Compositions for blood storage and infusion

Abstract

Formulations comprising one or more active agents for blood storage and infusion are provided. Methods of extending blood viability and enhancing blood transfusion efficacy are also provided.

Description

[0001] Cross - Reference to Related Applications

[0002] This application is a partial continuation of U.S. application Ser. No. 18 / 051,266, filed Oct. 31, 2022, which is a divisional application of U.S. Pat. No. 11,484,493, which is a continuation of International Application No. PCT / US2021 / 058611, filed Nov. 9, 2021, which claims the benefit of priority of Provisional Application No. 63 / 111,160, filed Nov. 9, 2020, Provisional Application No. 63 / 161,696, filed Mar. 16, 2021, and Provisional Application No. 63 / 235,880, filed Aug. 23, 2021, the disclosures of all of which are incorporated herein by reference in their entirety. Technical Field

[0003] Disclosed herein are formulations comprising curcuminoid compounds for blood storage and infusion. Also provided are methods for prolonging blood viability and enhancing transfusion efficacy. Background of the Invention

[0005] Due to bioavailability issues, the delivery of many therapeutic agents remains a challenge because bioavailability issues impede achieving therapeutic levels systemically or locally at the target site. Low bioavailability is caused by multiple factors, including low solubility, low intestinal absorption rate, and rapid elimination from the circulation due to rapid liver breakdown (first - pass limitation). Thus, many promising therapeutic agents with appropriate activity cannot be directly delivered to diseased sites, such as the lungs, arthritic joints, inner ear, sinuses, etc., in an effective manner. At the same time, oral or IV administration routes often cannot provide sufficient therapeutic levels at the target local site.

[0006] Nitric oxide (NO) has known systemic benefits, including reversing inflammation, preventing and reversing endothelial dysfunction, repolarizing activated macrophages, inactivating activated platelets, protecting / restoring the vascular endothelial lining (glycocalyx), and antimicrobial / antiviral activity. Pro-inflammatory injury leads to increased production of reactive oxygen species (ROS), which drives the development and persistence of endothelial dysfunction. Elevated ROS levels trigger multiple events leading to vascular system dysregulation. ROS degrades the glycocalyx, resulting in loss of vascular integrity, enhanced ability of circulating cells (monocytes, erythrocytes, neutrophils, and platelets) to enter and adhere to the endothelial surface, loss of shear stress-mediated NO production, and loss of superoxide dismutase that limits ROS. Enhanced ROS causes endothelial nitric oxide synthase (eNOS) to stop producing NO in the endothelium and start generating more ROS. Increasing NO levels by direct supplementation or enhanced production can significantly reverse this cycle and restore vascular homeostasis. However, systemic delivery of NO is challenging due to the short lifespan of the NO molecule and limited routes of entry into the circulation.

[0007] CD38 plays a key role in multiple organ systems and tissues. CD38 is the major enzyme for nicotinamide adenine dinucleotide (NAD) degradation in mammalian cells. Reduced NAD levels are closely associated with metabolic syndrome and age-related diseases. In the vascular system and in the treatment of endothelial dysfunction, reducing CD38 levels can significantly alleviate angiotensin II (Ang II)-induced vascular remodeling in mice, manifested as reduced blood pressure; reduced vascular media thickness, media-to-lumen ratio, and collagen deposition; and restored elastin expression. Reducing CD38 levels can significantly mitigate Ang II-induced vascular senescence by inhibiting the biogenesis, secretion, and internalization of senescence-associated small extracellular vesicles (SA-sEVs, which promote the senescence of neighboring unimpaired VSMCs). In addition, the protective effect of CD38 deficiency on VSMC senescence is related to the restoration of lysosomal dysfunction, particularly to the maintenance of sirtuin-mediated mitochondrial homeostasis and the activation of the mitochondrial-lysosomal axis in VSMCs. Therefore, CD38 activity and the resulting associated intracellular NAD decline are crucial for Ang II-induced VSMC (vascular smooth muscle cell) senescence and vascular remodeling, and thus CD38 and its associated intracellular NAD decline are crucial for Ang II-induced VSMC senescence and vascular remodeling (which can lead to cardiovascular disease and renal failure). Similar results have been reported for 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 is partly attributed to any of a variety of pro-inflammatory triggers, including the accumulation of senescent cells, leading to its overexpression in activated immune cells (such as macrophages and microglia) and the activated endothelial lining of all blood vessels. SUMMARY OF THE INVENTION

[0008] The present invention provides a transdermal preparation for delivering one or more active agents to a subject for treating a disease or condition associated with elevated CD38 levels, which in turn leads to a decrease in NAD+ levels. Low NAD+ promotes mitochondrial dysfunction and limits the activity of SIRTUIN. Sirtuin activity is necessary to maintain NO production in the endothelium and to switch off oxidative and nitrosative stress generated by activated immune cells (such as the M1 population of macrophages and microglia). CD38 is expressed on the surface of macrophages, microglia, and other immunocompetent white blood cells, as well as on the vascular endothelial lining. Elevated CD38 levels are triggered by diseases or conditions such as trauma, inflammation, infection, radiation, chemotherapy, and an excess of senescent cells. The enhanced CD38 levels in turn further promote cellular senescence, inflammation, and oxidative stress, which further stimulate the production of more CD38, thus forming a persistent inflammatory cycle that is difficult to break.

[0009] The transdermal preparation disclosed herein results in a decrease in CD38 extracellular enzyme activity (such as NADase activity); thereby increasing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. By doing so, this approach provides a new method for preventing and treating various diseases, including those caused by endothelial dysfunction after acute or chronic pro-inflammatory insult, age-related physical and cognitive decline, age-related cardiac tissue changes, vascular hypertrophy, osteoarthritis, peripheral neuropathy, and long COVID. In addition, this approach overcomes the negative impact of excessive CD38 production on the ability of stem cells to differentiate into mature cells, which at least in part contributes to chronic inflammatory anemia and the insufficient efficacy of native stem cells and stem cell therapies in repairing damaged tissues.

[0010] The topical delivery formulations disclosed herein can be directly delivered into plasma and blood cells, thereby bypassing the first-pass elimination by the gut and liver. In addition, red blood cells carrying an active agent can act as stealth delivery vehicles that cannot be detected by the immune system and the liver. Further, uptake of the deliverable by circulating macrophages and other immunocompetent white blood cells carrying activated CD38 (M1 population) results in rapid repolarization (back to the M2 population), thereby rapidly reducing CD38 levels and the production of pro-inflammatory cytokines. This strategy should also reduce the senescent cell burden. By combining a potent anti-inflammatory agent (curcuminoids) (which repolarizes M1 macrophages (thereby reducing CD38 levels) while promoting sirtuin activity) with anti-aging agents (such as quercetin, apigening, fisetin, luteolin), the prospects of breaking the chronic disease inflammation cycle and promoting tissue repair by allowing stem cell differentiation and proliferation are significantly enhanced.

[0011] One aspect of the present application discloses a transdermal formulation for enhancing systemic nitric oxide (NO) through the CD38 / NAD+ pathway, comprising:

[0012] (a) an effective amount of one or more curcuminoids and / or an effective amount of at least one anti-aging flavonoid, wherein the curcuminoids include, for example, one or more of curcumin, demethoxycurcumin, and bisdemethoxycurcumin, and the flavonoids from the anti-aging agents include one or more of quercetin, fisetin, apigenin, luteolin, and rapamycin;

[0013] (b) a polyol in an amount sufficient to dissolve the effective amount of curcuminoids and flavonoids; and optionally

[0014] (c) a fatty acid, wherein the ratio of the polyol to the fatty acid is in the range of about 10:1 to about 50:1 by weight,

[0015] wherein the amounts of the polyol and the fatty acid are selected such that the transdermal formulation delivers an effective amount of the active agent by transdermal or transmucosal delivery after topical application.

[0016] In some embodiments, the curcuminoid compound is curcumin. In some embodiments, the other active agent is at least one flavonoid selected from the group consisting of quercetin, apigenin, fisetin, luteolin, or additional small molecule biocompatible anti-aging agents (such as rapamycin). In some embodiments, the formulation comprises a combination of a curcuminoid compound and an anti-aging agent (such as quercetin and apigenin).

[0017] The formulations and kits disclosed herein can be applied to treat a variety of conditions or diseases. Examples include transdermal treatment of acute and chronic inflammatory conditions, transdermal prevention and reversal of endothelial dysfunction, reducing the risk of cytokine storm or cytokine storm phenomenon (release of abnormal or higher than normal levels of pro-inflammatory cytokines) in patients suffering from diseases that cause potential endothelial dysfunction (such as COVID-19 or SARS-CoV-2 infection), topical treatment of skin diseases, anti-aging skin treatment, treatment of ophthalmic diseases, aerosol-based treatment of pulmonary diseases, topical treatment of infections, treating red blood cells to improve storage characteristics and reverse storage damage thereby enhancing the safety and effectiveness of stored red blood cells, loading red blood cells with therapeutic agents prior to transfusion, stabilizing red blood cells by intravenous (IV) delivery of RBC stabilizers, IV intervention to treat cytokine storm phenomenon and related acute inflammatory crises, aerosol treatment and prevention of acute respiratory distress syndrome (ARDS) and other diseases that damage lung tissue through excessive oxidative damage and subsequent inflammation, loading medical sponges with therapeutic agents for use in, for example, the ear, nose, mouth, rectum, and vagina. Sustained transdermal systemic delivery can also be achieved to harness the therapeutic potential of NO for various chronic diseases and conditions.

[0018] The formulation may additionally comprise polyphenols, flavonoids, stilbenoids, secosteroids, and other phytochemicals and natural products that promote NO formation. In some embodiments, the NO enhancer comprises at least one agent selected from polyphenols, flavonoids, stilbenoids, secosteroids, and similar natural products. In some embodiments, the curcuminoid compound comprises at least one of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, and vitamin D.

[0019] 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.

[0020] In some embodiments, the formulation provides for the sustained release of an NO enhancer or NO releasing agent from an NO precursor (e.g., an S-nitrosothiol-containing molecule) over a period of about 8, about 10, about 15, about 20, about 24, or about 48 hours.

[0021] 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 petroleum jelly, cocoa butter, or a polyalkylene glycol having a molecular weight greater than 2 kDa.

[0022] Another aspect of the invention provides a kit or a transdermal delivery system that includes the transdermal formulation disclosed herein. In some embodiments, the transdermal delivery system is a kit or a nebulizer.

[0023] Another aspect of the invention provides a method for preparing a blood sample for retarding the occurrence or progression of ex vivo red blood cell storage lesions in the sample as compared to an untreated reference sample during storage. The method includes mixing the red blood cells with a solution that includes an effective amount of a curcuminoid compound and optionally a flavonoid compound. 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.

[0024] In some embodiments, an effective amount is selected such that inflammation, hemolysis, or microparticle formation is reduced by at least 20% as compared to an untreated reference sample over the same period of time. In some embodiments, an effective amount is selected such that the levels of adenosine triphosphate (ATP) and / or 2,3-diphosphoglycerate (2,3-DPG) are reduced by less than 5%, less than 10%, less than 20%, less than 30%, or less than 40% over the specified period of time. In some embodiments, an effective amount is selected such that the levels of adenosine triphosphate (ATP) and / or 2,3-diphosphoglycerate (2,3-DPG) are more than 5%, more than 10%, more than 20%, more than 30%, or more than 40% higher than an untreated reference sample or a control over the same period of time. In some embodiments, the specified period of time is 10, 15, 20, 30, 40, 50, 60, 80 days or longer.

[0025] 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.

[0026] In some embodiments, prior to mixing with red blood cells, the concentration of curcuminoid compounds 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. In some embodiments, the pH of the solution ranges from 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 comprises a fatty acid. In some embodiments, the fatty acid is myristic acid.

[0027] In some embodiments, the concentration of curcuminoid compounds in the sample ranges from about 0.2 to about 20 mM, about 0.5 to about 10 mM, about 1 to about 10 mM, or about 1 to about 5 mM.

[0028] In some embodiments, the method comprises consuming oxygen and / or carbon dioxide in the sample.

[0029] In some embodiments, the solution comprises a solvent selected from 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 comprises polyethylene glycol. In some embodiments, the solvent consists essentially of PEG. In some embodiments, the weight ratio of the solvent to the curcuminoid compounds ranges from 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. In some embodiments, the curcuminoid compound is curcumin. In some embodiments, the solution is substantially free of water.

[0030] Another aspect of the invention provides an ex vivo blood sample comprising red blood cells, an agent comprising curcuminoid compounds in a solvent and optionally flavonoid compounds, wherein the amount of the agent is effective to extend the viability of red blood cells in the sample or slow the occurrence or progression of ex vivo red blood cell storage lesions, and wherein the solvent is sufficient to dissolve the agent 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 mM, about 0.5 to about 10 mM, about 1 to about 10 mM, or about 1 to about 5 mM. In some embodiments, the agent comprises curcumin. In some embodiments, the agent comprises curcumin and at least one flavonoid compound.

[0031] A related aspect provides a method of transfusing a subject in need thereof. The method comprises administering the blood sample disclosed herein to the subject.

[0032] Another aspect provides a method of enhancing the efficacy of a transfusion, comprising administering a transdermal preparation to a subject in need thereof, wherein the transdermal preparation comprises:

[0033] (a) An effective amount of a curcuminoid compound, wherein the curcuminoid compound is selected from curcumin, demethoxycurcumin, and bisdemethoxycurcumin;

[0034] (b) A polyol in an amount sufficient to dissolve the effective amount of the curcuminoid compound; and optionally

[0035] (c) A fatty acid, wherein the weight ratio of the polyol to the fatty acid ranges from about 10:1 to about 50:1,

[0036] wherein the amounts of the polyol and the fatty acid are selected such that an effective amount of the NO enhancer is delivered transdermally upon administration.

[0037] In some embodiments, the curcuminoid compound is curcumin. In some embodiments, the transdermal preparation further comprises at least one flavonoid compound selected from quercetin, apigenin, fisetin, luteolin, and rapamycin. In some embodiments, the transdermal preparation comprises curcumin and further comprises at least one of quercetin and apigenin. In some embodiments, the transdermal preparation comprises curcuminoid compounds in a weight percentage range of about 3% to about 10%.

[0038] In some embodiments, the transdermal preparation further comprises at least one flavonoid compound, wherein the ratio of the curcuminoid compound to the at least one flavonoid compound ranges from about 1:1 to about 10:1. In some embodiments, the transdermal preparation comprises quercetin and apigenin in a ratio ranging from about 1:1 to about 10:1.

[0039] In some embodiments, the transdermal preparation comprises myristic acid. In some embodiments, the polyol is selected from polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, and glycerol. In some embodiments, the polyol is polyethylene glycol having a molecular weight range of 200 to about 600. In some embodiments, the ratio of the polyol to the curcuminoid compound ranges from about 8:1 to about 12:1.

[0040] In some embodiments, the blood is an ex vivo blood sample disclosed herein that exhibits extended red blood cell viability or a slowed onset or progression of storage damage. In some embodiments, the transdermal preparation is administered prior to blood transfusion. Detailed Description

[0041] Embodiments of the present invention provide transdermal formulations and methods for enhancing systemic NO levels. Also provided are formulations and methods for enhancing ex vivo red blood cell viability and improving transfusion efficacy. Since known systemic benefits of NO include the ability to reverse inflammation, prevent and reverse endothelial dysfunction, repolarize activated macrophages, inactivate activated platelets, protect the vascular endothelial lining, and have antimicrobial / antiviral activity, the formulations of the present invention can be applied to the treatment of various diseases and conditions. In some embodiments, the treatment is based on transdermal delivery of an agent that can improve nitric oxide production in the endothelium or can directly release NO from a suitable S-nitrosothiol-containing molecule. The formulations disclosed herein are capable of enhancing endothelial NO production and reducing ROS levels by reducing CD38 levels.

[0042] Compared to traditional oral administration of active agents, the transdermal formulations described herein have the advantage of reduced side effects. For example, oral administration of curcumin can cause gastrointestinal discomfort (hypermotility, increased acid production in the stomach), especially when used long-term. Delivery of an agent through the skin or mucosa bypasses the gastrointestinal tract, achieving the desired therapeutic effect while minimizing side effects as much as possible.

[0043] Although specific embodiments of formulations, kits, or methods related to the treatment or prevention of diseases may be cited or exemplified below, it is not intended to limit the scope of the formulations, kits, or methods to such specific citations or examples. Those skilled in the art can make various modifications based on practical and economic considerations, such as the specific form of the formulation and the dosage or frequency of the formulation used to treat or prevent a disease or condition.

[0044] Unless otherwise specified, the article "a" or "an" as used herein refers to "one or more" or "at least one". That is, referring to any element or component of an embodiment with the indefinite article "a" or "an" does not exclude the possibility of there being more than one element or component.

[0045] The term "about" as used herein refers to the recited numerical value plus or minus 10% of the recited numerical value. In some embodiments, "about" refers to the recited numerical value plus or minus 5% of the recited numerical value.

[0046] The term "agent" or "active agent" refers to a molecule or compound that prevents, alleviates, or improves the symptoms of a disease, prolongs the survival of the treated subject, or achieves a desired / acceptable medical or hygienic condition. The agent in an NO enhancer increases the systemic production of NO in a subject. The agent in an NO precursor releases NO or reacts to form another agent that releases NO after transdermal delivery to the bloodstream.

[0047] The term "body cavity" includes any opening on a subject's body and / or the surface area within the opening. Non-limiting examples of body cavities include the nose, sinuses, mouth, ear, rectum, vagina, open wound, sore, buccal cavity, and mucosal surfaces (such as the gums).

[0048] The term C 1-30 Alkyl includes branched or unbranched alkyl groups having any number of carbons in the range of 1 to 30. Non-limiting examples include methyl, ethyl, propyl, and butyl.

[0049] The term "cytokine storm" refers to the dysregulated and abnormal systemic release of pro-inflammatory cytokines that cause disease, and is also known as "cytokine release syndrome" or "inflammatory cascade". Typically, a cytokine storm or cascade is referred to as part of a cohort because one cytokine often causes the production of multiple other cytokines, which can enhance and amplify the immune response. Typically, these pro-inflammatory mediators are divided into two subgroups: early mediators and late mediators. Early mediators, such as tumor necrosis factor, interleukin-1, and interleukin-6, are not sufficient treatment targets for re-establishing homeostatic balance because they have been resolved within the time frame in which the patient presents to the clinic for medical care. In contrast, the so-called "late mediators" are treatment targets because it is during this later "inflammatory cascade" that the patient becomes aware that they are ill.

[0050] In the context of a biomaterial (such as blood, tissue, or an organ), the term "ex vivo" refers to a biomaterial that is located outside of a living organism.

[0051] 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 condition, host tissue damage, or loss of tissue function. "Inflammatory disease" also refers to a pathological condition mediated by the influx of granulocytes and / or neutrophil chemotaxis, as well as transient inflammatory conditions including "brain fog" and leaky gut syndrome caused by chemotherapy.

[0052] "Long COVID" refers to side effects or symptoms attributed to COVID that manifest a long time after seemingly recovering from the initial infection. Non-limiting examples of long COVID symptoms include, but are not limited to, brain fog, fatigue, achiness, blood clotting problems, myocarditis, and edema.

[0053] The term "NO booster" refers to an agent or mixture of agents that increases the production of NO systemically in a subject. An NO booster does not itself release NO.

[0054] The term "NO precursor" refers to an agent or mixture that releases NO directly or indirectly through derivatives. The NO precursor can be or include an agent containing an NO-releasing moiety and is transdermally delivered into the bloodstream of a subject before releasing NO. Non-limiting examples of such NO-releasing agents include S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Alternatively, the NO precursor can include an agent that results in 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 result in derivatives releasing NO include glutathione, N-acetylcysteine (NAC), N-acetylpenicillamine, and cysteine, which can be nitrosated at the thiol group to produce derivatives containing S-nitrosothiols.

[0055] The term "semi-solid" refers to a flexible and deformable solid form. Free-flowing liquids and rigid solid forms do not belong to semi-solids. Non-limiting examples include gels, ointments, creams, emulsions, microemulsions, nanoemulsions, pastes, balms, lotions, and mousses.

[0056] The term "subject" includes any animal, but preferably a mammal, such as a human, non-human primate, dog, cat, horse, cow, or rodent. More preferably, the subject is a human.

[0057] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound of the present invention that has the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or acid addition salts formed with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropanoic 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 monocarboxylic and dicarboxylic acids, aliphatic sulfates, aromatic sulfates, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropanoic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, dodecylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propanoic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present are capable of reacting 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 specific anion or cation forming part of any salt of the present invention is not critical, so long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and their methods of preparation and use are given in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0058] The term "therapeutically effective amount" or "effective amount" refers to the amount of an active agent that is effective in preventing, alleviating, or ameliorating the symptoms of a disease, prolonging the survival of the subject being treated, or achieving a desired / acceptable medical or hygienic condition. Determination of a therapeutically effective amount or effective amount is entirely within the capabilities of those skilled in the art, particularly based on the detailed disclosure provided herein. In the context of blood storage or infusion, the term "effective amount" as used herein also refers to the amount of an agent that maintains the viability or function of a biological sample (such as a cell, tissue, or organ).

[0059] In some embodiments, the term "treat" or "treatment" of any disease or condition refers to ameliorating the disease or condition (i.e., arresting or reducing the development of the disease or at least one of its clinical symptoms). In some embodiments, "treat" or "treatment" refers to ameliorating at least one physical parameter that may not be discernible to the subject. In some embodiments, "treat" or "treatment" refers to modulating the disease or condition, whether physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. In some embodiments, "treat" or "treatment" refers to delaying the onset of a disease or disorder, or even preventing the onset of a disease or disorder. For example, treatment can be "preventive treatment", which is construed to mean 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.

[0060] The term "transdermal" or "transdermally" means the delivery, administration, or application of a formulation containing an active agent by direct contact with the skin or mucosa followed by the transport of the agent across the skin or mucosa into the bloodstream of the subject. Such delivery, transport, administration, or application is known to also include dermal, percutaneous, transmucosal, and buccal routes. As used herein, "dermal" includes the skin and mucosa, including oral, buccal, nasal, rectal, and vaginal mucosa. In some embodiments, the term also refers to the delivery and transport of an agent through a cell wall (e.g., a red blood cell wall) into a cell.

[0061] The term "transdermal formulation" means a composition or formulation of an agent that, when applied to the skin or mucosa, delivers the agent or a derivative of the agent (e.g., an S-nitrosothiol molecule derived from a thiol-containing molecule) across the skin or mucosa (or any other surface as described above). A transdermal formulation 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 containing a transdermal formulation can be in the form of, for example, a patch, swab, nebulizer, sprayer, sponge, or pouch.

[0062] The terms "living cells", "living tissues", and "living organs" refer, respectively, to one or more cells, tissues, and / or organs containing at least a first population of living cells capable of surviving and substantially maintaining their existing biological functions, provided that the first population of living cells is harvested, stored, maintained, cultured, transported, and / or transplanted under the necessary biological conditions (e.g., nutrients, culture temperature, etc.) effective to maintain the viability of such cells, tissues, or organs sufficient for implantation into a suitable recipient host. When x% (e.g., 60% or 80%) of the blood cells in a processed blood sample remain viable after a certain period of time, this percentage value refers to the blood cells in the original total blood cell population that maintain their ex vivo viability. Alternatively, when x% (e.g., 60% or 80%) of the blood cells remain in a processed blood sample after a specified period of time, this percentage refers to the infused cells that remain in circulation during the specified period.

[0063] Transdermal preparation

[0064] One aspect of the present invention provides a transdermal preparation that transdermally delivers a therapeutically effective amount of an agent. The preparation generally includes:

[0065] (a) An effective amount of an active agent, such as a CD38 inhibitor, a sirtuin-1 (SIRT1) activator, a NO enhancer, and / or a NO precursor, wherein the NO enhancer increases the systemic production of NO, and wherein the NO precursor includes a NO-releasing agent or a derivative NO-releasing agent,

[0066] (b) A solvent in an amount sufficient to dissolve the effective amount of the NO enhancer or NO precursor; and optionally

[0067] (c) A fatty acid,

[0068] wherein, after administration, a therapeutically effective amount of the active agent (e.g., a sirtuin-1 (SIRT1) activator, a NO enhancer, and / or a NO-releasing agent) is transdermally delivered. In some embodiments, the active agent is delivered into the blood vessels such that the active agent enters the systemic circulation from an artery or a vein. In some embodiments, the active agent is delivered into the deep layer of the skin (e.g., the upper epidermal layer or the lower epidermal layer beneath the stratum corneum).

[0069] In some embodiments, the agent in the transdermal preparation is a flavonoid compound or a curcuminoid compound, or a combination thereof. One or more flavonoid compounds and / or one or more curcuminoid compounds may be included in the preparation. For example, the preparation may contain one or two flavonoid compounds, such as quercetin and apigenin, optionally in combination with curcumin, wherein each ingredient or component is present in a therapeutically effective amount.

[0070] In some embodiments, the transdermal preparation comprises one or more flavonoid compounds or curcuminoid compounds. 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 comprises one or more flavonoid compounds or one or more curcuminoid compounds, the weight ratio between each flavonoid compound and each curcuminoid compound 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 such 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 above values. In some embodiments, the combination comprises curcumin; and one or both of quercetin and apigenin, wherein the mass ratio between curcumin and 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.

[0071] When the preparation contains two or more flavonoids (such as quercetin, apigenin, fisetin, luteolin and rapamycin), the weight ratio between 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 such 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 above values. In some embodiments, the preparation includes 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.

[0072] The NO level of a subject can be measured using known techniques such as those disclosed in U.S. Patent Nos. 9,044,182 and 8,425,428. In some embodiments, an effective amount is an amount sufficient to produce a measurable effect on a disease, including, for example, hypertension, inflammation, osteoarthritis, endothelial dysfunction, dermatological condition, ophthalmological condition, bacterial infection, viral infection, ischemia reperfusion injury, hypoxiareoxygenation injury, cytokine storm phenomena, cerebral malaria, Chagas disease, and hemoglobinopathies such as Sickle Cell Disease and HbE / beta Thalassemia, type 2 diabetes, and Lupus. In some embodiments, an effective amount is an amount sufficient to produce 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 locally observed at the site of topical application. In some embodiments, an effective amount is an amount sufficient to produce a measurable effect on an inflammatory disease (such as inflammatory skin diseases, inflammatory bowel diseases, and inflammation of the systemic vasculature including the blood-brain barrier caused by chemotherapy), as demonstrated by appropriate clinical parameters (such as improvement in the physician's overall assessment after treatment with the formulation). In some embodiments, an effective amount is an amount sufficient to achieve a systemic or local nitric oxide level that produces the desired effect, as demonstrated by appropriate clinical parameters, such as a measurable positive effect on blood flow and / or vasodilation, a measurable negative effect on blood pressure, and / or a measurable effect on an inflammatory skin disease (such as inflammatory skin diseases).

[0073] Non-limiting examples of SIRT1 activators include polyphenols, flavonoids, stilbenes, seco-steroids, and other phytochemicals or natural products that promote NO formation.

[0074] After transdermal delivery of an NO enhancer or NO precursor, NO is generated in the subject. The amount of the NO enhancer or NO precursor effectively increases NO systemically or locally to a high enough level to achieve the purpose of treating a disease or condition. In some embodiments, the NO enhancer includes polyphenols, flavonoids, stilbenoids, seco-steroids, or natural products that promote NO production. In some embodiments, the NO precursor includes a molecule containing S-nitrosothiol, or a molecule containing thiol and a source of nitrite. In some embodiments, the NO enhancer includes one or more of curcuminoids, flavonoids, berberine, resveratrol, a source of vitamin D, and pharmaceutically acceptable salts and derivatives thereof. Curcuminoids are linear diarylheptanoids, including, for example, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Flavonoids have a 3-hydroxyflavone backbone, including, for example, apigenin, 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 and any precursors of vitamin D. Non-limiting examples of polyphenols include plant extracts, brazilin, and theaflavins (such as theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3)).

[0075] In some embodiments, the NO enhancer consists essentially of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, a source of vitamin D, and any combination thereof.

[0076] In some embodiments, the formulation contains both an NO enhancer and an NO precursor. For example, a combination of curcumin and an NO-releasing agent (such as a molecule containing S-nitrosothiol or a thiol-containing agent) in a polyol / fatty acid system can be a potent formulation for treating local inflammation and infection, while providing the systemic benefit of curcumin in controlling systemic inflammation.

[0077] Other 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 enhancer is curcumin, or synthetic curcumin, which is 80%, 85%, 90%, or 98% pure di-feruloylmethane.

[0078] In some embodiments, the transdermal preparation comprises one or more curcuminoids, and optionally, one or more of polyphenols, flavonoids, stilbenes, seco-steroids, or natural products that promote NO production, as the active ingredient for treating a disease or condition. In some embodiments, the transdermal preparation comprises one or both of curcumin and quercetin, and optionally, one or more of polyphenols, flavonoids, stilbenes, and seco-steroids.

[0079] The NO precursor is a molecule containing S-nitrosothiol, or a mixture of a thiol-containing molecule and a nitrite source. When in contact with an acid source, the nitrite source produces nitrous acid, which can then nitrosate the active thiol in the thiol-containing molecule. The molecule containing S-nitrosothiol releases NO to a subject in need.

[0080] A variety of thiol-containing molecules can be used as precursors. Examples include glutathione, N-acetylcysteine (NAC), N-acetylpenicillamine, cysteine, and their derivatives. The amino group of cysteine or NAC can be acetylated with an acetyl group or other carbonyl groups of different carbon lengths (e.g., COC 2-30 alkyl). By adjusting the length of the carbon chain, the solubility and lipophilicity of the molecule can be changed. Similarly, the carboxyl 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 with an NR2 moiety (where each R is independently H or other substituted or unsubstituted C 3-30 alkyl). The change in the carbon chain can regulate the properties of the molecule.

[0081] A variety of inorganic compounds can be used as a source of nitrite. Non-limiting examples of nitrite sources include alkali metal nitrites, alkaline earth metal nitrites, transition metal nitrites, 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. Nitrite 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 include nitrite-loaded nanoparticles.

[0082] Nitrite-loaded nanoparticles can be prepared by techniques known in the art, including, for example, the method reported in U.S. Patent No. 8,333,997, the entire disclosure of which is incorporated herein by reference. To limit the release of NO from the nanoparticles during the production process, the pH of the medium should be maintained above about 7.5 throughout the preparation process. The nitrite-loaded nanoparticles can then be mixed with a solvent system of a polyol and a fatty acid (such as PEG400 and myristic acid) and remain stable until exposed to an aqueous environment. In the presence of a thiol-containing molecule, when the mixture is exposed to an acid source or a mildly acidic aqueous environment on the skin, the nitrite-loaded nanoparticles will allow the formation of S-nitrosothiols.

[0083] The use of nitrite-loaded nanoparticles allows for the use of a high concentration of nitrite under hydrophobic conditions. The combination of nitrite-loaded nanoparticles with a solvent system of a polyol and a fatty acid (such as PEG400 and myristic acid) (regardless of other included deliverables) allows for the formation of a stable mixture that will release NO and S-nitrosothiol when exposed to an aqueous environment. No nitrite is released or NO is produced in the viscous solvent before the introduction of water or an acid source.

[0084] The acid source can be packaged separately from the mixture containing the thiol-containing molecule and the nitrite source and mixed with the nitrite before application. For example, the nitrite source and the acid source can be separately encapsulated in permeable or frangible sachets. 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 the acid include acetic acid, oxalic acid, and citric acid.

[0085] Non-limiting examples of molecules containing S-nitrosothiol include S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Similar to the thiol-containing molecules, these molecules containing S-nitrosothiol can be modified by changing the carbon chain of their respective ester, amide, or N-acyl moieties to fine-tune their properties.

[0086] Solvents (such as polyols) suitable for the delivery system allow high concentrations of poorly soluble pharmaceutical agents. In addition, it should be biocompatible and have properties that are safe for biomedical applications. Additionally, ideally, it will facilitate skin and mucosal penetration to allow 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.

[0087] Other examples of solvents for the formulations disclosed herein can include ethylene glycol, 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 can include PEG and one or more of acetone, tetrahydrofuran, and n-hexane. In a further example, the solvent can be a combination of acetone and tetrahydrofuran, a combination of acetonitrile and tetrahydrofuran, or a combination of n-hexane and tetrahydrofuran.

[0088] In some embodiments, the solvent of the formulation consists essentially of a polyol. In some embodiments, the formulation can include one or more additional solvents. Non-limiting examples include mineral oil, petrolatum, castor oil, essential oils such as eugenol, menthol, cineole, or rose oil, n-methylpyrrolidone, vegetable oil, oleyl alcohol, dipropylene glycol, polyoxyethylene derivatives of sorbitan esters, saturated 8-10 polyglycolated C 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 fatty acids, and mixtures thereof. In addition to dissolving the pharmaceutical agent, these solvents can also be used as plasticizers to make the formulation flexible, stretchable, plastic, and / or otherwise skin-friendly.

[0089] In some embodiments, the polyol solvent is a low molecular weight polyethylene glycol (PEG) having a molecular weight in the range of from about 50 to about 2000, from about 50 to about 1000, from about 100 to about 1000, from about 100 to about 800, from about 100 to about 700, from about 100 to about 600, from about 200 to about 800, from about 200 to about 600, or from about 200 to about 400 daltons. Non-limiting examples of the molecular weight of the polyol solvent include about 100, including about 200, including about 300, including about 400, including about 500, including about 600, including about 800, and including about 1000. Short-chain PEG molecules that are liquid at room temperature (e.g., PEG200 and PEG400) are particularly useful.

[0090] Other solvents that can be used alone or in combination with polyols 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 that can also be used in combination include derivatives of the following: organic acids (e.g., emalic acid, proline, betaine), choline chloride (e.g., choline chloride, d(-) fructose; choline chloride, A-l rhamnose; choline chloride, lactic acid), different sugars (fructose, sucrose; glucose, sucrose, fructose), and other combinations (betaine, sucrose; betaine, d-(+) glucose, proline).

[0091] The solvent can also be based on colloidal delivery systems, which include, for example, micelles (e.g., produced by surfactants having a hydrophobic core for encapsulating curcumin), microemulsions (e.g., produced by a water phase and an organic phase stabilized by surfactants suitable for introducing curcumin into the hydrophobic core), conventional emulsions and nanoemulsions (the organic phase (oil) is surrounded by a hydrophilic emulsifier; curcumin is encapsulated in the core oil phase), Pickering emulsions (oil-in-water emulsions stabilized by nanoparticles suitable for encapsulating curcumin in the core oil), multilayered emulsions (e.g., prepared by layer-by-layer deposition (lbl) of charged emulsifiers around an internal oil phase introducing curcumin), solid lipid nanoparticles / microparticles (SLNs / SLMs) (e.g., produced by cooling an oil-in-water emulsion to form crystalline lipid particles that can introduce lipophilic curcumin), liposomes and nanoliposomes (e.g., particles having a hydrophilic core and a hydrophobic shell prepared with phospholipids that can encapsulate curcumin), hydrogels (micro / nanogels) (e.g., porous networks of biopolymer networks suitable for encapsulating curcumin).

[0092] 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, glycerol, propylene carbonate, ethanol, acetone, ethyl acetate, acetonitrile, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, and any combination thereof. The above solvents can be used alone or in combination, and their amounts can be easily adjusted using conventional procedures without excessive experimentation.

[0093] Fatty acids are used as penetration enhancers. Non-limiting examples of fatty acids include myristoleic acid, palmitoleic acid, cis-6-hexadecenoic acid (sapienic acid), oleic acid, elaidic acid, trans-11-octadecenoic acid (vaccenic acid), linoleic acid, elaidic linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, octanoic acid, decanoic 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-sized fatty acids such as myristic acid and / or other fatty acids of comparable size / molecular weight are particularly useful. In some embodiments, the formulation does not contain fatty acids or contains only trace or minute amounts of fatty acids.

[0094] In some embodiments, the penetration enhancer consists essentially of a fatty acid or its ester. In some embodiments, the formulation may contain one or more additional penetration 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 methanol, ethanol, isopropanol, 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, pyrrolidones, transcarbam, capsaicin derivatives, dimethyl amino acid esters, peptides, iminosulfuranes, dicarboxylic acid esters, nanocarriers, triglycerides, hydrocarbons, phospholipids, alone or in combination.

[0095] By adjusting the amounts and ratios of polyol, fatty acid, and NO promoter or NO precursor, the solubility of the active agent in the fatty acid and NO promoter 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 between the polyol and the fatty acid affects the form of the solution and is generally in the range of 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 is in the range of 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.

[0096] The ranges and amounts of polyol and fatty acid are as described above and can be modified by those of ordinary skill in the art according to actual needs without undue experimentation. In some embodiments, the fatty acid is selected from myristic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, elaidic acid, trans-11-octadecenoic acid, linoleic acid, linolelaidic 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 polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, glycerol, and any combination 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 ratio of polyol to fatty acid is in the range of about 5:1 to about 100:1 by weight.

[0097] The content range of fatty acids can be 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 enhancer or NO precursor, polyol and fatty acid (if present) or the total weight of the formulation. Non-limiting examples of the amount of fatty acids include about 1%, about 3%, about 5%, about 7%, about 8% or about 10% by weight.

[0098] Water can cause aggregation and particle formation. In some embodiments, the transdermal formulation is anhydrous or substantially water-free. Reducing or eliminating water from the formulation as much as possible can help maintain the uniform distribution of fatty acids and / or active ingredients (such as NO enhancers or NO precursors) in the polyol (such as PEG). In some embodiments, the water in the transdermal formulation is less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or less than 0.01% by weight.

[0099] In some embodiments, the transdermal formulation includes at least one water repellent, also known as a water avoidance agent. Examples of water repellents include silicones, such as cyclomethicone, dimethicone, simethicone, C 26-28 alkyl dimethicone, C 26-28 alkyl methicone, polyphenylsisquioxane, trimethylsiloxysilicate, and a cross-linked polymer of cyclopentasiloxane and dimethicone / vinyltrimethylsiloxysilicate, and mixtures thereof. The water repellent may be particularly useful in embodiments where the topical carrier is used with a water reactive agent, such as a nitric oxide releasing agent (such as diazeniumdialate or sodium nitrite) that releases nitric oxide in the presence of water. In other cases, such as when the active agent is not sensitive to water, the water repellent may or may not be included.

[0100] Depending on the therapeutic objective of the formulation, the active agent in the formulation (such as a CD38 inhibitor, an NO enhancer or an NO precursor, or a mixture thereof) accounts for 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 enhancer or NO precursor, polyol, and fatty acid (if present) or the total weight of the formulation. Non-limiting examples of the amount of the active agent in the formulation include about 1%, about 3%, about 5%, about 7%, about 8%, about 10%, about 12%, and about 15% by weight in the formulation. In some embodiments, the amount of each active agent in the formulation or in a dosage unit of the formulation is independently 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 the dosage unit. The dosage unit can be in a physically separate package form (such as a capsule, a patch, a vial). The dosage unit can also be a predetermined portion of the formulation for each individual administration. For example, an appropriate amount can be taken from a container as the dosage unit of the formulation for direct topical application, or loaded onto a patch or any suitable carrier and then topically applied. The amount or size of the dosage unit can be easily adjusted according to the intended use and the application area.Non-limiting examples of the amount of each active agent in a dosage unit independently 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 independently ranges from about 0.01 to about 1 mg, from about 0.01 to about 0.5 mg, or from about 0.1 to about 0.5 mg. In some embodiments, the active agent is a curcuminoid compound selected from one, two, or three of curcumin, demethoxycurcumin, and bisdemethoxycurcumin, optionally in combination with one or more of the foregoing flavonoid compounds. The dosage unit may be administered once, twice, three times a day, or as needed. In some embodiments, the dosage unit is administered once a day, every two days, every three days, every four days, every five days, every six days, every seven days, or every ten days.

[0101] In some embodiments, the ratio between each active agent and the polyol independently ranges from about 1:5 to about 1:100, from about 1:5 to about 1:50, from about 1:5 to about 1:30, from about 1:5 to about 1:20, from about 1:8 to about 1:15, or from 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 to be 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 a liquid, gel, or other suitable form. Other agents can be added to control the physical state of the formulation.

[0102] In some embodiments, the transdermal formulation contains PEG, myristic acid, and / or other fatty acids of comparable 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 comparable 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 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 between PEG and myristic acid (and / or other fatty acids of comparable 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 between PEG and a NO enhancer (e.g., curcumin) is in the range of about 5:1 to about 100:1 by weight. Non-limiting examples of the ratio between PEG and a NO enhancer include 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 above 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 an individual active ingredient in the formulation is in the range of about 0.01M to about 1M, about 0.05M to about 0.5M, about 0.05M to about 0.3M, or about 0.1M to about 0.2M. Non-limiting examples of the concentration of an active ingredient (e.g., curcumin) in a polyol (e.g., PEG) include about 0.06M, about 0.08M, about 0.1M, about 0.12M, about 0.14M, about 0.16M, about 0.18M, about 0.20M, about 0.25M, about 0.30M, about 0.40M, about 0.60M, and about 0.80M.

[0103] The transdermal preparation has an extended shelf life and as little decomposition of the active ingredient as possible. In some embodiments, the active ingredient of the preparation remains stable at over 95% or over 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 preparation comprises one or more curcuminoids, myristic acid and PEG. The amount of the one or more curcuminoids in the preparation ranges from about 2% to about 10%, from about 3% to about 8% or from about 4% to about 6% by weight. The amount of myristic acid in the preparation 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. The amount of PEG in the preparation 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 some embodiments, the PEG is PEG400.

[0104] In some embodiments, the formulation further comprises a gelling or thickening agent that keeps the formulation in a semi-solid or solid form. Non-limiting examples of gelling or thickening agents include carbomers, methylcellulose, hydroxypropyl methylcellulose, poloxamer, polyacrylic acid, alginate, chitosan, xanthan gum, gellan gum, xyloglucan, paraffin, silicone, petrolatum, cocoa butter, and high molecular weight polyalkylene glycols. Other examples include polyethylene oxide, ammonium methacrylate, carrageenan, aqueous solution of cellulose acetate phthalate (e.g., CAPNF from Eastman), sodium carboxymethyl cellulose, carboxy polymethylene, cellulose, cellulose acetate (microcrystalline), cellulose polymers, divinylbenzene styrene, ethyl cellulose, ethylene vinyl acetate, silicone, polyisobutene, shellac (FMC BioPolymer), guar gum, guar rosin, cellulose derivatives (e.g., hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, and methylcellulose), hydroxypropyl methylcellulose phthalate (hydroxypropyl methylcellulose phthalate), methyl acrylate, microcrystalline wax, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetate phthalate (e.g., Suretic from Colorcon), PVP ethyl cellulose, polyvinylpyrrolidone (PVP), acrylate, PEG / PVP, trimethylsiloxysilicate, maleic acid / anhydride copolymer, polacrilin, poloxamer, polylactic acid / poly-l-lactic acid, terpene resin, locust bean gum, zein, acrylic copolymer, polyurethane dispersion, gelatin (type A and type B from various sources such as pigs, cows, and fish), dextrin, starch, polyvinyl alcohol-polyethylene glycol copolymer, methacrylic acid-ethyl acrylate copolymer (e.g., Kollicoat polymer from BASF), polymers based on methacrylic acid and methacrylates (e.g., poly(methacrylic acid) copolymer and methyl methacrylate copolymer, including Eudragit polymers (Eudragit (E, L, NE, RL, RS, S100)) from Rohm and Haas), esters of polyvinyl methyl ether / maleic anhydride copolymer (e.g., Gantrez ES-425, Gantrez ES-225 available from ISP), and mixtures thereof. Non-limiting examples of high molecular weight polyalkylene glycols include PEG and polypropylene glycol (PPG). The molecular weight of the polyalkylene glycol can be greater than 1k, greater than 2k, greater than 3k, greater than 4k, greater than 6k, greater than 8k, greater than 10k, greater than 15k, greater than 20k, greater than 25k, or greater than 30k daltons. Without intending to limit the scope, the semi-solid formulation can be an ointment, gel, cream, emulsion, paste, lotion, or liposomal formulation.

[0105] In some embodiments, the formulation comprises a combination of small and large polyalkylene glycols having a molecular weight difference in the range of 500 to 5000, 1000 to 3000, 1000 to 2000, or 1500 to 2000 Daltons. By adjusting the ratio between two or more polyalkylene glycols, the viscosity as well as the rate / extent of skin penetration and systemic absorption can be controlled. For example, the combination may include one or both of PEG and PPG, each having a molecular weight in the range of 100 to 2000, 200 to 2000, 400 to 1000, or 500 to 800 Daltons. The combination may also include one or both of PEG and PPG, each having a higher MW in the range of 800 to 5000, 1000 to 3000, or 1000 to 2000 Daltons. In a further exemplary embodiment, the MW of one polyalkylene glycol is 100, 200, 400, 600, or 800, and the MW of the other polyalkylene glycol is 1000, 1500, 2000, 2500, or 3000. In some embodiments, the combination comprises PEG of 400 Daltons and PEG of 2000 Daltons. In some embodiments, the ratio of the low molecular weight polyalkylene glycol to the high molecular weight 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. Other exemplary ratios between the low molecular weight polyalkylene glycol (e.g., PEG and / or PPG) and the high molecular weight 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.

[0106] In some embodiments, the formulation does not include additional therapeutic agents other than the NO enhancer or NO precursor. In some embodiments, the active agent in the formulation consists essentially of the NO enhancer and / or NO precursor described herein. In some embodiments, the formulation may include additional therapeutic agents, including, for example, antioxidants, antibiotics, antiviral agents, and / or antifungal agents.

[0107] The formulation may include other components, including, for example, solubilizers, skin immersion enhancers, surfactants, cosolvents, thickeners or viscosity increasing agents, preservatives, isotonizing agents, isoosmotizing agents, absorption promoters for the medicament, mucoadhesive polymers, non - mucoadhesive polymers, chelating agents, stabilizers, antioxidants, and mixtures thereof.

[0108] In some embodiments, the thickening agent is selected from one or more of carbomer, methylcellulose, hydroxypropyl methylcellulose, poloxamer, polyacrylic acid, alginate, chitosan, xanthan gum, gellan gum, xyloglucan, paraffin, silicone, petrolatum, and cocoa butter.

[0109] Non-limiting examples of solubilizers include, but are not limited to, diethylene glycol monoethyl ether (ethoxydiglycol, commercially available under the trade name ) and diethylene glycol monoethyl ether oleate commercially available under the trade name Poly(TM)); polyethylene castor oil derivatives such as polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil; polyethylene glycol, especially low molecular weight polyethylene glycol; polyethylene glycol derivatives such as glycerol caprylate / caprate (commercially available under the trade name ); alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also be used as absorption promoters. A single solubilizer can be added to the formulation, or a mixture of solubilizers can be added to the formulation.

[0110] Skin penetration enhancers help promote the passage of therapeutic levels of active agents through a suitably sized area of unbroken skin. Suitable enhancers are well known in the art and include, for example, lower alcohols such as methanol, ethanol, and 2-propanol; alkyl methyl sulfoxides such as dimethyl sulfoxide (DMSO), decyl methyl sulfoxide (C10MSO), and tetradecyl methyl sulfoxide; urea; 2-pyrrolidone, N-methyl-2-pyrrolidone, and N-pyrrolidone such as N-(2-hydroxyethyl)pyrrolidone, N,N-diethyl-m-toluamide; C2-C6 alkylene glycols; dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and various solvents such as tetrahydrofurfuryl alcohol; and 1-substituted azacycloheptan-2-ones, especially 1-n-dodecylazacycloheptan-2-one (laurocapram, which is commercially available from Whitby Research Incorporated, Richmond, Va. under the trade name and is commercially available).

[0111] Examples of surfactants can include, but are not limited to, for example, polyethoxylated glycerol esters, polysorbates, poloxamers, sodium dodecyl sulfate, phospholipids such as phosphatidylcholine or phosphatidylglycerol and their derivatives, polyoxethylated hydrogenated castor oil, polyoxethylated fatty acids, mixtures of mono-, di-, and triglycerides of optionally polyoxethylated fatty acids, and mixtures thereof.

[0112] Examples of preservatives can include, but are not limited to, for example, benzalkonium chloride, boric acid, benzoic acid, C 1-4 alkyl esters of p-hydroxybenzoic acid, chlorobutanol, benzyl alcohol, phenethyl alcohol, organometallic derivatives of mercury, polyquaternium such as polyquaternium 1, and mixtures thereof.

[0113] Examples of isotonic agents and isosmotic agents can include, but are not limited to, for example, inorganic salts such as sodium chloride, glucose, trehalose, mannitol, amino acids, and mixtures thereof.

[0114] Examples of mucoadhesive polymers can include, but are not limited to, 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. Examples of non-mucoadhesive polymers can include, but are not limited to, for example, polyvinyl alcohol. Examples of chelating agents can include, but are not limited to, for example, disodium edetate and sodium cromoglycate. Examples of antioxidants can include, but are not limited to, for example, sodium metabisulfite, sodium bisulfite, acetylcysteine, ascorbic acid, and mixtures thereof.

[0115] By adjusting the amounts and ratios of the polyol, fatty acid, and one or more active agents, the solubility of the fatty acid and the active agent, as well as the physical state of the formulation and the release profile of the active agent, can be controlled. In some embodiments, the ratios of the polyol, fatty acid, active ingredient, and other necessary components are configured such that the formulation has a rapid onset of action within about 5 minutes, about 10 minutes, about 15 minutes, or about 30 minutes.

[0116] The transdermal formulations disclosed herein can provide extended or sustained release of a medicament (such as a CD38 inhibitor, an NO enhancer, or an S-nitrosothiol-containing molecule). In some embodiments, the formulation provides extended release of the medicament (transdermally delivered into the bloodstream) over 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. By selecting the polyol solvent and fatty acid in appropriate ratios, the release rate can also be controlled. In some embodiments, one, two, or three of the following parameters can be achieved for the formulation:

[0117] (a) Within about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours, less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% of the medicament is delivered into the bloodstream;

[0118] (b) within about 6 hours, about 8 hours, about 10 hours, about 12 hours, or about 14 hours, 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 agent is delivered into the bloodstream; and

[0119] (c) within about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, or about 48 hours, more than 60%, more than 70%, or more than 80% of the agent is delivered into the bloodstream.

[0120] In some embodiments, the active agent and the carrier (e.g., polyol or fatty acid) and their amounts in the formulation 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, wherein the plasma concentration of the active agent changes less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 40% during this 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 administration of the formulation.

[0121] The formulation may comprise a second agent. Examples of second agents include anti - hypertensive agents, anti - microbial agents, anti - inflammatory agents, analgesics, anesthetics, anti - histamines, preservatives, immunosuppressive agents, anti - hemorrhagic agents, vasodilators, wound healing agents, anti - biofilm agents, and mixtures thereof. Alternatively, the second agent may be present in a separate formulation and / or administered separately from the transdermal formulation described herein.

[0122] 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, 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, halobetasol proprionate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, desoximetasone, triamcinolone acetonide, mometasone furoate, fluticasone proprionate, betamethasone dipropionate, triamcinolone acetonide, fluticasone proprionate, desonide, fluocinolone acetonide, hydrocortisone vlaerate, prednicarbate, triamcinolone acetonide, fluocinolone acetonide, hydrocortisone, and other drugs known in the art, prednisolone, dexamethasone, fluocinolone acetonide, hydrocortisone acetate, prednisolone acetate, methylprednisolone, dexamethasone acetate, betamethasone, betamethasone valerate, flumetasone, fluorometholone, beclomethasone diproprionate, fluocinonide, topical corticosteroids, which can be one of the low-potency corticosteroids, such as 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, prednisolone or methylprednisolone, or can be high-potency corticosteroids,For example, clobetasol propionate, betamethasone benzoate, betamethasone dipropionate, diflorasone diacetate, fluocinonide acetate, mometasone furoate, triamcinolone acetonide.

[0123] In some embodiments, the formulation contains an antiviral agent, such as acyclovir, trifluridine, idoxuridine, penciclovir, famciclovir, cidofovir, gancyclovir, valacyclovir, podofilox, 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. Antiviral therapy can be used to treat local and systemic viral infections, such as COVID-19, cold sores or genital herpes.

[0124] Examples of antimicrobials include penicillins and related drugs, carbapenems, cephalosporins and related drugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, fusidate sodium, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines, vanomycin, teicoplanin, streptogramins, anti-folates (including sulfonamides), trimethoprim and its combinations and pyrimethamine, synthetic antibacterial agents including nitrofurans, methenamine mandelate and methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin, glycopeptide, glyclyclycline, ketolides, oxazolidinone;Imipenen, Amikacin, Netilmicin, Fosfomycin, Gentamycin, Ceftriaxone, Ziracin, Linezolid, Synercid, Aztreonam and Metronidazole, Epiroprim, Sanfetrinem sodium, Biapenem, Dynemicin, Cefluprenam, Cefoselis, Sanfetrinem celexetil, Cefpirome, Mersacidin, Rifalazil, Kosan, Lenapenem, Veneprim, Sulopenem, ritipenam acoxyl, Cyclothialidine, Micacocidin A, Carumonam, Cefozopran and Cefetamet pivoxil.;

[0125] Examples of antihistamines include diphenhydramine hydrochloride, diphenhydramine salicylate, diphenhydramine, chlorpheniramine hydrochloride, chlorpheniramine maleate, isothipendyl hydrochloride, tripelennamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride, etc. Examples of local anesthetics include dibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, 2-(diethylamino)ethyl p-aminobenzoate hydrochloride, procaine hydrochloride, tetracaine, tetracaine hydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonine, and dyclonine hydrochloride.

[0126] Examples of preservatives include alcohols, quaternary ammonium compounds, boric acid, chlorhexidine and chlorhexidine derivatives, iodine, phenol, terpenes, fungicides, disinfectants including thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, chlorhexidine, povidone iode, cetylpyridinium chloride, eugenol, and trimethylammonium bromide.

[0127] Examples of analgesics include alfentanil, benzocaine, buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone, indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine, pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil, tetracaine, and tramadol.

[0128] Examples of anesthetics include alcohols such as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol; pramoxine; resorcinol; troclosan; procaine drugs such as benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine; dexivacaine; diamocaine; dibucaine; etidocaine; hexylcaine; levobupivacaine; lidocaine; mepivacaine; oxethazaine; prilocaine; procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; rodocaine; ropivacaine; tetracaine; and their derivatives such as pharmaceutically acceptable salts and esters, including bupivacaine hydrochloride, chloroprocaine hydrochloride, diamocaine cyclamate, dibucaine hydrochloride, dyclonine hydrochloride, etidocaine hydrochloride, levobupivacaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, pramoxine hydrochloride, prilocaine hydrochloride, procaine hydrochloride, proparacaine hydrochloride, propoxycaine hydrochloride, ropivacaine hydrochloride, and tetracaine hydrochloride.

[0129] Examples of anti - hemorrhagic agents include thrombin, phytonadione, protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome, carbaxochrome sodium sulfanate, rutin, and hesperidin. Other examples of the second or additional agent include chemotherapeutic drugs (tyrosine kinase inhibitors, immune checkpoint inhibitors, VEGF inhibitors, etc.) and hypoglycemic drugs (such as Metformin).

[0130] Other examples of the second agent include daunorubicin (daunorubicin or daunomycin), dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, maphosphamide, ifosfamide, cytosine arabinoside, bischloroethyl-nitrosourea, Busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxypregesterone, testosterone, tamoxifen, dacarbacine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, Melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide,VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol. In some embodiments, second agents that can be used in combination with or incorporated into the same transdermal formulation as those disclosed herein 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, salicylate esters, and PDE5 inhibitors (e.g., sildenafil (Viagra), tadalafil (Cialis), vardenafil (Levitra), and avanafil (Stendra)).,

[0131] Any of the second agents described herein can be incorporated into the same transdermal formulation. Alternatively, in some embodiments of any of the methods disclosed herein, the second agent can be administered separately from the transdermal formulation by any suitable route, including oral, transdermal, and parenteral routes.

[0132] In some embodiments, 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 together, can lead to earlier symptom recovery (fever, cough, sore throat, and breathlessness), less disease progression, and fewer red flag signs in patients with a viral infection (e.g., COVID-19).

[0133] Transdermal delivery system

[0134] A kit or transdermal delivery system can contain any combination of the components described herein in an amount sufficient for at least one agent, and can also include instructions for use of the components recorded in a tangible form. In some applications, one or more components can be provided in a pre-measured single-use amount in an individual (usually disposable) patch, tube, or equivalent container.

[0135] The formulations disclosed herein can be incorporated into transdermal delivery systems or kits and used as patches, swabs, aerosols, creams, sponges, nebulizers, atomizers, or by other suitable means. The transdermal delivery system can also include an instruction manual regarding the administration of the formulation and one or more treatment methods disclosed herein. The liquid or semi-solid formulation can be applied directly to the skin using a swab or sponge, etc. Alternatively, the transdermal delivery system can include a layer coated with 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 can also include an adhesive component for attaching it to the skin.

[0136] The transdermal delivery system or kit can have any shape suitable for application to a subject in need. For example, a sponge carrying the formulation disclosed herein can have a round, 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 coated with a hydrophilic or hygroscopic layer that exhibits an affinity for aqueous solutions, especially for moisture (such as moisture from the site of a placement reservoir or a body cavity). The sponge with absorption properties can be made of natural or synthetic materials, such as including polyester, polyurethane, and plant cellulose.

[0137] In some embodiments, the formulation is incorporated into a liquid reservoir. The reservoir can be used independently or connected to a sponge or partially or fully enclosed within a sponge. Alternatively, after necessary treatment or mixing with additional agents, the contents of the liquid reservoir can be loaded into a sponge for application. In formulations involving a source of nitrite that requires an acid to generate nitrous acid and NO, the acid can be added to the nitrite-containing reservoir prior to administration. Alternatively, a dual-liquid reservoir system can be employed. For example, one sachet contains a thiol-containing molecule and nitrite in a polyol solvent system, while another sachet contains a source of acid. Optionally, additional sachets 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 sachets are mixed to initiate the reaction between the acid and nitrite, followed by nitration of the thiol-containing molecule. The sachets used to encapsulate the NO precursor or acid source are typically fragile or permeable containers that do not contact each other or are separated by an impermeable and removable barrier prior to administration of the formulation. Upon administration, after removal of the barrier, the acid and NO precursor can ooze out from their respective sachets under the pressure applied by the user and mix with each other. The acid and NO precursor can also be simply mixed by rupturing the sachets during or prior to administration. In another exemplary embodiment, the acid and NO precursor are mixed in a container prior to administration. The resulting mixture is absorbed with a swab, sponge, or absorbent patch and then applied to the skin.

[0138] In some embodiments, the sachet has a permeable or semi-permeable membrane surface that is optionally coated with an adhesive for securing the membrane to the skin. In addition to using an adhesive coating, the sachet can be secured to the skin by placing the sachet on the skin and then covering the sachet with an adhesive patch or seal. Commercially available pouchstock materials (e.g., from DuPont) can also be used for the liquid reservoir. Other examples include co-extruded ethylene acrylic acid / low density polyethylene (EAA / LDPE) materials, or (acrylonitrile-methyl acrylate) from INEOS.

[0139] In some embodiments where the NO precursor is a mixture of a sulfur-containing molecule and nitrite, the formulation can be incorporated into a patch. One layer of the patch is impregnated with the NO precursor in a polyol and fatty acid solvent system, while the acid source is provided in another layer. These two layers do not come into contact with 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 includes an impermeable barrier between the two layers, and removal of the barrier prior to administration allows the nitrite and acid to mix.

[0140] In some embodiments, the formulation is a solid and contains a thickening or solidifying material, such as cocoa butter. In some embodiments, the formulation is a solid or semi-solid and contains petrolatum. The solid or semi-solid formulation can be applied to the skin or melted upon vigorous rubbing of the skin.

[0141] In some embodiments, the formulation is loaded into an atomizer or nebulizer and the medicament is delivered as an aerosol to the nose, mouth, or lungs of a subject in need. Carbon dioxide or other suitable gas can be used as a propellant.

[0142] 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 that include certain S-nitrosothiol-containing molecules in a liquid or gel, they can be provided and maintained at a suitable temperature or about 0°C.

[0143] The kit or system can also include instructions and packaging materials for containing the container or combination of containers. Instructions (e.g., written instructions or video demonstrations detailing the use of the transdermal formulation to treat target diseases and conditions) can be included in the kit or system. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, etc.) that contain the components in any of a variety of configurations (e.g., in sachets, tubes, etc.).

[0144] Such kits or systems can also include information such as scientific literature references, package inserts, clinical trial results, and / or summaries of these, which indicate or establish the activity and / or advantages of the composition, and / or which describe administration, dosage, side effects, drug interactions, or other information useful to a healthcare provider. Such information can be based on the results of various studies, e.g., studies using experimental animals involving 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, prescribing officers, etc. In some embodiments, the kits can also be sold directly to consumers.

[0145] Regarding the formulations disclosed herein and their effects on blood, one aspect of the present application provides a biological material sample (e.g., a cell sample, tissue sample, organ sample, and blood sample) that contains biological material treated with the formulations disclosed herein. The sample can be prepared for testing, storage, infusion, or any other suitable purpose. Kits are also provided herein that include biological material stored in a container. The container can be configured for storage, transportation, infusion, or any other use of the sample.

[0146] A biological sample can be prepared by, for example, washing, rinsing the biological sample with the formulations disclosed herein, or mixing the biological sample with the formulations disclosed herein. In some embodiments, the biological sample comprises a formulation and ex vivo biological material. In some embodiments, the biological material is blood.

[0147] Biological material can be recovered from one animal and then implanted into an animal of the same species (allograft) or an animal of another species (xenograft). Tissues can be from whole or part of an organ, such as a heart valve or aorta, or from a specific location of an animal, such as cartilage or tendon of the knee joint.

[0148] 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, islet cells, osteoblasts, nerve cells, bone cells, bone marrow cells, adipocytes, fibroblasts, muscle cells, blood, blood components, stem cells, and embryonic stem cells. In some embodiments, the biological material is blood.

[0149] Exemplary types of mammalian tissues that can be recovered, stored, and / or transported according to the present invention include, but are not limited to: skin, cartilage, tendon, ligament; fascia, tibia, patella, and other bones, heart valve, semitendinosus tissue, blood vessel, intervertebral disc, cornea, lens, meniscus, hair, adipose tissue, fibrous tissue, nerve tissue, connective tissue, and striated, smooth, or cardiac muscle tissue. Cells or tissues can be recovered from human or animal subjects and then processed and / or cryopreserved (frozen) for later implantation. Allograft tissues, including but not limited to heart valves and partial heart valves, aortic root, aortic wall, connective tissues including fascia and dura mater, vascular grafts (including arterial conduits, venous conduits, and biological conduits), and orthopedic soft tissues (e.g., boned or non-boned tendons or ligaments directly connected to bone), are typically cryopreserved. In this way, these valuable tissues can be made available at any time for later implantation into mammals, especially humans. In addition, viable xenograft tissues from transgenic animals or tissues developed from human or non-human cells (which may include differentiated cell types, stem cells, or genetically modified cells from various sources) can be appropriately processed, cryopreserved, and stored for later implantation. Other examples include engineered cells of tissues or tissue-engineered constructs.

[0150] Transplanted animal tissues, cell populations, and recovered mammalian organs stored or maintained by any method or process disclosed herein, or any transplanted mammalian cells, tissues, or organs stored in one or more of the disclosed compositions, are preferably suitable for implantation into a selected recipient animal, particularly a selected recipient mammal. Examples of mammalian species into which transplanted tissue can be transplanted include, but are not limited to, humans, cattle, horses, sheep, pigs, goats, rabbits, dogs, cats, and non-human primates.

[0151] In some embodiments of any method disclosed herein, cell types can include chondrocytes, chondroblasts, osteochondrocytes, islet cells, osteoblasts, nerve cells, osteocytes, 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, striated 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.

[0152] Cell populations, tissues, and organs prepared by the methods provided herein can be from any source, but are preferably those of animal origin, particularly mammalian origin. Exemplary transplanted biomaterials can be obtained from one or more animals, including but not limited to bovines, canines, caprines, equines, felines, avians, humans, lapines, leporines, lupines, murines, ovines, porcines, vulpines, or non-human primates.

[0153] Due to the high affinity for red blood cells, curcuminoids (such as curcumin) and / or flavonoids are expected to also act as therapeutic agents when loaded into red blood cells by enhancing the production and release of nitric oxide or small molecules that produce / release nitric oxide (nitrosothiols and ferro-NO free heme). At the same time, curcuminoids (and other botanicals) loaded into red blood cells are expected to exhibit improved pharmacokinetics as they are not readily cleared by the immune system or the liver.

[0154] Since the formulation is directly mixed with the blood cells in the blood sample, the amount of the solvent will accordingly increase to maintain the curcuminoid compound and / or flavonoid compound dissolved in the mixture or to prevent precipitation of the medicament. As described above, the solvent can contain one or more components. The ratio range between the solvent and one or more medicaments can be 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 above ratios. In some embodiments, the concentration of each medicament in the mixture or sample is independently 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 / m. 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 above concentrations. In some embodiments, before mixing with the blood sample, the formulation disclosed herein has a concentration of curcuminoid compound in 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.

[0155] The (one or more) medicaments, the solvent and their amounts are selected such that more than 40% of the red blood cells remain 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. In some embodiments, the medicament is curcumin or consists essentially of curcumin.

[0156] In some embodiments, the (one or more) agents, solvents, and their amounts are selected such that, compared to a reference sample not treated with the formulation under the same test conditions, a blood sample shows an increased enhanced circulation time after being re-infused into a subject. The increase can range from about 2% to about 30%, from about 5% to about 20%, or from about 8% to about 15%. Non-limiting examples of the increased 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 above values. Thus, when a blood sample is infused into a patient with an inflammation-related comorbidity (including, for example, hemorrhagic shock), the blood sample can act both as a physiological agent (oxygen delivery) and as a therapeutic agent (curcumin delivery).

[0157] In animal studies, histopathology has shown a significant reduction in vascular congestion and iron deposition in the spleen of male HbSS compared to the vehicle, and a significant reduction in the % area involved in liver infarction and liver iron deposition. In summary, the reduced hemolysis and increased Hb, hematocrit, and ATP indicate red blood cell stabilization, improved mitochondrial metabolism, and reduced oxidative stress and organ damage resulting from the action of the formulations disclosed herein.

[0158] Other biomarkers affected by the formulation include increased ATP levels, reduced protein carbonylation, reduced vascular congestion and / or iron deposition, reduced serum amyloid P, a biomarker of overall inflammation, reduced inflammatory cytokines in the skin secretory proteome (with or without reduced interleukin), reduced monocyte chemoattractant protein 1 (MCP-1), reduced interferon-γ (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 by 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 composition but not mixed with or treated with the formulation disclosed herein. Non-limiting examples of the increase or decrease in 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 above values.

[0159] These biomarkers are associated with various diseases or conditions. For example, MCP-1 causes neuropathic pain and inflammation; GM-CSF stimulates granulocyte differentiation and growth. Both RANTES and MCP-1 can also activate mast cells. Thus, compared to a vehicle or reference sample, the formulations disclosed herein result in a significant reduction in skin mast cell degranulation in subjects with sickle cell disease. The formulations disclosed herein provide an anti-inflammatory effect by transdermal administration or by mixing with ex vivo blood, through targeted cytokine release and inhibition of granulocyte activity.

[0160] Attributable to the mixing of the formulation with ex vivo blood, the above-described effects on various biomarkers can also be achieved by administering the transdermal formulations disclosed herein to a subject. Administration of the transdermal formulations disclosed herein and infusion of the treated ex vivo blood samples can both 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 improvement and anti-nociceptive effects.

[0161] The mixture or blood sample can contain other components, such as stabilizers, antibacterial agents, and anti-inflammatory agents. For example, myristic acid and other medium-chain fatty acids with potential health benefits can be added as additives. Saturated fatty acids, such as lauric acid, which have been demonstrated to have antibacterial and anti-inflammatory properties against Propionibacterium acnes (P. acnes), can also be added to the blood sample.

[0162] Method of use

[0163] Due to the local and / or systemic introduction of high levels of flavonoid compounds, curcuminoid compounds, and / or other potent anti-inflammatory agents and / or antioxidants by the transdermal formulation, it can rapidly intervene in various diseases and conditions.

[0164] One physiological and natural way to enhance endothelial NO production and reduce ROS levels is by reducing CD38 levels. High levels of CD38 result in low levels of NAD+, which in turn lead to: i) mitochondrial dysfunction, resulting in excessive ROS production; and ii) reduced Sirtuin protein activity. These CD38 consequences together lead to reduced endothelial NO production, as well as excessive production of ROS and peroxynitrite by activated macrophages and microglia, promoting endothelial dysfunction. In addition, there is an interaction between senescent cells and CD38, each promoting the accumulation of the other. The accumulation of senescent cells in the endothelium not only prevents the repair of the vascular endothelial lining but also promotes persistent endothelial dysfunction, including pathological vascular remodeling and a continuous reduction in NO production.

[0165] Formulations for sustained transdermal delivery of an active agent can reduce CD38 extracellular enzyme activity (e.g., NADase activity); thereby increasing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. By doing so, this approach provides a new method for preventing and treating various diseases, such as those caused by endothelial dysfunction after acute or chronic pro-inflammatory injury, age-related physical and cognitive decline, age-related cardiac tissue changes, vascular hypertrophy, osteoarthritis, peripheral neuropathy, and long COVID. In addition, this approach overcomes the negative impact of CD38 overproduction on the ability of stem cells to differentiate into mature cells, which at least in part contributes to chronic inflammatory anemia and the insufficient efficacy of native stem cells and stem cell therapies in repairing damaged tissues.

[0166] The transdermal formulation can be delivered by any suitable route to deliver the active ingredient through the skin, mucosa, or membrane of a subject's body. Non-limiting examples of suitable routes include, for example, topical routes (e.g., instillation and mucosal routes, including vaginal and rectal delivery), pulmonary routes (e.g., by inhalation or insufflation of powders or aerosols, including via nebulizers), intratracheal routes, intranasal routes, 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.

[0167] Without being bound by any particular theory, it is presumed that the transdermal formulations disclosed herein will transdermally deliver the active agent to a subject and increase the systemic or local NO level in the subject. In some embodiments, the formulation contains an effective amount of a NO enhancer to increase the systemic or local NO level in the subject. In some embodiments, the formulation contains an effective amount of a NO precursor, wherein the method converts the NO precursor into, for example, an S-nitrosothiol-containing molecule that releases NO upon transdermal delivery to the subject. In some embodiments, the formulation can contain both a NO enhancer and a NO precursor. In some embodiments, the formulation can also contain a second agent as defined above.

[0168] The transdermal formulation achieves NO elevation through pathways including upregulating endothelial nitric oxide synthase (eNOS), enhancing eNOS activity, and reducing ROS levels. For example, ROS scavenges NO, causing eNOS uncoupling, which leads to the cessation of eNOS-related NO synthase and instead eNOS further produces ROS. At the same time, the loss of endothelium results in: i) the loss of the flow-mediated mechanical transduction mechanism for controlling eNOS to produce NO; and ii) the loss of the acellular zone adjacent to the endothelium, which prevents hemoglobin in red blood cells from scavenging NO. The transdermal formulation of the present invention provides an effective amount of an active agent that can increase the NO level in the vascular endothelial lining by inhibiting ROS from scavenging NO and limiting the degradation of the endothelial glycocalyx lining.

[0169] Transdermal formulations can be applied to the body surface or body cavities of a subject. For example, methods for increasing systemic or local NO levels or treating a disease or condition can include inserting a sponge loaded with the formulations disclosed herein between the cheek and the gum.

[0170] In diseases or conditions associated with elevated CD38 levels, the transdermal formulations disclosed herein are capable of reducing CD38 levels / activity and restoring nicotinamide adenine dinucleotide (NAD), thereby preventing and reversing many of the consequences of CD38 augmentation. NAD+(NAD) is a key coenzyme present in every cell in the human body and is involved in hundreds of metabolic processes such as cellular energy and mitochondrial health. It is essential for the activity of SIRT1 and SIRT3, which are crucial for controlling inflammation, oxidative stress, and cell repair. CD38 is an enzyme that is primarily present on but not limited to the surface of macrophages and microglia. CD38 has NADase activity (breaking down NAD). The amount of CD38 on macrophages and microglia increases with aging and the onset of acute and chronic inflammation. Therefore, a decrease in NAD levels leads to energy loss, fatigue, an increase in inflammatory processes, and an inability to repair damaged cellular components including DNA. Since the transdermal formulations disclosed herein can effectively reduce CD38 levels with little or no side effects, they can be used to prevent and limit severe endothelial dysfunction after acute pro-inflammatory injury, limit age-related physical and cognitive decline, reverse age-related cardiac tissue changes, and stabilize red blood cells.

[0171] Certain flavonoids, such as apigenin and quercetin, have additional advantages over curcumin due to their anti-aging activity in addition to having many anti-inflammatory and antioxidant stress properties similar to curcumin. Senescent cells induce elevated CD38 levels. By eliminating senescent cells, CD38 levels can be reduced, thereby maintaining or restoring NAD levels. In addition, CD38 can be reduced when activated M1 macrophages repolarize to M2 macrophages. Both curcumin and flavonoids have the ability to repolarize macrophages. It is proposed that circulating M1 macrophages absorb these active substances through the transdermal / transmucosal route, which is a mechanism for rapidly reducing CD38 activity. A CD38 inhibitor can be an agent that repolarizes macrophages and then leads to a decrease in CD38 activity.

[0172] Diseases or conditions associated with elevated CD38 levels refer to those diseases or conditions in which a subject is detected to have CD38 levels higher than normal. Using well-known procedures and statistically acceptable analyses, the normal levels of CD38 can be easily obtained from healthy individuals.

[0173] A variety of diseases or medical conditions can be treated with the transdermal formulations disclosed herein. Non-limiting examples of such 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, severity, and persistence (long COVID), diabetes, hypertension, neuropathic pain, osteoarthritis, anemia of chronic illness, ALS, Parkinson's disease, ischemia-reperfusion injury, hypoxia-reoxygenation injury, transfusion-induced injury, radiation-induced injury (including dermatitis and neuroinflammation).

[0174] Other diseases or medical conditions that can be treated with the formulations disclosed herein include muscle structure disorder, neuronal activation disorder, muscle fatigue disorder, muscle mass disorder, metabolic diseases, vascular diseases, ocular vascular diseases, muscular eye disease, kidney diseases, hypertension, inflammation, endothelial dysfunction, skin diseases, ophthalmic diseases, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomenon, sickle cell disease, inflammatory consequences of acute sickle cell crisis, and other hemoglobinopathies including HbE / β-thalassemia, Chagas disease, type 2 diabetes, lupus, and transient inflammatory conditions including chemotherapy-induced "brain fog" and leaky gut syndrome.

[0175] In some embodiments, the disease or condition is 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 channel disorder, myotonic chondrodystrophy, myotonic dystrophy, myotubular myopathy, nemaline body disease, oculopharyngeal MD, or stress urinary incontinence; a neuronal activation disorder selected from amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Guillain-Barre syndrome, Lambert-Eaton syndrome, multiple sclerosis, myasthenia gravis, nerve lesion, peripheral neuropathy, spinal muscular atrophy, tardy ulnar nerve palsy, and toxic myoneural disorder; a muscle fatigue disorder,It is selected from chronic fatigue syndrome, diabetes (type I or type II), glycogen storage disease, fibromyalgia, Friedreich’s ataxia, intermittent claudication, lipid storage myopathy, MELAS, mucopolysaccharidosis, Pompe disease or thyrotoxic myopathy; muscle mass disorders, which are selected from cachexia, cartilage degeneration, cerebral palsy, compartment syndrome, critical illness myopathy, inclusion body myositis, polymyositis, muscular atrophy (disuse), sarcopenia, steroid myopathy and systemic lupus erythematosus; β-oxidation diseases, which are selected from systemic carnitine transporter, carnitine palmitoyl transferase (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 disorders of β-oxidation (RR-MADD); metabolic diseases,It is selected from hyperlipidemia, dyslipidemia, hyperchlolesterolemia, hypertriglyceridemia, HDL hypocholesterolemia, LDL hypercholesterolemia and / or HLD non-cholesterolemia, VLDLhyperproteinemia, dyslipoproteinemia, apolipoprotein A-I hypoproteinemia, atherosclerosis, arteriosclerosis disease, cardiovascular system disease, cerebrovascular disease, peripheral circulation disease, metabolic syndrome, syndrome X, obesity, diabetes (type I or type II), hyperglycemia, insulin resistance, impaired glucose tolerance, hyperinsulinism, diabetic complication, cardiac insufficiency, cardiac infarction, cardiomyopathy, hypertension, Non-alcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis,NASH), thrombus, Alzheimer disease, neurodegenerative disease, demyelinating disease, multiple sclerosis, adrenal leukodystrophy, dermatitis, psoriasis, acne, skin aging, trichosis, inflammation, arthritis, asthma, hypersensitive intestine syndrome, ulcerative colitis, Crohn's disease, and pancreatitis; cancer, selected from colon cancer, colorectal cancer, skin cancer, breast cancer, prostate cancer, ovarian cancer, and lung cancer; vascular diseases, selected from peripheral vascular insufficiency, peripheral vascular disease, intermittent claudication, peripheral vascular disease (PVD), peripheral artery disease (PAD), peripheral artery occlusive disease (PAOD), and peripheral obliterative arteriopathy; ocular vascular diseases, selected from age-related macular degeneration (AMD), Stargardt disease, hypertensive retinopathy, diabetic retinopathy, retinopathy, macular degeneration, retinal haemorrhage, and glaucoma; myo-ocular diseases,It is selected from strabismus, progressive external ophthalmoplegia, esotropia, exotropia, disorder of refraction and accommodation, hypermetropia, myopia, astigmatism, anisometropia, presbyopia, disorders of accommodation, and internal ophthalmoplegia; kidney diseases, which 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's syndrome.,

[0176] In some embodiments, the disease or condition is selected from genetic 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 genetic 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.

[0177] In some embodiments, the disease or condition is selected from Alpers’s Disease, Chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayra Syndrome (KSS), Leber Hereditary Optic Neuropathy (LHON), MELAS - Mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like episodes, Myoclonic epilepsy and ragged-red fiber disease (MERRF), NARP - neurogenic muscle weakness, ataxia and retinitis pigmentosa, Pearson Syndrome, platinum-based chemotherapy induced ototoxicity, Cockayne syndrome, xeroderma pigmentosum A, Wallerian degeneration, and HIV-induced lipodystrophy.

[0178] In some embodiments, the disease or condition is a neurodegenerative disease, including dementia, Alzheimer's disease (AD), Parkinson's disease.

[0179] 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.

[0180] 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).

[0181] 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).

[0182] In further exemplary embodiments, the transdermal formulations and systems can be used as transdermal therapies for preventing, managing, and reversing the clinical consequences of inflammatory diseases (including diabetes, COVID-19 infection, and sickle cell disease), providing topical treatment of hypertension or topical treatment of osteoarthritis, reversing the acute inflammatory cascade (cytokine storm), improving the safety and efficacy of stored red blood cells for transfusion, treating cerebral malaria or Chagas disease, or treating other early acute inflammatory diseases.

[0183] Phytochemicals (such as curcuminoids) have been shown to have antiviral activity. For example, recent studies have shown that the glycocalyx can prevent viruses from entering the ACE2 binding receptor on endothelial cells, thereby limiting viral uptake and replication. Potential endothelial dysfunction degrades the glycocalyx, thereby increasing the opportunity for viruses to enter the ACE2 binding site. Curcumin and many other phytochemicals protect and preserve the glycocalyx by reducing ROS production and enhancing endothelial NO production (see below). These phytochemicals also reduce pro-inflammatory damage caused by diet and obesity by normalizing lipid and glucose metabolism (including insulin production and utilization). For treating inflammation caused by toxic chemicals and metals, curcumin and other phytochemicals can chelate and eliminate toxins in the blood. It can also limit the inflammatory response to inhaled toxic substances, thereby reducing the tendency to progress to ARDS. Phytochemicals stabilize red blood cells and thus minimize hemolysis caused by toxic agents, and hemolysis is a powerful inducer of inflammation.

[0184] The formulations disclosed herein are capable of addressing pro-inflammatory damage, including acute inflammatory damage caused by certain viral infections (such as SARS CoV2, dengue, and influenza), obesity, and glucose-induced inflammatory triggers, as well as inflammation triggered by exposure to toxic metals and chemicals. For example, in patients with long COVID, side effects attributed to COVID that manifest long after apparent recovery from the primary infection include brain fog, fatigue, pain, coagulation problems, myocarditis, edema, etc. Most of these long COVID symptoms can be attributed to a persistent imbalance between pro-inflammatory and anti-inflammatory factors, which promotes the development and persistence of endothelial dysfunction. The formulations and methods disclosed herein can be applied to treat these clinical manifestations of long COVID.

[0185] In some embodiments of the treatment methods, the transdermal formulations disclosed herein are used to treat diseases or conditions commonly 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., cytokine storm in brain cells), graft versus host disease (GVHD), or the result of treatment with activated immune cells, e.g., IL-2-activated T cells, T cells activated with anti-CD19 chimeric antigen receptor (CAR) T cells. In addition to the effect on endothelial function, a sufficient concentration of the active agent (e.g., curcumin) can also effectively block the binding of the spike protein on SARS CoV 2 to the ACE2 binding sites on endothelial cells and lung epithelial cells, thereby inhibiting viral replication in susceptible subjects.

[0186] In some embodiments of the treatment methods disclosed herein, a 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, especially sepsis, lupus, irritable bowel disease, inflammatory bowel disease, and inflammation of the systemic vascular system including the blood-brain barrier caused by chemotherapy. Vascular leakage is characterized by hypotension, peripheral edema, and hypoalbuminemia. Vascular leakage may also be associated with diseases caused by pathogens, especially viruses and bacteria.

[0187] In some embodiments of the therapeutic methods disclosed herein, a transdermal preparation 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 a key role in cardiovascular homeostasis by regulating blood fluidity and fibrinolysis, vascular tone, 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 diseases (CAD), such as angina and myocardial infarction (commonly known as heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.

[0188] In some embodiments, the amount / dose of the active agent and / or the administration schedule is selected such that the method increases or decreases the level of a biomarker associated with cardiovascular disease in the subject 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 (not treated with the transdermal preparation) or the level prior to treatment with the transdermal preparation 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 has an abnormal level of one or more biomarkers associated with cardiovascular disease prior to treatment, wherein 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 the normal level or the level of a healthy subject.

[0189] Further examples of infectious diseases commonly associated with "cytokine storms" or vascular leakage include, but are not limited to, coronavirus (COV, including COVID-19 / (SARS-CoV-2) coronavirus infection), malaria, avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS). Certain specific infectious agents include, but are not limited to, Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever (CCHF) virus, South American hemorrhagic fever virus, dengue virus, yellow fever virus, Rift Valley fever virus, Omsk hemorrhagic fever virus, Kyasanur Forest virus, Junin virus, Machupo virus, Sabiá virus, Guanarito virus, Garissa virus, Ilesha virus 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 (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, i.e., a chronic inflammatory response to an infectious disease.

[0190] 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 surgery-related pain, and orthopedic or arthritic pain. For example, pain associated with periodontal or other dental surgery can be treated by inserting a sponge into a suitable location in the subject's mouth (e.g., between the cheek and the gum). The transdermal preparation can be administered before or after the onset of pain. For example, the preparation can be administered to the subject before a surgical procedure as a prophylactic method for reducing pain.

[0191] Traditional drugs for neuropathic pain have varying degrees of side effects. The transdermal preparations disclosed herein can be used alone or in combination with traditional drugs, including, for example, gabapentinoids, tricyclic antidepressants, and / or selective serotonin-norepinephrine reuptake inhibitors as first-line drugs, lidocaine, capsaicin, and / or tramadol as second-line drugs, and morphine, oxycodone, botulinum toxin-A, and other opioids as third-line treatments. Thus, the transdermal preparations have the benefit of reducing dependence on traditional painkillers and minimizing side effects.

[0192] The transdermal preparations can also be applied to postoperative pain management. For individuals undergoing surgery (including bypass surgery, thoracic surgery, coronary artery surgery, inguinal hernia repair, and leg amputation), the transdermal preparations can serve as alternative therapeutic agents for treating the above-mentioned pain conditions.

[0193] As described above, the preparation can be administered in any form suitable for the methods disclosed herein. In some embodiments of any of the preparations or methods disclosed herein, the preparation is in semi-solid or solid form and is applied by spreading or rolling on the skin or mucosal surface of the subject. In some embodiments, the patch is coated or impregnated with a preparation in liquid, semi-solid, or solid form. In some embodiments, one or more of the NO enhancer, NO precursor, and acid source are in a liquid reservoir before administration. In some embodiments, the preparation is administered by a nebulizer or atomizer. In some embodiments, the subject is human. In some embodiments, the presence of symptoms, signs, and / or risk factors of the disease or condition to be treated is determined before the start of administration of the preparation.

[0194] The transdermal preparation of the present invention can be administered to activate NAD-dependent deacetylase sirtuin-1 (SIRT1) in a subject. Accordingly, various diseases or conditions associated with SIRT1 dysfunction can be treated. Sirtuins are a class of NAD+-dependent protein deacetylases that regulate a variety of cellular activities, promoting cell survival and extending lifespan in response to environmental stress. Sirtuins exert their function by removing acetyl groups from certain target proteins in the presence of oxidized nicotinamide adenine dinucleotide (NAD+). For example, the yeast sirtuin enzyme Sir2 (silent information regulator 2) was initially discovered for its role in silencing DNA transcription and has also been shown to promote cell survival in response to calorie restriction. Similarly, in Caenorhabditis 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. It has been reported that SIRT1 regulates various pathways, including, for example, restoring angiogenic function and the secretion of angiogenic factors in endothelial progenitor cells. Pioneering papers have shown that SIRT1 is involved in preventing excessive inflammation and oxidative stress by deacetylating NFκB and Forkhead box O transcription factors. In addition, SIRT1 can inhibit cellular senescence, promote keratinocyte differentiation, and prevent ultraviolet-induced DNA damage. Multiple studies have also shown that downregulated or dysfunctional SIRT1 is associated with various diseases (e.g., in a diabetic context), while SIRT1 overexpression improves glucose intolerance and insulin sensitivity and prevents diabetes. Thus, sirtuins appear to be activated as part of a beneficial cellular response to stress, leading to cell survival and extended lifespan.

[0195] Accordingly, sirtuin activators can be beneficial for influencing fundamental cellular processes, protecting cells from stress, preventing or treating various diseases or conditions, and extending healthspan.

[0196] Transdermal delivery of an active agent (e.g., an NO enhancing and SIRT1 activating therapeutic agent) allows for easy combination with oral therapies that target other relevant disease pathways not effectively addressed by the agent delivered transdermally. The method includes administering to a subject in need thereof a transdermal formulation disclosed herein. In some embodiments, the formulation includes (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 enhancing agents described above. In some embodiments, the SIRT1 activator includes one or more of curcuminoids, berberine, quercetin, resveratrol, and fisetin. The amount of the activator can be adjusted according to the nature of the activator and the disease or condition to be treated. In some embodiments, the weight percentage of the activator in the formulation ranges from about 0.05% to about 40%. In some embodiments, the formulation provides for sustained release of the activator over about 15 hours.

[0197] Treatment of acute and chronic diseases or other conditions can benefit from increased 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.

[0198] In some embodiments, the method enhances the SIRT1 activity of 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 a 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 curcuminoids, berberine, quercetin, resveratrol, and fisetin. The amount of the activator can be adjusted according to the nature of the activator and the disease or condition to be treated. In some embodiments, the weight percentage of the activator in the formulation ranges from about 0.05% to about 40%. 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 infection, bacterial infection, and fungal infection. In some embodiments, the subject is diagnosed with endothelial dysfunction or a disease or condition associated with endothelial dysfunction. In some embodiments, the subject has been diagnosed with a disease or condition selected from neurodegenerative diseases, diabetic nephropathy, diabetes, cardiovascular diseases, endothelial dysfunction, muscular dystrophy, pain, neuropathic conditions, abnormal vascular homeostasis, and lupus.

[0199] The transdermal formulations of the present invention can be administered to facilitate a therapeutic effect or reduce adverse events of another therapy. In some embodiments of any of the methods disclosed herein, the transdermal formulations of the present invention can be administered before, simultaneously with, or after another therapy, which includes, for example, oral administration of a drug, intravenous infusion, intramuscular infusion, topical drug treatment, and / or surgery. In some embodiments, the transdermal formulation is administered before an additional therapy for the disease or condition. For example, topical pretreatment with the formulations disclosed herein before blood transfusion can maximize tissue perfusion and minimize transfusion-related inflammation. Topical pretreatment with the formulation or administration simultaneously with another therapy can also reduce adverse events associated with that therapy (e.g., side effects associated with hypoglycemic drugs such as metformin, rashes, chemotherapy-related stomatitis / stomatitis).

[0200] Transdermal formulations can also enhance the endothelial function of a subject. Accordingly, various diseases or conditions associated with endothelial dysfunction or imbalance can be treated. The endothelium has two main interconnected elements that are crucial for vascular homeostasis: the glycocalyx and endothelial nitric oxide synthase (eNOS). The hair-like projections from the endothelium, known as the glycocalyx, are responsible for: i) maintaining vascular integrity, thereby limiting vascular leakage as well as macrophage and lipid entry into the deep layers of the vessel wall (a trigger for plaque formation); ii) controlling the excessive production of reactive oxygen species (ROS) by acting as a reservoir for the potent antioxidant superoxide dismutase (SOD); iii) regulating blood flow in response to physiological demands by controlling eNOS production of nitric oxide in response to shear stress; iv) limiting the entry of blood-borne cells (red blood cells, monocytes, white blood cells), platelets, and infectious agents into and their binding to the endothelium; v) limiting platelet activation; vi) preventing blood stasis; vii) ensuring continuous NO production by eNOS by preventing eNOS uncoupling due to excessive ROS. In the uncoupled state, eNOS no longer generates NO but instead produces more inflammation-generating ROS; viii) maintaining a cell-free zone along the endothelium, thereby preventing red blood cells from scavenging endothelium-generated NO in close proximity to the endothelium. At the same time, nitric oxide produced by endothelial nitric oxide synthase (eNOS) is crucial for vascular homeostasis. Key functions of eNO include: i) maintaining tissue perfusion / oxygenation; ii) preventing blood stasis; iii) preventing a procoagulant environment; iv) repolarizing activated macrophages, thereby promoting tissue repair and limiting tissue damage; v) regulating pro-inflammatory and anti-inflammatory processes (balancing pro-inflammatory iNOS activity that generates destructive peroxynitrite with anti-inflammatory eNOS activity that produces eNO), activating SIRT-1; vi) preventing inflammatory damage caused by ischemia-reperfusion and hypoxia-reoxygenation; vii) preventing ROS-induced damage, including lipid peroxidation and glycocalyx degradation; viii) establishing a reservoir of stored nitrosothiols within the endothelium and the surrounding vascular layers, which can rapidly provide NO in situations where elevated NO levels are required (such as during extreme muscle activity).

[0201] Endothelial dysfunction is a physiological impairment of the normal biochemical processes carried out by the endothelium (the cells lining the inner surface of blood vessels). A hallmark of endothelial dysfunction is impaired endothelium-dependent vasodilation, which is mediated by nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS), a constitutive form of NOS that is predominantly expressed in endothelial cells. In a healthy vascular system, NO produced by the endothelium diffuses into vascular smooth muscle cells (VSMCs), where it activates guanylate cyclase and stimulates the production of cyclic guanosine monophosphate (cGMP), promoting VSMC relaxation and thereby 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 detected clinically, for example, by an increase in the number of circulating endothelial cells (CECs).

[0202] Endothelial dysfunction is associated with a variety of diseases, including, for example, hypertension, coronary artery disease, heart failure, stroke, peripheral artery disease, diabetes, chronic kidney failure, abnormal proliferation of vascular smooth muscle cells and other cardiovascular diseases, type 2 diabetes, insulin resistance and other metabolic syndromes, lupus, HIV, inflammation caused by radiation and drug therapy (such as chemotherapy), hemoglobinopathies (sickle cell disease, HbE / β-thalassemia), cytokine storm-related diseases caused by viral diseases (such as SARS CoV 2, dengue, influenza, hemorrhagic shock, hemorrhagic fever), erectile dysfunction secondary to inflammation caused by surgery, and inflammation associated with an increased population of senescent cells that typically occurs with aging. In addition, endothelial dysfunction is considered a key event in the development of atherosclerosis and occurs many years before clinically apparent vascular pathology. Endothelial dysfunction has also been shown to be predictive in the prediction of vascular events, including stroke and myocardial infarction. Furthermore, endothelial dysfunction has been shown to be associated with inflammation, infection, immune system dysfunction, sleep apnea, sepsis, chronic obstructive pulmonary disease, and exposure to pro-inflammatory agents.

[0203] The methods disclosed herein are applicable to treating both 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 treating acute consequences, including, for example, cytokine storm and associated physical activity- or diet-induced hypoxic / ischemic organ injury (e.g., heart attack due to insufficient tissue perfusion / oxygenation), stroke, microembolism and macroembolism, pulmonary embolism, ischemia-reperfusion injury, hypoxia-reoxygenation injury, and long COVID, which is a consequence of persistent chronic inflammation / endothelial dysfunction. In some embodiments, the methods are applicable to treating chronic consequences, including, for example, cardiovascular disease (CVD), coronary artery disease (CAD), kidney failure, enhanced susceptibility to cognitive decline and dementia, hypertension, sexual dysfunction, slow wound healing, accelerated stent failure / closure, coronary artery bypass graft failure, slow wound healing, reduced physical activity tolerance due to mitochondrial dysfunction, accelerated age-related diseases, osteoarthritis, transient ischemic events, diabetic retinopathy, reduced insulin production due to inflammation-induced pancreatic beta cell injury, HIV-induced CVD, sleep apnea, and / or CAD and CVS secondary to the persistent cyclic episodes of blood stasis (e.g., sickle cell disease). Other applications of the method include blood transfusion (including red blood cells or hemoglobin-based oxygen carriers (HBOCs)) and renal dialysis.

[0204] Transdermal formulations can be administered to treat various local conditions associated with endothelial dysfunction. For example, local conditions such as slow healing of leg ulcers and erectile dysfunction are associated with underlying and often severe endothelial dysfunction that restricts blood flow to the damaged tissue. Methods for treating slow healing of leg ulcers include continuous local delivery of nitric oxide to eliminate biofilms and infections that impede the therapeutic efficacy of agents aimed at accelerating wound closure. At the same time, transdermal delivery of an active agent (e.g., curcumin) will normalize the systemic vascular system, thereby promoting tissue oxygenation and allowing stem cell migration and development. The transdermal formulation can be administered in combination with an antibiotic agent or any suitable wound healing agent. For erectile dysfunction, a transdermally delivered agent (e.g., curcumin) can be used alone or in combination with local nitric oxide and / or an oral PD5 inhibitor to restore systemic vascular health and reduce systemic inflammation. Given that systemic NO enhancers (e.g., curcumin) can enhance NO production in the endothelium and oral supplementation with PD5 inhibitors can prolong the action of NO, the combination of a transdermally delivered active agent and an oral PD5 inhibitor will accelerate endothelial recovery in patients with endothelial dysfunction, including long COVID and cytokine storm.

[0205] The transdermal formulations disclosed herein can also be administered to a subject in need thereof to reduce ROS production, peroxynitrite production (by inactivating iNOS activity) and / or increase eNO production in the endothelium. Without being bound by any particular theory, it is presumed that the formulation provides a pleiotropic effect of upregulating and / or activating multiple anti-inflammatory and antioxidant enzymes and signaling pathways, including, for example, Sirtuin1 (SIRT1) and other inflammatory regulatory sirtuins, PPAR(γ) (peroxisome proliferator-activated receptor-γ), peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α (which is a member of the transcriptional coactivator family and plays a central role in regulating cellular energy metabolism). AMP-activated protein kinase (AMPK) is a phylogenetically conserved fuel-sensing enzyme that is present in all mammalian cells. When activated, AMPK stimulates energy-producing processes such as glucose uptake and fatty acid oxidation and reduces energy-consuming processes such as protein and lipid synthesis. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is the main regulator of antioxidant and cytoprotective genes and is mainly activated in response to oxidative stress. The SIRT1 / PGC-1α / PPAR-γ pathway, enhanced eNOS-mediated eNO production, PPARP, Nrf2, heme oxygenase, AMPK, and ACE2 (angiotensin-converting enzyme 2), or the ACE2 "receptor", which provides an entry point for coronaviruses to attach to and infect a variety of human cells. The formulation can also be used to downregulate or inhibit TLR4 (toll-like receptor 4, part of the inflammatory trigger mechanism), NADPH oxidase (NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) is a membrane-bound enzyme complex that faces the extracellular space and generates reactive oxygen species), and ACE (ACE (angiotensin I-converting enzyme)).

[0206] The transdermal formulations described herein can be applied to treat and manage acute pro-inflammatory injuries and can promote the chronic consequences of endothelial dysfunction (ED) or many pro-inflammatory conditions 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 wound healing, hypertension, stroke, microembolism, edema, sexual dysfunction, retinopathy, neuropathy, and neuropathic pain.

[0207] For acute diseases or conditions, the formulation is capable of rapidly initiating overall anti-inflammatory and antioxidant activities, thereby shortening the cascade and limiting the progression of severe consequences leading to extremely severe ED. For example, a suitable patch or sponge loaded with the formulation provides a very high concentration of NO stimulators (such as curcumin) and is inserted between the gum and cheek for a time to be determined to ensure rapid and sustained delivery of therapeutic levels of curcuminoids or other anti-inflammatory agents, antioxidants, and NO stimulators. This safe approach eliminates concerns about direct NO overdose and additionally activates a full set of host-based anti-inflammatory and antioxidant pathways. The use of a transdermal delivery of a systemic or topical active agent (such as curcuminoids) can stimulate NO production in the vascular system and / or reduce the excessive production of reactive oxygen species (ROS). This combination of enhancing endothelium-generated NO and shutting down ROS production is designed to prevent, limit, and reverse ED and its consequences.

[0208] The transdermal formulation of the present invention can be administered to a subject in need thereof for treating diabetes and related inflammation and other conditions. Inflammation in adipose tissue promotes insulin resistance and hyperglycemia, both of which contribute to and prolong endothelial dysfunction. In addition, chronic untreated endothelial dysfunction generated and enhanced by hyperglycemia, excessive ROS production, and other diabetes-related factors is a common pathway by which type 2 diabetes and other pro-inflammatory factors trigger end-stage clinical symptoms, including, for example, cardiovascular disease, renal failure, hypertension, stroke, and microembolism, slow wound healing, sexual and bladder dysfunction, neuropathic pain, and cognitive decline. By reducing blood glucose, reversing insulin resistance, lowering elevated blood glucose levels or surgically-induced excessive and persistent hyperglycemia, reducing ROS levels, increasing NO levels in the endothelium, and restoring vascular homeostasis, the transdermal formulation of the present invention can minimize the negative and / or adverse consequences associated with surgery, blood transfusion, stent implantation, dialysis, and any other invasive procedures 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 allowing the recovery of damaged endothelium, treating endothelial dysfunction, and preventing the onset or progression of endothelial dysfunction). In addition, it limits the enhanced blood glucose response after surgery in diabetic patients and shortens the recovery time of elevated glucose levels. In addition, it can limit the prolonged and excessive stress-induced glucose spikes in diabetic patients under stress conditions (such as injury, surgery, and blood transfusion), as well as the inflammatory consequences of stress, including urogenital dysfunction after prostatectomy.

[0209] Accordingly, a transdermal preparation can be administered to limit the negative clinical consequences associated with inflammatory triggers in subjects having a pre-existing condition or at risk of developing a condition that promotes hyperglycemia and subsequent endothelial dysfunction. In some embodiments, the preparation is administered prophylactically to subjects at risk of developing hyperglycemia. In some embodiments, the subject has been determined to have hyperglycemia.

[0210] In some embodiments, the amount / dose of the active agent and / or the administration schedule is selected 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 (not treated with the transdermal preparation) or the level prior to treatment with the transdermal preparation disclosed herein. In some embodiments, the subject has been diagnosed with diabetes (e.g., type 2 diabetes), has undergone an invasive procedure (e.g., surgery, blood transfusion, stent implantation, and dialysis), or has suffered an injury. In some embodiments, the subject has an abnormal blood glucose level prior to treatment, wherein 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 the normal level or the level of a healthy subject.

[0211] The transdermal formulations disclosed herein are capable of reducing the levels of pro-inflammatory cytokines. In some embodiments of the methods disclosed herein, the amount / dose of the active agent and / or the administration schedule is selected such that one or more biomarkers are reduced or altered 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 control or the levels prior to treatment with the transdermal formulation. Non-limiting examples of biomarkers that can be reduced or altered by the transdermal formulations disclosed herein include TNF-α, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, IL-10, IL-12, 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 red blood cells, selectins, laminin and Cahedrin, immunoglobulin receptors, chondroitin sulfate, syndecan-1, IL-1a / b, TNF-a, IL-6, D-dimer and other markers reflecting abnormal tendency of blood coagulation, embolism formation, thrombosis, C-reactive protein (CRP), Nrf2, NFκB, glutathione peroxidase (GPx), superoxide dismutase (SOD), Syndecan-1, HMW-hyaluronic acid (1,000 - 6,000 kDa), disintegrin and metalloprotease with thrombospondin type 1 repeats-13, protein C, von Willebrand factor, chondroitin sulfate and sP-selectin. Other examples include markers related to blood pressure, vasodilation, hemodynamics, vascular leakage / edema, M1 / M2 macrophage polarization, soluble platelet selectin, heparan sulfate, and cell and tissue oxygenation. In some embodiments, the subject has an abnormal level of one or more biomarkers or pro-inflammatory cytokines prior to treatment, wherein the abnormal level of the biomarker or one or more cytokines 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% or at least about 100% higher or lower than the normal level or the level of a healthy subject.

[0212] In some embodiments of the methods disclosed herein, the subject has been diagnosed with a disease or condition selected from aging, chronic and acute inflammatory conditions, chemically induced vascular and / or pulmonary inflammation, viral infections, bacterial infections, and fungal infections.

[0213] Further examples of diseases or conditions that can be treated with the methods disclosed herein include neurodegenerative diseases, diabetic nephropathy, 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 intracerebral hemorrhage, hypertension, inflammation, osteoarthritis, rheumatoid arthritis, endothelial dysfunction, skin diseases, ophthalmic diseases, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomenon, cerebral malaria, Chagas disease, hemoglobinopathies, type 2 diabetes, coronavirus, skin / dermatological conditions, acne, inflammatory skin diseases, Raynaud's disease, postherpetic lesions, shingles, skin infections, wounds, burns, leg ulcers, sickle cell, diabetes, onychomycosis, peripheral vascular diseases, infected and / or inflamed mucosal tissues, erectile dysfunction, female sexual dysfunction, vaginal infections / inflammation, catheter-associated urinary tract infections, sinusitis, cystic fibrosis, acute respiratory distress syndrome, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, pulmonary infections (including tuberculosis), pulmonary arterial hypertension, burns and other open wounds, inner ear infections, outer ear infections, gastrointestinal diseases, and acute vasculitic conditions. Further examples of diseases that can be treated with the methods described herein include infectious diseases selected from the group consisting of coronavirus (including SARS-CoV-2), Ebola virus, dengue, hemorrhagic shock, endotoxin shock, acellular hemoglobin toxicity due to hemolysis and / or use of blood substitutes (HBOC) containing acellular hemoglobin, Rift Valley fever, Marburg virus, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Omsk hemorrhagic fever virus, Kyasanur Forest virus, Junin virus, Machupo virus, Sabia virus, Guanarito virus, Garissa virus, Ilesha virus, and Lassa fever virus.

[0214] Further examples of diseases or conditions treatable with the formulations disclosed herein include neurodegenerative diseases or conditions (such as Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and conditions caused by polyglutamine aggregation); skeletal muscle diseases (such as Duchenne muscular dystrophy, skeletal muscle atrophy, Becker muscular dystrophy, or myotonic dystrophy); metabolic disorders (such as insulin resistance, diabetes, obesity, impaired glucose tolerance, hypercholesterolemia, hyperglycemia, dyslipidemia, and hyperlipidemia); adult-onset diabetes, diabetic nephropathy, neuropathy (such as, sensory neuropathy, autonomic neuropathy, motor neuropathy, retinopathy); skeletal diseases (such as osteoporosis), blood diseases (such as leukemia); liver diseases (such as due to alcohol abuse or hepatitis); obesity; bone resorption, age-related macular degeneration, AIDS-related dementia, ALS, Bell's palsy, atherosclerosis, heart diseases (such as arrhythmia, chronic congestive heart failure, ischemic stroke, coronary artery disease, and cardiomyopathy), chronic degenerative diseases (such as cardiomyopathy), chronic renal failure, type 2 diabetes, ulcers, cataracts, presbyopia, glomerulonephritis, Guillain-Barré syndrome, hemorrhagic stroke, rheumatoid arthritis, inflammatory bowel disease, SLE, Crohn's disease, diseases or conditions associated with osteoarthritis, osteoporosis, chronic obstructive pulmonary disease (COPD), pneumonia, skin aging, urinary incontinence, mitochondrial dysfunction (such as mitochondrial myopathy, encephalopathy, Leber's disease, Lee encephalopathy, Pearson Disease, lactate acidosis, "mitochondrial encephalopathy, lactate acidosis and stroke-like symptoms" (MELAS), muscle diseases, including neuromuscular diseases, such as muscular dystrophy and myopathy, and diseases or conditions associated with neuronal death, aging, or other conditions characterized by unwanted cell loss).In some embodiments, the disease or condition is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, neurodegenerative consequences of traumatic brain injury or intracerebral hemorrhage, sickle cell disease (including pain associated with sickle cell disease), thalassemia (such as HbE / β-thalassemia), diabetic retinopathy, glaucoma, dry eye disease, surgery-induced inflammation, aging, chronic and acute inflammatory conditions, chemically induced vascular and / or pulmonary inflammation, viral infections, bacterial infections, fungal infections, diabetic kidney disease, diabetes, cardiovascular disease, endothelial dysfunction, muscular dystrophy, pain, neuroinflammatory 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), surgical-induced inflammatory sequelae (urogenital system dysfunction), transfusion-induced inflammation, inhibition of stent restenosis, inflammatory consequences of restricted dialysis, inflammation and pain after dental surgery, neuropathic pain caused by any pro-inflammatory injury (including peripheral neuropathy, spinal neuropathy, arthritis pain), childhood cognitive dysfunction due to cerebrovascular injury caused by sickle cell disease, cytokine storms caused by coronavirus, dengue, Ebola virus, Rift Valley fever and influenza, cerebral malaria, tumor metastatic spread through dysfunctional blood vessels, systemic consequences of psoriasis, dementia (including Alzheimer's disease and Pick's disease, traumatic brain injury and hemorrhagic shock).

[0215] 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, 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, bullous skin disease, erythema multiforme, contact dermatitis, psoriasis, allergic diseases, asthma, allergic rhinitis, atopic dermatitis, foodhypersensitivity), urticaria, pulmonary immune diseases, eosinophilic pneumonias, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, systemic lupus erythematosus, scleroderma and arthritis.

[0216] Non-limiting examples of neurological diseases that can be treated or have their progression limited by the methods of the present invention include neurodegenerative diseases, including 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), Friedreich's ataxia, periventricular leukomalacia (PVL), the ALS-Parkinson's-Dementia complex of Guam, Wilson's disease, cerebral palsy, progressive supranuclear palsy (Steel-Richardson syndrome), bulbar and pseudobulbar palsy, diabetic retinopathy, multi-infarct dementia, macular degeneration, Pick's disease, diffuse Lewy body disease, prion diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, Kuru, and fatal familial insomnia, primary lateral sclerosis, degenerative ataxias, Machado-Joseph disease / spinocerebellar ataxia type 3, and olivopontocerebellar degeneration, spinal and bulbar muscular atrophy (Kennedy's disease), familial spastic paraplegia, Wohlfart-Kugelberg-Welander disease, Tay-Sach'sdisease), multisystem degeneration (Shy-Drager syndrome), Gilles De La Tourette's disease, familial dysautonomia (Riley-Day syndrome), Kugelberg-Welander disease, subacute sclerosing panencephalitis, Werdnig-Hoffmann disease, synucleinopathies (including multiple system atrophy), Sandhoff disease, cortical basal degeneration, spastic paraparesis, primary progressive aphasia, progressive multifocal leukoencephalopathy, striatonigral degeneration, familial spastic disease, chronic epilepsy associated with neurodegenerative diseases, Binswanger's disease, and dementia (including all potential causes of dementia).

[0217] Insulin resistance conditions treatable by the methods of the present invention include any disease or condition caused or contributed to by insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin resistance syndrome, syndrome X, insulin resistance, hypertension, hypertensive disease, hypercholesterolemia, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic diseases including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including coronary heart disease, angina, congestive heart failure, stroke, cognitive function in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, certain types of cancer (e.g., endometrial cancer, breast cancer, prostate cancer and colon cancer), pregnancy complications, poor female reproductive health (e.g., irregular menstruation, infertility, irregular ovulation, polycystic ovary syndrome (PCOS)), lipodystrophy, cholesterol-related conditions such as gallstones, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and breathing problems, osteoarthritis, and prevention and treatment of bone loss, such as osteoporosis. Further applications of the methods of the present invention include promoting wound healing, e.g., can also be used to promote wound healing and diabetic - poor wound healing.

[0218] In some embodiments of any of the methods disclosed herein, it further includes the step of determining that the subject has SIRT1 downregulated or dysregulated compared to a normal standard or reference. In some embodiments, the methods disclosed herein further include diagnosing the subject with endothelial dysfunction or a disease or condition associated with endothelial dysfunction before administering the transdermal preparation to the subject.

[0219] In some embodiments of the methods disclosed herein, it includes the step of determining that the systemic NO level or plasma nitrite and / or nitrate level of the subject 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.

[0220] In any of the methods disclosed herein, the treatment regimen can be administered after the clinical manifestation of symptoms or the appearance of a disease or condition is clinically observed. Alternatively, the method can be used prophylactically before the onset of any clinical symptoms or the observation of any clinical symptoms. For example, the methods disclosed herein are capable of addressing pro-inflammatory insults, including acute inflammatory insults triggered by certain viral infections (such as SARS-CoV-2, dengue, and influenza), obesity and glucose-induced inflammatory triggers, and inflammation triggered by exposure to toxic metals and chemicals. The treatment can be administered when symptoms have been identified or before the onset or observation of any symptoms. In a further example, a transdermal formulation can be administered prophylactically to prevent the inflammatory consequences of surgery, including pain or erectile dysfunction caused by postoperative inflammation.

[0221] The methods disclosed herein can increase NO levels systemically or locally. In some embodiments of any transdermal formulation or method of the invention, the amount of the active ingredient (such as a NO enhancer and / or NO precursor, polyol, optional fatty acid, and / or other components) in the transdermal formulation is selected such that the systemic or local NO level or plasma nitrite and / or nitrate level of the subject is increased 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 nitrite and / or nitrate level before the administration of the formulation. In some embodiments, the desired increase or change can be achieved within about 1 hour, about 2 hours, about 3 hours, about 5 hours, or about 8 hours. In some embodiments, the increase or change can be maintained for about 1 day, about 3 days, about 5 days, about 7 days, about 10 days, about 15 days, about 30 days, or longer. A variety of methods can be used to measure the NO level or plasma nitrite and / or nitrate level, such as colorimetry and chemiluminescence using Griess reagent.

[0222] In some embodiments of any transdermal formulation or method of the invention, the amount of the active ingredient (such as a NO enhancer 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 is decreased by at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40 or more mmHg over a 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 nitrite and / or nitrate level before the administration of the formulation.

[0223] In some embodiments of any of the methods disclosed herein, the subject has been diagnosed with hypertension or is 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 embodiments, formulations in the form of patches, creams, or gels are administered once every 1, 3, 5, 7, or 10 days.

[0224] Another aspect of the invention provides a method of reducing CD38 activity or reducing elevated CD levels, comprising contacting a transdermal formulation disclosed herein with cells that overexpress CD38. The desired effect can be optimally achieved through a combination of macrophage / monocyte / microglia repolarization from the M1 to the M2 phenotype, reduction of the senescent cell population, and anti-inflammatory / antioxidant activity. In some embodiments, CD38 is located in the cell (on the cell surface or intracellularly). In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo.

[0225] Another aspect of the invention provides a method of incorporating an agent into cells by contacting the cells with a formulation disclosed herein. Due to increased NO levels, the method can stabilize the cells, improve storage characteristics, and reverse storage damage. In some embodiments, the cells are red blood cells. In some embodiments, the formulation is in liquid form. In some embodiments, the formulation comprises a molecule containing S-nitrosothiol.

[0226] Related aspects provide a method of prolonging or enhancing the ex vivo viability of a biomaterial (such as a cell, tissue, organ, body fluid sample, or blood). The method comprises contacting (such as rinsing, mixing, perfusing, lavaging, and / or infusing) the biomaterial with a formulation disclosed herein. The range and amount of the solvent, curcuminoids, optional flavonoids, and other components in the formulation are as described above.

[0227] In addition to mixing or contacting the ex vivo biomaterial with a formulation disclosed herein, the formulation (adjusted for concentration and form) can also be administered (such as transdermally) to a donor subject (while the subject is alive or after the subject has died) prior to harvesting the cells, tissue, organ, or blood. In some embodiments, the disclosed formulation is used as a washing solution to clean freshly recovered biomaterial 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 curcuminoids. In some embodiments, the method is intended to prolong the ex vivo viability of red blood cells by mixing or contacting the red blood cells with the solution.

[0228] In the practice of the present invention, it is often necessary to maintain cells, tissues or organs in a composition essentially from immediately after harvest until the explant material is ready to be transplanted into a recipient subject. During the interval between harvest and implantation or infusion, it is also necessary to monitor and control environmental conditions and storage parameters to maintain the integrity, viability and biochemical activity of the recovered biomaterial.

[0229] During storage, red blood cells (RBCs) undergo a variety of biochemical and biomechanical changes that reduce the survival rate of RBCs and impair the oxygen delivery function of the blood. These changes are collectively referred to as storage lesion, which is typically characterized by increased inflammation, hemolysis, microparticle formation, and / or shortened circulation time. Storage lesion may result in reduced RBC survival rate and impaired oxygen delivery function of the blood. The solution formulations disclosed herein are capable of slowing the occurrence of storage lesion phenomena in RBCs and / or slowing the progression of changes associated with storage lesion. Compared with a control or untreated reference sample (collected simultaneously with and / or from the same source as the target sample except not treated with the formulations disclosed herein) over the same time period, the target sample treated with the formulation is characterized by a reduction in one or more of inflammation, hemolysis, and microparticle formation, and an increase in circulation time, independently increased 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%, at least 90%, at least 100%, at least 120%, at least 150%, at least 200%, or any range between any two of the above percentage values. In some embodiments, the weight ratio of the biomaterial (e.g., blood sample) to the formulation or solution is 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 biomaterial (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 above values.

[0230] The solution formulation is capable of stabilizing red blood cells, preventing hemolysis of unstable red blood cells, and generating circulating red blood cells that exhibit biochemical and biophysical signs of stabilization. Thus, the red blood cells treated with the formulation exhibit significantly slowed or reduced oxidative damage that limits both the shelf life and efficacy of red blood cells.

[0231] 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.

[0232] In any of the embodiments disclosed herein, the viability of a cell, tissue, or organ can be determined by assays known to those skilled in the relevant art. For example, the viability of a cell can be readily determined by a microscopy assay commonly referred to in the art as a "live / dead assay." In one such assay, the biologic dyes 5-chloromethylfluorescein diacetate and propidium iodide (which differentially stain live and non-live cells) are used and evaluated by a microscopy-based assay. Such dyes are typically fluorescent, and fluorescence can be detected and used to generate a two-parameter fluorescence histogram, most typically using fluorescence microscopy techniques to distinguish live and non-live cells, where live and non-live cells fluoresce at distinctly different wavelengths.

[0233] In some embodiments, to determine the % viability of stored cells or tissue over time, the viability of a biological sample can first be determined (usually within 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, or 72 hours after harvest from a donor subject) to determine the "initial viability." Then, a subsequent viability determination of the tissue is performed after a period of time to determine the "current viability." Thus, the % viability can be determined at any time after harvest using the following formula:

[0234] [(Current viability) / (Initial viability)] × 100 = Viability percentage

[0235] If desired, multiple samples can be analyzed and averaged during the initial determination and / or subsequent analysis to determine the "average viability" of the recovered tissue. Alternatively, the determination of tissue, cell, or organ viability can also include one or more biochemical or anatomical assays known in the art that provide qualitative and / or quantitative evidence of the biological activity or functionality of the transplanted tissue after it is introduced into a recipient animal. U.S. Patent No. 9,737,071 provides additional assays and detection methods, the entire disclosure of which is incorporated herein by reference.

[0236] In some embodiments, treating (e.g., mixing or contacting as described above) blood (freshly collected or aged) with the formulations disclosed herein allows for the delivery and delivery of the loaded therapeutic agent (e.g., curcuminoid compounds) to red blood cells. Utilizing red blood cells as a stealth pharmacological carrier, the therapeutic agent (e.g., medicament) can be delivered to a subject in need by infusion and restore or improve important parameters such as microvascular blood flow, permeability, glycocalyx, and oxygenation. The blood can be treated prior to infusion, e.g., 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 any two of the above values. Treatment can be performed at one or more time points (one or more times) prior to infusion. Thus, blood samples prepared from the blood treatment method can be applied to treat various diseases and conditions.

[0237] In some embodiments disclosed herein, the components and their amounts of the formulation are configured such that the formulation can reduce 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 above percentage values (relative to a control or untreated sample).

[0238] In some embodiments, the method includes adding one or more additional components, such as stabilizers, antibacterial agents, and anti-inflammatory agents, to the biological material (e.g., blood sample). The additional components can 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 with potential health benefits.

[0239] In some embodiments, the biological sample is stored in a temperature range of about -30°C to about 37°C. Non-limiting examples of the temperature 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 above temperatures.

[0240] In some embodiments, the blood is collected from a healthy individual. In some embodiments, the blood is collected from an individual and then injected back into the individual after treatment with the formulations described herein and other necessary medical procedures.

[0241] In some embodiments, the pH of the solution is in the range of about 5.5 to about 7.0 before or after mixing with the blood.

[0242] In some embodiments, the method includes consuming oxygen and / or carbon dioxide in the sample.

[0243] In some embodiments, the agent, solvent, and their amounts are selected such that the levels of adenosine triphosphate (ATP) and / or 2,3-diphosphoglycerate (2,3-DPG) 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 compared to a control or untreated sample.

[0244] In some embodiments, the concentration of curcuminoids in the solution prior to mixing with the sample ranges from 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 curcuminoids 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 above values. In some embodiments, the concentration of curcuminoids in the solution prior to mixing with the sample ranges from 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. Non-limiting examples of the 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 above values. In some embodiments, the concentration of curcuminoids in the blood 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. Non-limiting examples of the concentration of curcuminoids 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 above values.

[0245] Treatment of cells (e.g., red blood cells) with the formulations described herein can deliver NO boosters (e.g., curcumin) and optionally other anti-inflammatory / antioxidant agents into the lipid membrane or beyond the cytosol, should be able to stabilize the cells for storage, and convert the infused cells into long-acting (circulating) anti-inflammatory agents that can slowly deliver these agents to the endothelial lining of capillaries and other small-diameter blood vessels. Similarly, treatment of cells with a formulation containing a high concentration of NO precursors (e.g., lipophilic S-nitrosothiols and S-NO derivatives of alkyl ester derivatives of NAC) should be able to effectively transnitrosylate thiols intracellularly and on the cell. Nitrosylation of key thiols on and in red blood cells, including the thiols of β93 on Hb and 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 to deliver NO to thiols on the endothelium.

[0246] On the other hand, a method of transfusing blood to a subject in need thereof is provided. The method includes transfusing a blood sample disclosed herein, the blood sample being stored in a container or kit as described above. The blood sample is prepared in the same manner as the method for extending the viability of ex vivo red blood cells disclosed herein. Red blood cells loaded with curcumin are well-suited to deliver curcumin to the capillary bed and smaller blood vessels, such as arterioles. These are suitable sites for treating a number of vascular disorders caused, for example, by endothelial dysfunction, including, for example, ischemia / reperfusion injury and vaso-occlusive crises. Related aspects provide a method of treating a disease or condition associated with endothelial dysfunction. The method includes administering to a subject in need thereof an effective amount of a blood sample disclosed herein. Since red blood cells act as carriers for active agents (e.g., curcumin, other curcuminoids, or flavonoids), a therapeutic effect can be achieved when the agent is delivered to an appropriate site, such as a stenotic blood vessel, such as an arteriole and a capillary bed, where curcumin can be released / redistributed to the inner wall of the vascular endothelium due to the very close proximity of red blood cells to the inner wall of the blood vessel. Release of curcumin will reduce or eliminate endothelial dysfunction and restore the production of NO within the endothelium, which is required for normal endothelial function. Non-limiting examples of diseases or conditions associated with endothelial dysfunction include those diseases or conditions disclosed above in this application.

[0247] In some embodiments of any of the methods disclosed herein, the active agent (e.g., curcuminoids or flavonoids) is loaded into red blood cells and white blood cells, such as macrophages, monocytes, microglia, and neutrophils. Uptake of curcuminoids and / or flavonoids by activated pro-inflammatory white blood cells can lead to a rapid reversal of cytokine storm-type phenomena, for example, in acute inflammatory diseases or conditions, under the treatment methods disclosed herein.

[0248] Another aspect related to the blood samples disclosed herein is a method of maintaining or restoring microcirculation, or reducing or reversing the microcirculatory consequences of a disease or condition (such as hemorrhagic shock), by administering the blood samples to a subject in need thereof. The blood samples can be treated with the formulations disclosed herein prior to infusion (e.g., 1, 2, 3, 4, 6, or 8 hours prior to infusion).

[0249] In some embodiments, the method is applicable to the treatment or prevention of hemorrhagic shock (HS). Traumatic hemorrhage leads to HS. HS is a leading cause of death in civilian and military settings, accounting for one-third of all trauma-related deaths and 80% of potentially survivable battlefield injuries. Almost 50% of the deaths attributed to hemorrhage occur within 3 to 6 hours after injury. Studies conducted in various species and models have clearly established that the microvasculature is crucial in the pathophysiology of HS and is associated with organ damage attributed to reduced tissue perfusion and oxygenation, as well as damage caused by excessive production of reactive oxygen species (ROS). The intense inflammatory response associated with HS leads to reduced tissue perfusion, upregulation of interleukins / cytokines, activation of microvascular endothelium and neutrophils, ROS generation, and increased permeability also caused by glycocalyx degradation. HS / trauma disrupts the balance between the microvasculature and various blood components, sometimes leading to coagulopathy, which also contributes to the morbidity and mortality associated with HS. The maintenance and restoration of microcirculation are involved in all therapeutic attempts aimed at reversing or minimizing the adverse consequences of HS. Thus, the blood samples disclosed herein can be infused to deliver therapeutic agents that can improve the outcome of hemorrhagic shock. Aged red blood cells may be repurposed to treat combat casualties predisposed to or suffering from HS. The ability to use red blood cells (including aged / stored red blood cells) as therapeutic agents that effectively treat the underlying pathogenic mechanisms driving HS represents a significant advancement in combat care treatment. In some embodiments, infusion of the blood sample restores or improves one or more important parameters, such as microvascular blood flow, permeability, glycocalyx, and oxygenation.

[0250] The related aspects provide a method for enhancing tissue perfusion in a subject by administering or transfusing a blood sample treated with the formulations disclosed herein to a subject in need, or by administering to the subject a transdermal formulation disclosed herein (before or during blood transfusion). Adequate tissue perfusion and oxygenation are essential for all metabolic processes in cells and are the main factors affecting tissue repair and resistance to infectious organisms. Tissue perfusion is analogous to blood flow, oxygen delivery, or a combination of blood flow and nutrient supply (including oxygen supply). Enhanced tissue perfusion can be determined by measuring the levels of biomarkers, including, for example, serum lactate, central venous oxygen saturation, reticulocytosis, 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 after / and or before and after blood transfusion. Biomarkers of untreated blood samples can also be collected as a reference to show the improvement of the blood samples treated with the formulations disclosed herein. Monitoring peripheral circulation can also allow for the evaluation of changes in tissue perfusion. For detection purposes, well-known methods (such as near-infrared spectroscopy (NIRS) oximetry) can be used.

[0251] In some embodiments, the amount of curcuminoids in the formulation or in the treated blood sample is selected such that tissue perfusion is enhanced and / or one or more of the above-mentioned 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 above percentage values, relative to a control or untreated sample (collected at the same time and / or from the same source).

[0252] Another aspect provides a method for improving transfusion safety and / or efficacy by administering to a subject in need a transdermal formulation disclosed herein before, during, or shortly after an infusion procedure, transfusing a blood sample disclosed herein to the subject, or contacting cells or fluids to be transfused with a formulation disclosed herein. For example, a formulation containing an NO enhancer or NO precursor can be administered transdermally (e.g., in the form of a patch or paste) to a subject before, during, or shortly after a blood transfusion to protect red blood cells and enhance the therapeutic effect. In some embodiments, the method includes administering a transdermal formulation to the subject or contacting the formulation with cells or fluids 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or more than 4 hours before a blood transfusion is performed. In some embodiments, the method involves transfusing a blood sample disclosed herein.

[0253] When in direct contact with isolated cells or fluids (such as the blood samples disclosed herein), the formulation can enhance the viability of, for example, red blood cells and / or load therapeutic agents (such as curcuminoid compounds) into the cells, such that subsequent infusion into a subject can achieve better efficacy and safety. In some embodiments, the blood samples disclosed herein are infused into a subject who administers the transdermal formulation disclosed herein before, during, or after the infusion.

[0254] Related aspects provide a method for transdermally delivering an active agent to a subject in need thereof. The method includes administering to the subject the transdermal formulation or blood sample disclosed herein. Rapid and prolonged therapeutic effects can be achieved by locally or systemically delivering an effective amount of the active agent. The range of target diseases or conditions is as described above. In some embodiments, the transdermal formulation includes one or more curcuminoid compounds as the active agent or ingredient for treating a disease or condition, and optionally one or more of polyphenols, flavonoids, stilbenes, seco-steroids, or natural products that promote NO production. In some embodiments, the transdermal formulation includes one or both of curcumin and quercetin, and optionally one or more of polyphenols, flavonoids, stilbenes, and seco-steroids. Thus, in addition to playing its basic physiological role of oxygen delivery in the body, red blood cells can act as natural biopharmacodynamic carriers (e.g., releasing curcumin in the vascular system), which are capable of avoiding the normal mechanisms of drug elimination from the circulation. This discovery has the potential to change the paradigm of drug delivery.

[0255] In any embodiment of the above method, the formulation of the present invention can be added to the blood sample at any suitable interval to slow down the occurrence of storage damage phenomena or achieve other desired results (e.g., less inflammation, less hemolysis, less particle formation, or longer circulation time compared to a control or untreated sample). For example, after treating the blood sample on day 1, the formulation can be processed or added one, two, three, four, or more times. The intervals between two consecutive treatments / additions are each independently 1, 2, 4, 7, 8, 10, 14, 20, 25, or 30 days. In some embodiments, the formulation is administered in a single-dose regimen, including, for example, a single-dose administration only on the first day of storage and a single-dose administration on any intermediate day between day 1 and day X (such as day 10, day 20, day 30, day 40, or day 50), including day X (or any final storage day to be determined). In some embodiments, the formulation is administered in a multiple-dose regimen, including, for example, sequential administration starting at any time point during storage, with dosing intervals ranging from every 7 days, every 14 days, every 21 days, every 28 days, where the starting date ranges from day 1 to any intermediate time point. Administration on the last day of storage is also an option independent of the interval regimen.

[0256] The formulations can be delivered in a variety of ways. In some embodiments, the formulations disclosed herein can be loaded into a storage bag for a single-dose regimen on day 1 or day X. In some embodiments, the formulation is delivered into the storage bag by injection through an injection port on the bag using a syringe. In some embodiments, the formulation is delivered directly into the bag as needed. In some embodiments, pre-filled frangible sachets are incorporated into the wall of the storage bag and can be "ruptured" using finger pressure to release a dose of the formulation.

[0257] Manufacturing method

[0258] Another aspect of the present invention provides a method of manufacturing the formulations or blood samples disclosed herein. The method includes, for example, preparing a solution of a polyol solvent and an optional fatty acid penetration enhancer or additive, and then adding a medicament (NO enhancer or NO precursor). If desired, the solution can be heated to a suitable temperature to dissolve the fatty acid and / or the medicament. In some embodiments, the active agent (such as an NO enhancer or NO precursor or a source of nitrite) is saturated in the solution. After cooling the solution, the formulation can become a gel and the precipitated excess active agent can be easily removed. It is also possible to vary the conditions or order of mixing or adding different ingredients / components, provided that the NO enhancer or NO precursor is appropriately distributed in the formulation to achieve the desired therapeutic effect.

[0259] In some exemplary embodiments, the medicament dissolved in the solution is loaded into a container (such as a nebulizer or atomizer; a permeable or frangible sachet as described above) or is immersed in a dispensing carrier (such as the absorbent layer of a swab, sponge, or patch). When the NO precursor mixture contains a source of nitrite, the acid source can be stored, for example, in a separate sachet or a separate layer of the 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.

[0260] In some embodiments, the formulation is in the form of a gel or semi-solid. A gelling agent or thickening agent can be added to adjust the formulation form. The formulation is then loaded into a suitable container and then dispensed in the form of a gel, ointment, cream, emulsion, microemulsion, nanoemulsion, paste, balm, or other suitable form. The semi-solid formulation can also be coated on a backing member (such as the support layer of a patch).

[0261] Depending on the amount and nature of the thickening agent, the formulation can also be made in solid form. For example, a solution of an NO enhancer (such as curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine) can be mixed with melted pure cocoa butter and then cooled. The resulting solid formulation will melt when rubbed vigorously on human skin.

[0262] The general procedures for mixing reagents and conducting the manufacturing process can be obtained through common knowledge well-known to those skilled in the art or pharmaceutical technical manuals. For example, Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins, Philadelphia, 2000, or in the review article Souza et al, Topical ocular delivery of therapeutics: carrier systems and physical methods, J. Pharm. Pharmacol., 2013, 66, 507-530.

[0263] Dosing regimen

[0264] The effective amount of an agent (NO enhancer or NO precursor) that effectively enhances the systemic NO level in the formulations or kits described herein will depend on the route of delivery, the type of subject being treated (including humans), and the physical characteristics of the particular subject under consideration. The dose or amount can be adjusted to achieve the desired effect, but will depend on body weight, diet, concurrent medications, and other factors that will be recognized by those skilled in the medical arts. More specifically, the effective amount or therapeutically effective amount refers to the amount of the agent that effectively increases systemic NO to a level that prevents, alleviates, or improves the symptoms of the disease or prolongs the survival of the subject being treated. The delivery of the formulation can be adjusted to provide an optimal therapeutic response or a prolonged beneficial effect. For example, the formulation can be delivered topically more than twice or more than three times a day. Or, if desired, the amount or frequency of delivery can be reduced. Those skilled in the art are fully capable of determining the effective amount, especially in view of the detailed disclosure provided herein.

[0265] In non-human animal studies, the application of the potential product starts at a higher dose level and the dose is gradually reduced until the desired effect is no longer achieved or the adverse side effects disappear. The dose range can be wide, depending on the desired effect and the therapeutic indication. Generally, the effective amount or dose of the agent in the formulation can be from about 10 μg / kg body weight to about 100 mg / kg body weight, preferably from about 100 μg / kg body weight to about 10 mg / kg body weight. Alternatively, the dose can be calculated based on the patient's body surface area, as understood by those skilled in the art.

[0266] 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.

[0267] Individual physicians can select the exact formulation, route of administration, and dosage of the pharmaceutical composition according to the condition of the patient. (See, for example, Fingl et al. 1975, “The Pharmacological Basis of Therapeutics”, which is incorporated herein by reference in its entirety, particularly with reference to page 1 of Chapter 1).

[0268] It should be noted that the attending physician will know how and when to terminate, interrupt, or adjust the administration in response to toxicity or organ dysfunction. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. When managing the condition of interest, the range of the administered dose will vary with the severity of the condition to be treated and the route of administration. For example, the severity of the condition can be evaluated in part by standard prognostic assessment methods. In addition, the dose and dose frequency may also vary according to the age, weight, and response of the individual patient. Procedures equivalent to the above procedures can be used in veterinary medicine.

[0269] Known methods can be used to evaluate the efficacy and toxicity of the formulations disclosed herein. For example, the toxicology of the formulation can be established by determining the in vitro toxicity to cell lines (such as mammalian, preferably human cell lines). The results of such studies can generally predict the toxicity in animals (such as mammals, or more specifically humans). Alternatively, known methods can be used to determine the toxicity in animal models (such as mice, rats, rabbits, or monkeys). Several recognized methods (such as in vitro methods, animal models, or human clinical trials) can be used to determine the efficacy of a specific compound. There are recognized in vitro models for almost every disease type. Similarly, acceptable animal models can be used to determine the efficacy of a chemical in treating such diseases. When selecting a model to determine efficacy, those skilled in the art can select an appropriate model, dose, route of administration, and protocol according to the guidance of the prior art. Of course, human clinical trials can also be used to determine the efficacy of the active agent of the transdermal formulation in humans.

[0270] If desired, the formulations or kits described herein can be presented in the form of a package or dispenser device, which can contain one or more unit dosage forms containing the formulated medicament. For example, the package can include a metal or plastic foil, such as a blister pack. The package or dispenser device can be accompanied by instructions for administration. The package or dispenser can also be accompanied by a notice associated with the container, the form of which is prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and which reflects the approval of the agency for the form of the drug for human or veterinary administration. For example, the notice can be a prescription drug label approved by the U.S. Food and Drug Administration, or an approved product insert.

[0271] All references cited herein are incorporated herein by reference in their entirety.

[0272] Example

[0273] Example 1

[0274] Preparation of Transdermal Preparation

[0275] Prepare carriers (vehicles) for loading active ingredients as shown in Samples 1-4. After heating the mixture in a warm water bath, myristic acid (solid flakes) was dissolved in PEG400. Oscillation accelerated the formation of the resulting clear solution. Solutions of myristic acid (MA) in PEG400 at the following four concentrations were prepared and evaluated. When the samples reached room temperature, Samples 1, 2, 3, and 4 were all clear solutions.

[0276] a. 30 ml PEG400 + 1.5 g MA (Sample 1)

[0277] b. 30 ml PEG400 + 3 g MA (Sample 2)

[0278] c. 30 ml PEG400 + 0.75 g MA (Sample 3)

[0279] d. 30 ml PEG400 + 2.25 g MA (Sample 4)

[0280] Load NO enhancer into the samples as carriers. Load 95% pure curcumin (curcumin 95) as a NO enhancer into the sample carrier preparation [or solvent system] to test the loading potential. Add 1.5 g of curcumin 95 to Samples 1, 2, and 3. The resulting curcumin 95-loaded samples were heated and shaken until the maximum amount of curcumin was dissolved, and then cooled back to room temperature. Almost all of the added curcumin was dissolved in all three samples, forming a strongly colored dark solution. Sample 3 provided higher solubility and remained liquid for several weeks. Samples 1 and 2 were initially liquid but formed a uniform solid gel within a few hours. Sample 3 was prepared by dissolving 0.75 g of MA in 30 ml of PEG400 and then heating in a water bath (about 60 - 80 °C) for about 15 minutes. Sample 4 was prepared by dissolving 2.25 g of MA in 30 ml of PEG400 and then heating in a water bath (about 60 - 80 °C) for about 15 minutes. After the heating cycle ended, both Sample 3 and Sample 4 were homogeneous solutions. After cooling, Sample 3 remained liquid while Sample 4 formed a uniform gel.

[0281] After determining the loading potential, a sample transdermal preparation containing curcugen as a NO enhancer was prepared. Curcugen is a product containing curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). These three curcuminoids account for approximately 50% by weight in curcugen. Sample preparations V3.3 and V4.3 were made by adding curcugen to sample 3 and sample 4 carriers, respectively (3 g of curcugen dissolved in 30 ml of the corresponding solvent). The resulting mixtures were heated and shaken for about 15 minutes, after which both appeared as dark chestnut-colored homogeneous solutions. After cooling, V3.3 (3 g of curcugen / 30 ml of sample 3 carrier) remained liquid; while V4.3 (3 g of curcugen / 30 ml of sample 4 carrier) became a homogeneous gel with the same dark chestnut color (the same color as V3.3 when heated).

[0282] No loss of color was observed in the samples stored at room temperature for at least three months. In sharp contrast, the same volume of water containing the same added amount of curcumin had little color in the liquid phase and a large amount of undissolved material.

[0283] V4.3 was loaded into a syringe while warm and was thus in a liquid state. The liquid in the syringe gelled upon cooling to room temperature but could be extruded from the syringe as a gel and slowly melted on the skin. The applied gel was easily covered and trapped by a waterproof and leak-proof transparent dressing (such as Mepitel transparent film dressing). The dressing prevented the loss of the applied preparation and could remain in situ for several days without loss of the preparation. When the Mepitel dressing was removed after several days, there was no adherent preparation on the dressing, and all the preparation was in the skin, which was reflected by no staining on the tissue of the colored skin under the dressing when wiped vigorously. The color completely disappeared after 7 - 8 days.

[0284] Adding water to the PEG400 / MA-based preparation produced a non-uniform emulsion, which caused difficulties for topical use. The presence of water limited the solubility of curcumin, curcugen, quercetin, and berberine in PEG. As a 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 had little color in the liquid phase and contained a large amount of undissolved material.

[0285] Using a higher concentration of MA solution with PEG as the base solvent, a concentrated saturated solution of low-solubility NO-enhancing active substances (such as curcumin, curcugen, quercetin, berberine, and related molecules) can be easily prepared by adding an excess of active agent to a heated high-MA PEG / MA solvent system (such as V4.3) and then allowing the mixture to cool. The resulting homogeneous gel is fully saturated with the active agent, and the undissolved excess material remains at the bottom of the tube in solid form. Thereafter, the homogeneous saturated gel can be easily removed and separated from the undissolved material.

[0286] The loading potential of the sample 3 solvent system as a carrier for the following low-solubility reagents was also tested using the above method: quercetin, berberine, N-acetylcysteine amide (NACA), and N-acetylcysteine ethyl ester (NAC-ethyl ester). In all cases, the solvent system allowed a large amount of these poorly soluble agents to dissolve. Comparing the aqueous solution with the sample 3 solution loaded with quercetin and berberine, it was found that only a very small amount of the dissolved substance was present in the aqueous sample, while the degree of dissolution in sample 3 was very high.

[0287] Similar results were also obtained for quercetin in sample 3 and sample 4 as carriers, but the solubility of quercetin in these solvents was lower than that of curcugen (about 200 - 300 mg of quercetin in 30 ml of sample 3 or sample 4). Like curcumin and curcugen, the sample 3 loaded with quercetin did not form a gel at room temperature, while the sample 4 loaded with quercetin formed a gel at room temperature.

[0288] The PEG400 / MA solution was mixed with other delivery carriers such as petrolatum. Sample 3 containing curcumin was mixed with petrolatum to give a gel with a uniform color that remained stable (no color loss) for over two months. Sample 3 containing dissolved NAC amide and an SNO derivative of NAC was readily mixed with petrolatum to produce a stable pink jelly. The stability may stem from the low water activity and high viscosity, which inhibit the loss of NO from the thiol groups. Samples 3 specimens of different sizes (all containing curcumin and curcugen) were mixed with melted pure cocoa butter and then cooled. The optimized mixture produced a uniform yellow / orange cocoa butter solid block / tube that remained solid at room temperature. When rubbed against human skin with pressure, the solid material melted. Similar results were obtained using coconut oil, but the lower melting point of coconut oil made it difficult to apply to human skin without smudging and dripping. Combinations of the two oils were also tested. PEG400 / MA showed the most promising properties as a topical delivery carrier suitable for cosmetic and dermatological applications when used with cocoa butter due to the more appropriate hardness of the resulting curcumin-loaded cocoa butter. Increasing the amount of PEG400 / MA solvent added to the cocoa butter eventually produced a gel-like substance with a stable color.

[0289] Solutions of the two lipophilic NAC derivatives were prepared using PEG400 saturated with sodium nitrite to give clear solutions. The lipophilic NAC derivatives readily dissolved under conditions where the solubility of NAC was limited. NAC is soluble in water while the two derivatives are sparingly soluble in water. The two solutions were then treated with a few drops of acetic acid to trigger the formation of nitrous acid from the nitrite, which could then nitrosate the reactive thiols on the two NAC derivatives. The solutions turned pink, indicating the formation of S-nitrosothiols. The solutions remained pink for several days. The corresponding aqueous solutions lost color within hours. Using PEG400 saturated with nitrite enabled the formulation to be used to generate NO and S-nitrosothiols by mixing with a reagent that acidified the mixture. Two fragile sachets in the patch could be used for NO sustained delivery carriers suitable for topical and transdermal applications.

[0290] Example 2

[0291] The effect of formulation V2.3 (2 grams curcugen / 30 ml sample 3 vehicle) on rat blood pressure (BP) was tested. The formulation was topically applied to the shaved abdomen of 5 Sprague Dawley rats. A Q-tip saturated with V2.3 was rubbed onto the shaved abdomen of the rats. The systemic blood pressure of all rats (N = 5) decreased by 20% within 15 to 20 minutes after application of V2.3. The decreased blood pressure was maintained for a three-hour observation window without signs of recovery. A second laboratory conducted a similar test on 6 rats, and the results (N = 6) showed similar results (a 20% decrease in blood pressure). Similar results for V3.3 were consistently obtained after application of V3.3 to the forearm of a human subject (∼0.05 ml). A similar decrease in BP occurred within 15 to 20 minutes after application, and the decreased BP persisted for many hours, gradually returning to a higher initial value after 12 to 24 hours. Similar results were obtained using V4.3 on the same human subject. Sample 3 (V1.3) doped with cocoa butter also showed similar physiological consequences when applied to a single human test subject. Control sample 3 without one or more curcuminoids or curcugen did not cause any physiological consequences when applied to rats or humans.

[0292] V3.3 was applied to a flap with an optical window on a healthy hamster. The optical window can monitor the blood vessels in the dermis layer beneath the site of V3.3 application. A dose-dependent increase in the blood vessel diameter at the site of topical application was observed within minutes of application.

[0293] Effect on plasma NO levels. After topical application of V3.3 to 4 rats, the plasma levels of NO degradation products (nitrite / nitrate) were measured. The results showed that the plasma levels of NO degradation products (nitrite / nitrate) increased by 15% in two of the tested rats, which was consistent with the decrease in blood pressure and was due to an increase in nitric oxide production, which is due to the known ability of curcumin to upregulate endothelial nitric oxide synthase (eNOS) and thus upregulate NO production. Two control animals did not show this increase under the same conditions. The results indicate that transdermal curcuminoids can increase systemic NO levels.

[0294] Blood pressure decreased within 15 to 20 minutes after local application of the curcumin-containing sample, indicating that curcumin was being transdermally delivered and that therapeutically effective levels were present within a short time. The blood pressure decrease and the increase in plasma nitrite / nitrate levels were consistent with the known action of curcumin, namely upregulating nitric oxide production by eNOS in the endothelium. The physiological response could last for many hours, consistent with the sustained delivery of topically delivered curcumin / curcugen into the blood circulation. In contrast, due to the rapid conversion of curcumin by the liver into inactive agents, the circulation time of curcumin delivered into the circulation via the oral route or IV was only 2 hours. Such a distinct and long-lasting physiological response was not observed even with oral administration of a large dose of curcumin.

[0295] The physiological responses of the transdermal formulations of curcuminoids in animals were also tested for V3.3 (Curcugen 9 g, myristic acid 2.25 g, PEG 400 90 ml) and V4.3 (Curcugen 9 g, myristic acid 6.75 g, PEG 400 90 ml).

[0296] After local application of the transdermal formulations of curcuminoids in rodents, blood pressure was measured. The BP of both rats and mice showed a decrease of up to 20%, and V3.3 was more effective than V4.3 both in terms of onset time and the magnitude of BP decrease.

[0297] It was also observed that local application of V3.3 or V4.3 led to an increase in plasma nitrite and nitrate levels within the three-hour monitoring window after a single local dose. During the same time period, the BP continued to decrease (within the three-hour window). After local application of V3.3 in rats (N = 3), a continuous increase in plasma curcuminoids at detectable concentrations was observed within the three-hour window. Oral curcumin plasma levels peaked within one hour and dropped to near-undetectable levels within three hours.

[0298] Example 3

[0299] Systemic inflammation is controlled by transdermal delivery of NO boosters or NO precursors.

[0300] The inhibitory effect of the topically applied formulation V3.3 on the development of severe vascular leakage in an acute inflammation rat model was tested. The following lipopolysaccharide (LPS)-induced cytokine storm protocol was used in this study:

[0301] a. LPS (10 mg / kg) was intraperitoneally infused every 24 hours to initiate and maintain the acute inflammatory response.

[0302] b. Starting from the first LPS treatment, local application (0.1 ml) of formulation V3.3 was performed on the LPS-treated rats every 24 hours for three days.

[0303] c. Measure physiological parameters obtained from blood sampling every 24 hours

[0304] d. After three days, the animals are anesthetized and the cavity is opened surgically to perform in vivo fluorescence imaging of the large blood vessel system and the microvascular system.

[0305] i. Fluorescently labeled albumin and dextran (500 kDa) preparations are infused IV, and fluorescence-derived images of the vascular system and the surrounding tissue are used to determine the rate of albumin and dextran leakage from the vascular system into the surrounding tissue.

[0306] Local application of the carrier (PEG400 / MA) to LPS-treated rats showed a rapid and extremely severe leakage pattern for both albumin and larger dextran, which was consistent with what was observed under severe acute inflammatory conditions. However, daily local application of V3.3 significantly reduced the amount of albumin leakage. The low leakage levels observed were essentially the same as those observed in control animals. Similar results were also observed for dextran. Extensive vascular leakage is a potentially fatal consequence of cytokine storm, regardless of the cause (COVID-19, Ebola, dengue fever, hemorrhagic shock, endotoxin shock, Rift Valley fever, etc.). The transdermal preparation provided significantly positive intervention results with profound clinical implications.

[0307] Example 4

[0308] This study evaluated the potential therapeutic efficacy of a transdermal formulation of the present invention (V4.3: curcugen 9 g, myristic acid 6.75 g, PEG 400 90 ml) in a murine model of acute vascular inflammation. Three cohorts, each with three subjects, were studied and compared. In cohort 1, the subjects were infused with LPS but received no local treatment (untreated). In cohort 2, the subjects were infused with LPS and received local treatment (0.1 ml of V4.3). In cohort 3, local treatment (0.1 ml of V4.3) was administered four hours after LPS infusion. Endotoxemia was induced by infusing 10 mg / kg LPS (lipopolysaccharide from Escherichia coli serotype O128:B12, Sigma Aldrich St. Louis, MO). This procedure was the same as that described in earlier 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).

[0309] It was observed that topical application of V4.3 (a curcuminoid compound dissolved in a PEG400 / myristic acid mixture) could serve as a prophylactic and interventional treatment for lipopolysaccharide (LPS)-induced cytokine storm. The results included the temporal changes in the vascular consequences and cytokine profiles of three different groups of LPS-treated mice: i) no topical treatment with V4.3; ii) pre-treatment with topical V4.3 before LPS treatment; and iii) topical treatment with V4.3 after the onset of LPS-induced cytokine storm. In terms of the response of the microvascular system to LPS treatment in the three groups, topical V4.3 treatment both restricted arterial dilation (which indicates shock-induced vascular collapse) and maintained arterial blood flow. It was also observed that topical V4.3 treatment could prevent the sharp decline in functional capillary density (FCD) that accompanies LPS-induced endotoxemia. V4.3 could serve as both a prophylactic and an interventional treatment in preventing the sharp decline in FCD relative to baseline (BL). FCD is related to survival because it reflects the ability to maintain tissue perfusion and deliver oxygen to tissues. In addition, it was found that pre-treatment and interventional treatment with topical V4.3 could both restrict the production of pro-inflammatory cytokines. These results confirmed the efficacy of the transdermal preparation in restricting the inflammatory consequences when topically administered as a prophylactic agent or as an active therapeutic agent after the onset of inflammation.

[0310] Example 5

[0311] This study evaluated the prevention of LPS-induced vascular leakage by topical administration of a curcuminoid compound using the transdermal preparation of the present invention in a rat model of LPS endotoxemia. LPS (Escherichia coli O26:B6) was inoculated into the rat model (10 mg / kg / day). The curcumin transdermal preparation (V3.3: curcugen 9 g, myristic acid 2.25 g, PEG400 90 ml) or vehicle control was applied daily for three days (0.1 ml / dose). On day 3, the animals were surgically prepared for in vivo and fluorescence microscopy. It was observed that treatment with the curcumin transdermal preparation could prevent early leakage (the first 4 hours) induced by LPS. In the LPS-induced inflammation pattern, vascular leakage was significantly reduced after topical application of V3.3. Meanwhile, topical application of V4.3 (curcugen 9 g, myristic acid 6.75 g, PEG 400 90 ml) before and after LPS-induced inflammation in mice (N = 3) both reduced the increase in LPS-induced pro-inflammatory cytokine levels.

[0312] Combined with the results of earlier studies, this experiment showed that the topical transdermal curcumin preparation of the present invention could restrict inflammation-induced vascular leakage, which is characteristic of cytokine storm and other inflammation-induced conditions.

[0313] Example 6

[0314] The skin permeability of the transdermal preparation of the present invention was studied. The skin permeability of the following transdermal preparations of the present invention was studied using a confocal microscope. These preparations differed only in the amount of myristic acid:

[0315] a. Lot number 43: V4.3 Curcugen 9 g, myristic acid 6.75 g, PEG 400 90 ml

[0316] b. Lot number 39: V3.3 Curcugen 9 g, myristic acid 2.25 g, PEG 400 90 ml

[0317] c. Lot number 38: V0.3 Curcugen 9 g, PEG 400 90 ml

[0318] When the temperature approaches zero degrees Celsius, the preparation solidifies. When the temperature rises above room temperature, the viscosity of all samples decreases significantly. The viscosities of the preparation and its components are shown below:

[0319] Water 0.9 cP; PEG 400 (100%) 99 cP; PEG 400 / water 90%, 80 cP; V3.3: 149.8 cP; PEG 400 + myristic acid, V3.3: 149.8 cP; PEG400 + myristic acid, V4.3: 4220 cP.

[0320] Curcuminoids have a broad absorption 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 skin barrier integrity. The penetration of curcuminoids was visualized using a DAPI filter. All images were taken under the same settings.

[0321] All three preparations showed the penetration of curcuminoids into the skin. At the earliest one-hour time point, the penetration of curcuminoids into the stratum corneum was observed in all three preparations.

[0322] In the formulations of batch number 38, in two donors, broadening of the fluorescent band occurred between 3 and 6 hours. In the formulations of batch numbers 39 and 43, broadening of the fluorescent band was not seen until the 24th hour. The broadening indicates that curcuminoids penetrated into the upper epidermal layer just beneath the stratum corneum. In the formulation of batch number 39, in two donors, the amount of curcuminoids decreased between 3 and 6 hours. However, in the formulations of batch numbers 38 and 43, a decrease in the intensity of curcuminoids was observed after 6 hours. In the formulation of batch number 38, fluorescent spots were seen beneath the stratum corneum at early time points. However, when using the formulations of batch numbers 39 and 43, no fluorescent spots were seen above the stratum corneum. The trans-epidermal water loss (TEWL) values were comparable for all formulations and all time points, indicating that the barrier integrity of all human skin samples was the same.

[0323] Fluorescence decreased over time in the three formulations, indicating that curcuminoids had penetrated into the deeper layers of the skin. (Due to the relatively high autofluorescence of untreated skin, it may not be possible to see and / or quantify active substances with low penetration amounts). Based on this, the formulation of batch number 39 showed that curcuminoids penetrated into the skin earliest. The depth of fluorescence was also measured. The measured values were between 5 and 25 microns. The formulation of batch number 38 showed the fastest broadening of the fluorescent band, indicating that curcuminoids penetrated into the skin more rapidly.

[0324] Example 7

[0325] The role of the transdermal formulation of the present invention in restricting or preventing the onset of cardiovascular-induced inflammation in a ZDSD diabetic rat model was tested. Three rats were fed a normal diet for 60 days without symptoms of diabetes. Starting from about the 65th day, the rats were fed a high-fat diet, resulting in a slow increase in blood glucose. Throughout the test period, the animals were topically treated with formulation V4.3 every two days until day 80. The cytokine profile showed an onset of inflammation at about the 75th day. Treatment with formulation V4.3 restricted the increase in pro-inflammatory cytokines observed in the sham controls at the 75th day. In particular, the IL-18 profile clearly demonstrated that the formulation could prevent the increase in IL-18, which is a marker of the tendency for cardiovascular consequences in diabetic patients.

[0326] Example 8

[0327] The therapeutic effect of formulation V4.3 was tested on mice with severe advanced endothelial dysfunction. 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 with a 12 / 12 - hour day / night cycle and had free access to food and water. The study was conducted for 4 weeks. The first week was used for acclimation as the animals were divided into different experimental groups. Two groups received L - NAME (50 mg / kg) from their drinking water for two weeks (weeks 2 and 3) to chronically induce nitric oxide synthase (NOS) inhibition. One L - NAME - treated group received the topical formulation V4.3, administered at 0.1 ml per day for 10 days, and then was characterized. The group not treated with L - NAME served as a sham control group.

[0328] Local treatment was initiated after the onset of the condition. The results showed direct evidence of treatment, namely the restoration of elements of normal endothelial function (decreased oxidative stress in plasma and red blood cells, decreased leukocyte adhesion to the endothelium, indicating restoration of the glycocalyx, and increased functional capillary density). Compared with the sham control group, the transdermal formulation - treated group showed lower levels of TNF - α, TGFβ, MCP - 1, IL - 1α, IL - 1β, IL - 6, IL - 10, and IL - 12. Better results were also observed in the treatment group in terms of micro - hemodynamic changes, cell adhesion, vascular responses of isolated aortic vessels, changes in red blood cell and plasma antioxidants, hypoxia, reoxygenation, and systemic hemodynamic changes.

[0329] Table 1: Changes in body weight, relative tissue weight, and water intake at the end of the study.

[0330]

[0331] Table 2: Changes in red blood cell and plasma antioxidants at the end of the study

[0332]

[0333] Example 9

[0334] This experiment demonstrated that pretreatment with oral curcumin a few days before hemorrhage significantly reduced the inflammatory and oxidative factors of HS. This indicates that transdermal delivery of curcumin (in the form of formulation 4.3) restored vascular homeostasis in a guinea pig model of septic shock. Additionally, infusion studies showed that red blood cells can act as stealth pharmacological carriers for transporting and delivering curcumin. Overall, these studies and many other published studies suggest that curcumin can mitigate the consequences of HS.

[0335] This experiment tested the following hypothesis: Packed red blood cells loaded with curcumin can be used to enhance the therapeutic efficacy of the HS model in guinea pigs. The resuscitation ability of fresh red blood cells, old red blood cells, and old red blood cells pretreated with formulation 4.3 before infusion after an HS attack was compared. Formulation 4.3 restored i) functional capillary density (FCD), which is the microvascular functional parameter most closely related to survival, ii) blood flow, and iii) other indicators of cardiovascular function. The R120 FCD results showed that the red blood cells treated with formulation 4.3 performed best in most vascular health indicators, even exceeding fresh red blood cells.

[0336] Example 10

[0337] The following steps were used to evaluate the effect of formulation 4.3:

[0338] · On day 1 of a 14-day storage period, a single dose of formulation 4.3 was used to treat red blood cells (freshly packed) suspended in PBS-glucose (resulting in 1 mM curcuminoid compounds in the stored red blood cell bags)

[0339] · The 24-hour survival rate of treated and untreated stored red blood cells (7 days and 14 days) with and without a single dose of formulation 4.3 treatment on day 1. Small aliquots were taken on days 1, 7, and 14 for analysis of storage damage markers.

[0340] · Starting from the first dose on day 28, the stored red blood cells were administered multiple times (3 times) (29-day storage period)

[0341] · Evaluate the stability of circulating unstable red blood cells (after local application of formulation 4.3 in a humanized sickle cell mouse model)

[0342] It was observed that adding a single dose of the formulation at the start of storage limited the loss of ATP levels and oxidative damage.

[0343] Relative to the ATP level on day 1

[0344] · Day 1: 100%

[0345] · Day 14: Control group 28%, formulation 4.3-treated group 45%

[0346] Oxidative damage (multiple increase relative to the value on the first day)

[0347] ■ Intracellular ROS

[0348] ○ Day 14: Control group increased 9-fold vs formulation-treated sample increased 5-fold

[0349] ■ Protein carbonyl content

[0350] · Day 14

[0351] ○ The control group increased by 5 - fold vs the formulation - treated sample increased by 3 - fold

[0352] ■ Lipid hydroperoxide content

[0353] · On the 14th day

[0354] ○ The control group increased by 7 - fold vs the formulation - treated sample increased by 5 - fold

[0355] ■ Formation of ferryl hemoglobin on the 14th day (Ferryl hemoglobin is highly reactive and can trigger oxidative stress, ROS, and peroxynitrite production). The ferryl hemoglobin content is reflected by the SulfHb concentration in μM.

[0356] ● The carrier control group was 4.9 vs the sample treated with the formulation was 3.8

[0357] Example 11

[0358] This experiment shows that the circulation time of the infused red blood cells stored with the addition of formulation 4.3 at day 1 increased compared to the carrier control after 14 days and 21 days of storage.

[0359] Blood storage and processing. Guinea pigs weighing between 350 and 400 grams were used. The guinea pigs were fed a regular diet (ND; Envigo TD.2040). The guinea pigs were anesthetized and a femoral artery catheter was implanted. The animals were bled freely to CP2D, and AS - 3 was added at the recommended concentration by the manufacturer. The red blood cells were passed through a neonatal leukocyte reduction filter. The red blood cell units were stored for three weeks. The red blood cells of formulation 4.3 were stored in the presence of 1 mM of the formulation.

[0360] Recovery was performed 24 hours after infusion. After 2 weeks and 3 weeks of storage, the red blood cells of each group were radio - labeled with technetium - 99 (Tc99). 200 μL of Tc99 - radio - labeled blood (about 2% of BV) was delivered to the anesthetized guinea pigs through the femoral vein catheter, and samples were drawn through the femoral artery catheter at 5 minutes, 30 minutes, and 24 hours after injection. All samples were radio - detected on a Cobra II gamma counter. The following percentage results of the recovery rate of the infused red blood cells were observed 24 hours after infusion: Fresh red blood cells were 88%; two weeks was 75% vs two weeks with formulation treatment was 80%; three weeks was 67% vs three weeks with formulation 4.3 treatment was 75%.

[0361] Therefore, adding the formulation at the beginning of the storage process can extend the circulation time of the infused old red blood cells. In addition, the circulation time reflects the overall condition of the red blood cells. These results are consistent with the direct stability studies, which show that adding the formulation limits the development of storage damage of old red blood cells.

[0362] Example 12

[0363] This experiment examined the effect of formulation 4.3 on human red blood cells (RBCs) starting from day 28 of storage. Finally, a significant enhancement of the RBC profile was observed, and the cold storage shelf life was extended.

[0364] Human red blood cells were collected and stored in the presence of a standard additive (AS-3: α-d-glucopyranose, trisodium citrate, sodium chloride, phosphoric acid, monosodium salt, citric acid, adenine). The experiment included the following steps:

[0365] 1. 50 ml of whole blood was used for each experimental group.

[0366] 2. After removing the white blood cell layer and plasma, red blood cells were isolated from the leukocyte-depleted blood.

[0367] 3. The packed red blood cells were stored in a standard red blood cell storage bag after mixing with an AS-3 solution (11 ml).

[0368] 4. On day 28 of storage, the red blood cells in the AS-3 solution were divided into 3 equal parts and stored in separate storage bags (15 ml, 50 μM in each bag).

[0369] 5. Further additions were repeated on days 35 and 42 of storage, respectively.

[0370] 6. Samples were collected and analyzed on days 28, 35, 42, and 49 of storage, respectively.

[0371] On day 28 of storage, the samples were divided into 3 separate storage bags (50 ml each) as follows:

[0372] a. Control - no additional additives

[0373] b. Formulation 4.3 - 8 μl of formulation 4.3 was added, such that the bag contained 50 μmol of curcuminoids.

[0374] c. Vehicle - 8 μl of the solvent system was added, resulting in 0 μmol of curcuminoids in the bag.

[0375]

[0376] Results: The shelf life of red blood cells treated with formulation 4.3 was extended, which was reflected in a statistically significant improvement in the markers of storage damage. An increase in ATP levels, a decrease in Band 3 protein phosphorylation, and a reduction in oxidative damage were observed. The formulation showed a convincing efficacy as a shelf life extender for stored human red blood cells, which are one of the most commonly used blood transfusion materials.

[0377] ATP level increased (percentage of starting control value) compared to the control group

[0378] · Day 35: Control group 60, vehicle group 50, formulation 4.3 group 80

[0379] · Day 42: Control group 38, vehicle group 40, formulation 4.3 group 55

[0380] · Day 49: Control group 32, vehicle group 30, formulation 4.3 group 45

[0381] Reduction in Band 3 protein phosphorylation (percentage increase relative to control at starting time)

[0382] · Day 35: Control group 3.5, vehicle group 3.3, formulation 4.3 group 2.0

[0383] · Day 42: Control group 4.8, vehicle group 5.0, formulation 4.3 group 3.0

[0384] · Day 49: Control group 5.8, vehicle group 6.3, formulation 4.3 group 5.5

[0385] Restriction of protein oxidative damage (carbonyl formation), expressed as nmol / ml rbc

[0386] · Day 35: Control group 40, vehicle group 40, formulation 4.3 group 22

[0387] · Day 42: Control group 60, vehicle group 65, formulation 4.3 group 45

[0388] · Day 49: Control group 75, vehicle group 75, formulation 4.3 group 58

[0389] Example 13

[0390] Topical application of formulation 4.3 stabilizes circulating red blood cells: Compared to the control group (vehicle), three - week alternate - day topical treatment with formulation 4.3 (4 mg per dose) enhanced red blood cell stability in a humanized sickle - cell mouse model (highly unstable red blood cells).

[0391] Briefly, formulation 4.3 or vehicle was topically applied every other day for 21 days. Red blood cells were isolated on day 21 and analyzed for ATP content and protein oxidation. Blood was taken on day 21 and hemolysis markers (LDH, lactate dehydrogenase) were evaluated. Then PK data and the following data were obtained:

[0392] · ATP content (nmol / ml rbc): Vehicle group 30, formulation 4.3 group 50

[0393] · Protein oxidation (carbonylation) nmol / ml rbc: 25 in the vehicle group, 25 in the formulation 4.3 group

[0394] · LDH level (units / ml): 2.2 in the vehicle group, 0.5 in the formulation 4.3 group

[0395] · Multiple PK studies conducted on rats and mice showed that topical application of formulation 4.3 led to the sustained presence of curcumin in both plasma and blood cells.

[0396] Thus, topical application of formulation 4.3 led to the transdermal systemic stabilization of circulating red blood cells, accompanied by: i) evidence that red blood cells obtained from treated animals showed improved redox status and increased ATP content; and ii) in vivo evidence of enhanced red blood cell stability transdermally during a 21-day test period, as manifested by a decrease in the levels of key markers of hemolysis, compared to the use of the vehicle control alone.

[0397] Example 14

[0398] This experiment used a hemorrhagic shock model (hamster), in which the therapeutic efficacy of stored red blood cells (identical to the protocol in the human red blood cell study) infused and treated with a single dose of formulation 4.3 at the start of storage was compared with the results of fresh red blood cells or untreated stored red blood cells. The treated stored red blood cells performed better than fresh red blood cells and untreated stored red blood cells in maintaining functional capillary density, which is a major determinant of tissue perfusion and clinical outcome.

[0399] In this preliminary study, the effects of infusing old red blood cells (RBCs) stored with formulation 4.3 on systemic and microvascular parameters during the resuscitation of severe hemorrhagic shock were evaluated, and the results were compared with those of conventionally stored red blood cells. The experiments were performed on unanesthetized hamsters equipped with a dorsal skinfold chamber that allowed real-time visualization of microvascular responses. The experimental protocol consisted of inducing hemorrhage equivalent to 50% of the total blood volume of the hamster, followed by 1 hour of hypovolemic shock. Subsequently, fluid resuscitation corresponding to 50% of the blood loss was administered. This animal model and experimental design were chosen to closely mimic the situation of severe hemorrhagic shock and its clinical management.

[0400] The results showed that the resuscitation with aged red blood cells treated with formulation 4.3 was not inferior to that with fresh red blood cells and was superior compared to aged red blood cells stored conventionally. The assessment of systemic parameters, including blood pressure, showed that red blood cells treated with formulation 4.3 had a good response, highlighting their effectiveness in restoring hemodynamic stability during the critical resuscitation phase. In addition, the study of microvascular parameters, including microhemodynamics and functional capillary density, provided insights into the tissue-level response to the infusion. After using formulation 4.3-stored red blood cells, the functional capillary density, which is a key indicator of the balance between enhanced oxygen delivery and tissue perfusion, showed a significant improvement.

[0401] Those skilled in the art will recognize that the invention described herein is not limited to what has been particularly shown and described. Instead, the scope of the invention is defined by the claims that follow. It should also be understood that the foregoing description represents only illustrative examples of embodiments. The specification has not attempted to exhaustively list all possible variations. Alternative embodiments may not be given for specific components of the composition or steps of the method, and may be produced by different combinations of the said ingredients, or other PEG alternative embodiments that may be used in the formulation, kit, or method should not be regarded as not claiming protection for these alternative embodiments. It will be recognized that many un-described embodiments are covered within the literal scope of the appended claims and that other embodiments are equivalent.

Claims

1. A method for preparing a blood sample that delays the onset of storage damage to ex vivo red blood cells in the sample or slows the progression of storage damage to ex vivo red blood cells in the sample during storage, the method comprising mixing the sample with a solution comprising an effective amount of a curcuminoid compound, wherein the concentration of the curcuminoid compound 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 the red blood cells remain substantially viable for 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 time period.

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

5. The method according to claim 1, wherein the levels of adenosine triphosphate (ATP) and / or 2,3-diphosphoglycerate (2,3-DPG) in the sample are reduced by less than 20% over a 50-day time period.

6. The method according to claim 1, wherein the solution comprises a solvent selected from polyethylene glycol, ethanol, acetone, ethyl acetate, acetonitrile, DMF, THF, DMSO, isopropanol, 1-butanol, xylene, n-hexane, n-heptane, and any combination thereof.

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

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

9. The method according to claim 8, wherein the weight ratio of the solvent to the curcuminoid compound is in the range of about 5:1 to about 20:

1.

10. The method according to claim 8, wherein the molecular weight of the PEG is in the range of 200 to about 600.

11. The method according to claim 8, wherein the ratio of PEG to the curcuminoid compound is in the range of about 8:1 to about 30:

1.

12. The method according to claim 1, wherein the weight ratio of the sample to the solution is in the range of about 8000:1 to about 3000:

1.

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

14. The method according to claim 1, wherein the curcuminoid compound in the solution is in the range of about 3% to about 15% by weight.

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

16. The method according to claim 1, wherein the solution further comprises at least one flavonoid compound.

17. The method according to claim 16, wherein the flavonoid compound is selected from quercetin, apigenin, fisetin, luteolin, and rapamycin.

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

19. A method for transfusing a subject in need, comprising transfusing the subject with the blood sample according to claim 18.

20. A method for preparing a blood sample that delays the occurrence of storage damage to ex vivo red blood cells in the sample or slows the progression of storage damage to ex vivo red blood cells in the sample during storage, the method comprising mixing the sample with a solution comprising an effective amount of a curcuminoid compound, wherein the effective amount is selected such that the red blood cells remain substantially viable for at least about 60 days.

Images