and upregulation and / or activation of signaling pathways; PPAR(γ) (peroxisome proliferator-activated receptor γ), peroxisome proliferator-activated receptor γ coactivator (PGC)-1α are members of a family of transcriptional coactivators that play a central role in the regulation of cellular energy metabolism. AMP-activated protein kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme present in all mammalian cells. When activated, AMPK stimulates energy-generating processes such as glucose uptake and fatty acid oxidation and reduces energy-consuming processes such as protein and lipid synthesis. Transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), a major regulator of antioxidant and cytoprotective genes, is mainly activated in response to oxidative stress. The SIRT1 / PGC-1α / PPAR-γ pathway, eNOS-mediated enhancement of eNO production, PPARP, Nrf2, heme oxygenase, AMPK, and ACE2 (angiotensin-converting enzyme 2, or ACE2 “receptor”, the protein provides an entry point for coronaviruses to hook onto and infect a wide range of human cells. The formulations can also be applied to downregulation or inhibition of (the toll-like receptor 4 moiety of the inflammatory induction mechanism) TLR4, NADPH oxidase (NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) is a membrane-bound enzyme complex that generates reactive oxygen species facing the extracellular space), and ACE (ACE (angiotensin I-converting enzyme)).

[0189] The transdermal formulations described herein can be applied to the treatment and management of acute inflammatory injuries and chronic sequelae of many pro-inflammatory conditions that can promote or be associated with endothelial dysfunction (ED). Non-limiting examples of diseases or conditions associated with endothelial dysfunction (ED) include cardiovascular disease, renal failure, cognitive decline, slow-healing wounds, hypertension, stroke, microemboli, edema, sexual dysfunction, retinopathy, neuropathy, and neuropathic pain.

[0190] For acute diseases or conditions, the formulation can rapidly initiate overall anti-inflammatory and antioxidant activities to prevent and limit progression leading to the severe consequences of extreme ED. For example, a suitable patch or sponge loaded with the formulation is inserted between the gum and cheek to provide a very high concentration of NO stimulant such as curcumin and to determine the period for ensuring rapid and sustained delivery of therapeutic levels of curcuminoids or other anti-inflammatory agents, antioxidants, and NO stimulants. This safe approach eliminates the concern of direct over-ingestion of NO and further activates the complete repertoire of host-based anti-inflammatory and antioxidant pathways. The use of active agents delivered transdermally, either systemically or locally, such as curcuminoids, can stimulate NO production in the vasculature and / or reduce the overproduction of reactive oxygen species (ROS). This combination of enhanced endothelial NO production and cessation of ROS production is designed to prevent, limit, and reverse ED and its consequences.

[0191] The transdermal formulation of this patent document can be administered to a subject in need thereof for treating diabetes and related inflammation and other conditions. Inflammation of adipose tissue promotes insulin resistance and hyperglycemia, both of which cause and exacerbate endothelial dysfunction. Furthermore, chronic untreated endothelial dysfunction caused and enhanced by hyperglycemia, excessive ROS production, and other diabetes-related factors is a common pathway by which type 2 diabetes and other inflammation-induced conditions, such as cardiovascular disease, renal failure, hypertension, stroke, and microemboli, slow-healing wounds, sexual dysfunction, and bladder dysfunction, neuropathic pain, and cognitive decline, cause end-stage clinical symptoms. By reducing blood glucose, reversing insulin resistance, reducing elevated or surgically induced exacerbated persistent hyperglycemia, reducing ROS levels, increasing NO levels in the endothelium, and restoring vascular homeostasis, the transdermal formulation of this patent document minimizes the negative and / or undesirable outcomes associated with surgery, blood transfusion, stent implantation, dialysis, and any other invasive procedure that can promote systemic inflammation. The formulation can upregulate Nrf2 and related antioxidant enzymes, including the potent antioxidant heme oxygenase (HO-1), and reduce oxidative stress (thereby enabling the recovery of damaged endothelium, treating endothelial dysfunction, and preventing the onset or progression of endothelial dysfunction). Furthermore, it limits the glucose response enhanced by diabetes after surgery and shortens the recovery time of elevated glucose levels. Additionally, it can limit stress-induced glucose spikes in stressed (e.g., injured, surgically treated, and transfused) diabetic patients and inflammatory outcomes of stress, including urogenital dysfunction after radical prostatectomy.

[0192] Accordingly, the transdermal formulation can be administered to limit the negative clinical outcomes associated with inflammation-inducing factors in a subject having an existing condition or at risk of developing a condition that promotes hyperglycemia and subsequent endothelial dysfunction. In some embodiments, the formulation is administered prophylactically to a subject at risk of developing hyperglycemia. In some embodiments, the subject is determined to have hyperglycemia.

[0193] In some embodiments, the amount / dosage of the active agent(s) is selected and / or the dosing schedule is configured such that the method restores insulin sensitivity and / or reduces elevated blood glucose by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% compared to a control (no treatment with the transdermal formulation) or a level prior to treatment with the transdermal formulation disclosed herein. In some embodiments, the subject is diagnosed with diabetes (e.g., type 2 diabetes) and has undergone an invasive procedure such as surgery, blood transfusion, stent implantation, and dialysis or has been injured. In some embodiments, the subject prior to treatment has an abnormal level of blood glucose, and the abnormal level is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least 80% or at least 100% higher or lower than a normal level or the level of a healthy subject.

[0194] The transdermal formulations disclosed herein can reduce the levels of inflammatory cytokines. In some aspects of the methods disclosed herein, the amount / dosage of the active agent(s) is selected and / or the dosing schedule is configured such that one or more of the biomarkers are reduced or modified by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 100% compared to the levels before treatment with the control or the transdermal formulation. Non-limiting examples of markers that can be reduced or modified by the transdermal formulations disclosed herein include TNF-α, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, IL-10, IL-1, IL-18, MIF, TNF-β, MMP9, HIF-1, GLUT1, Hemox, PDK1, VEGF, CD11, EMR1, CXCR4, CCR5, IL-8, receptor for advanced glycation end products (RAGE), hsCRP, total antioxidant capacity (TAC), prostaglandins, leukotrienes, substance P, phosphatidylserine surface presentation on RBCs, selectins, laminin and cadherin, immunoglobulin receptors, chondroitin sulfate, syndecan-1, IL-1a / b, TNF-a, IL-6, D-dimer, and other markers that reflect a tendency towards abnormal blood clotting, embolism, thrombosis, C-reactive protein (CRP), Nrf2, NFκB, glutathione peroxidase (GPx), superoxide dismutase (SOD), syndecan-1, high molecular weight hyaluronic acid (1,000 - 6,000 kDa), a disintegrin and metalloprotease with thrombospondin type 1 repeats-13, protein C, von Willebrand factor, chondroitin sulfate, and sP-selectin. Additional examples include blood pressure, vasodilation, blood flow dynamics, vascular leakage / edema, M1 / M2 macrophage polarization, soluble platelet selectin, heparan sulfate, and markers related to cell and tissue oxygenation.In some embodiments, the subject prior to treatment has one or more biomarkers or inflammatory cytokines at abnormal levels, and the abnormal levels of the biomarker or one or more cytokines are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 100% higher or lower than normal levels or levels in healthy subjects.

[0195] In some embodiments of the methods disclosed herein, the subject is diagnosed as having a disease or condition selected from the group consisting of aging, chronic and acute inflammatory conditions, chemically induced vascular and / or lung inflammation, viral infections, bacterial infections, and fungal infections.

[0196] Additional examples of diseases or conditions that can be treated by the methods disclosed herein include neurodegenerative diseases, diabetic kidney disease, diabetes, cardiovascular diseases, endothelial dysfunction, muscular dystrophy, pain, neuropathic conditions, abnormal vascular homeostasis, lupus, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, neurodegenerative consequences of traumatic brain injury or cerebral hemorrhage, hypertension, inflammation, osteoarthritis, rheumatoid arthritis, endothelial dysfunction, dermatological conditions, ophthalmological conditions, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomenon, cerebral malaria, Chagas disease, abnormal hemoglobinopathies, type 2 diabetes, coronavirus, skin / dermatological conditions, urticaria, inflammatory skin conditions, Raynaud's disease, post-herpetic lesions, shingles, skin infections, wounds, burns, lower extremity ulcers, sickle cell, diabetic, onychomycosis, peripheral vascular disease, infectious and / or inflammatory mucosal tissues, erectile dysfunction, female sexual dysfunction, vaginal infections / inflammation, catheter-related urinary tract infections, rhinitis, cystic fibrosis, acute respiratory distress syndrome, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, pulmonary infections including tuberculosis, pulmonary hypertension, as well as burns and other open wounds, inner ear infections, outer ear infections, gastrointestinal diseases, and acute vasculitic conditions. Further examples of diseases treatable by the methods described herein include infectious diseases selected from the group consisting of coronavirus (including SARS-CoV-2), Ebola, dengue fever, hemorrhagic shock, endotoxic shock, cell-free hemoglobin toxicity resulting from the use of hemolysis and / or cell-free hemoglobin-based blood substitutes (HBOC), Rift Valley fever, Marburg, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Funin, Machupo, Sabia, Guanarito, Garissa, Iresh, and Lassa fever virus.

[0197] Additional examples of diseases or conditions treatable with the formulations disclosed herein include neurodegenerative diseases or disorders (e.g., Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and disorders caused by polyglutamine aggregation); skeletal muscle diseases (e.g., Duchenne muscular dystrophy, skeletal muscle atrophy, Becker muscular dystrophy, or myotonic dystrophy); metabolic disorders (e.g., insulin resistance, diabetes, obesity, impaired glucose tolerance, hypercholesterolemia, hyperglycemia, dyslipidemia, and hyperlipidemia); adult-onset diabetes, diabetic nephropathy, neuropathy (e.g., sensory neuropathy, autonomic neuropathy, motor neuropathy, retinopathy); bone diseases (e.g., osteoporosis), blood diseases (e.g., leukemia); liver diseases (e.g., due to alcohol abuse or hepatitis); obesity; bone resorption, age-related macular degeneration, AIDS-related dementia, ALS, Bell's palsy, atherosclerosis, heart diseases (e.g., arrhythmia, chronic congestive heart failure, ischemic stroke, coronary artery disease, and cardiomyopathy), chronic degenerative diseases (e.g., myocardial diseases), chronic renal insufficiency, type 2 diabetes, ulcers, cataracts, presbyopia, glomerulonephritis, Guillain-Barré syndrome, hemorrhagic stroke, rheumatoid arthritis, inflammatory bowel disease, SLE, Crohn's disease, diseases or disorders related to osteoarthritis, osteoporosis, chronic obstructive pulmonary disease (COPD), pneumonia, skin aging, urinary incontinence, mitochondrial dysfunction (e.g., mitochondrial myopathy, encephalopathy, Leber's disease, Leigh encephalopathy, Pearson's disease, lactic acidosis, "mitochondrial encephalopathy, lactic acidosis, stroke-like symptoms" (MELAS), neuromuscular diseases such as muscular dystrophy and myopathy, muscle diseases, as well as diseases or disorders related to other conditions characterized by neuronal cell death, aging, or unwanted cell loss are included).In some embodiments, the disease or condition is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and neurodegenerative consequences of traumatic brain injury or cerebral hemorrhage, sickle cell disease (including pain associated with sickle cell disease), thalassemia (e.g., HbE / beta thalassemia), diabetic retinopathy, glaucoma, dry eye syndrome, and inflammation caused by surgery, aging, chronic and acute inflammatory conditions, chemically induced vascular and / or lung inflammation, viral infections, bacterial infections, fungal infections, diabetic kidney disease, diabetes, cardiovascular disease, endothelial dysfunction, muscular dystrophy, pain, neuropathic conditions, abnormal vascular homeostasis, lupus, retinopathy including diabetic retinopathy, macular degeneration, peripheral vascular disease, long-term systemic consequences of chemotherapy and radiotherapy, brain fog, rheumatoid arthritis, soft tissue injuries (muscles, tendons and ligaments), surgery-induced inflammatory sequelae (urogenital dysfunction), inflammation induced by blood transfusion, suppression of stent restenosis, limitation of inflammatory consequences of dialysis, inflammation and pain after dental treatment, neuropathic pain from any inflammation-induced injury including peripheral neuropathy, spinal cord neuropathy, arthritic pain, cognitive dysfunction in children due to cerebrovascular injury resulting from sickle cell disease, cytokine storm resulting from coronavirus, dengue, Ebola, Rift Valley fever and influenza, cerebral malaria, metastatic spread of tumors via dysfunctional blood vessels, systemic consequences of psoriasis, dementia including Alzheimer's disease and Pick's disease, traumatic brain injury and hemorrhagic shock.

[0198] Autoimmune and immune-related disorders and diseases can also be treated or prevented by the methods described herein. Exemplary autoimmune diseases and immune-related disorders include systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis, idiopathic inflammatory myopathy, Sjogren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central or peripheral nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive enteropathy, Whipple's disease, autoimmune or immune-mediated skin diseases, vesicular skin diseases, erythema multiforme, contact dermatitis, psoriasis, allergic diseases, asthma, allergic rhinitis, atopic dermatitis, food allergies, urticaria, immune diseases of the lung, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, systemic lupus erythematosus, scleroderma, and arthritis.

[0199] Non-limiting examples of neurological diseases that can be treated or progression restricted by the methods of this patent document include neurodegenerative disorders, such as stroke, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewy body disease, chorea-acanthocytosis, primary lateral sclerosis, multiple sclerosis (MS), and Friedreich's ataxia, periventricular leukomalacia (PVL), Guam ALS-Parkinson's disease-dementia complex, Wilson's disease, cerebral palsy, progressive supranuclear palsy (Steele-Richardson syndrome), bulbar and pseudobulbar palsy, diabetic retinopathy, multi-infarct dementia, macular degeneration, Pick's disease, diffuse Lewy body disease, Creutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease, kuru and fatal familial insomnia and other prion diseases, primary lateral sclerosis, degenerative ataxia, Machado-Joseph disease / spinocerebellar ataxia type 3 and olivopontocerebellar atrophy, spinal and bulbospinal muscular atrophy (Kennedy disease), familial spastic paraplegia, Wolfram-Kugelberg-Welander disease, Tay-Sachs disease, multisystem atrophy (Shy-Drager syndrome), Gilles de la Tourette syndrome, familial dysautonomia (Riley-Day syndrome), Kugelberg-Welander disease, subacute sclerosing panencephalitis, Werdnig-Hoffmann disease, synucleinopathy (including multisystem atrophy), Sandhoff disease, corticobasal degeneration, spastic paraplegia, primary progressive aphasia, progressive multifocal leukoencephalopathy, striatonigral degeneration, familial spastic disorders, chronic epilepsy states associated with neurodegeneration, Binswanger's disease, and dementia (including all underlying etiologies of dementia).

[0200] Insulin resistance disorders treatable by the method of this patent document include any disease or condition caused by or contributing to insulin resistance. Examples include diabetes, obesity, metabolic syndrome, insulin resistance syndrome, syndrome X, insulin resistance, hypertension, hypertensive disease, hypercholesterolemia, dyslipidemia, hyperlipidemia, dyslipidemia, stroke, atherosclerotic diseases including coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive function of dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, certain cancers (e.g., endometrial, breast, prostate, and colon), pregnancy complications, poor female reproductive health (e.g., irregular menstruation, infertility, ovulation disorder, polycystic ovary syndrome (PCOS)), lipodystrophy, cholesterol-related disorders, e.g., gallstones, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and prevention and treatment of bone loss, e.g., osteoporosis. Further applications of the method of this patent document include promotion of wound healing, and it can also be used, for example, to promote wound healing and diabetic wound healing.

[0201] In some aspects of any method disclosed herein, the method also includes determining that the subject has downregulated or dysfunctional SIRT1 compared to a normal standard or reference. In some aspects, the methods disclosed herein further include diagnosing that the subject has endothelial dysfunction or a disease or condition associated with endothelial dysfunction prior to administering the transdermal formulation to the subject.

[0202] In some aspects of the methods disclosed herein, determining that a subject has a systemic NO level or plasma nitrite and / or nitrate level that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% lower than a normal level or a healthy reference standard is included.

[0203] In any of the methods disclosed herein, the treatment regimen can be administered after symptoms are clinically observed or after clinical symptoms of a disease or condition have occurred. Alternatively, the method can be used prophylactically before the onset or observation of any clinical symptoms. For example, the methods disclosed herein can address inflammatory injuries, including acute inflammatory injuries caused by specific viral infections (e.g., SARS CoV2, dengue, and influenza), obesity and glucose-induced inflammatory triggers, and inflammation caused by exposure to toxic metals and chemicals. Treatment can be administered when symptoms are identified or before the onset or observation of symptoms. In a further example, a transdermal formulation can be administered prophylactically to prevent inflammatory outcomes of surgery, including postoperative inflammatory pain or erectile dysfunction.

[0204] The methods disclosed herein can increase NO levels either systemically or locally. In some aspects of any transdermal formulation or method of this patent document, the amount of the active ingredient (e.g., NO booster and / or NO precursor, polyol, optional fatty acid, and / or other components) in the transdermal formulation is selected such that it increases the systemic or local NO level or plasma nitrous and / or nitric acid level of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60% or more compared to the control or the NO level or plasma nitrous and / or nitric acid level before administration of the formulation. In some aspects, the desired increase or change can be achieved within about 1 hour, within about 2 hours, within about 3 hours, within about 5 hours, or within about 8 hours. In some aspects, the increase or change is maintained for a period of about 1 day, about 3 days, about 5 days, about 7 days, about 10 days, about 15 days, about 30 days or more. Various methods can be used to measure the NO level or plasma nitrous and / or nitric acid level, such as colorimetric methods and chemiluminescence methods using, for example, Griess reagent.

[0205] In some aspects of any transdermal formulation or method of this patent document, the amount of the active ingredient (e.g., NO booster and / or NO precursor, polyol, optional fatty acid, and / or other components) in the transdermal formulation is selected such that the systolic blood pressure and / or diastolic blood pressure and / or mean arterial pressure of the subject decreases by at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, about 18 (abou 18), at least 20, at least 25, at least 30, at least 35, at least 40 mmHg or more over the above period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more compared to the control or the NO level or plasma nitrous and / or nitric acid level before administration of the formulation.

[0206] In some aspects of any of the methods disclosed herein, the subject is diagnosed with hypertension or at risk of developing hypertension. The transdermal formulations disclosed herein can be administered once, twice, three times, or as needed over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. In some aspects, formulations in the form of, for example, patches, creams, or gels are administered once every 1, 3, 5, 7, or 10 days.

[0207] Another aspect of the disclosure provides a method of reducing CD38 activity or reducing elevated CD levels, comprising contacting a transdermal formulation disclosed herein with a cell that overexpresses CD38. The desired effect is best achieved through a combination of macrophage / monocyte / microglia repolarization from the M1 phenotype to the M2 phenotype, a reduction in the senescent cell population, and anti-inflammatory / antioxidant activity. In some aspects, CD38 is present on (on the surface of or within) the cell. In some aspects, the contact occurs in vitro. In some aspects, the contact occurs in vivo.

[0208] Another aspect of the disclosure provides a method of incorporating a drug into a cell by contacting the cell with a formulation disclosed herein. As a result of enhanced NO levels, the method can stabilize the cell, improve storage characteristics, and reverse storage damage. In some aspects, the cell is a red blood cell. In some aspects, the formulation is in liquid form. In some aspects, the formulation comprises an S-nitrosothiol-containing molecule.

[0209] A related aspect provides a method for extending or enhancing the ex vivo viability of biological materials (e.g., cells, tissues, organs, body fluid samples, or blood). The method comprises contacting (e.g., rinsing, mixing, perfusing, washing, and / or injecting) the biological material with a formulation disclosed herein. The range and amount of the solvent, curcuminoid, any flavonoid, and other components of the formulation are as disclosed above.

[0210] In addition to mixing or contacting the formulations and biological materials disclosed herein ex vivo, the formulations (formulations adjusted in concentration and form) can also be administered (e.g., transdermally) to a donor subject prior to collection of cells, tissues, organs, or blood (while the subject is alive or after death). In some embodiments, the disclosed formulations are used as a washing solution for washing freshly collected biological materials from a donor subject prior to long-term storage, transportation, or transplantation. In some embodiments, the formulation is a solution containing an effective amount of curcuminoid. In some embodiments, the method relates to extending the viability of red blood cells ex vivo by mixing or contacting the red blood cells with the solution.

[0211] In the practice of the present invention, it is often desirable to maintain cells, tissues, or organs in a composition from essentially the time of collection until the explanted material is ready for transplantation into a recipient subject. It is also desirable to monitor or control environmental conditions and storage parameters between collection and transplantation or transfusion to maintain the integrity, viability, and biochemical activity of the collected biological material.

[0212] During storage, red blood cells (RBCs) undergo several biochemical and biomechanical changes that reduce RBC survival and impair the oxygen-carrying function of the blood. These changes are collectively referred to as storage lesions and are often characterized in terms of increased inflammation, hemolysis, microparticle formation, and / or shorter circulation times. Storage lesions may contribute to reduced RBC survival and impaired oxygen-carrying function of the blood. The solution formulations disclosed herein have the ability to slow the occurrence of storage lesion phenomena in RBCs and / or slow the progression of changes associated with storage lesions. Compared to a control or untreated reference sample (collected at the same time point and / or from the same source as the target sample, except that it has not been treated with the formulations disclosed herein) over the same period, target samples treated with the formulations are independently characterized by a decrease in inflammation, hemolysis, and microparticle formation, and an increase in circulation time of at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150%, at least 200%, or any range between any two of the foregoing percentage values. In some embodiments, the biological material (e.g., blood sample) and the formulation or solution are in a ratio by weight in the range of about 10,000:1 to about 5:1, about 10,000:1 to about 50:1, about 10,000:1 to about 500:1, about 10,000:1 to about 1000:1, about 8,000:1 to about 500:1, or about 5,000:1 to about 500:1. Non-limiting examples of the weight ratio between the biological material (e.g., blood sample) and the formulation or solution include about 10,000:1, about 5,000:1, about 2,000:1, about 1,000:1, about 500:1, about 400:1, about 300:1, about 200:1, about 100:1, about 80:1, about 50:1, about 10:1, and any range between any two of the foregoing values.

[0213] The solution formulation can stabilize red blood cells, prevent hemolysis of unstable red blood cells, and generate circulating red blood cells that exhibit stabilized biochemical and biophysical evidence. Thus, red blood cells treated with the formulation exhibit a significant delay or reduction in oxidative damage that limits both the storage life and effectiveness of red blood cells.

[0214] In some embodiments, the agent, solvent, and their amounts are selected such that more than 40% of the biological material (e.g., red blood cells) remains substantially viable for at least about 24 hours. In some embodiments, at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, or at least 99% of the red blood cells remain viable for at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 20 days, at least 30 days, at least 45 days, at least 2 months, at least 3 months, at least 4 months, or at least 6 months.

[0215] In any of the embodiments disclosed herein, the viability of cells, tissues, or organs can be determined by assays known to those of skill in the art. For example, the viability of cells can be readily determined by using a microscopy assay commonly referred to in the art as a "live / dead assay." In one such assay, the biological dyes 5-chloromethylfluorescein diacetate and propidium iodide (which stain live and non-live cells differentially) are utilized and evaluated by a microscopy-based assay. Such dyes are typically fluorescent, and the fluorescence can be detected and used, most typically, to create a dual-parameter fluorescence histogram using fluorescence microscopy techniques for distinguishing live cells from non-live cells, where live cells and non-live cells fluoresce at distinctively different wavelengths, respectively.

[0216] In some embodiments, to determine the % viability of stored cells or tissues as a function of time, a biological sample is first assayed for viability (typically within 10 minutes, within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 8 hours, within 12 hours, within 24 hours, within 48 hours, or within 72 hours of collection from the donor subject) to determine an “initial viability”. The tissue is then assayed for subsequent viability over a period of time to determine a “current viability”. Thus, the % viability can be determined at any point in time after collection using the following formula. [(Current viability) / (Initial viability)] × 100 = Percent viability

[0217] Optionally, multiple samples may be analyzed and averaged at both the time of the initial assay and / or during subsequent analysis to determine the “average viability” of the recovered tissue. Alternatively, determination of the viability of a tissue, cell, or organ may include one or more biochemical or anatomical assays known in the art, and this provides qualitative and / or quantitative evidence of the biological activity or functionality of the explanted tissue once introduced into the recipient animal. Further assays and detection methods are provided in U.S. Patent No. 9,737,071, the entire disclosure of which is incorporated herein by reference.

[0218] In some embodiments, the treatment (e.g., mixing or contacting as described above) of blood (freshly collected or aged) with the formulations disclosed herein enables the transport and delivery of therapeutics (e.g., curcuminoids) loaded onto red blood cells. By functioning as a stealth pharmacological vehicle, red blood cells can deliver therapeutics, such as drugs, to a subject in need thereof via transfusion, and can restore or improve important parameters, such as microvascular blood flow, permeability, glycocalyx, and oxygen load. Blood can be treated prior to transfusion, for example, for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 15 hours, about 24 hours, about 48 hours, about 3 days, about 4 days, about 6 days, or any range between the foregoing values. The treatment can be performed one or more times (once or multiple times) at one or more time points prior to transfusion. Accordingly, blood samples prepared from the blood treatment approach can be applied to the treatment of various diseases and conditions.

[0219] In any of the embodiments disclosed herein, the components of the formulation and their amounts are configured such that the formulation can decrease or increase a parameter or biomarker by at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or any range between any two of the foregoing percentage values, with reference to a control or untreated sample.

[0220] In some embodiments, the method includes adding one or more additional components, such as stabilizers, antibacterial agents, and anti-inflammatory agents, to a biological material (e.g., a blood sample). The additional components may also be dissolved in the formulation solution prior to mixing with the blood. In some embodiments, the additional components are myristic acid and other medium-chain fatty acids that may be beneficial to health.

[0221] In some embodiments, the biological sample is stored at a temperature in the range of about -30°C to about 37°C. Non-limiting examples of temperatures include about -30°C, about -20°C, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, and any range between any two of the foregoing temperatures.

[0222] In some embodiments, the blood is taken from a healthy individual. In some embodiments, the blood is taken from an individual, processed with the formulations and other necessary medical procedures described herein, and then re-infused into the individual.

[0223] In some embodiments, the solution has a pH in the range of about 5.5 to about 7.0, either before or after mixing with the blood.

[0224] In some embodiments, the method includes a step of depleting oxygen and / or carbon dioxide in the sample.

[0225] In some embodiments, the agent, solvent, and their amounts are selected such that, compared to a control or untreated sample, the adenosine triphosphate (ATP) and / or 2,3-diphosphoglycerate (2,3-DPG) levels are reduced by less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 80% over a period of 1, 2, 4, 6, 8, 10, 14, or 20 days.

[0226] In some embodiments, the concentration of curcuminoid in the solution before mixing with the sample is in the range of about 1.0 to about 500, about 1.0 to about 350, about 1.0 to about 200, about 5.0 to about 300, about 10 to about 300, about 20 to about 300, about 50 to about 200, about 100 to about 200 μg / mL. Non-limiting examples of the concentration of curcuminoid in the solution include about 1.0, about 2.0, about 5.0, about 10, about 20, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 500 μg / mL, and any range between any two of the foregoing values. In some embodiments, the concentration of curcuminoid in the solution before mixing with the sample is in the range of about 0.05 to about 30, about 0.1 to about 10, about 0.1 to about 5, about 0.1 to about 1, about 0.5 to about 1 mM, and non-limiting examples of such concentration include about 0.1, about 0.2, about 0.5, about 0.6, about 0.8, about 1.0, about 1.5, about 2.0, about 3.0, about 5.0, about 8.0, about 10.0, about 15 mM, and any range between any two of the foregoing values. In some embodiments, the concentration of curcuminoid in the blood sample is in the range of about 0.2 to about 20, about 0.5 to about 10, about 1 to about 10, or about 1 to about 5 mM in the sample. Non-limiting examples of the concentration of curcuminoid in the blood sample include about 0.1, about 0.2, about 0.5, about 0.6, about 0.8, about 1.0, about 1.5, about 2.0, about 3.0, about 5.0, about 8.0, about 10.0, about 15 mM, and any range between any two of the foregoing values.

[0227] When cells (e.g., red blood cells) are treated with the formulations described herein, NO boosters (e.g., curcumin) and optionally other anti-inflammatory / antioxidants can be delivered into the lipid membrane or beyond the cytosol, stabilizing the cells for storage and converting the transfused cells into long-lasting (circulating) anti-inflammatory agents that can gradually deliver these agents to the endothelial lining of capillaries and other small-diameter blood vessels. Similarly, treating cells with formulations containing high concentrations of NO precursors such as lipophilic S-nitrosothiols and S-NO derivatives of alkyl ester derivatives of NAC will be effective in the transnitrosation of thiols intracellularly and on the cell surface. Nitrosation of important thiols on and in red blood cells (including the thiols of beta93 on Hb and the thiol of the band 3 protein) has been shown to stabilize red blood cells against oxidative damage, microparticle formation, and hemolysis. Thus, such red blood cells can also be used as a vehicle for delivering NO to thiols on the endothelium.

[0228] Another aspect provides a method of transfusing blood to a subject in need thereof. The method includes the step of transfusing a blood sample as disclosed herein, which is stored in a container or kit as described above. The blood sample can be prepared in the same manner as the methods disclosed herein for ex vivo extending the viability of red blood cells. Curcumin-loaded red blood cells are ideally suited to deliver curcumin to the capillary bed and smaller blood vessels, such as arterioles. These are appropriate sites for treating a number of vascular disorders resulting from endothelial dysfunction, including, for example, ischemia / reperfusion injury and vaso-occlusive crises. A related aspect provides a method of treating a disease or condition associated with endothelial dysfunction. The method includes the step of administering to a subject in need thereof an effective amount of a blood sample as disclosed herein. Since red blood cells function as a vehicle for an active agent (e.g., curcumin, other curcuminoids, or flavonoids), when the agent is delivered to an appropriate site, including small blood vessels such as arterioles and the capillary bed, where the RBCs are in proximity to the endothelium, curcumin can be released / redistributed to the endothelial layer of the blood vessel, and a therapeutic effect can be achieved. The release of curcumin reduces or eliminates endothelial dysfunction and restores NO production from within the endothelium required for normal endothelial function. Non-limiting examples of diseases or conditions associated with endothelial dysfunction include those disclosed above in this patent document.

[0229] In some embodiments of any of the methods disclosed herein, an active agent (e.g., a curcuminoid or flavonoid) is loaded into red blood cells and white blood cells (leukocytes), such as macrophages, monocytes, microglia, and neutrophils. Uptake of curcuminoids and / or flavonoids by activated pro-inflammatory leukocytes may cause a rapid reversal of the cytokine storm-type phenomenon, for example, in acute inflammatory diseases or conditions, under the treatment methods disclosed herein.

[0230] Another aspect related to the blood samples disclosed herein is a method of maintaining or restoring microcirculation or reducing or reversing microcirculatory consequences in a disease or condition (e.g., hemorrhagic shock) by administering the blood sample to a subject in need thereof. The blood sample can be treated with the formulations disclosed herein prior to transfusion (e.g., 1, 2, 3, 4, 6, or 8 hours prior to transfusion).

[0231] In some embodiments, the method is applicable for treating or preventing hemorrhagic shock (HS). Traumatic bleeding results in HS. HS is a major cause of death in both civilian and military situations, accounting for one-third of all trauma-related deaths and 80% of potentially survivable battlefield injuries. Almost 50% of deaths due to bleeding occur within 3 - 6 hours of injury. Studies conducted in various species and models have clearly established that the microvasculature is extremely important in the pathophysiology of HS and is associated with organ damage due to reduced tissue perfusion and oxygen loading as well as damage due to excessive production of reactive oxygen species (ROS). The strong inflammatory response associated with HS also leads to increased permeability caused by reduced tissue perfusion, upregulation of interleukins / cytokines, activation of microvascular endothelium and neutrophils, ROS generation, and glycocalyx degradation. HS / trauma disrupts the balance between the microvasculature and multiple blood components, sometimes resulting in coagulopathy which also contributes to HS-related morbidity and mortality. Preservation and restoration of the microcirculation are involved in all therapeutic attempts to reverse or minimize the deleterious consequences of HS. Thus, the blood samples disclosed herein can be transfused to deliver therapeutic agents that can improve hemorrhagic shock outcomes. Aged RBCs can also be aimed at treating combat casualties who are vulnerable to or suffering from HS. The ability to use RBCs, including aged / stored RBCs, as a therapeutic agent effective in treating the underlying mechanisms causing HS represents a significant advancement in combat care. In some embodiments, transfusion of the blood sample restores or improves one or more of the important parameters such as microvascular blood flow, permeability, glycocalyx, and oxygen loading.

[0232] The related aspect provides a method of enhancing tissue perfusion in a subject in need thereof by administering or transfusing a blood sample treated with a formulation disclosed herein, or by administering to the subject (before or during transfusion) a transdermal formulation disclosed herein. Sufficient tissue perfusion and oxygen loading are essential for all metabolic processes in cells and are major influencers of tissue repair and resistance to infectious organisms. Tissue perfusion is similar to blood flow, oxygen delivery, or a combination of nutrient supply including blood flow and oxygen supply. Enhanced tissue perfusion can be determined, for example, by measuring the levels of biomarkers including serum lactate, central venous oxygen saturation, reticulocyte increase, lactate dehydrogenase, unconjugated bilirubin, haptoglobin, hematocrit, cell adhesion molecules (CAMs), E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), syndecan-1. These biomarkers can be measured as a function of time post-transfusion / or before and after transfusion. Biomarkers of untreated blood samples can also be collected as a reference to show the improved profile of blood samples treated with a formulation disclosed herein. Monitoring of peripheral circulation also enables the evaluation of changes in tissue perfusion. Well-known approaches such as near-infrared spectroscopy (NIRS) oxygen measurements can be used for detection purposes.

[0233] In some embodiments, the amount of curcuminoid in the formulation or in the treated blood sample is selected such that tissue perfusion is enhanced and / or one or more of the aforementioned biomarkers increase or decrease by at least 2%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or any range between any two of the aforementioned percentage values, with reference to a control or untreated sample (collected simultaneously and / or from the same source).

[0234] Another aspect provides a method of improving the safety and / or efficacy of a transfusion by administering to a subject in need thereof a transdermal formulation disclosed herein before, during, or immediately after a transfusion procedure, transfusing a blood sample disclosed herein into the subject, or contacting cells or body fluids to be transfused with a formulation disclosed herein. For example, a formulation containing an NO booster or NO precursor can be administered transdermally (e.g., by patch or paste) to a subject before, during, or immediately after a transfusion to protect red blood cells and enhance the therapeutic effect. In some embodiments, the method includes administering a transdermal formulation to a subject 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or more than 4 hours before a transfusion is performed, or contacting a formulation with cells or body fluids. In some embodiments, the method relates to the transfusion of a blood sample disclosed herein.

[0235] Contacting cells or body fluids ex vivo directly (e.g., a blood sample disclosed herein) with a formulation enhances, for example, the viability of red blood cells and / or loads cells with a therapeutic agent (e.g., curcuminoid), such that subsequent transfusion into a subject achieves greater efficacy and safety. In some embodiments, a blood sample disclosed herein is transfused into a subject to whom a transdermal formulation disclosed herein is administered before, during, or after a transfusion.

[0236] The related aspect provides a method for transdermally delivering an active agent to a subject in need thereof. The method includes administering to the subject a transdermal formulation or a blood sample disclosed herein. By delivering an effective amount of the active agent locally or systemically, a rapid and extensive therapeutic effect can be achieved. The range of target diseases or conditions is as described above. In some embodiments, the transdermal formulation includes one or more curcuminoids, and optionally, one or more of polyphenols, flavonoids, stilbenoids, seco-steroids, or natural products that promote NO production, as the active agent or active ingredient for treating a disease or condition. In some embodiments, the transdermal formulation includes one or both of curcumin and quercetin, and optionally, one or more of polyphenols, flavonoids, stilbenoids, and seco-steroids. As a result, red blood cells can also function as a natural biological pharmacokinetic vehicle that can avoid the normal mechanism for drug elimination from the circulation, apart from their role of essential physiological oxygen delivery in the body (e.g., release of curcumin in the vasculature). This finding has the potential for a paradigm shift in drug delivery.

[0237] In any aspect of the above method, the formulation of this patent document is added to a blood sample at any appropriate interval to delay the occurrence of storage damage phenomena or achieve other desirable results (e.g., a reduction in inflammation, a reduction in hemolysis, a reduction in particulate formation, or an extension of circulation time) compared to a control or untreated sample. For example, after treating the blood sample on day 1, treatment or addition with the formulation can be performed one, two, three, or more than four times. Each of the intervals between two consecutive treatments / additions is independently 1, 2, 4, 7, 8, 10, 14, 20, 25, or 30 days. In some aspects, the formulation is administered in a single-dose protocol that includes, for example, administering a single dose only on day 1 of storage and administering a single dose on any intermediate day (e.g., day 10, day 20, day 30, day 40, or day 50) between day 1 and day X (or any day determined to be the last day of storage). In some aspects, the formulation is administered in a multiple-dose schedule that includes continuous administration starting at any point during the storage process with a dosing interval in the range of, for example, every 7, 14, 21, or 28 days, where the start date ranges from day 1 to any intermediate time point. Administering on the last day of storage is also an option not dependent on the interval protocol.

[0238] Various means can be used for the delivery of the formulation. In some aspects, for the single-dose protocol on day 1 or day X, the formulation disclosed herein can be filled into a storage bag. In some aspects, the formulation is transported into the storage bag via syringe injection into an injection port on the bag. In some aspects, the formulation is transported directly into the bag as needed. In some aspects, a pre-filled frangible pouch that can be "ruptured" using finger pressure and thereby release a single dose of the formulation is incorporated within the wall of the storage bag.

[0239] Manufacturing method Another aspect of the present disclosure provides a method of manufacturing a formulation or a blood sample disclosed herein. The method includes, for example, preparing a solution of a polyol solvent and an optional fatty acid permeation enhancer or additive, followed by adding a drug (NO booster or NO precursor). Optionally, the solution can be heated to a temperature appropriate for dissolving the fatty acid and / or the drug. In some embodiments, the active drug (e.g., NO booster or NO precursor or nitrite source) is saturated in the solution. After cooling the solution, the formulation can turn into a gel, and the precipitated excess active drug can be easily removed. Variations in the conditions or order of mixing or adding different components are also possible as long as the NO booster or NO precursor is properly distributed in the formulation to achieve the desired therapeutic effect.

[0240] In some exemplary embodiments, the drug dissolved in the solution is filled into a container (e.g., a nebulizer or a nebuliser; a permeable or frangible pouch as described above) or immersed in a dispensing vehicle (e.g., the absorbent layer of a cotton swab, a sponge, or a patch). When the NO precursor mixture contains a nitrite source, the acid source can be stored, for example, in a separate pouch or a separate layer of a patch. The NO precursor mixture and the acid source can also be separated by a removable barrier disposed between two different compartments of the container or between two layers of the patch.

[0241] In some embodiments, the formulation is in the form of a gel or a semi-solid. A gelling agent or a thickening agent can be added to adjust the form of the formulation. The formulation is then filled into a suitable container and then dispensed as a gel, an ointment, a cream, an emulsion, a microemulsion, a nanoemulsion, a paste, a balm, or other suitable forms. The semi-solid formulation can also be coated on a backing material (e.g., the support layer of a patch).

[0242] Depending on the amount and nature of the thickener, the formulation can also be prepared in solid form. For example, a solution of NO boosters (e.g., curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine) can be mixed with molten pure cocoa butter and then cooled. The resulting solid formulation will dissolve when rubbed into human skin while applying pressure.

[0243] General procedures for mixing reagents and handling the manufacturing process are available to those skilled in the art through common general knowledge or pharmaceutical technology handbooks, such as Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins, Philadelphia, 2000, or the review article Souza et al, Topical ocular delivery of therapeutics: carrier systems and physical methods, J. Pharm. Pharmacol., 2013, 66, 507-530.

[0244] Dosing regimen The effective amount of the agent (NO booster or NO precursor) in the formulations or kits described herein for effectively enhancing the systemic NO level depends on the route of administration, the type of subject including the human being being treated, and the physical characteristics of the particular subject under consideration. The dosage or amount can be adjusted to achieve the desired effect, but will depend on factors such as body weight, diet, concomitant medication, and other factors that would be recognized by one of ordinary skill in the medical arts. More specifically, the effective amount or therapeutically effective amount means the amount of the agent effective to increase systemic NO to a level that prevents, alleviates or improves the symptoms of the disease, or extends the survival period of the subject being treated. Administration of the formulation can be adjusted to provide an optimal therapeutic response or long-term beneficial effect. For example, the formulation can be administered topically more than twice or three times a day. Alternatively, the amount or frequency of administration may be reduced as needed. Determination of the effective amount is well within the ability of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

[0245] In non-human animal studies, the application of the potential product is started at a higher dosage level and the dosage is reduced until the desired effect is no longer achieved or the adverse side effects disappear. The dosage can range widely depending on the desired effect and the therapeutic indication. Typically, the effective amount of the agent or the dosage in the formulation can be from about 10 micrograms / kg to about 100 mg / kg body weight, preferably from about 100 micrograms / kg to about 10 mg / kg body weight. Alternatively, the dosage can be calculated based on the surface area of the patient, as understood by one of ordinary skill in the art.

[0246] In an exemplary embodiment, the formulation is administered once a day, twice a day, three times a day, once every two days, once every three days, once a week, once every two weeks or once a month.

[0247] The exact formulation, route of administration and dosage of the pharmaceutical compositions can be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl et al. 1975, “The Pharmacological Basis of Therapeutics”, which is hereby incorporated by reference in its entirety, and particularly see Chapter 1, page 1).

[0248] It should be noted that the attending physician will know how and when to discontinue, interrupt or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician also knows to adjust the treatment to a higher level (so as not to cause toxicity) if the clinical response is inappropriate. The magnitude of the dosage in the management of the disorder of interest will vary depending on the severity of the condition being treated and the route of administration. The severity of the condition can be evaluated, at least in part, by standard prognostic assessment methods. Furthermore, the dosage and perhaps the dosing frequency will also vary depending on the age, weight and response of the individual patient. Programs comparable to those discussed above can be used in veterinary medicine.

[0249] The formulations disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a formulation can be established by determining its in vitro toxicity against mammalian, preferably human cell lines such as cell lines. The results of such studies are often used to predict toxicity in animals such as mammals, more specifically in humans. Alternatively, toxicity in animal models such as mice, rats, rabbits, or monkeys can be determined using known methods. The efficacy of a particular compound can be established using several recognized methods such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for almost all classes of conditions. Similarly, acceptable animal models can be used to establish the efficacy of a chemical for treating such conditions. When selecting a model to determine efficacy, one of ordinary skill in the art can be guided by state-of-the-art techniques to select an appropriate model, dose, and route of administration, and regimen. Of course, human clinical trials can also be used to determine the efficacy of the active agent of transdermal f in humans.

[0250] The formulations or kits described herein can optionally be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the formulating agent. The pack can include, for example, a metal or plastic foil such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the form of the drug for human or veterinary administration. Such notice can be, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert.

[0251] All references cited herein are hereby incorporated by reference in their entirety.

Examples

[0252] Example 1 Preparation of Transdermal Formulation Vehicles for loading the active ingredient were prepared as shown in Samples 1 to 4. Myristic acid (solid flakes) dissolved in PEG400 when the mixture was warmed in a warm water bath. Shaking accelerated the formation of the resulting clear solution. The following four concentrations of myristic acid (MA) in PEG400 were prepared and evaluated. Samples 1, 2, 3, and 4 were all clear solutions when the samples reached ambient temperature. a. 30 ml of PEG400 + 1.5 grams of MA (Sample 1) b. 30 ml of PEG400 + 3 grams of MA (Sample 2) c. 30 ml of PEG400 + 0.75 grams of MA (Sample 3) d. 30 ml of PEG400 + 2.25 grams of MA (Sample 4)

[0253] Samples as vehicles were loaded with an NO enhancer. The sample carrier formulation [or solvent system] was loaded with 95% pure curcumin (curcumin 95) as an NO boosting agent, and the loading potential was tested. 1.5 grams of curcumin 95 was added to Samples 1, 2, and 3. The resulting curcumin 95-loaded samples were heated and shaken until what seemed to be the maximum dissolution amount of curcumin occurred, and then cooled back to ambient temperature. All three samples were able to dissolve almost all of the added curcumin, resulting in a dark solution with a strong color tone. Sample 3 provided a higher solubility and remained liquid for several weeks. Samples 1 and 2 were initially liquid but formed a homogeneous gel of solid over a period of several hours. Sample 3 was prepared by dissolving 0.75 gram of MA in 30 ml of PEG400 and heating in a water bath (about 60 - 80 °C) for about 15 minutes. Sample 4 was prepared by dissolving 2.25 grams of MA in 30 ml of PEG400 and heating in a water bath (about 60 - 80 °C) for about 15 minutes. Both Sample 3 and Sample 4 were homogeneous solutions at the end of the heating cycle. Upon cooling, Sample 3 remained liquid while Sample 4 formed a homogeneous gel.

[0254] Once the loading ability was established, a transdermal preparation of a sample containing curcugen as a NO enhancer was prepared. Curcugen is a product containing curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). The three curcuminoids account for approximately 50% by weight in curcugen. Sample preparations V3.3 and V4.3 were prepared by adding curcugen to sample 3 and sample 4 vehicles, respectively (3 grams of curcugen in 30 ml of either solvent). The resulting mixtures were heated and shaken for about 15 minutes and then both appeared as dark maroon homogeneous solutions. Upon cooling, V3.3 (3 grams of curcugen per 30 ml of sample 3 vehicle) remained liquid; while V4.3 (3 grams of curcugen per 30 ml of sample 4 vehicle) became a homogeneous gel with the same dark maroon color (the same color as V3.3 when heated).

[0255] For samples stored at ambient temperature, no discoloration was observed over a period of at least three months. In contrast, when the same amount of curcumin was added to the same volume of water, there was little color in the liquid phase and a significant amount of undissolved material was seen.

[0256] V4.3 was loaded into a syringe when warmed and was thus in a liquid state. The liquid in the syringe gelled upon cooling to ambient temperature but could be extruded from the syringe as a gel that slowly melted on the skin. The applied gel was easily covered and captured with a waterproof and leak-proof transparent dressing (e.g., Mepitel transparent film dressing). The dressing was able to prevent loss of the applied preparation and keep the preparation in place at the desired location for several days without loss. When the Mepitel dressing was removed after several days, there was no preparation adhering to the dressing; all of the preparation was within the skin as reflected by no staining of the tissue that vigorously wiped the colored skin under the dressing. The color was completely lost after 7 - 8 days.

[0257] The addition of water to PEG400 / MA-based formulations can result in a non-uniform emulsion, which poses difficulties for topical use. The presence of water limits the solubility of curcumin, curcugen, quercetin, and berberine in PEG. For comparison, the same amount of curcumin was mixed with the same volume of water (i.e., 3 g of curcumin in 30 ml of water). The resulting mixture showed little color in the liquid phase and contained a significant amount of undissolved material.

[0258] The use of a high-concentration MA solution with PEG as the base solvent facilitated the preparation of a concentrated saturated solution of poorly soluble NO-enhancing active substances such as curcumin, curcugen, quercetin, berberine, and related molecules by simply adding an excess of the active agent to a heated high-MA PEG / MA solvent system (e.g., V4.3) and cooling the mixture. The resulting homogeneous gel was completely saturated with the active agent, while the undissolved excess remained solid at the bottom of the tube. The homogeneous saturated gel was then easily removed and separated from the undissolved material.

[0259] The sample 3 solvent system was also tested as a vehicle for loading capacity using the following poorly soluble reagents using the method described above: quercetin, berberine, N-acetylcysteine amide (NACA), and N-acetylcysteine ethyl ester (NAC-ethyl ester). In all cases, the solvent system enabled substantial dissolution of these difficult-to-solubilize drugs. Comparison of the aqueous solutions loaded with quercetin and berberine with the sample 3 solution revealed minimal dissolved material in the aqueous sample and high solubility in sample 3.

[0260] Similar results were obtained for quercetin in samples 3 and 4 as vehicles, although the solubility of quercetin in these solvents was lower than that of curcugen (about 200 - 300 mg of quercetin in either 30 ml of sample 3 or sample 4). Similar to curcumin and curcugen, samples 3 and 4 loaded with quercetin did not form a gel and formed a gel at ambient temperature, respectively.

[0261] The PEG400 / MA solution was combined with other delivery vehicles (e.g., petrolatum). Sample 3 containing curcumin was mixed with petrolatum, and a uniformly colored gel that remained stable (no discoloration) over a period of more than two months was obtained. Sample 3 in which the SNO derivatives of NAC amide and NAC were dissolved was easily combined with petrolatum to produce a stable pink jelly. The stability is likely due to the low water activity and high viscosity that inhibit the loss of NO from the thiol groups. Different aliquots of Sample 3 (containing both curcumin and curcugen) were mixed with molten pure cocoa butter and then cooled. The optimized mixture produced solid, uniformly yellow / orange-colored blocks / tubes of cocoa butter, which remained solid at ambient temperature. The solid material melted when pressure was applied and rubbed onto human skin. Similar results were obtained with coconut oil, but due to its low melting point, it was difficult to apply to human skin without soiling or dripping. Combinations of the two oils were also tested. The use of PEG400 / MA with cocoa butter showed the most promising properties as a topical delivery vehicle suitable for cosmetic and dermatological applications due to the more suitable hardness of the resulting curcumin-loaded cocoa butter. Increasing the amount of PEG400 / MA solvent added to the cocoa butter ultimately resulted in a gel-like substance that remained color-stable.

[0262] Solutions of two lipophilic NAC derivatives were prepared using sodium nitrite saturated PEG400, resulting in clear solutions. The lipophilic derivatives of NAC readily dissolved under conditions where NAC has limited solubility. NAC is water-soluble and the two derivatives are slightly soluble in water. The two solutions were then treated with a few drops of acetic acid to cause the formation of nitrous acid from nitrite, which can then nitrosate the reactive thiols on both NAC derivatives. The solution turned pink, indicating the formation of S-nitrosothiols. The solution remained pink for several days. The corresponding aqueous solution lost its color within a few hours. The use of sodium nitrite saturated PEG400 enables a formulation that can be used to generate NO and S-nitrosothiols by mixing the mixture with a reagent that acidifies it. The double flange frangible pouch incorporated into the patch can be used as a sustained NO delivery vehicle suitable for topical and transdermal applications.

[0263] Example 2 The effect of the V2.3 formulation (2 g of curcugen per 30 ml of sample 3 vehicle) on blood pressure (BP) in rats was investigated. The formulation was topically applied to the shaved abdomen of five Sprague-Dawley rats. A Q-tip saturated with V2.3 was rubbed onto the shaved abdomen of the rats. In all cases (N = 5), application of V2.3 resulted in a 20% decrease in systemic blood pressure within 15 - 20 minutes of application. The decrease in blood pressure was maintained during a 3-hour observation window without signs of recovery. Results from a second laboratory where six rats were similarly tested (N = 6) also showed similar results (a 20% decrease in blood pressure). Similar results to V3.3 were consistently obtained in one human subject when V3.3 was applied to the forearm (approx. 0.05 ml). An equivalent BP decrease occurred within 15 - 20 minutes after application, and the decreased BP persisted for several hours and gradually returned to the higher aforementioned initial value 12 - 24 hours later. Similar results were obtained using V4.3 in the same human subject. Cocoa butter doped with sample 3 (V1.3) also showed similar physiological results when applied to one human test subject. Control sample 3, which did not contain one or more curcuminoids or curcugen, did not induce physiological results when applied to either rats or humans.

[0264] V3.3 was applied to the skin flap with an optical window of a healthy hamster. The optical window enabled monitoring of the blood vessels in the subcutaneous layer under the administration site of V3.3. A dose-dependent increase in blood vessel diameter at the topical application site was observed within minutes of application.

[0265] Effect on NO plasma levels. The plasma levels of NO degradation products (nitrite / nitrate) were measured after topical application of V3.3 in 4 rats. The plasma levels of NO degradation products (nitrite / nitrate) in 2 of the tested rats were shown to increase by 15%, consistent with the decrease in blood pressure resulting from the enhanced production of nitric oxide due to the known ability of curcumin to upregulate endothelial nitric oxide synthase (eNOS) and thus NO production. The 2 control animals did not show such an increase under the same conditions. The results indicate that percutaneous curcuminoids can increase systemic NO levels.

[0266] The decrease in blood pressure within 15 - 20 minutes after topical application of the curcumin-containing sample indicates that curcumin is being delivered transdermally and that therapeutic effective levels are present within that short period. The decrease in blood pressure and the increase in nitrite / nitrate plasma levels are consistent with the known effect of curcumin on upregulation of nitric oxide production in the endothelium by eNOS. The physiological response persists for several hours, consistent with the locally delivered curcumin / curcugen being delivered into the circulation in a sustained manner. In contrast, curcumin delivered into the circulation via the oral route or IV has a circulation time of only 2 hours because the liver rapidly converts curcumin into an inactive agent. Even high doses of oral curcumin were not observed to produce this significant and long-lasting physiological response.

[0267] The physiological response in animals to percutaneous curcuminoid formulations was also investigated for V3.3 (curcugen 9 g, myristic acid 2.25 g, PEG400 90 ml), and V4.3 (curcugen 9 g, myristic acid 6.75 g, PEG400 90 ml).

[0268] Blood pressure was measured in rodents following topical application of the percutaneous curcuminoid formulation. BP decreased by up to 20% in both rats and mice, and V3.3 was more effective than V4.3 in both the time at which it occurred and the degree of BP decrease.

[0269] Furthermore, either V3.3 or V4.3 topical application was also observed to result in an increase in plasma nitrite and nitrate levels in a 3-hour monitoring window following a single topical administration. Over the same period, BP underwent a continuous decrease (over the 3-hour window). After topical application of V3.3 to rats (N = 3), a continuous accumulation of detectable concentrations of plasma curcuminoids was observed over a 3-hour window. Oral curcumin plasma levels peak within 1 hour and decline to undetectable levels within 3 hours.

[0270] Example 3 Control of systemic inflammation via transdermal delivery of NO boosters or NO precursors Inhibition of the development of severe vascular leakage in an acute inflammation rat model was investigated using the topically applied formulation V3.3. This study used the following lipopolysaccharide (LPS)-induced cytokine storm protocol: a. LPS (10 mg / kg) was injected IP every 24 hours to initiate and maintain the acute inflammatory response. b. LPS-treated rats were subjected to topical application (0.1 ml) of Vascarta formulation V3.3 starting at the time of the first LPS treatment and repeated every 24 hours for 3 days. c. Physiological parameters derived from the blood taken were measured every 24 hours. d. After 3 days, the animals were anesthetized and surgically incised to enable in vivo fluorescence imaging of the macrovasculature and microvasculature. i. Formulations of fluorescently labeled albumin and dextran (500 kDa) were injected IV, and the rates of extravasation into the extravascular space and into the surrounding tissue for both albumin and dextran were determined using fluorescence-derived images of the vasculature and surrounding tissue.

[0271] Local application of the vehicle (PEG400 / MA) to LPS-treated rats revealed a rapid and extreme pattern of leakage for both albumin and the much larger dextran, consistent with what was observed under conditions of severe acute inflammation. However, daily local application of V3.3 dramatically reduced the amount of leakage for albumin. The low levels of leakage observed were nearly the same as those observed for control animals. Similar results were observed for dextran. Widespread vascular leakage is a potentially lethal consequence of the cytokine storm, independent of the cause (such as COVID-19, Ebola, dengue fever, hemorrhagic shock, endotoxic shock, Rift Valley fever, etc.). The transdermal formulation provided dramatic positive intervention results with broad clinical implications.

[0272] Example 4 This study evaluated the potential therapeutic efficacy of a transdermal delivery transdermal formulation (V4.3: 9 g of curcugen, 6.75 g of myristic acid, 90 ml of PEG400) according to the present invention in an acute vascular inflammation mouse model. Three cohorts, each containing three subjects, were studied and compared. In cohort 1, subjects were injected with LPS but received no local treatment (untreated). In cohort 2, subjects were injected with LPS local treatment (0.1 ml of V4.3). In cohort 3, following LPS injection, local treatment (0.1 ml of V4.3) was performed 4 hours after LPS injection. Endotoxemia was induced by injection of 10 mg / kg of LPS (lipopolysaccharide from Escherichia coli (E. coli) serotype 0128:B12, Sigma Aldrich St. Louis, MO). The procedure was the same as that described in previously published studies (Williams AT, Muller CR, Govender K, Navati MS, Friedman AJ, Friedman JM, Cabrales P. Control of systemic inflammation through early nitric oxide supplementation with nitric oxide releasing nanoparticles. Free Radic Biol Med. 2020;161:15-22. Epub 2020 / 10 / 05. doi: 10.1016 / j.freeradbiomed.2020.09.025. PubMed PMID: 33011274; PMCID: PMC7529593 and references therein).

[0273] Locally applied V4.3 (curcuminoid dissolved in PEG400 / myristic acid mix) was observed to act as both a prophylactic and an interventional treatment for lipopolysaccharide (LPS)-induced cytokine storm. Results include vascular outcomes and cytokine profiles as a function of time for three different groups of LPS-treated mice: i) no local treatment with V4.3; ii) pretreatment with local V4.3 prior to LPS treatment, and iii) local treatment with V4.3 after the onset of the LPS-initiated cytokine storm. With respect to the microvascular response to LPS treatment in the three groups, treatment with local V4.3 limited arterial dilation, an indicator of shock-induced vascular collapse, and maintained arterial blood flow. Local V4.3 was also observed to prevent the sharp decline in functional capillary density (FCD) that occurs in LPS-induced endotoxemia. V4.3 was effective as both a prophylactic and an interventional treatment in the preventive decline of FCD compared to baseline (BL). FCD is correlated with survival in that it reflects the ability to maintain tissue perfusion and deliver oxygen to tissues. Furthermore, it was discovered that both pretreatment and interventional treatment with locally applied V4.3 limit the production of inflammatory cytokines. These results confirm the effectiveness of the transdermal formulation in limiting inflammatory outcomes when administered locally as a prophylactic or as an active therapeutic following the onset of inflammation.

[0274] Example 5 This study evaluated the prevention of LPS-induced vascular leakage via topically administered curcuminoids using the transdermal formulation according to the present invention in a LPS endotoxemia rat model. LPS (E. coli O26:B6) was inoculated into the rat model (10 mg / kg / day). A transdermal curcumin formulation (V3.3: 9 g of curcugen, 2.25 g of myristic acid, 90 ml of PEG400) or a vehicle control was applied daily for 3 days (0.1 ml / dose). On the third day, the animals were surgically prepared for in vivo and fluorescence microscopy. Treatment with the transdermal curcumin formulation was observed to prevent the leakage in the initial stage (the first 4 hours) induced by LPS. Vascular leakage decreased dramatically after topical application of V3.3 in the LPS-induced inflammation mode. On the other hand, when V4.3 (9 g of curcugen, 6.75 g of myristic acid, 90 ml of PEG400) was topically applied both before and after LPS-induced inflammation in mice (N = 3), the LPS-induced increase in inflammatory cytokine levels decreased.

[0275] This experiment, combined with the results of previous studies, shows that the topical transdermal curcumin formulation of this patent document can limit the inflammation-induced vascular leakage that is characteristic of cytokine storms and other inflammation-induced states.

[0276] Example 6 The skin permeability of the transdermal formulation of the present invention was studied. The skin permeability of the following transdermal formulations according to the present invention was studied using a confocal microscope. Only the amount of myristic acid differed in the formulations. a. Lot 43: V4.3 9 g of curcugen, 6.75 g of myristic acid, 90 ml of PEG400 b. Lot 39: V3.3 9 g of curcugen, 2.25 g of myristic acid, 90 ml of PEG400 c. Lot 38: V0.3 9 g of curcugen, 90 ml of PEG400

[0277] The formulation solidified as the temperature approached zero degrees Celsius. As the temperature rose above ambient, the viscosity visibly decreased in all samples. The viscosity of the formulation and its components are as follows: Water 0.9 cP; PEG400 (100%) 99 cP; PEG400 / water 90%, 80 cP; V3.3: 149.8 cP; PEG400 + myristic acid, V3.3: 149.8 cP; PEG400 + myristic acid, V4.3: 4220 cP.

[0278] Curcuminoids have a broad absorbance spectrum with a maximum absorbance at 425 nm. The integrity of all human skin samples was measured using TEWL data, and all samples used had good integrity of the skin barrier. DAPI filter was used to visualize the penetration of curcuminoids. All images were taken with the same settings.

[0279] All three formulations showed the penetration of curcuminoids into the skin. At the earliest time point of 1 hour, the penetration of curcuminoids into the stratum corneum was observed for all three formulations.

[0280] In the formulation of Lot 38, the expansion of the fluorescent band occurred at 3 - 6 hours in both donors. In the formulations of Lot 39 and 43, no band expansion was seen up to 24 hours. This expansion suggests the penetration of curcuminoids into the upper epidermal layer just below the stratum corneum. In the formulation of Lot 39, the amount of curcuminoids decreased in both donors between 3 - 6 hours. However, the decrease in intensity for curcuminoids in the formulations of Lot 38 and 43 was observed after 6 hours. In the formulation of Lot 38, fluorescent spots were seen under the stratum corneum at the initial time points. However, in the formulations of Lot 39 and 43, no fluorescent spots were seen on the stratum corneum. The trans-epidermal water loss (TEWL) values were equivalent for all formulations and all time points, suggesting that the skin barrier integrity of all human skin samples was the same.

[0281] The decrease in fluorescence over time in the three formulations is an indicator of the penetration of curcuminoids into the deeper layers of the skin. (Since the autofluorescence of untreated skin is high, it is possible that the amount of active substance penetration was not visualized and / or quantified due to low penetration). Based on this, the formulation of Lot 39 shows the fastest penetration of curcuminoids into the skin. The depth of fluorescence was also determined. This was measured at values from 5 microns to 25 microns. The formulation of Lot 38 showed the fastest expansion of the fluorescence band, suggesting faster skin penetration of curcuminoids.

[0282] Example 7 The effect of the transdermal formulation of this patent on restricting or preventing the onset of cardiovascular-induced inflammation in a diabetic rat model was investigated in a ZDSD diabetic rat model. Three rats were on a normal diet for 60 days without symptoms of diabetes. From approximately day 65, the rats were started on a high-fat diet and their blood glucose gradually increased. The animals were topically treated with formulation V4.3 every two days over the entire test period up to 80 days. The cytokine profile indicated the onset of inflammation at approximately day 75. Treatment with formulation V4.3 restricted the increase in inflammatory cytokines seen in the sham at day 75. In particular, the profile of IL-18 showed clear evidence of the formulation in preventing the increase in IL-18, a marker for the tendency to develop cardiovascular outcomes in diabetic patients.

[0283] Example 8 The effect of treatment of mice with severe progressive endothelial dysfunction was investigated using formulation V4.3. A total of 24 male C57BL / 6J mice, 6 - 8 weeks old, were used in the study. The mice were housed in an animal facility under a 12 / 12 hour light / dark cycle and with free access to food and water. The study was conducted over a period of 4 weeks. Since the animals were divided into experimental groups, the first week was used for adaptation. Two groups received L-NAME (50 mg / kg) in drinking water for 2 weeks (weeks 2 and 3) to chronically induce nitric oxide synthase (NOS) inhibition. One L-NAME-treated group received the topical formulation V4.3 at a daily dose of 0.1 ml for the last 10 days before characterization. The L-NAME untreated group was used as the sham group.

[0284] Local treatment was initiated after the onset of the disease state. The results showed direct evidence of restoring elements of normal endothelial function (decrease in oxidative stress in plasma and RBCs, decrease in leukocyte adhesion to endothelium indicating restoration of glycocalyx, and improvement in functional capillary density). The group treated with the transdermal formulation showed lower levels of TNF-α, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, IL-10, and IL-10 compared to the sham group. Better results from the treatment group were also observed in terms of microhemodynamic changes, cell adhesion, vascular responses of isolated aortic vessels, changes in erythrocyte and plasma antioxidants, hypoxia, reoxygenation, and systemic hemodynamic changes.

[0285] (Table 1) Changes in body weight, relative tissue weight, and water intake at the end of the study TIFF2025114001000001.tif52166

[0286] (Table 2) Changes in erythrocyte and plasma antioxidants at the end of the study TIFF2025114001000002.tif54138

[0287] Example 9 This experiment shows that pretreatment with oral curcumin over several days prior to hemorrhage dramatically reduced the inflammatory and oxidative elements of HS. Transdermal delivery of curcumin (in the form of formulation 4.3) has been shown to restore vascular homeostasis in a guinea pig model of septic shock. In addition, in a transfusion study, erythrocytes were shown to be able to function as a stealth pharmacological vehicle for the transport and delivery of curcumin. Overall, these studies and several other published studies suggest that curcumin can mitigate the outcomes of HS.

[0288] This experiment tested the hypothesis that packed RBCs loaded with curcumin can be used to enhance therapeutic efficacy in a guinea pig HS model. The ability to recover after HS was compared between fresh RBCs, aged RBCs, and aged RBCs pretreated with formulation 4.3 immediately before injection. Formulation 4.3 restored i) the functional capillary density (FCD), a parameter of microvascular functionality most closely correlated with survival, ii) blood flow, and iii) other measures of cardiovascular system function. The results of R120 FCD showed that RBCs treated with formulation 4.3 had the best performance in many measures of vascular health, even outperforming those of fresh RBCs.

[0289] Example 10 The following methods were used to evaluate the action of the formulation 4.3 prescription: · Single-dose treatment of RBCs (fresh packed) suspended in PBS-glucose with formulation 4.3 on day 1 of a 14-day storage period (resulting in 1 mM curcuminoid in the bag of stored RBCs) · 24-hour survival of treated and untreated stored RBCs (7 and 14 days) with and without single-dose treatment with formulation 4.3 on day 1. For analysis of markers of storage damage, small aliquots of samples were taken on days 1, 7, and 14. · Multiple (3 times) administration of stored RBCs (29-day storage period) starting the first administration on day 28 · Evaluation of the stability of circulating unstable RBCs (in a humanized sickle cell mouse model after topical application of formulation 4.3)

[0290] It was observed that a single dose of the formulation added at the start of storage limited the decrease in ATP levels and oxidative damage. ATP levels compared to day 1 · Day 1: 100% · Day 14: control 28%, formulation 4.3 treatment 45% Oxidative damage (fold increase compared to the value on day 1) · Intracellular ROS ·Day 14: 9-fold increase in the control vs. 5-fold increase in the sample treated with the formulation ·Protein carbonyl content ·Day 14 ·Day 14: 5-fold increase in the control vs. 3-fold increase in the sample treated with the formulation ·Lipid hydroperoxide content ·Day 14 ·Day 14: 7-fold increase in the control vs. 5-fold increase in the sample treated with the formulation ·Formation of ferryl hemoglobin on Day 14 (ferryl Hb highly reactive to induce oxidative stress, ROS and peroxynitrite production. Ferryl Hb content is reflected by SulfHb concentration μM). ·Vehicle control 4.9 vs. formulation treatment 3.8

[0291] Example 11 This experiment showed that transfused RBCs stored after adding formulation 4.3 on Day 1 had enhanced circulation time compared to the vehicle control after 14 and 21 days of storage.

[0292] Blood storage and treatment. Guinea pigs weighing 350 - 400 g were used. The guinea pigs were fed with normal solid feed (ND; Envigo TD.2040). The guinea pigs were anesthetized and a femoral artery catheter was implanted. The animals were bled massively into CP2D, AS-3 was added to the manufacturer-recommended concentration, and the RBCs were passed through a neonatal leukocyte removal filter. The RBC unit was stored for 3 weeks. Formulation 4.3 RBCs were stored with the formulation at 1 mM.

[0293] Recovery 24 hours after transfusion. RBCs from each group were radiolabeled with technetium 99 (Tc99) after 2 and 3 weeks of storage. 200 μL of Tc99 radiolabeled blood (approximately 2% of BV) was delivered via a femoral vein catheter to anesthetized guinea pigs, and samples were taken at 5 and 30 minutes and 24 hours after injection via a femoral artery catheter. All samples were assayed for radioactivity with a Cobra II gamma counter. The following results were observed for the percent recovery of transfused RBCs 24 hours after transfusion: 88% for fresh RBCs; 75% for 2-week-old vs 80% for 2-week-old treated with the formulation; 67% for 3-week-old vs 75% for 3-week-old treated with formulation 4.3.

[0294] Thus, addition of the formulation at the start of the storage process enhances the circulation time of aged transfused RBCs. In addition, the circulation time reflects the systemic state of the RBCs. These results are consistent with a direct stability test showing that addition of the formulation limits the occurrence of storage damage in aged RBCs.

[0295] Example 12 This experiment tested the effect of formulation 4.3 on human red blood cells (RBCs), starting after 28 days of storage. Finally, a substantial enhancement of the RBC profile and an extension of the refrigerated storage life were observed.

[0296] Human RBCs were collected and stored with standard additives (AS-3: α-d-glucopyranose, trisodium citrate, sodium chloride, phosphoric acid, monosodium salt, citric acid, adenine). The experiment included the following steps: 1. 50 ml of total whole blood was used in each set of experiments. 2. After removing the buffy coat and plasma, RBCs were separated from leukocyte-depleted blood. 3. After mixing with AS-3 solution (11 ml), the packed RBCs were stored in a standard RBC storage bag. 4. On day 28 of storage, the RBCs in AS-3 solution were divided into three equal parts and held in separate storage bags (15 ml, 50 μM each bag). 5. On the 35th and 42nd days of storage, additional additions were repeated respectively. 6. Samples were collected and analyzed on days 28, 35, 42, and 49 after storage respectively.

[0297] On the 28th day of storage, the sample was divided into three separate storage bags (50 ml / bag) as follows: a. Control - no other additives b. Formulation 4.3 - 8 microliters of Formulation 4.3 were added, so that the curcuminoids in the bag were 50 micromoles. c. Vehicle - 8 microliters of the solvent system were added, so that the curcuminoids in the bag were 0 micromoles.

[0298] (Table 3) TIFF2025114001000003.tif84167

[0299] Results: RBCs treated with Formulation 4.3 showed an extended storage life, as reflected in a statistically significant improvement in markers of storage damage. Enhancement of ATP levels, reduction of band 3 phosphorylation, and reduction of oxidative damage were observed. The formulation showed a compelling effect as an extender of the storage life of stored human RBCs, one of the most commonly used blood transfusion materials.

[0300] Enhanced ATP levels (percent of starting control value) compared to control · Day 35: Control 60, Vehicle 50, Formulation 4.3 80 · Day 42: Control 38, Vehicle 40, Formulation 4.3 55 · Day 49: Control 32, Vehicle 30, Formulation 4.3 45

[0301] Reduction of band 3 phosphorylation (percent increase compared to control at start) · Day 35: Control 3.5, Vehicle 3.3, Formulation 4.3 2.0 · Day 42: Control 4.8, Vehicle 5.0, Formulation 4.3 3.0 · Day 49: Control 5.8, Vehicle 6.3, Formulation 4.3 5.5

[0302] Limitation of protein oxidative damage (carbonyl formation) in nanomoles per ml of rbc. · Day 35: Control 40, Vehicle 40, Formulation 4.3 22 · Day 42: Control 60, Vehicle 65, Formulation 4.3 45 · Day 49: Control 75, Vehicle 75, Formulation 4.3 58

[0303] Example 13 Topically applied Formulation 4.3 can stabilize circulating red blood cells: Three-week alternate-day topical treatment (4 mg / dose) with Formulation 4.3 enhances RBC stability in a humanized sickle red blood cell mouse model (highly unstable RBC) compared to control (vehicle).

[0304] Briefly, either Formulation 4.3 or vehicle was applied topically every other day for 21 days. RBCs were isolated on day 21 and analyzed for ATP content and protein oxidation. Blood was withdrawn on day 21 and evaluated for markers of hemolysis (LDH, lactate dehydrogenase). PK data and the following were then obtained: · ATP content (nanomoles / ml rbc): Vehicle 30, Formulation 4.3 50 · Protein oxidation (carbonylation) nanomoles / ml rbc: Vehicle 25, Formulation 4.3 25 · LDH level (units / ml): Vehicle 2.2, Formulation 4.3 0.5 · Multiple PK studies in both rats and mice indicate that topical application of Formulation 4.3 results in the sustained appearance of curcumin in both plasma and blood cells

[0305] Thus, the results of topical application of Formulation 4.3 were percutaneous systemic stabilization of circulating RBCs, as evidenced by i) improved redox status and enhanced ATP content of RBCs obtained from treated animals, and ii) in vivo evidence of transcutaneous enhancement of RBC stability reflected by a decrease in the levels of significant hemolysis markers over a 21-day test period compared to vehicle-alone controls.

[0306] Example 14 This experiment used a hemorrhagic shock model (hamster), where at the start of the storage process, it was treated with a single dose of formulation 4.3 (same protocol as in the human RBC test), and the therapeutic efficacy of the transfused stored RBCs was compared to the outcome with either fresh RBCs or untreated stored RBCs. The treated stored RBCs were superior to both fresh RBCs and untreated stored RBCs with respect to maintaining the functional capillary density, which is a major determinant of tissue perfusion and clinical outcome.

[0307] In this preliminary study, the effects of transfusing aged red blood cells (RBCs) stored with formulation 4.3 on systemic and microvascular parameters during resuscitation from severe hemorrhagic shock were evaluated in comparison to the outcome with RBCs stored by conventional methods. The experiment was performed in anesthetized hamsters equipped with a subcutaneous fat window chamber that enabled real-time visualization of the microvascular response. The experimental protocol involved inducing hemorrhage corresponding to 50% of the total blood volume of the hamster, followed by inducing a 1-hour hypovolemic shock. Subsequently, infusion resuscitation corresponding to 50% of the shed blood volume was administered. The choice of this animal model and experimental design helped to closely simulate the state of severe hemorrhagic shock and its clinical management.

[0308] From these results, it was revealed that the resuscitation by the aged RBCs treated with formulation 4.3 was not inferior to the use of fresh RBCs and was significant compared to the aged RBCs stored by the conventional method. The evaluation of systemic parameters including blood pressure showed a favorable response to the RBCs treated with formulation 4.3, which emphasized its effectiveness in restoring hemodynamic stability during the critical resuscitation stage. Furthermore, investigations on microvascular parameters including microcirculation and functional capillary density provided insights into the tissue-level response to transfusion. The functional capillary density, a vital indicator of the balance between enhanced oxygen transport and tissue perfusion, showed significant improvement with the RBCs stored with formulation 4.3.

[0309] It will be understood by those skilled in the art that the invention described herein is not limited to what has been particularly shown and described. Rather, the scope of the invention is defined by the following claims. It should be further understood that the foregoing description is merely representative of exemplary embodiments. This description does not attempt to enumerate all possible variations. Alternative embodiments may not be presented for specific components of a composition or steps of a method, and it should not be regarded as a disclaimer of those alternative embodiments that they may arise from different combinations of the described components or that other unrecited PEG alternatives may be available for the formulation, kit or method. It will be understood that many of these unrecited embodiments are literally within the scope of the following claims and others are equivalent.

Claims

1. A method for preparing a blood sample in which the occurrence of ex vivo storage damage of red blood cells during storage is delayed or its progression is retarded, the method comprising the step of mixing a solution containing an effective amount of curcuminoid with the sample, wherein the concentration of curcuminoid in the sample is in the range of about 0.1 to about 1 mM.

2. The method according to claim 1, wherein the effective amount is selected such that red blood cells remain substantially viable over a period of at least about 60 days.

3. The method according to claim 1, wherein the effective amount is selected such that inflammation, hemolysis, or microparticle formation is reduced by at least 20% compared to an untreated reference over the same period.

4. The method according to claim 1, wherein the solution further contains myristic acid.

5. The method according to claim 1, wherein the adenosine triphosphate (ATP) level and / or 2,3-diphosphoglycerate (2,3-DPG) level in the sample decreases by less than 20% within a period of 50 days.

6. The method according to claim 1, wherein the solvent contains polyethylene glycol (PEG).

7. The method according to claim 1, wherein the solvent consists essentially of PEG.

8. The method according to claim 6, wherein the PEG and the curcuminoid are in a ratio in the range of about 5:1 to about 20:1 by weight.

9. The method according to claim 7, wherein the PEG has a molecular weight in the range of 200 to about 600.

10. The method according to claim 1, wherein the curcuminoid is curcumin.

11. The method according to claim 1, wherein the solution is substantially free of water.

12. An ex vivo blood sample prepared by the method according to claim 1.

13. A method of transfusing blood to a subject in need thereof, the method comprising the step of transfusing the blood sample according to claim 12 to the subject.

14. A method for preparing a blood sample in which the occurrence of ex vivo storage damage of red blood cells during storage is delayed or its progression is retarded, the method comprising the step of mixing a solution containing an effective amount of curcuminoid with the sample, wherein the effective amount is selected such that red blood cells remain substantially viable over a period of at least about 60 days.

15. An ex vivo blood sample comprising blood mixed with a solution, wherein the solution comprises an effective amount of curcuminoid to delay the occurrence or progression of storage damage of red blood cells, and the concentration of the curcuminoid in the blood sample ranges from about 0.1 to about 1 mM, said ex vivo blood sample.

16. Use of the ex vivo blood sample according to claim 15 for treating a disease or condition, wherein the ex vivo blood sample comprises blood mixed with a solution, and the solution comprises an effective amount of curcuminoid, said use.

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