Patent Application
20230233532
This disclosure relates to compounds for the prevention or inhibition of metastasis in cancer, and more particularly to sphingosine-1-phosphate receptor 1 agonists and compositions thereof.
Metastasis is the leading cause of cancer morbidity as well as mortality and is responsible for approximately 90% of cancer death (1-3). Metastasis is a general term to describe cancer cells traveling from primary sites to neighboring tissue or distant sites, which involves multiple steps including local invasion, intravasation, survival in circulation, extravasation, and proliferation in distant tissues (4). Each step can be rate-limiting since disruption of any of the multiple processes can retard metastasis (5).
The endothelial cell (EC) layer of vessels plays important roles during intravasation and extravasation in the metastatic cascade (6). While quiescent, non-activated ECs can reduce tumor cell transmigration and metastasis, inflammation increases these processes to promote cancer cell metastasis (7-9). The literature shows that melanomas and circulating cells secrete multiple factors, including thrombin, VEGF and monocyte chemoattractant protein 1 (MCP1/CCL2), which can disrupt endothelial integrity by triggering actin remodeling, cellular contractility and junctional disassembly (10-14).
Increases in vascular permeability mediated by inflammatory mediators can cause changes in EC junctions through modifications in adherens and tight junctions (15). Vascular endothelial (VE) cadherin at the edges of ECs plays a major role intracellularly linking actin filament in the cytoskeleton (16). Inflammatory factors can cause internalization of VE-cadherin, whose absence then disrupts the cellular interactions leading to gap formation. In addition, contractile forces mediated by actin and myosin further promotes gap formation (17).
The actin-myosin action depends on the phosphorylation status of myosin light chain (MLC) (18). Increased phosphorylated-MLC (pMLC) levels triggered by inflammatory mediators causes changes in the actin stress fiber leading to the formation of gaps in the EC layer (19).
Sphingosine-1-phosphate (S1P) is a natural lipid, and its levels in the plasma has been identified as a biomarker for vascular barrier integrity (20). Cell culture studies showed that S1P increased the levels of VE-cadherin and p-catenin at EC junctions and enhanced adherens and tight junctional assembly (21). Studies in intact microvessels revealed that the effect of S1P on maintaining microvessel permeability occurred through the activation of EC S1PR1, and the blockage of S1PR1 abolished the inhibitory effect of S1P on inflammatory mediator-induced increases in microvessel permeability (22). This disclosure translates these observations to cancer treatment by developing a bioavailable S1PR1 agonists to enhance the endothelium barrier integrity and reverse the effects of inflammatory mediators, thereby inhibiting the transit of metastatic cells across vascular walls.
Several S1P receptor agonists have been developed for treating inflammatory diseases but none are useful for clinical cancer therapy (23). The most well-known S1P analog, FTY720 (fingolimod) is FDA-approved for treating multiple sclerosis but is not useful in cancer because it acts as a broad spectrum S1PR1/3/4/5 agonist (24). This leads to S1PR1 degradation and lymphopenia making it unsuitable to strengthen the endothelial barrier and reduce metastasis (25). SEW2871, a S1PR1-selective agonist, has been shown to fully replicate the action of S1P on endothelium integrity in intact microvessels, and potently inhibited the inflammatory mediator-induced increases in vascular permeability (22). Unfortunately, SEW2871 is not bioavailable requiring solubilization in a DMSO containing solution, which limits its clinical utility, but is useful for research purposes. The chemical structure of SEW2871 also prevents it from being incorporated into a bioavailable nanoliposomal formulation.
There is a clear need for therapeutics that can be used in the treatment and prevention of metastasis.
The present disclosure provides a compound, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, that attenuates the metastasis of cancers in subjects in need thereof by selectively activating sphingosine-1-phosphate receptor 1 (S1PR1) to tighten the junctions between endothelial cells in the vascular endothelium. The presently disclosed compound can also more readily formulated as a liposome as compared to other S1PR1 agonists, allowing it to be more readily delivered to desired vascular tissue.
Thus in one aspect, Compound I is provided of the following formula
or a pharmaceutically acceptable salt, prodrug, or derivative thereof.
A pharmaceutical composition is also provided comprising Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, and a pharmaceutically acceptable carrier.
The present disclosure also provides a formulation comprising:
a plurality of nanoliposomes comprising a phospholipid and a PEG-phospholipid; and
Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, encapsulated within the plurality of nanoliposomes.
In some embodiments, the phospholipid comprises L-α-phosphatidylcholine (ePC). In some embodiments, the PEG-phospholipid comprises 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DPPE-PEG 2000).
In another aspect, a method of treating or preventing metastasis of a cancer in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof.
In another aspect, a method of treating or preventing diabetes, hyperglycemia, or complications arising therefrom in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof.
In another aspect, a method of decreasing vascular permeability in a subject in need thereof is provided, the method comprising administering a therapeutically effective amount of Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
The following description of the disclosure is provided as an enable teaching of the disclosure in its best, currently known embodiments. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. The following definitions are provided for the full understanding of terms used in the specification.
As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.
As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
Administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration”, “administration in combination”, “simultaneous administration” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.
As used here, the terms “beneficial agent” and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms “beneficial agent” or “active agent” are used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.
As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.
As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.
By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective’ amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
As used herein, a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term “therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
As used herein, “pharmaceutically acceptable salt” is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.
Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disuifonic, oxalic, isethionic, HOOC—(CH2)n- COOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).
Also, as used herein, the term “pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
As used herein, the term “subject” or “host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals.
Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human.
The present disclosure provides Compound I having the following formula:
or a pharmaceutically acceptable salt, prodrug, or derivative thereof. Compound I is also referred as ASR396 in various parts of the present disclosure.
Compound I may be prepared according using a process according to the synthetic scheme provided in
The compounds as used in the methods described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the active components described herein can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the active components of their compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art.
Compositions, as described herein, comprising an active compound and an excipient of some sort may be useful in a variety of medical and non-medical applications. For example, pharmaceutical compositions comprising an active compound and an excipient may be useful for the treatment or prevention of an infection with a Mycobacterium.
“Excipients” include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and/or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).
Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition or cosmetic composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and/or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.
Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.
Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.
Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof. Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and/or combinations thereof.
Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.
Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.
Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.
Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, varoius gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, polyglycolide, polyl(lactide-co-glycolide) and poly(epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid/acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxide-propylene oxide), and a Pluronic polymer, polyoxy ethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG-stearate, 1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000], 1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000], and 1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000]), copolymers and salts thereof.
Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof. In certain embodiments, the emulsifying agent is cholesterol.
Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles.
Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is admixed with an excipient and any needed preservatives or buffers as may be required.
The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.
The active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
The active ingredient may be administered by any route. In some embodiments, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
Useful dosages of the active agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
In some aspects, Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, may be prepared as a nanoliposomal formulation.
Thus in one aspect, a formulation is provided comprises:
a plurality of nanoliposomes comprising a phospholipid and a PEG-phospholipid; and
Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, encapsulated within the plurality of nanoliposomes.
In some embodiments, the phospholipid may be selected from a glycerophospholipid.
In some embodiments, the phospholipid may be selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine (PS), a phosphatidylinositol (PI), a phosphatidic acid (PA), a phosphatidylglycerol (PG), and a cardiolipid (CL). In some embodiments, the glycerophospholipid may be a dimyristoyl, a dipalmitoyl, or a distearoyl glycerophospholipid. The phospholipid may be derived from an animal tissue source (such as bovine brain or egg yolk) or a vegetable oil source (such as soybean, corn, cotton seed, sunflower, or rapeseed oil). In some embodiments, the phospholipid is derived from egg yolk. In other embodiments, the phospholipid is derived from soybean oil.
In some embodiments, the phospholipid may be selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dieurocoyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoryl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-3-phospho-L-serine (POPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1-stearoyl-2-lyso-sn-glycero-phosphocholine (S-LysoPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1-palmitoyl-2-lyso-sn-glycero-phosphocholine (P-LysoPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), 1,2-dilinoleol-sn-glycero-3-phosphoethanolamine (DLoPE), 1-myristoyl-2-lyso-sn-glycero-3-phosphocholine (M-LysoPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1-oleoyl-2-lyso-sn-glycerophosphocholine (O-LysoPC), salts thereof (such as, for example, sodium salts or ammonium salts), and combinations thereof.
In some embodiments, the PEG-phospholipid is selected from 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (DMG-PEG 2000 or DMG-PEG 5000), N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2000 or DSPE-PEG 5000), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG 2000 or DSG-PEG 5000), N-(methylpolyoxyethylene oxycarbonyl)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG 2000), N-(methylpolyoxyethylene oxycarbonyl)1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG 2000 or DPPE-PEG 5000), N-[2′,3′-bis(metylpolyoxyethylene oxy)propane-1′-oxycarbonyl]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-2arm-PEG 2000 or DSPE-2arm-PEG 5000), 1,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG 2000 or DPG-PEG 5000), 1,2-dioleoyl-rac-glycerol methoxypolyethylene glycol (DOG-PEG 2000 or DOG-PEG 5000), salts thereof (such as, for example, sodium salts or ammonium salts), or combinations thereof.
In some embodiments, the plurality of nanoliposomes have a molar % ratio of the phospholipid to the PEG-phospholipid greater than 50:50, for example a ratio of 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, or 90:10. In particular embodiments, the plurality of nanoliposomes have a molar % ratio of the phospholipid to the PEG-phospholipid of about 80:20.
In some embodiments, the present disclosure provides a formulation comprising: a plurality of nanoliposomes comprising L-α-phosphatidylcholine (ePC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]ammonium salt (DPPE-PEG 2000); and Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, encapsulated within the plurality of nanoliposomes.
In some embodiments, the plurality of nanoliposomes may have an average diameter from about 50 nm to about 90 nm, from about 55 nm to about 85 nm, or from about 60 nm to about 85 nm. In some embodiments, the nanoliposomes may have an average diameter of about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, or about 90 nm.
In some embodiments, the plurality of nanoliposomes may have a zeta potential of about 0.10 to about 0.40 mV, for example from about 0.20 to about 0.30 mV. In some embodiments, the plurality o nanoliposomes may have a zeta potential of about 0.10 mV, about 0.12 mV, about 0.14 mV, about 0.16 mV, about 0.18 mV, about 0.20 mV, about 0.22 mV, about 0.24 mV, about 0.26 mV, about 0.28 mV, about 0.30 mV, about 0.32 mV, about 0.34 mV, about 0.36 mV, about 0.38 mV, or about 0.40 mV
It is understood and herein contemplated that the compositions disclosed herein, for example, Compound I or a pharmaceutically acceptable salt, prodrug, or derivative thereof, either alone or as a pharmaceutical composition, in particular as a nanoliposomal formulation as described herein are effective in increasing vascular integrity/decreasing vascular permeability. Thus, in one aspect disclosed herein are methods of decreasing vascular permeability in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of Compound I or a pharmaceutically acceptable salt, prodrug, or derivative thereof, either alone or as a pharmaceutical composition, in particular as a nanoliposomal formulation as described herein.
Increased vascular permeability is a complication that can arise in diabetes and lead to complications such as diabetic retinopathy. Thus, in one aspect disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and/or preventing diabetes, hyperglycemia, and/or complications arising from diabetes and/or hyperglycemia in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of Compound I or a pharmaceutically acceptable salt, prodrug, or derivative thereof, either alone or as a pharmaceutical composition, in particular as a nanoliposomal formulation as described herein.
The present disclosure also provides for preventing metastasis of a cancer in a subject need thereof, the method comprising administering a therapeutically effective amount of Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, either alone or as a pharmaceutical composition, in particular as a nanoliposomal formulation as described herein.
In another aspect, the present disclosure provides a method for inhibiting metastasis of a cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Compound I, or a pharmaceutically acceptable salt, prodrug, or derivative thereof, either alone or as a pharmaceutical composition, in particular as a nanoliposomal formulation as described herein.
A non-limiting list of different types of cancers for which metastasis can be inhibited or prevented by the disclosed methods is as follows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general.
Representative examples cancers for which metastasis can be inhibited or prevented using the compound described herein include, but are not limited to: B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancers, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, AIDS-related lymphomas, or AIDS-related sarcomas.
Further examples of cancers for which metastasis can be inhibited or prevented by the disclosed methods include: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, and/or rectal cancers.
By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.
4-Phenyl-5-trifluoromethylthiophene-2-carboxylic acid (
3-(3-Methoxy-phenyl)-5-(4-phenyl-5-trifluoromethyl-thiophen-2-yl)-[1,2,4]oxadiazole (
3-[5-(4-Phenyl-5-trifluoromethyl-thiophen-2-yl)-[1,2,4]oxadiazol-3-yl]-phenol (Compound I) was synthesized as follows. To a stirred solution of intermediate 3 (0.166 g, 0.412 mmol) in CH2Cl2 under nitrogen was added BCl3 (6.18 mL, 6.18 mmol, 1M solution in CH2C12) slowly at 0° C. The resulting solution was stirred for 12 hours at room temperature.
After completion of the reaction as indicated by TLC, the reaction was quenched with slow addition of H2O (5 mL). The aqueous layer was extracted with CH2C12 (2×30 mL), and the combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4 and concentrated. The residue was purified by chromatography on silica gel using 7:3 v/v hexanes/ethyl acetate as the eluent to afford the pure Compound I as a white solid at a 92% (0.147 g) yield; m.p. 131-133° C.; 1H NMR (500 MHz, CDCl3): δ 5.40 (br s, OH, 1H), 7.05 (ddd, J=8.0, 2.5, 1.0 Hz, 1H), 7.41 (t, J=8. Hz, 1H), 7.47-7.50 (m, 5H), 7.64-7.65 (m, 1H), 7.75 (dt, J=8.0, 1.0 Hz, 1H), 7.92 (d, J=1.5 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 170.1, 168.8, 155.9, 145.3, 134.1, 133.1, 130.8, 130.5, 128.9, 128.6, 127.5, 126.6, 125.1, 120.2, 118.7, 118.6, 114.3; ESI-MS m/z 387 (M-H)+.
Compound melting points were recorded on a Fischer-Johns melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker Advance 500 instrument in CDCl3, operating at 500 and 125 MHz, respectively. Chemical shifts are reported in 6 values (ppm) and J values are reported in hertz (Hz). Experiments involving moisture and/or air sensitive components were performed under a nitrogen atmosphere in oven-dried glassware. Reagents, starting materials, and anhydrous solvents were purchased from commercial suppliers and were used as received. Reaction courses were monitored by thin-layer chromatography (TLC) on pre-coated silica gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany), and the spots were visualized under UV light. The crude reaction products were purified by silica gel column chromatography using silica gel 60 Å (Merck, 230-400 mesh).
In Silico Binding Assessment of Compound I to Ensure Retention of Interaction with S1PR1.
Crystal structure of S1PR1 receptor (PDB ID: 3V2W) was prepared and the active site pocket assigned as described previously (26). The crystal ligand in the active site pocket was examined with the various designed S1PR1 agonists and the docking interaction scores were calculated using the Glide module of the Schrodinger software.
The substructure-matching algorithm Erebus (erebus.doklab.org) was used to find proteins featuring a similar or identical binding site to S1PR1. Erebus is a substructure-matching platform that is based on a select query of inter-atomic distances, which define a structural motif on a bait protein, performs a search of the entire RCSB protein database for all proteins featuring a similar or identical scaffold (27). Using Erebus, off-target proteins from the RCSB database were identified by matching substructures with the same amino acids and atoms separated by the same distances (within a given tolerance) as the atoms of the query structure. The accuracy of a match was measured by the root-mean-square deviation (RMSD) or by the normal weight with a given variance.
A 80:20 mol % L-α-phosphatidylcholine (ePC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DPPE-PEG-2000) at a final concentration 25 mg/mL along with Compound I (in methanol at 5 mg/mL) was utilized for manufacturing a nanoliposomal formulation of Compound I (Avanti Polar Lipids) (28). The solvent from the mixture was evaporated under N2 gas and re-suspended in saline to make a final concentration of 5 mg/mL of the nanoliposomal formulation. The rehydrated mixture was heated at 60° C. for 60 minutes followed by sonication and extrusion through a 100 mm polycarbonate membrane.
Drug encapsulation efficiency was performed using a filter separation process as described previously (28). Compound I not encapsulated in the nanoliposomes was separated by centrifugation for 30 minutes using a 10 kDa filter. Drug from the purified nanoliposomal formulation was extracted by nanoparticle destruction addition of a 1:1 chloroform and methanol solution. Precipitated lipids were removed by centrifugation and the Compound I concentration was extrapolated from a standard curve using a UV-visible spectrophotometer with a λmax of 254 nm. Percentage of drug encapsulated was estimated as 100−(free drug after filter separation/total drug before separation)×100. Release kinetics were measured using dialysis through a 25 kDa membrane. Briefly, the nanoliposomal formulation was placed in a dialysis membrane and suspended in 1 L of a saline solution with constant stirring at 300 rpm for 120 hours. A 1-mL sample was taken from the dialysis bag at indicated time points, and the concentration of Compound I was measured by absorption at 254 nm after extracting with 1:1 chloroform:methanol.
Stability of the nanoliposomes containing Compound I was performed as described previously (28). The nanoliposomal formulation was stored at 4° C. for indicated times. The size and charge of the nanoliposomes were calculated using a Malvern Zetasizer. The nanoliposomes were considered stable if there was less than a 5% change in the size and charge of the nanoliposomes.
For vitrification, each TEM grid (QUANTIFOIL R 2/1, Electron Microscopy Sciences) with a thin layer of carbon film deposited over the holes was pre-treated by glow discharge. Samples (3.5 μl) were applied to the grid and vitrified using the vitrobot (Thermo Fisher) under the following condition: 4° C., 100% of humidity, and 3 seconds of blot time. Images were recorded by the electron microscope Arctica (Thermo Fisher) equipped with Ceta camera (4096×4096 pixels, Thermo Fisher). The imaging conditions was 92,000× in magnification, 4 in spot size, 2.7 mm in spherical aberration, at an accelerating voltage of 200 KV
Swiss-Webster mice were treated intravenously at indicated doses of the nanoliposomal formulation daily for 15-days and compared to control nanoliposomes lacking the agent. Changes in the animal body weights, motility and behavioral changes were monitored daily to measure body weight or behavioural related toxic effects (28). At the end of the 15-day treatment regimen, animals were euthanized and blood was collected to assess potential toxic effects. Serum samples were analyzed for levels of biomarkers that correspond to major organ functions as an assessment of toxic effects. Specifically, alanine aminotransferase, alkaline phosphatase, albumin, globulin, total protein, total bilirubin, blood urea nitrogen, glucose, creatinine, calcium and cholesterol were evaluated (29). Levels were compared to animals treated with nanoliposomes lacking Compound I, which served as the control.
The human melanoma cell lines, A375MGFP, UACC 903M-GFP and 1205Lu-GFP were maintained in DMEM (Invitrogen) supplemented with 5% FBS (Atlantic Biologicals) and GlutaMAX (Life Technologies). Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza and maintained on gelatin-coated plates in endothelial growth media (EGM) supplemented with 2% FBS (Lonza). HEK293A-S1P1-GFP cells were maintained in DMEM with 10% FBS and 200 μg/ml G418 (30). Melanoma cells were used within 20 passages; HUVEC cells were used between passages 2-5. SEW2871 was purchased from Cayman Chemical and W146 was from TOCRIS. Bradykinin (BK) was obtained from Millipore Sigma and vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) were from Peprotech.
HUVECs were seeded on coverslips coated with fibronectin (Millipore Sigma) and cultured to confluency. The inhibitors of VEGF (nintedanib), ICAM-1 (A286982), PDE-4 (roflumilast), and mTOR (tacrolimus), and S1PR1 agonists, SEW2871 (DMSO final concentration 0.05%), Compound I (DMSO final concentration: 0.05%), and the nanoliposomal formulation of Compound I (saline) were added to HUVECs at 5 μM for 30 minutes before cells were stimulated with 0.5 μM BK for 30 minutes, 100 ng/mL VEGF or 150 ng/mL IL-8 for 1 hour. HUVECs fixed with 2% cold paraformaldehyde (Electron Microscopy Science) and treated with 0.1% triton X-100 (Fisher) were exposed to a human VE-cadherin antibody (Santa Cruz) followed by the secondary antibody conjugated with Alexa488 (Invitrogen). Coverslips were mounted to slides using the antifade mounting medium with DAPI (Vector Laboratories).
HEK293A-21P1-GFP cells were plated on collagen-coated coverslips (30). Cells were cultured overnight in DMEM with 2% charcoal-stripped serum. Prior to the experiment, cells were cultured in serum-free DMEM for 1.5 hours. Cells were treated with 5 μM W146 for 30 minutes followed by 5 μM SEW2871 and Compound I. After washing with ice cold PBS, cells were fixed in 2% cold paraformaldehyde and mounted to slides using the antifade mounting medium with DAPI.
Experiments were undertaken on mesenteric venules from female Sprague-Dawley rats (2-3-month old, 220-250 g body weight, Sage Laboratory). Inactin hydrate was used for anesthesia through subcutaneous administration. The initial dosage of inactin was 180 mg/kg body weight and an additional 5 mg/dose given as needed. After the rat reached the appropriate plane of anesthesia, determined by the loss of the toe pinch response and the righting reflex, a midline surgical incision was made in the abdominal wall. A loop of the ileum was gently extended from the abdominal cavity and the mesentery was spread over a pillar attached to an animal tray. The upper surface of the mesentery was continuously perfused with mammalian Ringer's solution at 37° C. All experiments were carried out on venules with diameters ranging between 40 to 45 m. Each experiment was performed on a single vessel with one experiment per animal. Euthanasia was performed through bilateral thoracotomy while animals were under anesthesia.
Microvessel permeability was assessed by measuring one of the permeability coefficients, hydraulic conductivity, Lp, using the modified Landis technique. The assumptions and limitations of the method have been evaluated in detail (31-33). Briefly, a single venular microvessel was cannulated with a glass micropipette and perfused with albumin-Ringer solution (control) containing 1% (v/v) rat red blood cells as markers. A hydrostatic pressure (range 40-70 cm H2O), controlled by a water manometer, was applied through the micropipette to the microvessel lumen. The initial water flux (Jv) per unit area of microvessel wall (S), (Jv/S)0, was calculated from the velocity of the marker cell after the vessel was occluded, the vessel radius, and the length between the marker cell and the occlusion site. Microvessel Lp was calculated from the Starling equation, Lp=(Jv/S)/ΔP, where ΔP represents the pressure difference between the hydrostatic pressure applied to the microvessel and the effective oncotic pressure generated from the albumin in the perfusate, assuming the tissue hydrostatic and oncotic pressures are negligible (31). In each experiment, baseline Lp and the stimulus-induced Lp changes were measured in the same vessel, which allows the changes to be compared with its own control. If Lp is relatively constant throughout the time course, the mean Lp value for each perfusate was calculated from all of the occlusions during that perfusion period. If a transient increase in Lp is observed, Lp is reported as the means of peak and sustained values. Details are shown in
A monolayer of HUVEC cells following varying treatments were lyzed in RIPA buffer containing protease and phosphatase inhibitors (Pierce Biotechnologies). 20 μg of cell lysate was used for western blotting and probed with antibodies according to the suppliers' recommendations to assess off target effects on several major signaling pathways. Antibodies to p-ERK, ERK, p-PI3K, PI3K, p-FAK, FAK, p-PAK1, PAK1, p-PAK2, PAK2, p-MLC, p-NF-κB, NF-κB were used to probe western blots (Cell Signaling). α-Tubulin (Millipore Sigma) was used as a loading control.
Athymic Foxn1nu nude mice were intravenously injected with 15 mg/kg the nanoliposomal formulation of Compound I or nanoliposome lacking Compound I (Lipo) vehicle control, for 6, 12 or 18 hours prior to intravenous injection of 1 million A375M-GFP cells through the tail vein. For control comparison purposes, groups of nude mice were injected with 5 mg/kg SEW2871 or DMSO control intraperitoneally. For experiments using UACC 903M-GFP and 1205Lu-GFP cells, 15 mg/kg of the nanoliposomal formulation were given 6 hours before cancer cell injection. Body weight was monitored to ensure no significant toxicity during the experimental metastasis assay. On day 29, whole lungs were removed and imaged by fluorescent microscopy to quantify the numbers of metastatic nodules in a double-blinded manner.
All values of Lp and tumor metastasis were means±SE. In vitro results were shown as mean±SD. Paired t-test (Nonparametric Wilcoxon matched-paired signed rank test, two-tailed) was used for paired experiments conducted in the same vessel, and Mann-Whitney test (two-tailed) was used for data comparison between two groups. One- and two-way ANOVA with Tukey post hoc test was used to compare data among multiple groups. A probability value of P <0.05 was considered statistically significant. Each “n” of microvessel perfusion experiment represents one experiment conducted in each rat.
Compound I was synthesized as shown in
To predict whether the chemical modifications of SEW2871 led to any unexpected off-target interactions, the interaction plot of Compound I with S1PR1 was undertaken. This suggested that the binding scaffold of S1PR1 populated with R120, M124, F125, and V194 had significant interactions with Compound I. R1280 and F125 have been reported to be important for maintain the potency of SEW2871 (36). Hence, the side chain atoms of R120, M124, F125, and V194 engulfing Compound I in the binding pocket of S1PR1 were extracted and submitted as a query to Erebus. To precisely identify the most similar binding scaffolds to our query structure, a cut-off of RMSD≤5 Å to the query was imposed (Table 1). Erebus identified the query substructure itself from S1PR1 in the RCSB database (PDB IDs: 3V2Y, 3V2W) (
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To identify a nanoliposomal formulation enabling optimal loading of Compound I, several formulations were examined and results for size, zeta potential charge, membrane potential and surface hydration were evaluated (data not shown). The optimal liposomal formulation selected contained the PEGylated nanoliposomal formulation made of 80:20 mol % ePC: DPPE PEG-2000. The average size of the nanoliposomes in saline ranged between 68.14±4.42 nm with a zeta potential of 0.24±0.02 mV. Cryo-electron microscopy demonstrated the spherical structure of the nanoliposomes and that the nanoparticles were not aggregations (
The drug release kinetics of Compound I was measured by dialyzing the nanoliposomal formulation in saline over 120 hours, upon which the dialyzed nanoliposomes were lysed and the remaining drug measured by extrapolation off a standard curve (
Previous studies conducted in individually perfused intact microvessels demonstrated that S1P had a potent inhibitory effect on inflammatory mediator-induced increases in microvessel permeability, mediated through the activation of S1PR1 (22,38). In this example, the effect and specificity of the S1PR1 agonist nanoliposomal formulation was examined on platelet activating factor (PAF)-induced permeability increases and results were compared to SEW2871 under identical experimental conditions. Permeability was assessed by measuring hydraulic conductivity, Lp, in individually perfused intact rat mesenteric venules. Each vessel was first cannulated and perfused with the control perfusate (1% bovine serum albumin, BSA, in Ringer's solution) to measure baseline Lp. The mean baseline Lp of 6 vessels was 1.14±0.16×104 cm/s/cm H2O. Perfusion of vessels with SEW2871 (10 PM) for 30 minutes did not change the baseline Lp. However, when each of the vessels was perfused with PAF (10 nM) in the presence of SEW2871, the PAF-induced Lp increase was significantly inhibited with the mean Lp value at 1.73±0.15 times that of the control. To test the vessel normal response to PAF, each vessel was given a second application of PAF after washing out SEW2871 with albumin-Ringer perfusion for 40 minutes. Then each vessel showed a typical PAF response with the mean peak Lp increased to 7.50±0.63 times that of the baseline. The time course of the Lp changes from an individual experiment was shown in
The effect of the nanoliposomal formulation on PAF-induced permeability increases were examined following the same experimental procedures as those used for SEW2871. The mean baseline Lp of 4 vessels was 1.72±0.13×104′ cm/s/cm H2O. Perfusion of the nanoliposomal formulation (100 μM) did not alter baseline Lp but significantly inhibited PAF-induced Lp increase with a mean peak Lp at 2.92±0.14 times that of the control value. In contrast, perfusion of empty nanoliposome alone showed no effect on PAF-induced Lp increase. Overlay of two-individual experiments with the application of the nanoliposomal formulation and nanoliposome alone demonstrated that the significant inhibition of PAM-induced Lp increase was due to the effect of Compound I and not empty nanoliposome (
The action of the nanoliposomal formulation as a specific S1PR1 agonist was examined in vessels that were pretreated with S1PR1 antagonist W146 before the application of the nanoliposomal formulation and PA. Inhibition of S1PR1 with W146 (10 μM) abolished the effect of the nanoliposomal formulation on PAM-induced Lp increases. In four W146-pretreated vessels, the PAM-induced mean peak Lp in the presence of the nanoliposomal formulation was 7.12±1.41 times that of the control; insignificant from the Lp responses to PAM in the absence of the nanoliposomal formulation.
The effect of Compound I on endothelial permeability was further confirmed using a cultured EC monolayer model. As reported by other groups and shown in
Compound I efficacy for inhibiting gap formation by inflammatory mediators BK and IL-8 was also examined (
To show that Compound I was activating the S1PR1 to prevent gap formation, HUVECs were treated with W146 (S1PR1 antagonist) before treatment with Compound I and BK. W146 pretreatment abolished the inhibitory effect of Compound I on BK-induced gap formation (
A S1PR1 internalization assay using HEK293A-S1P1-GFP cells was undertaken to show that Compound I and SEW2871 similarly enhanced S1PR1 internalization, leading to its accumulation in the cytoplasm. Approximately 37% of cells in the DMSO treatment has internalized S1PR1 while more than 75% of cells in the Compound I (
MLC phosphorylation is critical in maintaining endothelial barrier integrity (18). HUVECs were treated with BK, VEGF or IL-8 and pMLC levels were compared by western blotting to treatment with Compound I. Compound I treatment alone did not increase pMLC levels but treatments of BK, VEGF or IL-8 significantly increased pMLC levels (
Compound I was able to inhibit gap formation mediated by inflammatory mediators VEGF (
To determine whether Compound I induced off-target effects in HUVEC cells, the effects of the compound were examined on several signaling pathways important in melanoma development, including the MAPK/ERK, PI3K/AKT, NF-1κB, and the vascular permeability regulation pathways. No significant changes in levels or activity of biomarker proteins in these pathways were detected between the control and Compound I treated ECS (
The Nanoliposomal Formulation Inhibits Metastasis Similar to that Occurring with Control SEW2871.
To test the efficacy of the nanoliposomal formulation for inhibiting melanoma metastasis, an experimental metastasis mouse model was used and the treatment protocol summarized in
S1P can potently inhibit inflammatory mediator-induced increases in microvessel permeability through the activation of S1PR1 (22,38). Based on those observations, we developed a new nanoparticle S1PR1 agonist, the nanoliposomal formulation of Compound I, to specifically reverse the cancer-associated vascular inflammation that promotes metastasis. Studies conducted in both intact microvessels and a cultured EC monolayer model demonstrated its potent efficacy for enhancing endothelial barrier function, inhibition of permeability increases, and EC gap formation mediated by a variety of inflammatory mediators as well as tumor cell secreted cytokines. Significantly, the effect of the nanoliposomal formulation inhibited the inflammatory mediator-induced permeability increases using a quantitative measure of the permeability coefficient, LP, in intact rat microvessels. The nanoliposomal formulation inhibited PAF-induced permeability increases similar to that observed with the SEW 2871 control. Furthermore, the action of the nanoliposomal formulation was abolished by pretreating vessels with a S1PR1 antagonist, W146, which confirmed the effect of the nanoliposomal formulation occur through S1PR1 activation in ECs. Most importantly, systemic application of the nanoliposomal formulation of Compound I inhibited lung metastasis by up to 80% in a melanoma metastasis animal model. Results demonstrated that the action of the nanoliposomal formulation not only recapitulated the effect of S1P on EC barrier integrity, but also minimized the potential confounding effects of S1P on the activation of other S1P receptors, suggesting its clinical potential for retarding the metastatic spread of cancer.
VEGF, BK and IL-8 are some of the inflammatory cytokines that are elevated in the serum of advanced-stage melanoma patients and in animal models containing melanomas (13). These cytokines can significantly alter the architecture of VE-cadherin and the phosphorylation status of MLC in endothelial cells leading to the detachment of these cells from one another to form gaps (39, 40). VEGF and BK are well-known permeability inducers and mechanisms mediating this process has been reviewed in detail (41). The IL-8 and CXCR1/2 complex has been shown to increase vascular permeability by transactivating VEGFR2 (42). It is almost impossible to target each individual inflammatory cytokine in order to prevent their effects on EC integrity. Therefore, our approach of directly enhancing the EC barrier integrity to prevent gap formation-mediated by a wide-spectrum of inflammatory cytokines could effectively reduce cancer cell metastasis for many cancer types and not just melanoma.
Compound I was developed to be loaded into a nanoliposome for bioavailable intravenous delivery. The chemical structure of Compound I was found to have better binding interaction in the S1PR1 active pocket with a higher docking score compared to SEW2871. Compound I was also confirmed to specifically interact in the active pocket of S1PR1 using the Erebus algorithm. Binding of Compound I to S1PR1 could be blocked using the S1PR1 antagonist W146, which functionally demonstrated that the new compound maintained its binding to the receptor. Thus, the data suggested that Compound I was a specific S1PR1 agonist.
A major advantage of Compound I is that it can be encapsulated into nanoliposomes while SEW2871 cannot. In recent years, nanotechnology has been used to more effectively deliver agents to treat cancer by reducing toxicity, increasing bioavailability and prolonging the time in the circulation (43). Nanoparticles can increase drug solubility, improve pharmacokinetics and minimize side-effects (43). Nanoliposomes, compared to the traditional drug delivery route, have some promising benefits, including better drug delivery, protection of active drugs from environmental factors, prevention of early degradation of the drug, cost-effective formulation for expensive agents and more efficient treatments with less toxicity (44). Moreover, the biodegradability and biocompatibility of nanoliposomes make them excellent therapeutic vehicles (44). Using a melanoma mouse model, we have previously demonstrated the low toxicity, high bioavailability and prolonged release of the nanoliposomal forms of anti-melanoma drugs, such as leelamine (45), arachidonyl trifluoromethyl ketone (46), aldehyde dehydrogenase inhibitors (47), celocoxib (48), and plumbagin (48).
The nanoliposomal formulation increased bioavailability by enabling direct injection into the vascular system wherein it would have immediate access to the ECs where it needed to act. The nanoliposomal formulation inhibited EC gap formation in the vascular system, which led to fewer melanoma lung metastases. We have previously shown that melanoma cells secrete IL-8 to attract neutrophils which tether them to the vascular endothelial cell layer to promote metastasis (49). The results of this study extend this process by showing that the cytokines secreted by these cells can further aid in this process by enabling gap formation in the EC layer through which the cancer cells can extravasate to promote metastasis. This process can be inhibiting using the bioavailable nanoliposomal formulation, which releases the S1PR1 agonist at the EC layer to enhance vascular barrier integrity. Thus, it is possible to counteract the effects of inflammatory cytokines non-specifically and provide better protection against these factors to maintain EC homeostasis to inhibit inflammation-mediated melanoma metastasis.
In normal endothelial cells, the S1P/S1PR1 complex enhances junctional assembly (50) while at high concentration of S1P, the S1P/S1PR3 complex in endothelial cells disrupts the barrier structure (51). An S1PR1/3 agonist, FTY720, has been developed but has the side-effects of inducing bradycardia (52), macular oedema, cardiovascular complications, and brain inflammation (53). This occurs following the activation of S1PR3 (52) or S1PR1 and S1PR3 (53). Since Compound I is a specific agonist to S1PR1, these toxic effects would not likely be observed.
S1P through the S1P receptors has been reported to trigger a possible epithelial-to-mesenchymal transition (EMT), leading to increase cell migration, invasion and metastasis in breast, liver and melanoma (54-56). In melanoma, the main S1P receptor regulating the S1P-mediated EMT and cell invasion has been reported to be S1PR2 (54), therefore, using the nanoliposomal formulation would likely avoid this concern. The S1PR1 pathway has been reported to be involved in S1P-mediated lymphangiogenesis, which indicated a potential role in lymphatic cancer metastasis (57). A recent study showed that S1PR1 plays a crucial role in high-molecular-weight-hyaluronan-mediated permeability decrease in cancer lymphatic endothelial cell monolayer (58). Effects, if any, of Compound I on melanoma lymphatic metastasis remains to be examined. However, since surgical cancer excision has potential to disseminate cancer around the patient's body (59), a single treatment of the naoliposomal formulation might be sufficient for use prior to melanoma surgery to enhance endothelial cell junctional integrity, in order to reduce surgery-mediated cancer spread. Since this treatment modality would only require a single administration of the nanoliposomal formulation, this approach could reduce the concerns related to potential EMT-mediated effects occurring with long term treatment.
In summary, this report suggests that multiple inflammatory cytokines secreted from melanoma cells can create gaps in the EC layer, which can subsequently promote metastasis. To combat vascular endothelial inflammation mediated by these factors, a novel S1PR1 agonist Compound I has been developed, which could reverse the collective effect of these proteins. Compared to SEW2871, Compound I exhibited several significant advantages including better association with S1PR1 and loadability into a nanoliposome. The nanoliposomal formulation of Compound I had an average size of 69 nm, neutral-charge in saline and was stable for 6 weeks when refrigerated. The nanoliposomal formulation significantly prevented permeability increases and gap formation in the vascular endothelium and an EC monolayer model induced by inflammatory cytokines. The nanoliposomal formulation enhanced endothelial cell layer integrity by decreasing the phosphorylation of MLC. As a result, melanoma lung metastasis development was inhibited. Endothelial homeostasis plays a key role in metastasis and enhancing the endothelial barrier integrity using the bioavailable S1PR1 agonist nanoliposomal formulation was a potentially effective strategy to reduce metastasis.
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Development of a novel multi-isoform ALDH inhibitor effective as an anti-melanoma agent.
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A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.
The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.
This application claims the benefit of priority to U.S. Provisional Application No. 63/044,515, filed Jun. 26, 2020, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant Nos. CA136667, CA138634, DK097391, and HL130363 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/029144 | 4/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63044515 | Jun 2020 | US |
