Transdermal delivery formulations

Abstract

The present invention provides a transdermal formulation for delivering one or more active agents to a subject. The formulations are useful in the treatment of various diseases or conditions associated with elevated CD38 levels.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a continuation-in-part of U.S. application No. 18 / 051,266, filed on October 31, 2022, which is a divisional application of U.S. Patent No. 11,484,493, which is a continuation-in-part of International Application No. PCT / US2021 / 058611, filed on November 9, 2021, and PCT / US2021 / 058611 claims priority to Provisional Application No. 63 / 111,160, filed on November 9, 2020, Provisional Application No. 63 / 161,696, filed on March 16, 2021, and Provisional Application No. 63 / 235,880, filed on August 23, 2021, all of which disclosures are incorporated herein by reference in their entirety. Technical Field

[0003] Disclosed herein are formulations for sustained transdermal delivery of active agents, which result in a decrease in CD38 extracellular enzyme activity (e.g., NADase activity); thereby increasing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. In this way, new methods for preventing and treating various diseases or conditions associated with elevated CD38 levels are provided. Background of the Invention

[0005] The delivery of many therapeutic agents remains a challenge due to bioavailability issues, which prevent them from achieving therapeutic levels systemically or locally at the target site. Low bioavailability is caused by a variety of factors, including low solubility, low intestinal absorption, and rapid elimination from the circulation due to rapid liver breakdown (first-pass limitation). Therefore, many promising therapeutic agents with appropriate activity cannot be delivered directly to the affected site, such as the lungs, arthritic joints, inner ear, sinuses, etc., in an effective manner. At the same time, oral or IV routes of administration are generally unable to provide adequate therapeutic levels at the target local site.

[0006] Nitric oxide (NO) has known systemic benefits, including reversal of inflammation, prevention and reversal of endothelial dysfunction, repolarization of activated macrophages, inactivation of activated platelets, protection / restoration of the vascular endothelial lining (glycocalyx), and antimicrobial / antiviral activity. Proinflammatory insults lead to increased production of reactive oxygen species (ROS), which drive the development and perpetuation of endothelial dysfunction. Increased levels of ROS initiate multiple events that lead to vascular system dysregulation. ROS degrade the glycocalyx, leading to loss of vascular integrity, enhanced ability of circulating cells (monocytes, erythrocytes, neutrophils, and platelets) to access and adhere to the endothelial surface, loss of shear stress-mediated NO production, and loss of superoxide dismutase, which limits ROS. Enhanced ROS causes endothelial nitric oxide synthase (eNOS) to stop producing NO in the endothelium and begin producing more ROS. Increasing NO levels by either directly supplementing or enhancing 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 access to the circulation.

[0007] CD38 plays a key role in multiple organ systems and tissues. CD38 is the main enzyme for the degradation of nicotinamide adenine dinucleotide (NAD) in mammalian cells. Reduced NAD levels are closely associated with metabolic syndrome and aging-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, as manifested by reduced blood pressure; reduced vascular media thickness, media-to-lumen ratio, and collagen deposition; and restored elastin expression. Reducing CD38 levels can significantly alleviate AngII-induced vascular senescence by inhibiting the biogenesis, secretion, and internalization of senescence-associated small extracellular vesicles (SA-sEVs, which promote the senescence of adjacent undamaged VSMCs). In addition, the protective effect of CD38 deficiency against VSMC senescence is associated with the restoration of lysosomal dysfunction, particularly with the maintenance of sirtuin-mediated mitochondrial homeostasis and the activation of the mitochondria-lysosome axis in VSMCs. Thus, CD38 activity and the resulting associated drop in intracellular NAD are critical for Ang II-induced VSMC (vascular smooth muscle cell) senescence and vascular remodeling, which can lead to cardiovascular disease and renal failure. Similar findings 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 has been attributed in part to multiple pro-inflammatory triggers, including the accumulation of senescent cells, leading to its overexpression in activated immune cells such as macrophages and microglia, as well as in the activated endothelial lining of all blood vessels. Summary of the invention

[0008] The present disclosure provides a transdermal formulation for delivering one or more active agents to a subject to treat a disease or condition associated with elevated CD38 levels, which in turn leads to decreased 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 shut down oxidative and nitrosative stress generated by activated immune cells (e.g., M1 population of macrophages and microglia). CD38 is expressed on the surface of macrophages, microglia, and other immunoactive leukocytes, as well as on the endothelial lining of blood vessels. Elevated CD38 levels are caused by diseases or conditions such as trauma, inflammation, infection, radiation, chemotherapy, and excessive senescent cells. The enhancement of CD38 levels in turn further promotes cellular senescence, inflammation, and oxidative stress, which further stimulates more CD38 production, thereby forming a persistent inflammatory cycle that is difficult to break.

[0009] The transdermal formulations disclosed herein result in a decrease in CD38 extracellular enzyme activity (e.g., NADase activity); thereby increasing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. In this way, 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 method overcomes the negative effects of excessive CD38 production on the ability of stem cells to differentiate into mature cells, which, at least to some extent, leads to chronic inflammatory anemia and the insufficient efficacy of intrinsic stem cells and stem cell therapies in repairing damaged tissues.

[0010] The topical delivery preparation disclosed herein can be delivered directly to plasma and blood cells, thereby bypassing the first-pass elimination of the intestine and liver. In addition, red blood cells loaded with active agents can act as invisible delivery vehicles, which cannot be found by the immune system and liver. In addition, the absorption of deliverables by circulating macrophages and other immune-active leukocytes loaded with activated CD38 (M1 group) will lead to rapid repolarization (back to the M2 group), thereby quickly reducing CD38 levels and the production of pro-inflammatory cytokines. This strategy should also reduce the burden of senescent cells. By combining potent anti-inflammatory agents (curcuminoids) (repolarizing M1 macrophages (thereby reducing CD38 levels) while promoting sirtuin activity) with anti-aging agents (such as quercetin, apigenin, fisetin, luteolin), the prospects for breaking the inflammatory cycle of chronic diseases and promoting tissue repair by allowing stem cell differentiation and proliferation can be significantly improved.

[0011] One aspect of the present application discloses a transdermal formulation for enhancing systemic nitric oxide (NO) via 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 an effective amount of the Curcuminoids and the flavonoids; and optionally

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

[0015] Wherein, the transdermal preparation is allowed to deliver an effective amount of the active agent through the skin or mucosa after topical application.

[0016] In some embodiments, the curcuminoid is curcumin. In some embodiments, the other active agent is at least one flavonoid selected from the following: quercetin, apigenin, fisetin, luteolin or another small molecule biocompatible anti-aging agent (e.g., rapamycin). In some embodiments, the formulation includes a combination of a curcuminoid and an anti-aging agent (e.g., quercetin and apigenin).

[0017] Preparations and kits disclosed herein can be applied to treat various conditions or diseases. Examples include transdermal treatment of acute and chronic inflammatory conditions, transdermal prevention and reversal of endothelial dysfunction, reduction of cytokine storm or cytokine storm phenomenon (releasing abnormal levels or higher than normal levels of proinflammatory cytokines) of patients with diseases (such as COVID-19 or SARS-CoV-2 infections) that cause potential endothelial dysfunction, local treatment of skin diseases, anti-aging skin treatment, ophthalmic disease treatment, aerosol-based treatment of lung diseases, local treatment of infection, treatment of red blood cells to improve storage characteristics and reverse storage lesions to improve the safety and effectiveness of stored red blood cells, loading red blood cells with therapeutic agents before transfusion, delivering RBC stabilizers by intravenous (IV) to stabilize red blood cells, 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 by excessive oxidative damage and subsequent inflammation, loading medical sponges with therapeutic agents for, for example, ears, nose, mouth, rectum and vagina, etc. Sustained transdermal systemic delivery can also be achieved to exploit the therapeutic potential of NO for various chronic diseases and conditions.

[0018] The formulation may additionally contain polyphenols, flavonoids, stilbenoids, secosteroids, and other phytochemicals and natural products that promote NO formation. In some embodiments, the NO enhancer includes at least one agent selected from polyphenols, flavonoids, stilbenoids, secosteroids, and similar natural products. In some embodiments, the NO enhancer includes 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 ranging from 100 to about 1000. In some embodiments, the fatty acid is myristic acid.

[0020] In some embodiments, the formulation provides sustained release of the NO enhancer or NO releaser from the NO precursor (eg, 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 comprises a thickener, which maintains the transdermal formulation in a semi-solid or solid form. In some embodiments, the thickener is petroleum jelly, cocoa butter, or a polyalkylene glycol having a molecular weight greater than 2 kDa.

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

[0023] Embodiments of the present disclosure provide transdermal formulations and methods for enhancing systemic NO levels. Since known systemic benefits of NO include the ability to reverse inflammation, prevent and reverse endothelial dysfunction, repolarize activated macrophages, inactivate activated platelets, protect vascular endothelial lining, and antimicrobial / antiviral activity, the formulations disclosed herein can be applied to treat a variety of diseases and conditions. Treatment is based on transdermal delivery of agents that can improve nitric oxide production in the endothelium or can release NO directly from appropriate S-nitrosothiol-containing molecules. The formulations disclosed herein can enhance endothelial NO production and reduce ROS levels by reducing CD38 levels.

[0024] Compared with traditional oral administration of active agents, the transdermal formulations described herein have the advantage of reducing side effects. For example, oral administration of curcumin can cause gastrointestinal discomfort (excessive movement, increased acid production in the stomach), especially when used for a long time. Delivery of agents through the skin or mucous membranes bypasses the gastrointestinal tract, achieving the desired therapeutic effect while minimizing side effects.

[0025] Although specific embodiments of the preparations, kits or methods related to the treatment or prevention of diseases may be cited or illustrated below, it is not intended that the scope of the preparations, kits or methods be limited to such specific citations or examples. Those skilled in the art may make various modifications, such as the specific form of the preparation and the amount or frequency of application of the preparation for the treatment or prevention of a disease or condition, based on practical and economic considerations.

[0026] Unless otherwise specified, the articles "a" and "an" used herein mean "one or more" or "at least one". That is, using the indefinite article "a" or "an" to refer to any element or component of an embodiment does not exclude the possibility that more than one element or component is present.

[0027] As used herein, the term "about" refers to the recited value plus or minus 10% of the recited value. In some embodiments, "about" refers to the recited value plus or minus 5% of the recited value.

[0028] The term "agent" or "active agent" refers to a molecule or compound that prevents, alleviates or ameliorates symptoms of a disease, prolongs the survival of a treated subject, or achieves a desired / acceptable medical or health condition. An agent in a NO booster increases systemic production of NO in a subject. An agent in a NO precursor releases NO or reacts to generate another agent that releases NO after transdermal delivery to the blood circulation.

[0029] The term "body cavity" includes any opening on a subject's body and / or a surface area within an opening. Non-limiting examples of body cavities include the nose, sinuses, mouth, ears, rectum, vagina, open wounds, sores, buccal cavities, and mucosal surfaces (e.g., gums).

[0030] Terminology C 1-30 Alkyl groups include branched or unbranched alkyl groups having any carbon number ranging from 1 to 30. Non-limiting examples include methyl, ethyl, propyl, and butyl.

[0031] The term “cytokine storm” refers to the dysregulated, abnormal systemic release of pro-inflammatory cytokines that leads to disease, also referred to as the “cytokine release syndrome” or “inflammatory cascade.” Often, a cytokine storm or cascade is referred to as part of a cohort because one cytokine often leads to the production of multiple other cytokines that 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, interleukin-6, are not adequate therapeutic targets to re-establish homeostatic balance because they are resolved in the time frame by which a patient presents to a clinic for medical care. In contrast, the so-called “late mediators” are therapeutic targets because it is during this late “inflammatory cascade” that patients realize they are ill.

[0032] The term "inflammatory disease or disorder" may refer to any disease, condition or syndrome in which an excessive or uncontrolled inflammatory response results in a transient inflammatory condition, host tissue damage or loss of tissue function. "Inflammatory disease" also refers to pathological conditions mediated by granulocyte influx and / or neutrophil chemotaxis, as well as transient inflammatory conditions including "brain fog" and leaky gut syndrome due to chemotherapy.

[0033] "Long COVID" refers to side effects or symptoms attributed to COVID that appear long after one has seemingly recovered from the initial infection. Non-limiting examples of long COVID symptoms include, but are not limited to, brain fog, fatigue, achiness, clotting problems, myocarditis, and edema.

[0034] The term "NO booster" refers to an agent or mixture of agents that increases systemic NO production in a subject. NO boosters do not release NO themselves.

[0035] The term "NO precursor" refers to an agent or mixture that releases NO directly or indirectly through a derivative. The NO precursor may be or include an agent containing a NO release portion, and is transdermally delivered to the subject's blood circulation before releasing NO. Non-limiting examples of such NO releasers include S-nitroso-glutathione (GSNO), S-nitroso-N-acetylcysteine ​​(SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Alternatively, the NO precursor may include an agent that causes a derivative containing a NO release portion, and the derivative releases NO after transdermal delivery to the blood circulation. Non-limiting examples of agents that cause the release of NO derivatives include glutathione, N-acetylcysteine ​​(NAC), N-acetylpenicillamine, and cysteine, which can be nitrosated at the thiol group to produce a derivative containing S-nitrosothiol.

[0036] The term "semisolid" refers to a solid form that is flexible and deformable. Free-flowing liquids and rigid solid forms are not considered semisolids. Non-limiting examples include gels, ointments, creams, emulsions, microemulsions, nanoemulsions, pastes, balms, lotions, and mousses.

[0037] 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.

[0038] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of the compound of the present invention having the desired pharmacological activity, and 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 with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic monocarboxylic acids and dicarboxylic acids, aliphatic sulfuric acid, aromatic The pharmaceutically acceptable salts include tert-butyl acetic acid and trimethyl acetic acid. Pharmaceutical ... Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine and N-methylglucamine. It should be appreciated that the specific anions or cations that form part of any salt of the present invention are not important, as long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and their preparation and use methods are provided in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0039] The term "therapeutically effective amount" or "effective amount" refers to an amount of an active agent that is effective in preventing, alleviating or ameliorating symptoms of a disease, prolonging the survival of a treated subject, or achieving a desired / acceptable medical or health condition. The determination of a therapeutically effective amount or effective amount is well within the capabilities of those skilled in the art, especially based on the detailed disclosure provided herein.

[0040] In some embodiments, the term "treating" any disease or condition refers to improving the disease or condition (i.e., preventing or reducing the development of the disease or at least one of its clinical symptoms). In some embodiments, "treating" refers to improving at least one physical parameter, which may not be discernible to the subject. In some embodiments, "treating" refers to regulating the disease or condition, whether physically (e.g., stabilizing discernible symptoms), physiologically (e.g., stabilizing physical parameters) or both. In some embodiments, "treating" refers to delaying the onset of a disease or condition, or even preventing the onset of a disease or condition. For example, treatment can be "prophylactic treatment", which is interpreted as any treatment modality used to prevent disease progression or for preventive purposes for people at risk of developing the condition.

[0041] The term "transdermal" or "transdermally" refers to the delivery, administration or application of a formulation containing an active agent by direct contact with the skin or mucous membranes, and then transporting the agent through the skin or mucous membranes into the blood circulation of the subject. Such delivery, transportation, administration or application is also known to include dermal, transdermal, transmucosal and buccal routes. As used herein, "dermal" includes skin and mucous membranes, including oral, cheek, 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.

[0042] The term "transdermal formulation" refers to a composition or formulation of a pharmaceutical agent that, when applied to the skin or mucosa, delivers the pharmaceutical agent or a derivative of the pharmaceutical agent (e.g., a molecule containing S-nitrosothiol derived from a molecule containing thiol) across the skin or mucosa (or any other surface described above). The 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 the transdermal formulation can be in the form of, for example, a patch, swab, atomizer, spray, sponge or pouch.

[0043] Transdermal preparations

[0044] One aspect of the present disclosure provides a transdermal formulation that delivers a therapeutically effective amount of a pharmaceutical agent transdermally. The formulation generally comprises:

[0045] (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 systemic production of NO, and wherein the NO precursor comprises a NO releaser or a derivative NO releaser,

[0046] (b) a solvent in an amount sufficient to dissolve an effective amount of a NO enhancer or a NO precursor; and optionally

[0047] (c) fatty acids,

[0048] Wherein, after administration, an effective amount of an active agent (e.g., a sirtuin-1 (SIRT1) activator, a NO enhancer, and / or a NO releaser) is delivered transdermally. In some embodiments, the active agent is delivered to a blood vessel so that the active agent enters the systemic circulation from an artery or vein. In some embodiments, the active agent is delivered to the deep layers of the skin (e.g., the upper epidermis or lower epidermis below the stratum corneum).

[0049] In some embodiments, the agent in the transdermal formulation is a flavonoid or a curcuminoid, or a combination thereof. One or more flavonoids and / or one or more curcuminoids may be included in the formulation. For example, the formulation may contain one or two flavonoids, such as quercetin and apigenin, optionally in combination with curcumin, wherein each ingredient or component is present in a therapeutically effective amount.

[0050] In some embodiments, the transdermal formulation comprises one or more flavonoids or curcuminoids. Non-limiting examples include a combination of quercetin and apigenin, a combination of quercetin and curcumin, a combination of curcumin and apigenin, and a combination of quercetin and apigenin and curcumin. Regardless of whether the combination comprises one or more flavonoids or one or more curcuminoids, the weight ratio between each flavonoid and each curcuminoid can be independently 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 this ratio include about 100:1, about 80:1, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 8:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:8, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:80, about 1:100, and any range between any two of the foregoing values. In some embodiments, the combination 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.

[0051] When the formulation contains two or more flavonoids (e.g., 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 this ratio 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 values. In some embodiments, the formulation includes quercetin and apigenin in a ratio 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.

[0052] The NO level of a subject can be measured using known techniques such as those disclosed in U.S. Pat. Nos. 9044182 and 8425428. 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 conditions, ophthalmological conditions, bacterial infections, viral infections, ischemia reperfusion injury, hypoxia reoxygenation 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 observed locally at the site of topical application. In some embodiments, an effective amount is an amount sufficient to produce a measurable effect on inflammatory diseases (e.g., inflammatory skin diseases, inflammatory bowel diseases, and chemotherapy-induced inflammation of the systemic vascular system including the blood-brain barrier), as confirmed by appropriate clinical parameters (e.g., improvement in the overall assessment of the physician after treatment with the formulation). In some embodiments, an effective amount is an amount sufficient to obtain a systemic or local nitric oxide level that produces a desired effect, 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 inflammatory skin diseases (e.g., inflammatory skin diseases).

[0053] Non-limiting examples of SIRT1 activators include polyphenols, flavonoids, stilbenes, secosteroids, and other phytochemicals or natural products that promote NO formation.

[0054] After the NO enhancer or NO precursor is delivered transdermally, NO is produced in the subject. The amount of the NO enhancer or NO precursor is effective to increase NO systemically or locally to a sufficiently high level to achieve the purpose of treating the disease or condition. In some embodiments, the NO enhancer includes polyphenols, flavonoids, stilbenes, secosteroids, or natural products that promote NO production. In some embodiments, the NO precursor includes a molecule containing S-nitrosothiol, or a thiol-containing molecule and a nitrite source. In some embodiments, the NO enhancer includes one or more of a curcuminoid, a flavonoid, berberine, resveratrol, a vitamin D source, and pharmaceutically acceptable salts and derivatives thereof. Curcuminoids are linear diaryl heptane compounds, including, for example, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Flavonoids have a 3-hydroxyflavone skeleton, 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 vitamin D sources include vitamin D2 and vitamin D3 and any precursors of vitamin D. Non-limiting examples of polyphenols include plant extracts, brazilin, and theaflavins (e.g., theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3)).

[0055] In some embodiments, the NO enhancer consists essentially of curcumin, demethoxycurcumin, bisdemethoxycurcumin, quercetin, berberine, resveratrol, a source of vitamin D, and any combination thereof.

[0056] In some embodiments, the formulation contains both a NO booster and a NO precursor. For example, the combination of Curcumin and a NO releaser (e.g., an S-nitrosothiol-containing molecule 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 benefits of Curcumin in controlling systemic inflammation.

[0057] 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-undeca-tetraene-5,7-dione. In some embodiments, the NO enhancer is curcumin, or a synthetic curcumin that is 80%, 85%, 90% or 98% pure diferuloylmethane.

[0058] In some embodiments, the transdermal formulation includes one or more Curcuminoids, and optionally, one or more of polyphenols, flavonoids, stilbenes, secosteroids, or natural products that promote NO production, as active ingredients for treating a disease or condition. In some embodiments, the transdermal formulation includes one or both of Curcumin and quercetin, and optionally, one or more of polyphenols, flavonoids, stilbenes, and secosteroids.

[0059] The NO precursor is an S-nitrosothiol-containing molecule, 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 S-nitrosothiol-containing molecule releases NO to a subject in need thereof.

[0060] A variety of thiol-containing molecules can be used as precursors. Examples include glutathione, N-acetylcysteine ​​(NAC), N-acetylpenicillamine, cysteine, and its derivatives. The amino group of cysteine ​​or NAC can be replaced with an acetyl group or other carbonyl groups of different carbon lengths (e.g., COC 2-30 By adjusting the length of the carbon chain, the solubility and lipophilicity of the molecule can be altered. 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 esters) or amides having NR2 moieties (wherein each R is independently H or other substituted or unsubstituted C 3-30 Alkyl). Changes in the carbon chain can adjust the properties of the molecule.

[0061] Various inorganic compounds can be used as nitrite sources. Non-limiting examples of nitrite sources include alkali metal nitrites, alkaline earth metal nitrites, transition metal nitrites and ammonium nitrites. 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 nanoparticles loaded with nitrite.

[0062] Nanoparticles loaded with nitrite 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 hereby incorporated by reference herein. In order 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 nanoparticles loaded with nitrite can then be mixed with a solvent system of a polyol and a fatty acid (e.g., PEG400 and myristic acid) and remain stable until exposed to an aqueous environment. In the presence of thiol-containing molecules, the nanoparticles loaded with nitrite will allow the formation of S-nitrosothiol when the mixture is exposed to an acid source or a slightly acidic aqueous environment on the skin.

[0063] The use of nitrite-loaded nanoparticles allows the use of high concentrations of nitrite under hydrophobic conditions. The combination of nitrite-loaded nanoparticles with a solvent system of polyols and fatty acids (e.g., PEG400 and myristic acid) (regardless of the other included deliverables) allows the formation of a stable mixture that will release NO and S-nitrite thiol when exposed to an aqueous environment. No nitrite is released or NO is generated in the viscous solvent until water or an acid source is introduced.

[0064] 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 administration. For example, the nitrite source and the acid source can be separately packaged in a permeable or frangible pouch. The amount and concentration of the acid can be adjusted according to the amount of other agents and the nature of the acid. Non-limiting examples of acids include acetic acid, oxalic acid, and citric acid.

[0065] Non-limiting examples of S-nitrosothiol-containing molecules include S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine ​​(SNAC), S-nitroso-N-acetylpenicillamine (SNAP), and S-nitroso-human serum albumin (SNO-HAS). Similar to thiol-containing molecules, these S-nitrosothiol-containing molecules can be modified by changing the carbon chains of the respective ester, amide or N-acyl moieties to fine-tune their properties.

[0066] Solvents suitable for delivery systems (e.g., polyols) allow for high concentrations of poorly soluble agents. In addition, it should have biocompatibility and have properties that are safe for biomedical applications. In addition, ideally, it will promote skin and mucous membrane penetration to allow transdermal delivery. The limiting examples of polyols include polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, and glycerol. In some embodiments, polyols are polyethylene glycols (PEG). In some embodiments, 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 preparation is substantially free of water.

[0067] In some embodiments, the solvent of the formulation consists essentially of a polyol. In some embodiments, the formulation may include one or more additional solvents. Non-limiting examples include mineral oil, petrolatum, castor oil, essential oils such as eugenol, menthol, eucalyptol or rose oil, n-methyl pyrrolidone, vegetable oils, oleyl alcohol, dipropylene glycol, polyoxyethylene derivatives of sorbitan esters, saturated polyethylene glycol C 8-10 Glycerides, polyoxyethylated fatty acid glycerides, oleic acid, dimethyl sulfoxide (DMSO), fatty alcohols, isopropyl myristate (IPM), triacetin, ethyl oleate, isostearic acid, medium chain fatty acids and other fatty acids, and mixtures thereof. In addition to dissolving the agent, these solvents can also be used as plasticizers to make the formulation flexible, stretchable, plastic and / or otherwise skin-friendly.

[0068] In some embodiments, the polyol solvent is a low molecular weight polyethylene glycol (PEG) having a molecular weight in the range of about 50 to about 2000, about 50 to about 1000, about 100 to about 1000, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 200 to about 800, about 200 to about 600, or about 200 to about 400 Daltons. Non-limiting examples of molecular weights of polyol solvents 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 (e.g., PEG200 and PEG400) that are liquid at room temperature are particularly useful.

[0069] Fatty acid is used as penetration enhancer.The non-limiting examples of fatty acid include myristic acid, palmitoleic acid, cis-6-hexadecenoic acid (sapienic acid), oleic acid, elaidic acid, trans-11-octadecenoic acid (vaccenic acid), linoleic acid, translinoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, sad, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristic acid, and any combination thereof.In some embodiments, fatty acid is myristic acid.Medium-sized fatty acids such as myristic acid and / or other fatty acids with considerable size / molecular weight are particularly useful.In some embodiments, preparation does not contain fatty acid or only contains trace or minute amount of fatty acid.

[0070] In some embodiments, the penetration enhancer is essentially composed of fatty acid or its ester. In some embodiments, the preparation may contain one or more additional penetration enhancers. Non-limiting examples include surfactants, alcohols, fatty alcohols and ethylene glycol, esters, fatty acid esters and fatty alcohol esters, esters of long-chain fatty acids and methanol, ethanol, isopropanol, esters of fatty alcohols and acetic acid, lactic acid and oleic acid, diethanolamine, essential oils, terpenes and terpenoids, amides, ureas, polyoxyethylene fatty alcohol ethers, polyoxyethylene fatty acid esters, sulfoxides, ether alcohols, pyrrolidone, transcarbam, capsaicin derivatives, dimethyl amino acid esters, peptides, iminosulfuranes, dicarboxylates, nanocarriers, triglycerides, hydrocarbons, phospholipids, alone or in combination.

[0071] By adjusting the amount and ratio of polyol, fatty acid and NO promoter or NO precursor, the solubility of the activating agent in fatty acid and NO promoter or NO precursor, as well as the physical state (e.g., liquid or gel or semisolid) of the preparation and the release profile of the activating agent can be controlled. The ratio between polyol and fatty acid affects the form of the solution, and is usually 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 fatty acid in the polyol is in the range of about 0.01M to about 1M, about 0.01M to about 0.8M, about 0.01M to about 0.6M, about 0.01M to about 0.4M, about 0.01M to about 0.2M, about 0.01M to about 0.15M, about 0.01M to about 0.1M, about 0.02M to about 0.2M, about 0.02M to about 0.1M, about 0.04M to about 0.08M, or about 0.06M to about 0.1M. In further exemplary embodiments, the concentration of fatty acid in the polyol is about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.06M, about 0.08M, about 0.1M, or about 0.12M. In some embodiments, fatty acid is saturated in the polyol. In some embodiments, the polyol is PEG. In some embodiments, the fatty acid is myristic acid.

[0072] The scope and dosage of polyols and fatty acids are as described above, and can be modified by those of ordinary skill in the art according to actual needs without too much experimentation. In some embodiments, fatty acids are selected from myristic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, elaidic acid, trans-11-octadecenoic acid, linoleic acid, trans-linoleic acid, alpha-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, polyols are selected from polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, glycerol, and any combination thereof. In some embodiments, polyols are polyethylene glycols with a molecular weight in the range of 200 to about 600. In some embodiments, the ratio of polyols and fatty acids is in the range of about 5:1 to about 100:1 by weight.

[0073] The fatty acid may be present in an amount ranging from about 0.1% to about 30%, about 0.5% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 2% to about 8%, or about 4% to about 8% of the total weight of the NO 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 acid include about 1%, about 3%, about 5%, about 7%, about 8%, or about 10% by weight.

[0074] Water may cause aggregation and particle formation. In some embodiments, the transdermal formulation is anhydrous or substantially free of water. Reducing or eliminating water as much as possible from the formulation can help maintain a uniform distribution of fatty acids and / or active ingredients (e.g., NO promoters or NO precursors) in the polyol (e.g., 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.

[0075] In some embodiments, the transdermal formulation includes at least one water repellent, also known as a water avoider. Examples of water repellents include silicones, such as cyclomethicone, dimethicone, simethicone, C 26-28 Alkyl dimethicone oil, C 26-28 Alkylmethicones, polyphenylsisquioxanes, trimethylsiloxysilicates, and cross-linked polymers of cyclopentasiloxane and dimethicone / vinyl trimethylsiloxysilicate, and mixtures thereof. Water repellents may be particularly useful in embodiments where the topical carrier is used with a water-reactive agent, such as a nitric oxide-releasing agent that releases nitric oxide in the presence of water (e.g., diazeniumdialate or sodium nitrite). In other cases, such as when the active agent is not water-sensitive, a water repellent may or may not be included.

[0076] Depending on the therapeutic goal of the formulation, the active agent (e.g., CD38 inhibitor, NO booster or NO precursor or mixture thereof) in the formulation comprises about 0.05% to about 80%, about 0.05% to about 50%, about 0.05% to about 35%, about 0.05% to about 30%, about 0.05% to about 20%, about 0.05% to about 10%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% to about 5%, about 1% to about 20%, about 1% to about 10%, or about 1% to about 5%, of the total weight of the NO booster or NO precursor, the polyol and the fatty acid (if present), or the total weight of the formulation. Non-limiting examples of the amount of active agent in the formulation include about 1%, 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 the formulation dosage unit 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. The dosage unit can be a physically separate package form (e.g., capsule, patch, vial). The dosage unit can also be a predetermined portion of a preparation for each separate application. For example, a suitable amount can be taken out from a container as a dosage unit of a preparation 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 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 In some embodiments, the active agent is a curcuminoid selected from one, two or three of curcumin, demethoxycurcumin and bisdemethoxycurcumin, optionally in combination with one or more flavonoids. The dosage unit can be administered once, twice, three times per day or as needed. In some embodiments, the dosage unit is administered once per day, every two days, every three days, every four days, every five days, every six days, every seven days or every ten days.

[0077] In some embodiments, the ratio between each active agent and the polyol is independently in the range of about 1:5 to about 1:100, about 1:5 to about 1:50, about 1:5 to about 1:30, about 1:5 to about 1:20, about 1:8 to about 1:15, or about 1:10 to about 1:15 by weight. In some embodiments, the polyol is PEG. In some embodiments, the fatty acid is myristic acid. In some embodiments, the preparation is substantially free of piperine. Depending on the disease or condition to be treated and the site of administration, the ratio and amount of PEG, fatty acid and active agent can be selected so that the resulting preparation is a liquid, gel or other suitable form. Other agents can be added to control the physical state of the preparation.

[0078] 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 (e.g., PEG200, PEG300, PEG400, or PEG500) with a molecular weight ranging from about 200 to about 500. 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 NO enhancer 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). 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 a single active ingredient in the formulation is in the range of about 0.01 M to about 1 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.3 M, or about 0.1 M to about 0.2 M. Non-limiting examples of concentrations of active ingredients (e.g., Curcumin) in polyols (e.g., PEG) include about 0.06 M, about 0.08 M, about 0.1 M, about 0.12 M, about 0.14 M, about 0.16 M, about 0.18 M, about 0.20 M, about 0.25 M, about 0.30 M, about 0.40 M, about 0.60 M, and about 0.80 M.

[0079] Transdermal preparations have an extended shelf life and as little decomposition of active ingredients as possible. In some embodiments, the active ingredients of the preparation remain stable for more than 95% or more than 99% over 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 includes one or more curcuminoids, myristic acid, and PEG. The amount of one or more curcuminoids in the preparation is in the range of about 2% to about 10%, about 3% to about 8%, or about 4% to about 6% by weight. The amount of myristic acid in the preparation is in the range of about 1% to about 10%, about 1% to about 8%, about 2% to about 8%, or about 4% to about 6% by weight. The amount of PEG in the preparation is in the range of about 60% to about 95%, about 70% to about 90%, about 80% to about 90%, or about 95% to about 90% by weight. In some embodiments, PEG is PEG400.

[0080] In some embodiments, the preparation also includes a gelling agent or thickening agent, which keeps the preparation in a semisolid or solid form. The non-limiting examples of gelling agents or thickening agents include carbomers, methylcellulose, hydroxypropyl methylcellulose, poloxamer, polyacrylic acid, alginate, chitosan, xanthan gum, gellan gum, xyloglucan, paraffin, silicone, vaseline, cocoa butter and high molecular weight polyalkylene glycol. Other examples include polyethylene oxide, ammonium methacrylate, carrageenan, cellulose acetate phthalate aqueous solution (e.g., CAPNF from Eastman), sodium carboxymethylcellulose, carboxypolymethylene, cellulose, cellulose acetate (microcrystals), cellulose polymers, divinyl styrene, ethylcellulose, ethylene vinyl acetate, silicone, polyisobutylene, shellac (FMC BioPolymer), guar gum, guar rosin, cellulose derivatives (e.g., hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, and methylcellulose), hydroxypropylmethylcellulose phthalate (hydroxypropylmethylcellulose phthalate), methyl acrylate, microcrystalline wax, polyvinyl alcohol, polyvinyl acetate, polyvinyl acetate phthalate (e.g., Suretic from Colorcon), PVP ethylcellulose, polyvinylpyrrolidone (PVP), acrylates, PEG / PVP, trimethylsiloxysilicate , maleic acid / anhydride copolymers, polacrilin, poloxamers, polylactic acid / poly-l-lactic acid, terpene resins, locust bean gum, Zein, acrylic acid copolymers, polyurethane dispersions, gelatin (type A and type B from various sources including porcine, bovine and fish), dextrins, starch, polyvinyl alcohol-polyethylene glycol copolymers, methacrylic acid-ethyl acrylate copolymers (e.g., Kollicoat polymers from BASF), methacrylic acid and methacrylate-based polymers (e.g., poly(methacrylic acid) copolymers and methyl methacrylate copolymers, including Rohmand Haas Eudragit polymers (Eudragit (E, L, NE, RL, RS, S100)), esters of polyvinyl methyl ether / maleic anhydride copolymers (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 may 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 semisolid formulation may be an ointment, gel, cream, emulsion, paste, lotion, or liposomal dosage form.

[0081] In some embodiments, the formulation includes a combination of small and large polyalkylene glycols, the molecular weight difference of which is 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 rate / degree of viscosity and skin penetration and circulation absorption can be controlled. For example, the combination can 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, respectively. The combination can 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 further exemplary embodiments, one polyalkylene glycol has a MW of 100, 200, 400, 600 or 800 and another polyalkylene glycol has a MW of 1000, 1500, 2000, 2500 or 3000. In some embodiments, the combination includes 400 Daltons of PEG and 2000 Daltons of PEG. In some embodiments, the ratio between the low molecular weight polyalkylene glycol and 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 low molecular weight polyalkylene glycols (e.g., PEG and / or PPG) and high molecular weight polyalkylene glycols (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.

[0082] 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, antivirals, and / or antifungals.

[0083] The formulations may include other components including, for example, solubilizers, skin immersion enhancers, surfactants, cosolvents, thickeners or viscosity enhancers, preservatives, isotonizing agents, isoosmotizing agents, absorption enhancers of the agent, mucoadhesive polymers, non-mucoadhesive polymers, chelating agents, stabilizers, antioxidants, and mixtures thereof.

[0084] 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.

[0085] 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 Polyethylene castor oil derivatives, such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil; polyethylene glycol, especially low molecular weight polyethylene glycol; polyethylene glycol derivatives, such as caprylic / capric glycerides (commercially available under the trade name Poly(TM)); polyethylene castor oil derivatives, such as polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil; polyethylene glycol, especially low molecular weight polyethylene glycol; polyethylene glycol derivatives, such as caprylic / capric glycerides (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 solubilizing agents can also be used as absorption enhancers. A single solubilizing agent can be added to the formulation, or a mixture of solubilizing agents can be added to the formulation.

[0086] Skin penetration enhancers help promote the passage of therapeutic levels of active agents through appropriately sized areas 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-(hydroxyethyl)pyrrolidone, N,N-diethyl-m-toluamide; C2-C6 alkanediols; dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and various solvents such as tetrahydrofurfuryl alcohol; and 1-substituted azepan-2-ones, particularly 1-n-dodecylazepan-2-one (laurocapram, which is available from Whitby Research Incorporated, Richmond, Va. under the trade name Commercially available).

[0087] Examples of surfactants may include, but are not limited to, for example, polyethoxylated glycerides, polysorbates, poloxamers, sodium lauryl sulfate, phospholipids such as phosphatidylcholine or phosphatidylglycerol and derivatives thereof, polyoxyethylated hydrogenated castor oil, polyoxyethylated fatty acids, mixtures of mono-, di- and triglycerides of optionally polyoxyethylated fatty acids, and mixtures thereof.

[0088] Examples of preservatives may include, but are not limited to, benzalkonium chloride, boric acid, benzoic acid, p-hydroxybenzoic acid, 1-4 Alkyl esters, chlorobutanol, benzyl alcohol, phenylethyl alcohol, organometallic derivatives of mercury, polyquaterniums such as polyquaternium 1, and mixtures thereof.

[0089] Examples of isotonic and isotonic agents may include, but are not limited to, inorganic salts such as sodium chloride, glucose, trehalose, mannitol, amino acids, and mixtures thereof.

[0090] Examples of mucoadhesive polymers may include, but are not limited to, for example, hyaluronic acid, polygalacturonic acid, polyacrylic acid, carboxymethyl amylose, carboxymethyl chitin, chondroitin sulfate, methylcellulose, gelatin, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, xanthan gum, chitosan, carbopol, polycarbophil, gellan gum, carrageenan, alginate, pectin, poloxamer, and mixtures thereof. Examples of non-mucoadhesive polymers may include, but are not limited to, for example, polyvinyl alcohol. Examples of chelating agents may include, but are not limited to, for example, disodium ethylenediaminetetraacetic acid and disodium cromoglycate. Examples of antioxidants may include, but are not limited to, for example, sodium metabisulfite, sodium bisulfite, acetylcysteine, ascorbic acid, and mixtures thereof.

[0091] By adjusting the amount and ratio of polyol, fatty acid and one or more activating agents, the solubility of fatty acid and activating agent and the physical state of the preparation and the release curve of the activating agent can be controlled. In some embodiments, the ratio of polyol, fatty acid, active ingredient and other essential components is configured to make the preparation take effect quickly in about 5 minutes, about 10 minutes, about 15 minutes or about 30 minutes.

[0092] Transdermal formulations disclosed herein can provide extended or sustained release of a pharmaceutical agent (e.g., a CD38 inhibitor, a NO booster, or a molecule containing S-nitrosothiol). In some embodiments, the formulation provides extended release of a pharmaceutical agent (transdermally delivered to the blood circulation) 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 a polyol solvent and a fatty acid in an appropriate ratio, the release rate can also be controlled. In some embodiments, one, two, or three of the following parameters can be achieved for the formulation:

[0093] (a) less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% of the agent is delivered to the blood circulation within about 30 minutes, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, or within about 5 hours;

[0094] (b) about 25% to about 90%, about 30% to about 85%, about 35% to about 70%, about 40% to about 70%, about 50% to about 60%, about 35% to about 50%, about 40% to about 60%, or about 35% to about 80% of the agent is delivered to the blood circulation within about 6 hours, about 8 hours, about 10 hours, about 12 hours, or about 14 hours; and

[0095] (c) greater than 60%, greater than 70%, or greater than 80% of the agent is delivered to the blood circulation within about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, or about 48 hours.

[0096] In some embodiments, the active agent and carrier (e.g., polyol or fatty acid) and their amounts in the formulation are selected so 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 by 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, within about 20 minutes, within about 30 minutes, within about 1 hour, or within about 2 hours after administration of the formulation.

[0097] The formulation may include a second agent. Examples of second agents include antihypertensives, antimicrobial agents, anti-inflammatory agents, analgesics, anesthetics, antihistamines, preservatives, immunosuppressants, 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.

[0098] Examples of anti-inflammatory agents include nonsteroidal 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 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 acetate acetonide, hydrocortisone valerate, prednicarbate, triamcinolone acetonide, fluocinolone acetate, hydrocortisone and other drugs known in the art, prednisolone, dexamethasone, fluocinolone acetate, hydrocortisone acetate, prednisolone acetate, methylprednisolone, dexamethasone acetate, betamethasone, betamethasone valerate, flumetasone, fluorometholone, beclomethasone dipropionate diproprionate), fluocinolone acetonide, a topical corticosteroid, which may 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 a high-potency corticosteroid,For example, clobetasol propionate, betamethasone benzoate, betamethasone dipropionate, diflorasone acetate, fluocinolone acetate, mometasone furoate, triamcinolone acetonide.

[0099] 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, nz), 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 localized and systemic viral infections, such as COVID-19, cold sores, or genital herpes.

[0100] Examples of antimicrobial agents 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, antifolates (including sulfonamides), trimethoprim and combinations thereof, and pyrimethamine, synthetic antibacterial agents including nitrofurans, methenamine mandelate and methenamine hippurate hippurate), nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin, glycopeptide, glyclyclycline, ketolides, oxazolidinone;Imipenem, amikacin, netilmicin, fosfomycin, gentamycin, ceftriaxone, Ziracin, Linezolid, Synercid, Aztreonam, 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 cefetametpivoxil. ;

[0101] Examples of the antihistamine include diphenhydramine hydrochloride, diphenhydramine salicylate, diphenhydramine, chlorpheniramine hydrochloride, chlorpheniramine maleate, isotipendyl hydrochloride, tripelennamine hydrochloride, promethazine hydrochloride, methdilazine hydrochloride and the like. Examples of local anesthetics include dibucaine hydrochloride, dibucaine, lidocaine hydrochloride, lidocaine, benzocaine, 2-(diethylamino)ethyl p-butylaminobenzoate hydrochloride, procaine hydrochloride, tetracaine, tetracaine hydrochloride, chloroprocaine hydrochloride, oxyprocaine hydrochloride, mepivacaine, cocaine hydrochloride, piperocaine hydrochloride, dyclonine, and dyclonine hydrochloride.

[0102] Examples of preservatives include alcohols, quaternary ammonium compounds, boric acid, chlorhexidine and chlorhexidine derivatives, iodine, phenols, terpenes, bactericides, disinfectants including thimerosal, phenol, thymol, benzalkonium chloride, benzethonium chloride, chlorhexidine, povidone iodine, cetylpyridinium chloride, eugenol and trimethylammonium bromide.

[0103] Examples of analgesics include alfentanil, benzocaine, buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine, dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone, indomethacin, lidocaine, levorphanol, meperidine, and phentermine. eridine, methadone, morphine, nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine, pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil, tetracaine, and tramadol.

[0104] Examples of anesthetics include alcohols such as phenol; benzyl benzoate; calamine; chloroxylenol; dyclonine; ketamine; menthol; pramoxine; resorcinol; troclosan; procaines such as benzocaine, bupivacaine, chloroprocaine; cinchocaine; cocaine; dexivacaine; diamocaine; dibucaine; etidocaine; ine; hexylcaine; levobupivacaine; lidocaine; mepivacaine; oxethazaine; prilocaine; procaine; proparacaine; propoxycaine; pyrrocaine; risocaine; rodocaine; ropivacaine; tetracaine; and derivatives thereof, such as pharmaceutically acceptable salts and esters, including bupivacaine hydrochloride, chloroprocaine hydrochloride, diamocaine cyclamate, 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.

[0105] Examples of anti-hemorrhagic agents include thrombin, phytonadione, protamine sulfate, aminocaproic acid, tranexamic acid, carbazochrome, carbaxochrome sodium sulfanate, rutin, and hesperidin. Other examples of second or additional agents include chemotherapeutic drugs (tyrosine kinase inhibitors, immune checkpoint inhibitors, VEGF inhibitors, etc.) and glucose-lowering drugs (e.g., metformin).

[0106] Other examples of second agents include daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, maphosphamide, ifosfamide, cytosine arabinoside, bischloroethyl-nitrosourea, Busulfan, mitomycin C, C), actinomycin D, mithramycin, prednisone, hydroxypregesterone, testosterone, tamoxifen, dacarbacine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide hamide), 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 (etoposide,VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol. In some embodiments, second agents that can be used in combination with the formulations disclosed herein or incorporated into the same transdermal formulation include anti-inflammatory agents, analgesics, antibacterial agents, antifungal agents, antibiotics, vitamins and antioxidants. In some embodiments, the second agent is selected from piperine, anthranilic acid, benzophenone, camphor derivatives, cinnamate (e.g., octyl methoxycinnamate), dibenzoylmethane (e.g., butyl methoxydibenzoylmethane), para-aminobenzoic acid (PABA) and its derivatives, salicylates, and PDE5 inhibitors (e.g., sildenafil (Viagra), tadalafil (Cialis), vardenafil (Levitra), and avanafil (Stendra).

[0107] Any second agent described herein can be incorporated into the same transdermal formulation. Alternatively, in some embodiments of any method disclosed herein, the second agent can be administered separately from the transdermal formulation by any suitable route, including oral, transdermal, and parenteral routes.

[0108] In some embodiments, the combination of active agents in the transdermal formulation produces a synergistic therapeutic effect. For example, Curcumin and PDE5 inhibitors, when incorporated into the same transdermal formulation or administered sequentially with each other, can lead to early symptom recovery (fever, cough, sore throat, and breathlessness), fewer exacerbations, and fewer red flag signs in patients with viral infections (e.g., COVID-19).

[0109] Transdermal delivery system

[0110] Kit or transdermal delivery system can be sufficient for at least one medicament to contain any combination of components described herein, and can also include component instructions for use recorded in a tangible form. In some applications, one or more components can be provided in individual (usually disposable) patches, tubes or equivalent containers with pre-measured single-use amounts.

[0111] Preparations disclosed herein can be incorporated into a transdermal delivery system or a kit and used as a patch, swab, aerosol, cream, sponge, sprayer, atomizer or by other suitable means. The transdermal delivery system can also include an instruction manual for the use of the preparation and one or more treatment methods disclosed herein. The liquid or semisolid preparation can be directly applied to the skin with a swab or sponge, etc. Alternatively, the transdermal delivery system can include a layer coated with or soaked with a liquid, semisolid or solid transdermal preparation. For example, a patch can have a layer soaked with a liquid preparation or coated with a semisolid or solid preparation. The transdermal delivery system can also include an adhesive component for attaching it to the skin.

[0112] Transdermal delivery system or kit can have any shape suitable for application to a subject in need. For example, the sponge carrying the preparation disclosed herein can have a circular, cylindrical, conical, planar, tubular and other symmetrical or asymmetrical shapes for insertion into a body cavity or attachment or application to a target location, and can include an applicator or 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 shows affinity for aqueous solutions, especially affinity for moisture (e.g., moisture from the position or body cavity where a reservoir is placed). The sponge with absorption properties can be made of natural or synthetic materials, such as polyester, polyurethane and plant cellulose.

[0113] In some embodiments, the preparation is incorporated into a liquid reservoir. The reservoir can be used independently, or it can be connected to a sponge or partially or completely enclosed in a sponge. Alternatively, after necessary treatment or mixing with another agent, the contents of the liquid reservoir can be loaded into a sponge for application. In preparations involving nitrite sources that require acid to generate nitrous acid and NO, acid can be added to a reservoir containing nitrite before application. Alternatively, a dual liquid reservoir system can be used. For example, one pouch contains molecules and nitrites containing thiol in a polyol solvent system, and another pouch contains an acid source. Of course, additional pouches can be used to encapsulate molecules containing thiol or nitrites or fatty acids, respectively. Before application or when in contact with the skin, the contents of the pouch are mixed to trigger a reaction between acid and nitrite, and then the molecules containing thiol are nitrated. The pouches used to encapsulate NO precursors or acid sources are usually fragile or permeable containers that do not contact each other or are separated by an impermeable and removable barrier before applying the preparation. During application, after removing the barrier, the acid and NO precursor can penetrate out of their respective pouches and mix with each other under the squeeze of the user. The acid and NO precursor can also be simply mixed by destroying the pouch during or before application. In another exemplary embodiment, the acid and NO precursor are mixed in a container before application. The resulting mixture is absorbed with a swab, sponge or absorbent patch and then applied to the skin.

[0114] In some embodiments, the pouch has a permeable or semipermeable membrane surface that is optionally coated with an adhesive for securing the membrane to the skin. In addition to using an adhesive coating, the pouch can also be secured to the skin by placing the pouch on the skin and then covering the pouch with an adhesive patch or seal. Commercially available pouch stock materials (e.g., DuPont's ) can also be used for liquid reservoirs. Other examples include coextruded ethylene acrylic acid / low density polyethylene (EAA / LDPE) materials, or (Acrylonitrile-methacrylate).

[0115] In some embodiments where the NO precursor is a mixture of a sulfur-containing molecule and a 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 disposed in another layer. The two layers do not come into contact with each other until the patch is attached to the skin or before application. 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 before application allows the nitrite and acid to mix.

[0116] In some embodiments, the formulation is solid, containing a thickener or solidifying material, such as cocoa butter. In some embodiments, the formulation is solid or semisolid, containing petrolatum. Solid or semisolid formulations can be applied to the skin or melt when rubbed vigorously on the skin.

[0117] In some embodiments, the formulation is loaded into a nebulizer or sprayer to deliver the agent to the nose, mouth or lungs of a subject in need thereof in the form of an aerosol. Carbon dioxide or other suitable gases may be used as a propellant.

[0118] The system or kit may contain any number of additional reagents or substances useful for practicing the methods of the invention. The kits or systems of the invention may be provided at any temperature. For example, for storage of kits including certain S-nitrosothiol-containing molecules in liquids or gels, they may be provided and maintained at a suitable temperature or about 0°C.

[0119] The kit or system may also include instructions and packaging materials for holding the container or combination of containers. Instructions (e.g., written instructions or videotaped demonstrations detailing the use of the transdermal formulation to treat target diseases and conditions) may 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 hold the components in any of a variety of configurations (e.g., in pouches, tubes, etc.).

[0120] Such kits or systems may also include information, such as scientific literature references, package inserts, clinical trial results, and / or summaries of these contents, etc., which indicate or determine the activity and / or advantages of the composition, and / or other information describing administration, application, side effects, drug interactions, or useful to medical care providers. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kits or systems described herein may be provided, sold, and / or promoted to medical service providers, including doctors, nurses, pharmacists, prescribing officials, etc. In some embodiments, the kits may also be sold directly to consumers.

[0121] How to use

[0122] Because transdermal formulations introduce high levels of flavonoids, curcuminoids, and / or other potent anti-inflammatory agents and / or antioxidants topically and / or systemically, they can rapidly intervene in a variety of diseases and conditions.

[0123] One physiological, natural way to enhance endothelial NO production and reduce ROS levels is through reduced CD38 levels. High levels of CD38 result in low levels of NAD+, which in turn leads to: i) mitochondrial dysfunction, resulting in excessive ROS production; and ii) reduced Sirtuin protein activity. Together, these CD38 consequences lead to reduced endothelial NO production, as well as excessive production of ROS and peroxynitrite by activated macrophages and microglia, promoting endothelial dysfunction. Furthermore, there is a cross-talk between senescent cells and CD38, with each promoting the accumulation of the other. The accumulation of senescent cells in the endothelium not only prevents the repair of the endothelial lining, but also promotes persistent endothelial dysfunction, including pathological vascular remodeling and persistent reductions in NO production.

[0124] Formulations for sustained transdermal delivery of active agents can reduce CD38 extracellular enzyme activity (such as NADase activity); thereby increasing NAD+ levels and promoting the activity of NAD+-dependent sirtuins. In this way, this approach provides a new method for preventing and treating various diseases, such as those caused by endothelial dysfunction after acute or chronic proinflammatory injury, age-related physical and cognitive decline, age-related cardiac tissue changes, vascular hypertrophy, osteoarthritis, peripheral neuropathy, and long-term COVID. In addition, this method overcomes the negative effects of excessive CD38 production on the ability of stem cells to differentiate into mature cells, which at least to some extent leads to chronic inflammatory anemia and the insufficient efficacy of intrinsic stem cells and stem cell therapies in repairing damaged tissues.

[0125] Transdermal formulations can be delivered by any suitable route to deliver the active ingredient across the skin, mucous membranes or membranes of the 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 by nebulizers), intratracheal routes, intranasal routes, and epithelial routes. In some embodiments, transdermal formulations include one or both of curcumin and quercetin, and optionally one or more of polyphenols, flavonoids, stilbenes, and secosteroids.

[0126] Without being bound by any particular theory, it is presumed that the transdermal formulations disclosed herein deliver active agents transdermally to a subject and increase the subject's systemic or local NO levels. In some embodiments, the formulation contains an effective amount of a NO enhancer to increase the subject's systemic or local NO levels. In some embodiments, the formulation contains an effective amount of a NO precursor, wherein the method converts the NO precursor into a molecule, such as an S-nitrosothiol-containing molecule, which releases NO when delivered transdermally to the subject. In some embodiments, the formulation may contain both a NO enhancer and a NO precursor. In some embodiments, the formulation may also contain a second agent as defined above.

[0127] The transdermal formulation achieves NO enhancement through pathways including upregulating endothelial nitric oxide synthase (eNOS), enhancing eNOS activity, and reducing ROS levels. For example, ROS scavenges NO, causing eNOS uncoupling, resulting in the cessation of eNOS-associated NO synthase, which is replaced by eNOS further producing ROS. At the same time, the loss of endothelium leads to: i) the loss of blood flow-mediated mechanical transduction mechanisms for controlling eNOS production of NO; ​​and the loss of acellular areas adjacent to the endothelium, which prevent hemoglobin in red blood cells from scavenging NO. The transdermal formulation of the present disclosure provides an effective amount of an active agent that can increase the NO level in the vascular endothelial lining by inhibiting ROS scavenging NO and limiting the degradation of the endothelial glycocalyx lining.

[0128] Transdermal formulations can be applied to a body surface or body cavity of a subject. For example, a method of increasing systemic or local NO levels or treating a disease or condition can include inserting a sponge loaded with a formulation disclosed herein between the cheek and gums.

[0129] 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 enhancement. 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 required for the activity of SIRT1 and SIRT3, which are essential for controlling inflammation, oxidative stress, and cell repair. CD38 is an enzyme that is primarily present on the surface of macrophages and microglia, but is not limited to it. CD38 has NADase activity (breaks down NAD). The amount of CD38 on macrophages and microglia increases with aging and the onset of acute and chronic inflammation, and therefore, decreased NAD levels lead to energy loss, fatigue, increased inflammatory processes, and an inability to restore damaged cellular components, including DNA. Because the transdermal formulation disclosed herein can effectively reduce CD38 levels with little or no side effects, it can be used to prevent and limit severe endothelial dysfunction after acute proinflammatory insults, limit age-related physical and cognitive decline, reverse age-related changes in cardiac tissue, and stabilize red blood cells.

[0130] Certain flavonoids, such as apigenin and quercetin, have additional advantages over curcumin due to their anti-aging activity, in addition to having many similar anti-inflammatory and anti-oxidative stress properties as 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. Absorption of these active substances by circulating M1 macrophages through transdermal / transmucosal routes is proposed to be a mechanism for the rapid reduction of CD38 activity. A CD38 inhibitor can be an agent that repolarizes macrophages and then causes a decrease in CD38 activity.

[0131] Diseases or conditions associated with elevated levels of CD38 are those in which a subject is detected to have higher than normal levels of CD38. Normal levels of CD38 can be readily obtained from healthy individuals using well-known procedures and statistically acceptable analyses.

[0132] Various diseases or conditions can be treated with the transdermal formulations disclosed herein. Non-limiting examples of the diseases and conditions include age-related physical and / or cognitive decline, lupus, rheumatoid arthritis, multiple sclerosis, leukemia and multiple myeloma, cardiovascular disease, neurodegenerative diseases, COVID-19 symptoms, severity and persistence (long-term COVID), diabetes, hypertension, neuropathic pain, osteoarthritis, anemia of chronic illness, ALS, Parkinson's disease, ischemia-reperfusion injury, hypoxia-reoxygenation injury, damage caused by blood transfusion, and damage caused by radiation (including dermatitis and neuroinflammation).

[0133] Other diseases or conditions that can be treated with the formulations disclosed herein include muscle structure disorders, neuronal activation disorders, muscle fatigue disorders, muscle mass disorders, metabolic diseases, vascular diseases, ocular vascular diseases, muscular eye diseases, kidney diseases, hypertension, inflammation, endothelial dysfunction, skin diseases, ophthalmic diseases, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomena, 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.

[0134] 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 myopathy, myotonic ... disease, oculopharyngeal MD, or stress urinary incontinence; a neuronal activation disorder selected from the group consisting of amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Guillain-Barre syndrome, Lambert-Eaton syndrome, multiple sclerosis, myasthenia gravis, nerve lesion, peripheral neuropathy, spinal muscular atrophy, tardyulnar nerve palsy, and toxic myoneural disorder; a muscle fatigue disorder,selected from chronic fatigue syndrome, diabetes (type I or II), glycogen storage disease, fibromyalgia, Friedreich's ataxia, intermittent claudication, lipid storage myopathy, MELAS, mucopolysaccharidosis, Pompe disease or thyrotoxic myopathy; a muscle mass disorder selected from cachexia, cartilage degeneration, cerebral palsy, compartment syndrome, critical illness myopathy, inclusion body myositis, polymyositis, muscular dystrophy, atrophy) (disuse), sarcopenia, steroid myopathy and systemic lupus erythematosus; beta oxidation disease selected from systemic carnitine transporter, carnitine palmitoyltransferase (CPT) II deficiency, very long chain acyl-CoA dehydrogenase (LCHAD or VLCAD) deficiency, trifunctional enzyme deficiency, medium chain acyl-CoA dehydrogenase (MCAD) deficiency, short chain acyl-CoA dehydrogenase (SCAD) deficiency and riboflavin-responsive disorders of b-oxidation (RR-MADD); metabolic disease,It is selected from hyperlipidemia, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, HDL hypocholesterolemia, LDL hypercholesterolemia and / or HLD non-cholesterolemia, VLDL hyperproteinemia, dyslipoproteinemia, apolipoprotein AI hypoproteinemia, atherosclerosis, arteriosclerotic diseases, cardiovascular system diseases, cerebrovascular diseases, peripheral circulation diseases, metabolic syndrome, syndrome X, obesity, diabetes (type I or type II), hyperglycemia, insulin resistance, impaired glucose tolerance, glucosetolerance), hyperinsulinism, diabetic complication, cardiac insufficiency, myocardial infarction, cardiomyopathy, hypertension, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NAFLD),The invention relates to a method of treating a patient suffering from NASH, thrombus, Alzheimer's 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; a cancer selected from the group consisting of colon cancer, colorectal cancer, skin cancer, breast cancer, prostate cancer, ovarian cancer and lung cancer; a vascular disease selected from the group consisting of peripheral vascular insufficiency, peripheral vascular disease, intermittent claudication, and spondylosis. ocular vascular disease selected from the group consisting of age-related macular degeneration (AMD), Stargardt disease, hypertensive retinopathy, diabetic retinopathy, retinopathy, macular degeneration, retinal haemorrhage and glaucoma; muscle-eye disease,It is selected from strabismus, progressive external ophthalmoplegia, esotropia, exotropia, disorders of refraction and accommodation, hypermetropia, myopia, astigmatism, anisometropia, presbyopia, disorders of accommodation and internal ophthalmoplegia; kidney disease is selected from glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, acute nephritis, recurrent hematuria, persistent hematuria, chronic nephritis. nephritis, rapidly progressive nephritis, acute renal failure, chronic renal failure, diabetic nephropathy, and Bartter's syndrome.

[0135] 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.

[0136] In some embodiments, the disease or condition is selected from Alpers' 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 fiberdisease (MERRF), NARP-neurogenic muscle weakness, ataxia and retinitis pigmentosa, Pearson Syndrome, platinum-based chemotherapy-induced ototoxicity (PAT), and scleroderma. inducedototoxicity, Cockayne syndrome, xeroderma pigmentosum A, Wallerian degeneration, and HIV-induced lipodystrophy.

[0137] In some embodiments, the disease or condition is a neurodegenerative disease, including dementia, Alzheimer's disease (AD), Parkinson's disease.

[0138] 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.

[0139] 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).

[0140] 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).

[0141] In further exemplary embodiments, the transdermal formulations and systems can be used as transdermal therapies to prevent, manage, and reverse the clinical consequences of inflammatory diseases (including diabetes, COVID-19 infection, and sickle cell disease), provide local treatment of hypertension or local treatment of osteoarthritis, reverse the acute inflammatory cascade (cytokine storm), improve the safety and efficacy of transfused stored red blood cells, treat cerebral malaria or Chagas disease, or treat other early acute inflammatory diseases.

[0142] Phytochemicals, such as curcuminoids, have been shown to have antiviral activity. For example, it has recently been shown that the glycocalyx can block viral access to ACE2 binding receptors on endothelial cells, thereby limiting viral uptake and replication. Underlying endothelial dysfunction degrades the glycocalyx, thereby increasing viral access to ACE2 binding sites. 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 the treatment of inflammation triggered by toxic chemicals and metals, curcumin and other phytochemicals can chelate and eliminate toxins from the blood. It can also limit the inflammatory response to inhaled toxic agents, thereby reducing the propensity to progress to ARDS. Phytochemicals stabilize red blood cells, thereby minimizing hemolysis caused by toxic agents, which is a powerful trigger for inflammation.

[0143] The formulations disclosed herein are capable of addressing pro-inflammatory injuries, including acute inflammatory injuries caused by certain viral infections (e.g., SARS CoV2, dengue fever, 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-term COVID, side effects attributed to COVID include brain fog, fatigue, pain, clotting problems, myocarditis, edema, etc., which appear long after seemingly recovering from the primary infection. Most of these long-term 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-term COVID.

[0144] In some embodiments of the method of treatment, the transdermal formulation disclosed herein is used to treat diseases or conditions commonly associated with "cytokine storms", including but not limited to: COVID-19 infection, sepsis, systemic inflammatory response syndrome (SIRS), cachexia, septic shock syndrome, traumatic brain injury (e.g., brain cytokine storm), graft versus host disease (GVHD), or the results 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, sufficient concentrations of active agents (e.g., curcumin) can effectively block the binding of the spike protein on SARS CoV 2 to the ACE2 binding site on endothelial cells and lung epithelial cells, thereby inhibiting viral replication in susceptible subjects.

[0145] In some embodiments of the methods of treatment 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 due to 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.

[0146] In some embodiments of the methods of treatment disclosed herein, a transdermal formulation is administered to treat or reduce the risk of cardiovascular disease 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 disease. Non-limiting examples of cardiovascular disease 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.

[0147] In some embodiments, the amount / dose of the active agent is selected and / or the administration schedule is set so that the method increases or decreases the level of biomarkers 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 the control (not treated with the transdermal formulation) or the level before treatment with the transdermal formulation disclosed herein. Non-limiting examples of biomarkers associated with cardiovascular disease include white blood cell count (WBC), erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), cardiac troponin, creatine kinase (CK), CK-MB, and myoglobin. In some embodiments, the subject has an abnormal level of one or more biomarkers associated with cardiovascular disease before treatment, wherein the abnormal level of 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.

[0148] Further examples of infectious diseases that are often associated with "cytokine storms" or vascular leakage include, but are not limited to, coronaviruses (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, bacteria, fungus, helminth, protozoa, 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 used to treat septic shock syndrome, a chronic inflammatory response to infectious diseases.

[0149] Methods disclosed herein can also be applied to treat 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, the pain associated with periodontal or other dental surgery can be treated by inserting a sponge into a suitable position (e.g., between the cheek and the gums) in the subject's mouth. Transdermal preparations can be applied before or after the onset of pain. For example, preparations can be applied to a subject before surgery as a preventive method to alleviate pain.

[0150] Traditional drugs for neuropathic pain have side effects to varying degrees. The transdermal preparation 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, morphine, oxycodone, botulinum toxin-A and other opioids as third-line treatments. Therefore, transdermal preparations have the benefits of reducing dependence on traditional analgesics and minimizing side effects.

[0151] Transdermal formulations can also be used for postoperative pain management. For individuals undergoing surgery (including bypass surgery, thoracic surgery, coronary artery surgery, inguinal hernia repair and leg amputation), transdermal formulations can serve as an alternative therapeutic agent for treating the above painful conditions.

[0152] As described above, the preparation can be applied in any form suitable for the methods disclosed herein. In some embodiments of any preparation or method disclosed herein, the preparation is in semi-solid form or solid form and is applied or rolled 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 application. In some embodiments, the preparation is applied by a sprayer 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 starting to apply the preparation.

[0153] The transdermal formulations of the present disclosure can be administered to activate the NAD-dependent deacetylase sirtuin-1 (SIRT1) of a subject. Thus, 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 effects 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), originally discovered for its role in silencing DNA transcription, has also been shown to promote cell survival in response to caloric restriction. Similarly, in C. elegans, the sirtuin enzyme SIR-2.1 has been shown to extend lifespan. In mammalian cells, the sirtuin enzyme SIRT1 (homolog of yeast Sir2 and Caenorhabditis elegans SIR-2.1 enzymes) deacetylates the tumor suppressor p53 to promote cell survival. SIRT1 has been reported to regulate various pathways, including, for example, restoration of angiogenic function and secretion of pro-angiogenic factors in endothelial progenitor cells. Seminal papers have shown that SIRT1 is involved in preventing excessive inflammation and oxidative stress by deacetylating NFκB and Forkheadbox O transcription factors. In addition, SIRT1 inhibits cellular senescence, promotes keratinocyte differentiation, and protects against UV-induced DNA damage. Several studies have also shown that downregulated or dysfunctional SIRT1 is associated with various diseases (e.g., in the setting of diabetes), while SIRT1 overexpression improves glucose intolerance and insulin sensitivity and prevents diabetes. Sirtuins therefore appear to be activated as part of a beneficial cellular response to stress, leading to cell survival and lifespan extension.

[0154] Therefore, sirtuin activators may beneficially affect fundamental cellular processes, protect cells from stress, prevent or treat various diseases or conditions, and extend healthy lifespan.

[0155] Transdermal delivery of active agents (e.g., NO enhancing and SIRT1 activating therapeutic agents) allows for easy combination with oral treatments that target other relevant disease pathways that cannot be effectively addressed by transdermal agents. The method comprises administering a transdermal formulation disclosed herein to a subject in need thereof. In some embodiments, the formulation comprises (a) a therapeutically effective amount of a SIRT1 activator; (b) a polyol solvent sufficient to dissolve the SIRT1 activator in an amount; and (c) a fatty acid. The SIRT1 activator may be one or more of the NO enhancers described above. In some embodiments, the SIRT1 activator comprises one or more of curcuminoids, berberine, quercetin, resveratrol, and fisetin. The amount of the activator may be adjusted according to the properties 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 releases the activator continuously over about 15 hours.

[0156] Treatment of acute and chronic diseases or other conditions may benefit from increased systemic nitric oxide levels in the endothelium and / or activation of 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 surgically induced inflammation.

[0157] In some embodiments, the method enhances SIRT1 activity in a subject. The scope and composition of the preparation are as described above. In some embodiments, the preparation includes (a) a therapeutically effective amount of a SIRT1 activator; (b) a polyol solvent sufficient to dissolve the SIRT1 activator; and optionally (c) a fatty acid. The SIRT1 activator may be one or more NO enhancers as described above. In some embodiments, the SIRT1 activator includes one or more of curcuminoids, berberine, quercetin, resveratrol, and fisetin. The amount of the activator may be adjusted according to the properties of the activator and the disease or condition to be treated. In some embodiments, the weight percentage of the activator in the preparation ranges from about 0.05% to about 40%. In some embodiments, the preparation continuously releases 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 a neurodegenerative disease, diabetic nephropathy, diabetes, cardiovascular disease, endothelial dysfunction, muscular dystrophy, pain, a neuroinflammatory condition, abnormal vascular homeostasis, and lupus.

[0158] The transdermal preparation of the present disclosure can be applied to promote the therapeutic effect or reduce the adverse events of another therapy. In some embodiments of any method disclosed herein, the transdermal preparation of the present disclosure can be applied before, simultaneously or after another therapy, and another therapy includes, for example, oral administration of drugs, intravenous infusion, intramuscular infusion, local drug treatment and / or surgery. In some embodiments, the transdermal preparation is applied before the additional therapy for the disease or condition. For example, before transfusion, local pretreatment with the preparation disclosed herein can maximize tissue perfusion and minimize transfusion-related inflammation. Local pretreatment with the preparation or simultaneous application with another therapy can also reduce the adverse events related to the therapy (for example, side effects related to hypoglycemic drugs such as metformin, rash, oral mucositis / stomatitis related to chemotherapy).

[0159] Transdermal preparations can also enhance the endothelial function of the subject. Therefore, various diseases or conditions related to endothelial dysfunction or imbalance can be treated. The endothelium has two interrelated main elements that are crucial to vascular homeostasis: glycocalyx and endothelial nitric oxide synthase (eNOS). The hair-like protrusions from the endothelium are called glycocalyx, which is responsible for: i) maintaining vascular integrity, thereby limiting vascular leakage and the entry of macrophages and lipids into the deep layers of the vascular wall (triggering factors 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 needs by controlling eNOS to produce nitric oxide through shear stress; iv) limiting the entry of hematopoietic cells (erythrocytes, monocytes, leukocytes), platelets and infectious substances into the endothelium and binding to the endothelium; v) limiting the activation of platelets; vi) preventing blood flow stagnation; vii) ensuring that eNOS continues to produce NO by preventing the decoupling of eNOS caused by excessive ROS. In the uncoupled state, eNOS no longer generates NO, but instead produces more inflammatory ROS; viii) maintains a cell-free zone along the endothelium, thereby preventing erythrocytes from scavenging endothelial-generated NO close to the endothelium. At the same time, nitric oxide generated by endothelial nitric oxide synthase (eNOS) is essential for vascular homeostasis. Key functions of eNO include: i) maintaining tissue perfusion / oxygenation; ii) preventing blood flow stasis; iii) preventing a procoagulant environment; iv) repolarizing activated macrophages, thereby promoting tissue repair and limiting tissue damage; v) regulating pro- and anti-inflammatory processes (balancing pro-inflammatory iNOS activity that generates damaging peroxynitrite with anti-inflammatory eNOS activity that produces eNO), activating SIRT-1; vi) preventing inflammatory injury caused by ischemia-reperfusion and hypoxia-reoxygenation; vii) preventing ROS-induced damage, including lipid peroxidation and glycocalyx degradation; and viii) establishing a reservoir of stored nitrosothiols within the endothelium and surrounding vascular layers that can rapidly provide NO when elevated NO levels are needed, such as during extreme muscle activity.

[0160] Endothelial dysfunction is a physiological dysfunction 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 primarily expressed in endothelial cells. In a healthy vasculature, endothelial-produced NO diffuses to vascular smooth muscle cells (VSMCs), where it activates guanylate cyclase and stimulates the production of cyclic guanosine monophosphate (cGMP), thereby promoting VSMC relaxation and, in turn, 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 a 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).

[0161] Endothelial dysfunction is associated with a variety of diseases, including, for example, hypertension, coronary artery disease, heart failure, stroke, peripheral artery disease, diabetes, chronic renal 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 (e.g., chemotherapy), hemoglobinopathies (sickle cell disease, HbE / β-thalassemia, cytokine storm-related diseases caused by viral diseases (e.g., SARS CoV 2, dengue fever, influenza, hemorrhagic shock, hemorrhagic fever), erectile dysfunction secondary to inflammation caused by surgery, and inflammation associated with an increase in the senescent cell population that usually occurs with aging. In addition, endothelial dysfunction is considered a key event in the development of atherosclerosis and precedes clinically evident vascular pathology by many years. Endothelial dysfunction has also been shown to have predictive significance in predicting vascular events including stroke and myocardial infarction. In addition, 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.

[0162] The methods disclosed herein are applicable to treating acute consequences as well as chronic consequences of endothelial dysfunction. Examples of chronic consequences of endothelial dysfunction include the above-mentioned diseases and conditions. In some embodiments, the methods are applicable to treating acute consequences, including, for example, cytokine storms and associated physical activity or diet-induced hypoxic / ischemic organ damage (e.g., heart attacks due to insufficient tissue perfusion / oxygenation), stroke, microembolism and macroembolism, pulmonary embolism, ischemia-reperfusion injury, hypoxia-reoxygenation injury, and long-term COVID, which are the consequences of persistent chronic inflammation / endothelial dysfunction. In some embodiments, the method is useful for treating chronic consequences, including, for example, cardiovascular disease (CVD), coronary artery disease (CAD), renal failure, increased susceptibility to cognitive decline and dementia, hypertension, sexual dysfunction, slow wound healing, accelerated stent failure / closure, failed coronary artery bypass grafts, slow wound healing, reduced tolerance to physical activity due to mitochondrial dysfunction, accelerated age-related disease, osteoarthritis, transient ischemic events, diabetic retinopathy, decreased insulin production due to inflammation-induced pancreatic beta cell damage, HIV-induced CVD, sleep apnea and / or CAD and CVS secondary to persistent periodic episodes of blood flow stasis (e.g., sickle cell disease). Other applications of the method include transfusions (including red blood cells or hemoglobin-based oxygen carriers (HBOCs)) and renal dialysis.

[0163] Transdermal preparations can be used 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 potential and usually severe endothelial dysfunction, which can limit blood flow to damaged tissues. The method for treating slow healing of leg ulcers includes continuous local delivery of nitric oxide to eliminate biofilms and infections that hinder the therapeutic efficacy of medicaments intended to accelerate wound closure. At the same time, transdermal delivery of active agents (such as curcumin) will normalize the systemic vascular system, thereby promoting tissue oxygenation and allowing stem cell migration and development. Transdermal preparations can be co-administered with antibiotic agents or any suitable wound healing agents. For erectile dysfunction, transdermal delivery of agents (such as curcumin) can be used alone or in combination with local nitric oxide and / or oral PD5 inhibitors to restore systemic vascular health and reduce systemic inflammation. Given that systemic NO boosters such as curcumin enhance NO production in the endothelium and oral supplementation with PD5 inhibitors prolongs the duration of NO action, the combination of transdermally delivered active agents with oral PD5 inhibitors will accelerate endothelial recovery in patients with endothelial dysfunction, including long-term COVID and cytokine storm.

[0164] The transdermal formulation disclosed herein can also be applied 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 limited by any particular theory, it is presumed that the formulation provides pleiotropic effects of upregulating and / or activating a variety of anti-inflammatory and antioxidant enzymes and signaling pathways, including, for example, Sirtuin 1 (SIRT1) and other inflammation-regulating sirtuins, PPAR (γ) (peroxisome proliferator-activated receptor-γ), peroxisome proliferator-activated receptor-γ coactivator (PGC) -1α (which is a member of the transcription 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-generating processes such as glucose absorption 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 a master regulator of antioxidant and cytoprotective genes and is primarily activated in response to oxidative stress. The SIRT1 / PGC-1α / PPAR-γ pathway, eNOS-mediated enhancement of eNO production, PPARP, Nrf2, heme oxygenase, AMPK and ACE2 (angiotensin converting enzyme 2) or ACE2 "receptor", the protein that provides an entry point for the coronavirus to attach to and infect a variety of human cells. The formulations can also be used to downregulate or inhibit TLR4 (toll-like receptor 4, part of the inflammatory triggering 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).

[0165] The transdermal formulations described herein can be applied to treat and manage acute proinflammatory insults as well as chronic consequences of many proinflammatory conditions that can promote or be associated with endothelial dysfunction (ED). Non-limiting examples of diseases or conditions associated with endothelial dysfunction (ED) include cardiovascular disease, renal failure, cognitive decline, slow wound healing, hypertension, stroke, microembolism, edema, sexual dysfunction, retinopathy, neuropathy, and neuropathic pain.

[0166] For acute diseases or conditions, the preparation can quickly start overall anti-inflammatory and antioxidant activity, thereby shortening the cascade and limiting the progress of the severe consequences that cause extremely severe ED. For example, a suitable patch or sponge loaded with the preparation provides a very high concentration of NO stimulant (such as curcumin), and is inserted between the gums and cheeks for a time to be determined to ensure that the curcuminoids or other anti-inflammatory agents, antioxidants and NO stimulants at therapeutic levels are delivered quickly and continuously. This safe method eliminates concerns about direct NO overdose, and additionally activates a full set of host-based anti-inflammatory and antioxidant pathways. Using systemic or local active agents (such as curcuminoids) delivered transdermally can stimulate the NO in the vascular system to produce and / or reduce the excessive production of reactive oxygen species (ROS). This combination of enhancing the NO produced by the endothelium and shutting down ROS production is intended to prevent, limit and reverse ED and its consequences.

[0167] The transdermal formulations of the present invention can be applied to subjects in need for the treatment of diabetes and related inflammation and other conditions. Inflammation in adipose tissue promotes insulin resistance and hyperglycemia, both of which can cause 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 for type 2 diabetes and other proinflammatory factors to 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 lowering blood sugar, reversing insulin resistance, reducing elevated blood sugar levels or excessive and sustained blood sugar elevation caused by surgery, reducing ROS levels, increasing NO levels in the endothelium and restoring vascular homeostasis, the transdermal formulations of the present invention can minimize the negative and / or adverse consequences associated with surgery, transfusion, stent implantation, dialysis and any other invasive surgery 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 recovery of damaged endothelium, treating endothelial dysfunction and preventing the onset or progression of endothelial dysfunction). In addition, it limits the enhanced glycemic response of diabetic patients after surgery and shortens the recovery time of elevated glucose levels. In addition, it can limit prolonged and excessive stress-induced glucose peaks in diabetic patients under stressful conditions (such as injury, surgery and transfusion), as well as the inflammatory consequences of stress, including urogenital dysfunction after prostatectomy.

[0168] The transdermal formulation can thus be administered to limit negative clinical consequences associated with inflammatory triggers in subjects who have a pre-existing condition or are at risk of developing a condition that promotes hyperglycemia and subsequent endothelial dysfunction. In some embodiments, the formulation is administered prophylactically to a subject at risk of developing hyperglycemia. In some embodiments, the subject has been determined to have hyperglycemia.

[0169] In some embodiments, the amount / dose of the active agent is selected and / or the administration schedule is set so 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 a transdermal formulation) or before treatment with a transdermal formulation 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 before 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 a normal level or the level of a healthy subject.

[0170] The transdermal formulations disclosed herein can reduce the levels of proinflammatory cytokines. In some embodiments of the methods disclosed herein, the amount / dose of the active agent is selected and / or the administration schedule is set so that one or more biomarkers are reduced or changed 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 level before the control or treatment with the transdermal formulation. Non-limiting examples of markers 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 erythrocytes, selectins. , laminin and cahedrin, immunoglobulin receptors, chondroitin sulfate, syndecan-1, IL-1a / b, TNF-a, IL-6, D-dimer and other markers reflecting abnormal blood coagulation tendency, embolism, thrombosis, C-reactive protein (CRP), Nrf2, NFκB, glutathione peroxidase (GPx), superoxide dismutase (SOD), Syndecan-1, HMW-hyaluronan (1,000-6,000kDa), disintegrin and metalloprotease with thrombospondin type 1repeats-13, protein C, von Willebrand factor, chondroitin sulfate and sP-selectin. Other examples include markers associated with 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 abnormal levels of one or more biomarkers or proinflammatory cytokines prior to treatment, wherein the abnormal levels of the biomarkers or one or more cytokines are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 100% higher or lower than the normal level or the level of a healthy subject.

[0171] 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 infection, bacterial infection, and fungal infection.

[0172] 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, neuroinflammatory conditions, abnormal vascular homeostasis, lupus, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, neurodegenerative consequences of traumatic brain injury or cerebral hemorrhage, hypertension, inflammation, osteoarthritis, rheumatoid arthritis, endothelial dysfunction, skin diseases, ophthalmic diseases, bacterial infections, viral infections, ischemia-reperfusion injury, hypoxia-reoxygenation injury, cytokine storm phenomenon, cerebral malaria, Chagas disease, hemoglobinopathy, type 2 diabetes, coronavirus, skin / dermatological conditions, acne, inflammatory skin diseases, Raynaud's disease, postherpetic lesions, lesions), shingles, skin infections, wounds, burns, leg ulcers, sickle cell, diabetes, onychomycosis, peripheral vascular disease, infected and / or inflamed mucosal tissue, erectile dysfunction, female sexual dysfunction, vaginal infection / inflammation, catheter-related urinary tract infection, sinusitis, cystic fibrosis, acute respiratory distress syndrome, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, lung infections (including tuberculosis), pulmonary hypertension, burns and other open wounds, inner ear infections, outer ear infections, gastrointestinal disorders, 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 coronaviruses (including SARS-CoV-2), Ebola virus, dengue fever, hemorrhagic shock, endotoxic shock, acellular hemoglobin toxicity caused by hemolysis and / or the use of acellular hemoglobin blood substitutes (HBOCs), Rift Valley fever, Marburg virus, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fevers, dengue fever, yellow fever, Omsk hemorrhagic fever virus, Kyosanur Forest virus, Junin virus, Machupo virus, Sabia virus, Guanarito virus, Garissa virus, Elesa virus, and Lassa fever virus.

[0173] Further examples of diseases or conditions that can be treated with the formulations disclosed herein include neurodegenerative diseases or conditions (e.g., Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, and disorders caused by polyglutamine aggregation); skeletal muscle diseases (e.g., Duchenne muscular dystrophy, skeletal muscle atrophy, Becker muscular dystrophy, or myotonic dystrophy); metabolic disorders (e.g., insulin resistance, diabetes, obesity, impaired glucose tolerance, hypercholesterolemia, hyperglycemia, dyslipidemia, and hyperlipidemia); adult-onset diabetes, diabetic nephropathy, neuropathy (e.g., sensory neuropathy, autonomic neuropathy, motor neuropathy, retinopathy); bone diseases (e.g., osteoporosis), blood diseases (e.g., leukemia); liver diseases (e.g., due to alcohol abuse or hepatitis); obesity; bone resorption, macular degeneration aging, AIDS-related dementia, ALS, Bell's palsy (Bell's palsy), atherosclerosis, heart disease (e.g., arrhythmias, chronic congestive heart failure, ischemic stroke, coronary artery disease, and cardiomyopathy), chronic degenerative disease (e.g., 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 (e.g., mitochondrial myopathy, encephalopathy, Leber's disease, Lee encephalopathy, Pearson Disease), lactate acidosis, "mitochondrial encephalopathy, lactate acidosis and stroke-like symptoms" (mitochondrial encephalopathy, lactate acidosis and stroke-like symptoms, etc.), 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 undesirable 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 cerebral hemorrhage, sickle cell disease, thalassemia (e.g., HbE / β-thalassemia), diabetic retinopathy, glaucoma, dry eye, surgically 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 radiation therapy, brain fog, rheumatoid arthritis, soft tissue damage (muscle, tendon and ligament), inflammatory sequelae of surgery (urogenital dysfunction), transfusion-induced inflammation, inhibition of stent restenosis, restenosis), inflammatory consequences that limit dialysis, inflammation and pain following dental procedures, neuropathic pain due to any proinflammatory insult (including peripheral neuropathy, spinal neuropathy, arthritis pain), cognitive impairment in children due to cerebrovascular damage from sickle cell disease, cytokine storms due to coronaviruses, dengue, Ebola, Rift Valley fever, and influenza, cerebral malaria, metastatic spread of tumors through dysfunctional blood vessels, systemic consequences of psoriasis, dementia (including Alzheimer's disease and Pick's disease), post-traumatic brain injury, and hemorrhagic shock.

[0174] Autoimmune and immune-related disorders and diseases can also be treated or prevented using 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, demyelinating polyneuropathy, hepatobiliary disease, 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 disease, bullous skin disease, erythema multiforme, contact dermatitis, psoriasis, allergic disease, asthma, allergic rhinitis, atopic dermatitis, food allergyhypersensitivity), urticaria, immune diseases of the lungs, eosinophilic pneumonias, idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, systemic lupus erythematosus, scleroderma, and arthritis.

[0175] Non-limiting examples of neurological diseases that can be treated or whose progression can be limited using the methods of the present disclosure 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), ALS-Parkinson's-Dementia complex of Guam, Wilson's disease, cerebral palsy, progressive supranuclear palsy, supranuclearpalsy (Steel-Richardson syndrome), bulbar and pseudobulbar palsy, diabetic retinopathy, multi-infarctdementia, macular degeneration, Pick's disease, diffuse Lewy body disease, prion diseases such as Creutzfeldt-Jakob, Gerstmann-Straussler-Scheinker disease, Kuru, and fatal familial insomnia, primary lateralsclerosis, degenerative ataxias, Machado-Joseph disease / spinocerebellar ataxia type 3, and olivopontocerebellar degeneration degeneration), spinal and spinobulbar muscular atrophy (Kennedy's disease), familial spastic paraplegia, Wohlfart-Kugelberg-Welander disease, Tay-Sachs diseasedisease), multisystem degeneration (Shy-Drager syndrome), Gilles De LaTourette'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 disorders, Binswanger's disease, and dementia (including all potential causes of dementia).

[0176] Insulin resistance disorders that can be treated with the methods of the present disclosure 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, hypertension, high cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, complications of diabetes including coronary heart disease, angina, congestive heart failure, stroke, cognitive function of dementia, retinal lesions, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, certain types of cancer (such as endometrial cancer, breast cancer, prostate cancer and colon cancer), pregnancy complications, poor female reproductive health (such as irregular menstruation, infertility, irregular ovulation, polycystic ovary syndrome (PCOS)), fat metabolism disorders, cholesterol-related conditions such as gallstones, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and prevention and treatment of bone loss, such as osteoporosis. Further applications of the methods disclosed herein include promoting wound healing, for example, it can also be used to promote wound healing and diabetic-wound healing disorders.

[0177] In some embodiments of any of the methods disclosed herein, further comprising the step of determining that the subject has downregulated or dysfunctional SIRT1 compared to a normal standard or reference. In some embodiments, the methods disclosed herein further comprise diagnosing the subject with endothelial dysfunction or a disease or condition associated with endothelial dysfunction prior to administering the transdermal formulation to the subject.

[0178] In some embodiments of the methods disclosed herein, the step of determining that the subject's systemic NO level or plasma nitrite and / or nitrate level 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 normal levels or healthy reference standards is included.

[0179] In any method disclosed herein, the treatment regimen can be administered after clinically observed symptoms or clinical manifestations of a disease or condition. Alternatively, the method can be used prophylactically before any clinical symptom onset or observation of any clinical symptom. For example, the method disclosed herein can address proinflammatory damage, including acute inflammatory damage caused by certain viral infections (such as SARSCoV2, dengue fever and influenza), inflammatory triggers induced by obesity and glucose, and inflammation caused by exposure to toxic metals and chemicals. Treatment can be administered when symptoms have been determined or before any symptom onset or observation. In a further example, a transdermal preparation can be administered prophylactically to prevent the inflammatory consequences of surgery, including pain or erectile dysfunction caused by postoperative inflammation.

[0180] Methods disclosed herein can increase NO levels systemically or locally. In some embodiments of any transdermal preparation or method disclosed herein, the amount of active ingredients (e.g., NO enhancers and / or NO precursors, polyols, optional fatty acids and / or other components) in the transdermal preparation is selected so that the NO level or plasma nitrite and / or nitrate level before the control or application of the preparation is compared to the systemic or local NO level or plasma nitrite and / or nitrate level of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60% or more. In some embodiments, the desired increase or change can be achieved within about 1 hour, within about 2 hours, within about 3 hours, within about 5 hours, or within about 8 hours. In some 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 more. A variety of methods can be used to measure NO levels or plasma nitrite and / or nitrate levels, including, for example, colorimetric methods using Griess reagent and chemiluminescent methods.

[0181] In some embodiments of any of the transdermal formulations or methods of the present disclosure, the amount of active ingredients (e.g., NO booster and / or NO precursor, polyol, optional fatty acid and / or other components) in the transdermal formulation is selected so that the subject's systolic and / or diastolic blood pressure and / or mean arterial pressure is reduced 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 the above 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days period as compared to a control or NO level or plasma nitrite and / or nitrate level before administration of the formulation.

[0182] 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 applied 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, the formulation, for example, in the form of a patch, cream, or gel, is applied once every 1, 3, 5, 7, or 10 days.

[0183] Another aspect of the present disclosure 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 best achieved by a combination of repolarization of macrophages / monocytes / microglia from M1 to M2 phenotypes, reduction of senescent cell populations, and anti-inflammatory / antioxidant activity. In some embodiments, CD38 is located in cells (on the cell surface or in cells). In some embodiments, the contact occurs in vitro. In some embodiments, the contact occurs in vivo.

[0184] Another aspect of the present disclosure provides a method of incorporating an agent into a cell by contacting the cell with a formulation disclosed herein. Due to increased NO levels, the method can stabilize the cell, improve storage characteristics, and reverse storage damage. In some embodiments, the cell is a red blood cell. In some embodiments, the formulation is in liquid form. In some embodiments, the formulation includes a molecule containing S-nitrosothiol.

[0185] Treatment of cells (e.g., erythrocytes) with the formulations described herein can deliver NO boosters (e.g., curcumin) and optional other anti-inflammatory / antioxidants into lipid membranes or beyond the cytosol, should 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 formulations containing high concentrations of NO precursors (e.g., lipophilic S-nitrosothiols and S-NO derivatives of alkyl ester derivatives of NAC) should be effective in transnitrosating thiols in and on cells. Nitrosation of key thiols on and in erythrocytes (including thiols of β93 and Band 3 proteins on Hb) has been shown to stabilize erythrocytes against oxidative damage, microparticle formation, and hemolysis. Therefore, such erythrocytes can also be used as vehicles to deliver NO to thiols on the endothelium.

[0186] In another aspect, the present invention provides a method for improving the safety and effectiveness of transfusion by contacting the cells or fluids to be transfused with a formulation before or during the transfusion process. For example, a formulation containing a NO enhancer or NO precursor can be administered before or during a transfusion to protect red blood cells and enhance the therapeutic effect. In some embodiments, the method comprises contacting the formulation with the cells or fluid 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or more than 4 hours before the transfusion occurs.

[0187] A related aspect provides a method for transdermal delivery of an active agent to a subject in need thereof. The method comprises administering a transdermal formulation disclosed herein to a subject. Rapid and prolonged therapeutic effects can be achieved by delivering an effective amount of an active agent locally or systemically. The scope of the target disease or condition is as described above. In some embodiments, the transdermal formulation comprises one or more curcuminoids as active ingredients for treating a disease or condition and one or more of polyphenols, flavonoids, stilbenes, secosteroids, or natural products that optionally promote NO production. In some embodiments, the transdermal formulation comprises one or both of curcumin and quercetin, and one or more of optional polyphenols, flavonoids, stilbenes, and secosteroids.

[0188] Manufacturing method

[0189] Another aspect of the present invention provides a method for manufacturing a preparation disclosed herein. The method generally includes preparing a solution of a polyol solvent and a fatty acid penetration enhancer, and then adding a medicament (NO enhancer or NO precursor). If necessary, the solution can be heated to a suitable temperature to dissolve the fatty acid and / or the medicament. In some embodiments, the active agent (e.g., NO enhancer or NO precursor or nitrite source) is saturated in the solution. After cooling the solution, the preparation can become a gel, and the excess active agent precipitated can be easily removed. Mixing or adding different ingredients / components or changes in the order of conditions or sequences are also feasible, as long as the NO enhancer or NO precursor is appropriately distributed in the preparation to achieve the desired therapeutic effect.

[0190] In some exemplary embodiments, the medicament dissolved in the solution is loaded into a container (e.g., a sprayer or atomizer; a permeable or frangible pouch as described above) or soaked in a distribution carrier (e.g., a swab, sponge, or absorbent layer of a patch). When the NO precursor mixture contains a nitrite source, the acid source can be stored, for example, in a separate pouch or in 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.

[0191] In some embodiments, the preparation is in the form of a gel or semisolid. A gelling agent or thickening agent can be added to adjust the preparation form. The preparation is then loaded into a suitable container and then distributed in a gel, ointment, cream, emulsion, microemulsion, nanoemulsion, paste, balm or other suitable form. The semisolid preparation can also be coated on a backing member (e.g., a support layer of a patch).

[0192] Depending on the amount and nature of the thickener, the formulation can also be made into a solid form. For example, a solution of a NO enhancer (e.g., 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 against human skin.

[0193] The general procedures for mixing reagents and carrying out the manufacturing process can be obtained by common sense or pharmaceutical technology manuals familiar to those skilled in the art, for example, Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins, Philadelphia, 2000, or in the review article Souza et al, Topicalocular delivery of therapeutics: carrier systems and physical methods, J. Pharm. Pharmacol., 2013, 66, 507-530.

[0194] Administration regimen

[0195] The effective amount of an agent (NO enhancer or NO precursor) in a formulation or kit described herein that effectively enhances systemic NO levels will depend on the route of delivery, the type of subject being treated (including humans), and the physical characteristics of the particular subject being considered. The dosage or amount can be adjusted to achieve the desired effect, but will depend on body weight, diet, concurrent medication, and other factors that will be recognized by those skilled in the art of medicine. More specifically, an effective amount or therapeutically effective amount refers to an amount of an agent that effectively increases systemic NO to a level that prevents, alleviates, or improves symptoms of a disease or prolongs the survival of a 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 locally more than twice or more than three times a day. Alternatively, the amount or delivery frequency can be reduced, if desired. Those skilled in the art are fully capable of determining an effective amount, especially in light of the detailed disclosure provided herein.

[0196] In non-human animal studies, the use of potential products is started 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 dosage range may 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 about 10 μg / kg body weight to about 100 mg / kg body weight, preferably about 100 μg / kg body weight to about 10 mg / kg body weight. Alternatively, the dose can be based on and calculated according to the patient's body surface area, as understood by those skilled in the art.

[0197] In exemplary embodiments, 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.

[0198] The exact formulation, route of administration and dosage of the pharmaceutical composition can be selected by the individual physician according to the patient's condition (see, e.g., Fingl et al. 1975, "The Pharmacological Basis of Therapeutics", which is incorporated herein by reference in its entirety, particularly with reference to Chapter 1, page 1).

[0199] It should be noted that the attending physician will know how and when to terminate, interrupt or adjust the administration to deal with toxicity or organ dysfunction. On the contrary, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. When managing the disease of interest, the amplitude 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 evaluation methods. In addition, dosage and dosage frequency may also vary according to the age, weight and response of individual patients. Programs comparable to the above-mentioned programs can be used in veterinary medicine.

[0200] The efficacy and toxicity of the formulations disclosed herein can be evaluated using known methods. For example, the toxicology of the formulation can be established by determining the in vitro toxicity to a cell line (e.g., a mammal, preferably a human cell line). The results of such studies can generally predict the toxicity in an animal (e.g., a mammal, or more specifically, a human). Alternatively, the toxicity in an animal model (e.g., a mouse, rat, rabbit, or monkey) can be determined using known methods. The efficacy of a particular compound can be determined using several recognized methods (e.g., in vitro methods, animal models, or human clinical trials). Almost every disease type has a recognized in vitro model. Similarly, acceptable animal models can be used to determine the efficacy of chemical drugs to treat such diseases. When selecting a model to determine the efficacy, those skilled in the art can select appropriate models, dosages, routes of administration, and regimens 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 a transdermal formulation in the human body.

[0201] If desired, the formulations or kits described herein may be presented in the form of a package or dispenser device that may contain one or more unit dosage forms containing a formulated medicament. For example, the package may include a metal or plastic foil, such as a blister pack. The package or dispenser device may be accompanied by instructions for administration. The package or dispenser may also be accompanied by a notice associated with the container, the form of which is specified by a government agency that manages the manufacture, use or sale of drugs, which reflects the agency's approval of the drug form for human or veterinary administration. For example, the notice may be a prescription drug label approved by the U.S. Food and Drug Administration, or an approved product insert.

[0202] All references cited herein are hereby incorporated by reference in their entirety.

[0203] Example

[0204] Example 1

[0205] Preparation of transdermal preparations

[0206] Vehicles for loading the active ingredient were prepared as shown in samples 1-4. Myristic acid (solid flakes) was dissolved in PEG400 after the mixture was heated in a warm water bath. Oscillation accelerated the formation of the resulting clear solution. The following four concentrations of myristic acid (MA) solutions in PEG400 were prepared and evaluated. When the samples reached room temperature, samples 1, 2, 3 and 4 were all clear solutions.

[0207] a.30ml PEG400+1.5g MA (sample 1)

[0208] b.30ml PEG400+3g MA (sample 2)

[0209] c.30ml PEG400+0.75g MA (sample 3)

[0210] d.30ml PEG400+2.25g MA (sample 4)

[0211] Loading NO enhancers into samples as carriers. 95% pure curcumin (Curcumin 95) was loaded as a NO enhancer into the sample carrier formulation [or solvent system] to test the loading potential. 1.5 grams of Curcumin 95 was added to samples 1, 2, and 3. The resulting Curcumin 95 loaded samples were heated and shaken until the amount of curcumin dissolved reached a maximum and then cooled back to room temperature. All three samples dissolved almost all of the added curcumin, forming intense dark solutions. 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 grams of MA in 30 ml of PEG400 and then heating in a water bath (~60-80°C) for about 15 minutes. Sample 4 was prepared by dissolving 2.25 grams 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, both sample 3 and sample 4 were homogeneous solutions. After cooling, sample 3 remained liquid, while sample 4 formed a homogeneous gel.

[0212] After determining the loading potential, a sample transdermal formulation containing curcugen as a NO enhancer was prepared. Curcugen is a product containing curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC). These three curcuminoids account for about 50% by weight in curcugen. Sample preparations V3.3 and V4.3 are made by adding curcugen to sample 3 and sample 4 carriers, respectively (3 grams of curcugen dissolved in 30ml of either solvent). The resulting mixture was heated and vibrated for about 15 minutes, after which both presented a dark chestnut uniform solution. After cooling, V3.3 (3 grams of curcugen / 30ml sample 3 carrier) remained liquid; while V4.3 (3 grams of curcugen / 30ml sample 4 carrier) became a uniform gel with the same dark chestnut color (the same color as V3.3 when heated).

[0213] No loss of color was observed for samples stored at room temperature for at least three months. In stark contrast, the same volume of water containing the same added amount of curcumin showed almost no color in the liquid phase and a large amount of undissolved material.

[0214] V4.3 is loaded into the syringe while warm and is therefore in a liquid state. The liquid in the syringe gels when cooled to room temperature, but can be squeezed out of the syringe as a gel and slowly melts on the skin. The applied gel is easily covered and captured by a transparent bandage that is waterproof and leakproof, such as the Mepitel transparent film dressing. The dressing prevents loss of the applied formulation and can remain in place for several days without loss of formulation. When the Mepitel dressing is removed after a few days, there is no adhered formulation on the dressing and all the formulation is in the skin, which is reflected by the lack of staining on the tissue of the colored skin under the dressing when rubbed vigorously. The color disappears completely after 7-8 days.

[0215] Adding water to PEG400 / MA-based formulations produces an inhomogeneous emulsion, which makes topical use difficult. The presence of water limits 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 grams of curcumin in 30 ml of water). The resulting mixture had almost no color in the liquid phase and contained a large amount of undissolved material.

[0216] Using higher concentration MA solutions with PEG as the base solvent, concentrated saturated solutions of low solubility NO enhancing actives (e.g., curcumin, curcugen, quercetin, berberine, and related molecules) can be easily prepared by adding excess active to a heated high MA PEG / MA solvent system (e.g., V4.3) and then allowing the mixture to cool. The resulting homogenous gel is completely saturated with the active, while the undissolved excess remains as a solid at the bottom of the tube. Thereafter, the homogenous saturated gel can be easily removed and separated from the undissolved material.

[0217] The Sample 3 solvent system was also tested for its loading potential as a carrier for the following low solubility agents: quercetin, berberine, N-acetylcysteine ​​amide (NACA), and N-acetylcysteine ​​ethyl ester (NAC-ethyl ester) using the above method. In all cases, the solvent system allowed a large amount of solubility of these poorly soluble agents. Comparison of the aqueous solution with the Sample 3 solution loaded with quercetin and berberine revealed that only a very small amount of dissolved material was found in the aqueous sample, while the degree of solubility in Sample 3 was very high.

[0218] Similar results were obtained with quercetin in samples 3 and 4 as carriers, but the solubility of quercetin in these solvents was lower than that of curcugen (about 200-300 mg quercetin in 30 ml of sample 3 or sample 4). Like curcumin and curcugen, sample 3 loaded with quercetin did not form a gel at room temperature, while sample 4 loaded with quercetin did form a gel at room temperature.

[0219] PEG400 / MA solution was mixed with other delivery vehicles (e.g., vaseline). Sample 3 containing curcumin was mixed with vaseline to obtain a uniformly colored gel that remained stable (no color loss) for more than two months. Sample 3 containing dissolved NAC amide and SNO derivatives of NAC was easily mixed with vaseline to produce a stable pink jelly. Stability may be due to low water activity and high viscosity, which inhibits the loss of NO in the thiol group. Samples of sample 3 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 rubbing human skin with pressure, the solid material melted. Similar results were obtained using coconut oil, but the melting point of coconut oil is lower, making it difficult to apply on human skin without getting dirty and dripping. A combination of the two oils was also tested. PEG400 / MA when used with cocoa butter showed the most promising properties as a topical delivery vehicle for cosmetic and dermatological applications due to the more suitable hardness of the resulting curcumin-loaded cocoa butter. Increasing the amount of PEG400 / MA solvent added in cocoa butter eventually produced a gel-like material whose color remained stable.

[0220] Solutions of the two aforementioned lipophilic NAC derivatives were prepared using PEG400 saturated with sodium nitrite to obtain clear solutions. Under conditions of limited NAC solubility, the lipophilic NAC derivatives are easily soluble. NAC is soluble in water, while the two derivatives are slightly soluble in water. The two solutions were then treated with a few drops of acetic acid to trigger the formation of nitrous acid from nitrite, which can then nitrosate the reactive thiols on the two NAC derivatives. The solution turns pink, indicating the formation of S-nitrosothiol. The solution remains pink for several days. The corresponding aqueous solution loses color within a few hours. The use of PEG400 saturated with nitrite enables the preparation to be used to generate NO and S-nitrosothiol by mixing with an agent that acidifies the mixture. The two frangible pouches in the patch can be used as a NO sustained delivery carrier, suitable for topical and transdermal applications.

[0221] Example 2

[0222] The effect of the V2.3 formulation (2 grams of curcugen / 30 ml of Sample 3 vehicle) on blood pressure (BP) in rats 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. All rats (N=5) experienced a 20% decrease in systemic BP within 15 to 20 minutes of application of V2.3. The reduced BP was maintained over a three hour observation window with no signs of recovery. A second laboratory performed similar tests on 6 rats and the results (N=6) showed similar results (20% decrease in BP). Similar results were consistently obtained for V3.3 after V3.3 was applied to the forearm of a human subject (~0.05 ml). A similar decrease in BP occurred within 15 to 20 minutes of application and the reduced BP persisted for many hours, gradually returning to the higher initial value after 12 to 24 hours. Similar results were obtained in the same human subject using V4.3. Cocoa butter adulterated with Sample 3 (V1.3) also exhibited similar physiological consequences when applied to a single human test subject. Control Sample 3, which did not contain one or more curcuminoids or curcugen, did not induce any physiological consequences when applied to rats or humans.

[0223] V3.3 was applied to a skin flap with an optical window on healthy hamsters. The optical window allowed monitoring of blood vessels in the dermis beneath the site of V3.3 application. Within minutes of application, a dose-dependent increase in the diameter of blood vessels at the site of topical application could be observed.

[0224] Effect on NO plasma levels. After topical application of V3.3 to four rats, plasma levels of NO degradation products (nitrite / nitrate) were measured. The results showed that plasma levels of NO degradation products (nitrite / nitrate) increased by 15% in two of the tested rats, which was consistent with a decrease in blood pressure, resulting from increased nitric oxide production due to curcumin's known ability to upregulate endothelial nitric oxide synthase (eNOS) and therefore NO production. Two control animals under the same conditions did not show this increase. The results show that transdermal curcuminoids can increase systemic NO levels.

[0225] The blood pressure dropped within 15 to 20 minutes after topical application of the curcumin-containing sample, indicating that curcumin is being delivered transdermally and that therapeutically effective levels exist in a short period of time. The drop in blood pressure and the increase in plasma levels of nitrite / nitrate are consistent with the known effects of curcumin, i.e., the nitric oxide production of eNOS in the endothelium is upregulated. The physiological response can last for many hours, which is consistent with the delivery of topically delivered curcumin / curcugen to the blood circulation in a sustained manner. In contrast, the circulation time of curcumin delivered to the circulation by oral route or IV is only 2 hours because the liver rapidly converts curcumin into an inactive agent. Even oral high-dose curcumin has not been observed to produce such a significant and lasting physiological response.

[0226] 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) were also tested for physiological responses of transdermal curcuminoid formulations in animals.

[0227] Blood pressure was measured after topical application of a transdermal curcuminoid formulation in rodents. Both rats and mice showed a decrease in BP of up to 20%, and V3.3 was more effective than V4.3 both in terms of onset time and magnitude of BP decrease.

[0228] It was also observed that topical application of V3.3 or V4.3 resulted in elevated plasma nitrite and nitrate levels within a three-hour monitoring window following a single topical dose. During the same time period, BP continued to decrease (within a three-hour window). Following topical application of V3.3 in rats (N=3), a sustained increase in plasma curcuminoids at detectable concentrations was observed within three hours. Oral curcumin plasma levels peaked within one hour and dropped to nearly undetectable levels within three hours.

[0229] Example 3

[0230] Control of systemic inflammation by transdermal delivery of NO boosters or NO precursors.

[0231] The inhibitory effect of topically applied Vascarta formulation V3.3 on the development of severe vascular leakage in a rat model of acute inflammation was tested. The following lipopolysaccharide (LPS)-induced cytokine storm protocol was used in this study:

[0232] a. LPS (10 mg / kg) was infused IP every 24 hours to initiate and maintain an acute inflammatory response.

[0233] b. Starting from the first LPS treatment, the LPS-treated rats were subjected to topical application of Vascarta formulation V3.3 (0.1 ml) every 24 hours for three days.

[0234] c. Physiological parameters measured by blood draw every 24 hours

[0235] d. Three days later, the animals were anesthetized and the cavity was surgically opened to allow for intravital fluorescence imaging of the macrovasculature and microvasculature.

[0236] i. Fluorescently labeled albumin and dextran (500 kDa) preparations were infused IV and fluorescent images of the vasculature and surrounding tissues were used to determine the rate at which albumin and dextran extravasated from the vasculature and entered the surrounding tissues.

[0237] Topical application of vehicle (PEG400 / MA) to LPS-treated rats demonstrated a rapid and extremely severe leakage pattern for both albumin and, to a greater extent, dextran, consistent with what is observed under conditions of severe acute inflammation. However, daily topical application of V3.3 significantly reduced the amount of albumin leakage. The low levels of leakage observed were essentially the same as those observed in control animals. Similar results were observed for dextran. Extensive vascular leakage is a potentially fatal consequence of a cytokine storm, regardless of the cause (COVID-19, Ebola, dengue, hemorrhagic shock, endotoxin shock, Rift Valley fever, etc.). The transdermal formulation provides a significant positive intervention outcome with far-reaching clinical implications.

[0238] Example 4

[0239] 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) delivered transdermally in a mouse model of acute vascular inflammation. Three cohorts, each with three subjects, were studied and compared. In cohort 1, subjects were infused with LPS but did not receive any topical treatment (untreated). In cohort 2, subjects were infused with LPS and topical treatment (0.1 ml of V4.3). In cohort 3, topical treatment (0.1 ml of V4.3) was performed four hours after LPS infusion. Endotoxemia was induced by infusion of 10 mg / kg LPS (lipopolysaccharide from Escherichia coli serotype O128:B12, Sigma Aldrich St. Louis, MO). The procedure was identical to that described in an earlier published study (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).

[0240] It was observed that topical application of V4.3 (a curcuminoid dissolved in a PEG400 / myristic acid mixture) can act as both a preventive and interventional treatment for lipopolysaccharide (LPS)-induced cytokine storm. Results included vascular consequences and changes in cytokine profiles over time in three different groups of LPS-treated mice: i) not treated topically with V4.3; ii) pretreated with topical V4.3 prior to LPS treatment; and iii) treated topically with V4.3 after the onset of LPS-induced cytokine storm. In terms of the response of the microvasculature to LPS treatment in the three groups, topical treatment with V4.3 both limited arterial dilation (indicating shock-induced vascular collapse) and maintained arterial blood flow. It was also observed that topical treatment with V4.3 prevented the acute drop in functional capillary density (FCD) that accompanies LPS-induced endotoxemia. V4.3 can act as both a preventive and interventional treatment in preventing the acute drop in FCD relative to baseline (BL). FCD is associated with survival because it reflects the ability to maintain tissue perfusion and deliver oxygen to tissues. In addition, topical application of V4.3 was found to limit the production of proinflammatory cytokines both pre-treatment and interventional. These results confirm the efficacy of transdermal formulations in limiting the consequences of inflammation when applied topically as a preventive agent or as an active treatment after the onset of inflammation.

[0241] Example 5

[0242] This study evaluated the use of the transdermal formulation of the present invention to prevent LPS-induced vascular leakage by topical application of curcuminoids in an LPS endotoxemia rat model. LPS (Escherichia coli O26: B6) was inoculated into the rat model (10 mg / kg / day). A transdermal curcumin formulation (V3.3: curcugen 9g, myristic acid 2.25g, PEG40090ml) or vehicle control was applied daily for three days (0.1ml / dose). On day 3, the animals were prepared for surgery for in vivo and fluorescence microscopy examinations. It was observed that treatment with a transdermal curcumin formulation prevented early leakage induced by LPS (the first 4 hours). In the LPS-induced inflammatory model, 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) both before and after LPS-induced inflammation in mice (N=3) reduced the LPS-induced increase in pro-inflammatory cytokine levels.

[0243] The present experiments, combined with the results of earlier studies, suggest that the topical transdermal curcumin formulations of the present disclosure can limit inflammation-induced vascular leakage, which is a hallmark of cytokine storms and other inflammation-inducing conditions.

[0244] Example 6

[0245] The skin permeability of the transdermal formulations of the present invention was studied. The skin permeability of the following transdermal formulations of the present invention was studied using confocal microscopy. These formulations differed only in the amount of myristic acid:

[0246] a. Batch No. 43: V4.3 Curcugen 9g, Myristic Acid 6.75g, PEG 40090ml

[0247] b. Batch No. 39: V3.3 Curcugen 9g, Myristic Acid 2.25g, PEG 40090ml

[0248] c. Batch No. 38: V0.3 Curcugen 9g, PEG 40090ml

[0249] When the temperature approaches zero degrees Celsius, the formulation solidifies. When the temperature rises above room temperature, the viscosity of all samples decreases significantly. The viscosity of the formulation and its components are shown below:

[0250] 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.

[0251] Curcuminoids have a broad absorption spectrum with a maximum absorbance at 425nm. 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.

[0252] All three formulations showed penetration of the curcuminoids into the skin. As early as the one-hour time point, penetration of the curcuminoids into the stratum corneum was observed for all three formulations.

[0253] In the formulation of batch 38, the widening of the fluorescence band occurred between 3 and 6 hours in both donors. The widening of the fluorescence band was not seen until the 24th hour in the formulations of batches 39 and 43. The widening indicates that curcumin penetrates into the upper epidermal layer just below the stratum corneum. In the formulation of batch 39, the amount of curcuminoids decreased between 3 and 6 hours in both donors. However, in the formulations of batches 38 and 43, a decrease in the intensity of curcuminoids was observed after 6 hours. In the formulation of batch 38, fluorescent spots were seen below the stratum corneum at early time points. However, when using the formulations of batches 39 and 43, no fluorescent spots were seen above the stratum corneum. The transepidermal water loss (TEWL) values ​​for all formulations and all time points were comparable, indicating that the barrier integrity of all human skin samples was the same.

[0254] The fluorescence decreased over time in the three formulations, indicating that the Curcuminoids had penetrated deeper into the skin. (Due to the high autofluorescence of untreated skin, low penetration of active ingredients may not be seen and / or quantified). Based on this, formulation No. 39 showed the earliest penetration of the Curcuminoids into the skin. The depth of fluorescence was also determined. The measurements were between 5 microns and 25 microns. The formulation No. 38 showed the fastest widening of the fluorescence band, indicating that the Curcuminoids penetrated into the skin more quickly.

[0255] Example 7

[0256] The effect of the transdermal formulation of the present disclosure in limiting or preventing cardiovascular-induced inflammatory episodes in a diabetic rat model was tested in a ZDSD diabetic rat model. Three rats were on a normal diet for 60 days and had no symptoms of diabetes. Starting from about the 65th day, the rats began a high-fat diet, which resulted in a slow rise in blood sugar. Throughout the test period, the animals were topically treated with formulation V4.3 every two days until 80 days. The cytokine profile showed an onset of inflammation at about the 75th day. Treatment with formulation V4.3 limited the increase in proinflammatory cytokines observed in the shams on the 75th day. In particular, the IL-18 profile clearly demonstrated that the formulation can prevent the increase in IL-18, which is a marker for the tendency of diabetic patients to develop cardiovascular consequences.

[0257] Example 8

[0258] The therapeutic effect of formulation V4.3 on mice with severe advanced endothelial dysfunction was tested. 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 free access to food and water. The study was conducted for 4 weeks. The first week was used for adaptation as the animals were divided into different experimental groups. Both groups received L-NAME (50 mg / kg) in 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, 0.1 ml per day for 10 days and then characterized. The group not treated with L-NAME served as a mock control group.

[0259] Topical treatment was initiated after the onset of the condition. Results showed direct evidence of treatment, i.e., restoration of elements of normal endothelial function (reduced oxidative stress in plasma and erythrocytes, reduced adhesion of leukocytes to the endothelium, indicating restoration of the glycocalyx, and improved functional capillary density). The transdermal treatment group showed lower levels of TNF-α, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, IL-10, and IL-12 compared to the mock control group. Better results were also observed for the treatment group in terms of microhemodynamic changes, cell adhesion, vascular reactivity of isolated aortic vessels, changes in erythrocyte and plasma antioxidants, hypoxia, reoxygenation, and systemic hemodynamic changes.

[0260] Table 1: Changes in body weight, relative tissue weights and water intake at the end of the study.

[0261]

[0262] Table 2: Changes in red blood cell and plasma antioxidants at the end of the study

[0263]

[0264] Those skilled in the art will recognize that the invention described herein is not limited to what has been specifically shown and described. On the contrary, the scope of the invention is defined by the claims that follow. It should also be understood that the above description represents only illustrative examples of embodiments. The specification does not attempt 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 ingredients, or other undescribed PEG alternative embodiments that may be used in formulations, kits or methods should not be considered as not claiming these alternative embodiments. It will be recognized that many undescribed embodiments are covered within the literal scope of the appended claims, and other embodiments are equivalent.

Claims

1. A transdermal preparation comprising: (a) an effective amount of a NO enhancer, comprising an effective amount of a curcuminoid and an effective amount of one or both of at least one flavonoid, wherein the curcuminoid is selected from curcumin, demethoxycurcumin and bisdemethoxycurcumin, wherein the at least one flavonoid is selected from quercetin, apigenin, fisetin, luteolin and rapamycin; (b) a polyol in an amount sufficient to dissolve an effective amount of the Curcuminoids and at least one flavonoid; and optionally (c) fatty acids, wherein the ratio of polyol to fatty acid is in the range of about 10:1 to about 50:1 by weight, in, The amounts of the polyol and fatty acid are selected so as to deliver an effective amount of the NO booster transdermally upon administration.

2. The transdermal preparation according to claim 1, wherein the curcuminoid compound is curcumin.

3. The transdermal preparation according to claim 1, wherein the preparation comprises curcumin; and at least one of quercetin and apigenin.

4. The transdermal formulation of claim 1, wherein the ratio of the Curcuminoids to the at least one flavonoid is in the range of about 1:1 to about 10:

1.

5. The transdermal formulation of claim 1, wherein the formulation comprises quercetin and apigenin in a ratio ranging from about 1:1 to about 10:

1. The transdermal preparation according to claim 1 , wherein the fatty acid is myristic acid.

7. The transdermal preparation according to claim 1, wherein the polyol is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol and glycerol.

8. The transdermal formulation of claim 1, wherein the polyol is polyethylene glycol having a molecular weight ranging from 200 to about 600.

9. The transdermal formulation of claim 1, comprising about 3 wt% to about 10 wt% of a Curcuminoid.

10. The transdermal formulation of claim 1, wherein the amounts of Curcuminoids and polyols are selected to provide sustained release of Curcuminoids over a period of about 15 hours.

11. The transdermal formulation according to claim 10, wherein the ratio of the polyol to the curcuminoid is in the range of about 8:1 to about 12:

1.

12. Use of a transdermal formulation in the preparation of a medicament for treating a disease or condition associated with elevated CD38 levels, wherein the transdermal formulation comprises: (a) an effective amount of a NO enhancer, comprising an effective amount of a Curcuminoid compound and an effective amount of at least one flavonoid compound or both, wherein the Curcuminoid compound is selected from the group consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin; (b) a polyol in an amount sufficient to dissolve an effective amount of the Curcuminoids and at least one flavonoid; and optionally (c) fatty acids, wherein the ratio of polyol to fatty acid is in the range of about 10:1 to about 50:1 by weight, in, The amounts of the polyol and fatty acid are selected so as to deliver an effective amount of the NO booster transdermally upon administration.

13. The use according to claim 12, wherein the disease or condition is selected from lupus, rheumatoid arthritis, multiple sclerosis, leukemia, multiple myeloma, neurodegenerative diseases, aging-related physical and cognitive decline, chronic and acute inflammatory conditions, renal failure, coronary artery disease, heart failure, stroke, peripheral arterial disease, myocardial infarction, diabetes, hypertension, neuropathic pain, osteoarthritis, anemia of chronic disease, ALS, Parkinson's disease, ischemia-reperfusion injury, hypoxia-reoxygenation injury, transfusion-induced injury, radiation-induced injury including dermatitis and neuroinflammation.

14. The use according to claim 12, wherein the disease or condition is selected from dementia, Alzheimer's disease (AD) and Parkinson's disease.

15. The use according to claim 12, wherein the disease or condition is selected from lupus, rheumatoid arthritis, multiple sclerosis, leukemia and multiple myeloma.

16. The use according to claim 12, wherein the disease or condition is type 2 diabetes.

17. Use of a transdermal preparation in the preparation of a medicament for reducing CD38 activity, wherein the transdermal preparation comprises: (a) an effective amount of a NO enhancer, comprising an effective amount of a Curcuminoid compound and an effective amount of at least one flavonoid compound or both, wherein the Curcuminoid compound is selected from the group consisting of curcumin, demethoxycurcumin and bisdemethoxycurcumin; (b) a polyol in an amount sufficient to dissolve an effective amount of the Curcuminoids and at least one flavonoid; and optionally (c) fatty acids, wherein the ratio of polyol to fatty acid is in the range of about 10:1 to about 50:1 by weight, in, The amounts of the polyol and fatty acid are selected so as to deliver an effective amount of the NO booster transdermally upon administration.

18. The use according to claim 17, wherein CD38 is located in cells.

19. The use according to claim 18, wherein the contacting occurs in vitro.

20. The use according to claim 18, wherein the contacting occurs in vivo.

21. The use according to claim 12, wherein the formulation comprises curcumin; and at least one of quercetin and apigenin.

22. The use according to claim 21, wherein the polyol is polyethylene glycol having a molecular weight in the range of 200 to about 600.

23. The use according to claim 17, wherein the formulation comprises curcumin; and at least one of quercetin and apigenin.

24. The use according to claim 23, wherein the polyol is polyethylene glycol having a molecular weight in the range of 200 to about 600.

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