Patent Application
20240408214
The disclosed technology is generally directed to a nanoparticulate composition. More particularly the technology is directed to a nanoparticle composition having a pharmaceutical application.
Melanoma (and malignant melanoma) is a form of skin cancer that develops from melanocytes, the pigment-producing cells. Melanocytes and melanoma cells are exclusively capable of synthesizing melanin pigment inside melanosomes, lysosome-like organelles. The first steps in melanin synthesis are the oxidation of the amino acid L-tyrosine to dopa-quinone, catalyzed by the enzyme tyrosinase and tyrosinase-related protein 1 (TRP1). As melanoma cells express melanosomal enzymes, targeting this melanogenic pathway by analogs of the melanin synthetic pathway provides a unique strategy for developing new treatments for melanoma.
Among the potential drug candidates in this approach are substituted phenols, which are structurally similar to tyrosine and can be converted to a product that is toxic to the host cells. As a model phenolic drug, monobenzyl ether of hydroquinone (MBEH), aka monobenzone, can indeed reduce melanoma growth when applied topically. However, melanomas rapidly metastasize, and for MBEH to exert therapeutic effects on tumors away from the skin, a systemic application is needed.
MBEH has poor bioavailability and melanoma cells are less sensitive to the free pro-drug than melanocytes. This necessitates high amounts of MBEH to be administered. However, such high amounts are toxic for the subject. As a result, there is a need for means of delivery for the systemic administration of concentrated phenolic drugs, such as MBEH, to treat melanoma.
The field of the invention relates to nanoparticulate monobenzone that is useful for the treatment of melanoma. In particular, the field of the invention relates to nanoparticulate monobenzone comprising monobenzone or conjugates thereof that are cytotoxic and also generate an antitumor immune response. The field of the invention relates to the use of nanoparticulate monobenzone in pharmaceutical compositions for treating melanoma.
One aspect of the technology provides for nanoparticulate monobenzone. Nanoparticulate monobenzone comprises monobenzone or a conjugate thereof embedded within a nanostructured carrier. The nanostructured carrier can be a liposome or a metal-organic framework. Besides the monobenzone conjugate, the nanostructured carrier may further comprise a detectable label, a stabilizing agent, a targeting agent, linking agent, or any combination thereof and tailored for particular uses.
Monobenzone is a compound with the formula:
Monobenzone conjugates may comprise an alkyl or aryl moiety covalently bound, directly or indirectly by a linking group, to monobenzone. Exemplary monobenzone conjugate include compounds of Formula I:
wherein R is an alkyl, aryl, —X-alkyl, or —X-aryl where X is a linking group such as —C(═O)—, —C(═O)O—, —C(═O)N(H)—, —O—, —N(H)—, or —N(alkyl)-.
Methods for the preparation of nanoparticulate monobenzone are also disclosed. The methods comprise embedding monobenzone or a conjugate thereof within the nanostructured carrier. In some embodiments, the nanostructured carrier is a liposome. In another aspect according to this disclosure, the nanostructured carrier is a metal-organic framework (MOF). When the nanostructured carrier is a liposome, the method may comprise the steps of preparing a composition comprising a lipid film and monobenzone or a conjugate thereof and forming a liposomal carrier comprising monobenzone or the conjugate thereof embedded within the liposomal carrier.
The disclosed monobenzone conjugates may be used to prepare and formulate pharmaceutical compositions for treating melanoma. As such, also disclosed herein are pharmaceutical compositions comprising an effective amount of any of the compounds or compositions disclosed herein, or pharmaceutically acceptable derivatives of any of the compounds or compositions disclosed herein, together with a pharmaceutically acceptable excipient, carrier, or diluent.
In some embodiments, the disclosed nanoparticulate monobenzone may be used for preparing a medicament for treating melanoma. In some embodiments, the disclosed nanoparticulate monobenzone may exhibit cytotoxicity and induce an immune response.
In some embodiments, the disclosed methods for treating melanoma may comprise administering to a subject in need thereof an effective amount of nanoparticulate monobenzone, or a pharmaceutical composition comprising an effective amount of the nanoparticulate monobenzone.
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
Disclosed herein are nanoparticular monobenzone compositions for use in the treatment of melanoma. Malignant melanoma is the most aggressive form of skin cancer and is responsible for most skin-cancer deaths. Although immunotherapeutics have recently been put to use as highly promising treatments, complete responders are still rare due to the high propensity for melanoma to metastasize and escape. Thus, there is a critical need for new drug candidates that can bring metastatic melanoma into total remission through novel strategies. Substituted phenols can target the melanogenic pathway, the primary mechanism for producing melanin pigment inside pigment-producing cells. In this pathway, the amino acid L-tyrosine is first oxidized to dopa-quinone by the enzymes tyrosinase and tyrosinase-related protein-1 (TRP-1), abundantly present in melanosomes, lysosome-like organelles that are uniquely present in melanocytes and melanoma cells. Aborting full maturation to melanin using phenol prodrugs that are structurally similar to tyrosine or intermediates of the melanogenic pathway as alternate substrates for melanogenic enzymes can result in reactive quinone products that are toxic to the host cells
Several factors have thus far prevented the translation of MBEH into systemic treatments. First, MBEH is highly insoluble in water, resulting in very low bioavailability. Second, melanoma cells are less sensitive to treatment with the free drug than melanocytes, attributed in part to the ability of tumor cells to purge toxic compounds through efflux transporters, upregulated during malignant transformation. Third, high concentrations of free MBEH that are toxic to the host would be necessary to mediate melanoma regression when administered systemically.
The nanoparticulate monobenzone composition described herein can overcome the aforementioned challenges by reducing the amount of the active ingredient necessary to inhibit the growth and proliferation of melanoma cells. The nanoparticulate monobenzone compositions described herein comprise nanostructured carriers embedded with substituted phenol active ingredients, such as MBEH or conjugates thereof. They are also more favorably taken up by tumors than by normal tissue cells; and their drug contents are preferentially released in the context of acidic melanosomes, thereby maximizing the prodrug exposure to melanogenic enzymes. This can result in a significant reduction in the amount of active ingredient necessary to demonstrate inhibition of growth or proliferation of tumor cells.
The term “nanostructured carrier” as contemplated herein means a carrier having dimensions less than a micron. Nanostructured carriers may have dimensions between 30 to 800 nm, 30 to 500 nm, 30 to 200 nm, 30 to 120 nm, 30 to 100 nm, or 30 to 80 nm. In some embodiments, the nanostructured has dimensions of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 nm, or any range therebetween.
The nanostructured carriers can be loaded with MBEH or conjugates thereof. The hydrophobic drugs may comprise between 1-50 mol %, such as between 5-25 mol % or 10 to 25 mol %, of the lipid content in the nanostructured carrier. In some embodiments, MBEH or a conjugate thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mol %, or any range therebetween of the lipid content in the nanostructured carrier. In some examples, the nanoparticulate monobenzone were synthesized with 1, 2.5, 5, 10, or 15 mol % of MBEH.
MBEH (monobenzone) is a compound with the formula
Other substituted phenols may be utilized in the compositions and methods described herein provided that they can be converted by tyrosinase to a product that is toxic to the host cells. In some embodiments, monobenzone conjugates are utilized in the compositions and methods described herein. As used herein, monobenzone conjugates comprise may comprise an alkyl or aryl moiety covalently bound, directly or indirectly by a linking group, to monobenzone, such as at the benzyl ether portion of monobenzone. The alkyl or aryl moiety may be in para-position relative to the methyl ethyl linking the phenyl to the phenol but may also be in ortho- or meta position as well. Exemplary monobenzone conjugates include compounds of Formula I:
wherein R is an alkyl, aryl, —X-alkyl, or —X-aryl where X is a linking group such as —C(═O)—, —C(═O)O—, —C(═O)N(H)—, —O—, —N(H)—, —N(alkyl)-.
The term “alkyl” as contemplated herein means a saturated or unsaturated, e.g., alkenyl or alkynyl, hydrocarbon radical in all of its isomeric forms, such as a straight or branched group. The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. In some embodiments, the alkyl may have between 1-28, 4-28, 6-22, 8-18 carbon atoms, which may be referred to as a C1-C28, C4-C28, C6-C22, or C8-C18 alkyl, respectively. In particulate embodiments, the alkyl group comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 carbon atoms or any range therebetween.
The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido or carboxamido, carboxylic acid, —C(O)alkyl, —CO2alkyl, carbonyl, carboxyl, carboxyamido, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, —CF3, —CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.
The alkyl or aryl groups may be selected to facilitate the loading of the monobenzone conjugate. This may include increased the amount of monobenzone conjugate that can be embedded within the nanostructured carrier. In some embodiments, the alkyl may be structurally similar to a fatty acid in number of carbon atoms, number of saturated or unsaturated carbon-carbon bonds, positioning of unsaturated carbon-carbon bonds. Exemplary fatty acids which the alkyls may bear structural similarity to include, without limitation, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, lignoceric, cerotic, myristoleic, palmitoleic, sapienic, oleic, elaidic, vaccenic, linoleic, linolaidic, α-linolenic, arachidonic, eicosapentaenoic, erucic, or docosahexaenoic acid.
Monobenzone conjugates may be prepared by the addition of hydroquinone to a substituted (bromomethyl)phenyl. For example, lauroyl MBEH (compound 3) may be prepared by adding potassium carbonate and 1-(4-(bromomethyl)phenyl)-1-dodecanone (compound 2) to a solution of hydroquinone. Exemplary methods for preparing monobenzone conjugates are provided in the Examples. Those of ordinary skill will appreciate that other synthetic methods may be used to prepared monobenzone conjugates.
Exemplary conjugated monobenzone include lauroyl MBEH and lauryl MBEH, which have the structures shown below.
In some embodiments, the nanostructured carrier is a liposomal carrier or liposome. Liposomes can be classified as unilamellar vesicles (ULVs), oligolamellar vesicles (OLVs), multilamellarvesicles (MLVs), and multivesicular liposomes (MVLs), depending on the compartment structure and lamellarity. ULVs can be further divided into small unilamellar vesicles (SUVs, 30-100 nm), large unilamellar vesicles (LUVs, >100 nm), and giant unilamellar vesicles (GUVs, >1000 nm). Liposomal carriers may be prepared that are highly biocompatible, easily modified, and tailored for specific applications. In one non-limiting example for use with MBEH conjugates, the liposomal carrier can be ˜100 nm diameter.
Liposomal carriers comprise a lipid bilayer that may be prepared from one or more amphiphilic components. The amphiphilic components may include a glycerolphospholipid (GP) and/or sphingomyelin (SM). The hydrophilic head group of the amphiphilic components may comprise phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS), phosphatidyl inositols (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin, or the like. The hydrophobic groups of the amphiphilic components may be selected from fatty acids, such as decanoic acid, lauric acid, palmitic acid, oleic acid, myristic acid, stearic acid, erucic acid, and the like, or sphingosine.
GPs and SM play a key role in formulation since they affect the biophysical properties of liposomes (e.g., drug encapsulation, stability, and drug release) and further influence the pharmacokinetic behavior and pharmacodynamics in vivo. The length, symmetry, inter- and intra-molecular interactions, branching, and unsaturation degree of hydrocarbon chains decide the thickness and fluidity of the bilayer, phase transition temperature, and drug release rate. Longer hydrocarbon chains can induce a tighter membrane packing and increase drug retention. A higher degree of unsaturation or branching of the hydrocarbon chain could result in looser membrane packaging.
Exemplary amphiphilic components include, without limitation, HSPC (hydrogenated soy phosphatidylcholine); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleoylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleoylphosphatidylserine); POPC (palmitoyloleoylphosphatidylcholine); SM (sphingomyelin); DMPC (dimyristoyl phosphatidylcholine); DMPG (dimyristoyl phosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleoly-sn-glycero-phosphoethanolamine); DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), and DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl), etc.
Steroids, such as cholesterol or cholesterol derivatives, may also be included in the liposomal carrier. Steroids, such as cholesterol, can promote the packing of lipid chains and bilayer formation, modulate the fluidity/rigidity of membrane, affect the drug release, stability of liposomes, and the kinetics of exocytosis. Optionally, the steroid may be conjugated, directly or indirectly, to another component of the liposomal carrier. The conjugated component may include a stabilizing agent, detectable label, targeting agent, or linking group.
Liposomal carriers can be prepared with detectable labels for detection of liposomal trafficking. Exemplary detectable labels, include, without limitation, fluorescent labels, near-IR labels, X-ray-active labels, radioactive labels, and magnetic-resonance-imaging labels.
Liposomal carriers can be prepared with stabilizing agents. To improve liposome stability and enhance their circulation times in the blood, sterically stabilized liposomes may be prepared. A hydrophilic polymer, such as polyethylene glycol (PEG), can be used to prepare sterically stabilized liposomes. The establishment of a steric barrier improves the efficacy of encapsulated agents by reducing in vivo opsonization with serum components, and the rapid recognition and uptake by the reticuloendothelial system. This reduces the elimination of drugs by prolonging blood circulation and providing accumulation at pathological sites while also attenuating side effects such as polyethylene glycol (PEG) oligomers on the exterior of the bilayer. Such PEG groups can include MPEG (methoxy polyethylene glycol). Alternatively, stabilizing agents such as steroids can be used to stabilize the lipid bilayer by embedding within the bilayer.
The outer surface of liposomal carriers can include targeting agents. Ligand-targeted liposomes offer potential for site-specific delivery of drugs to designated cell types or organs in vivo, which selectively express or over-express specific ligands (e.g., receptors or cell-adhesion molecules) at the site of disease. Many types of ligands are available, such as antibodies, peptides/proteins, carbohydrates, oligonucleotides such as DNA, RNA, small molecules, or combinations thereof. The coupling of antibodies, particularly monoclonal antibodies, to create immunoliposomes represents a versatile ligands that can be affixed to liposome surfaces
The liposomal nanocarrier may comprise a linking agent. As used herein, a “linking agent” is a compound or liposomal component having a functional group that is readily functionalized via a conjugation strategy generally suitable for biological molecule. Exemplary functional group include those that can participate via an amine reaction (e.g., NHS ester, imidoester, hydroxymethyl phosphine, guanidination of amine, fluorophenyl esters, carbodiimides, anhydrides, arylating agents, carbonates, aldehydes or glyoxals), thiol reactions (e.g., maleimide, haloacetyl, pyridyldisulfide, vinyl sulfone, or thiol-disulfide exchange), carboxylate reactions (e.g., carbodiimides), hydroxyl reactions (e.g., isocyanates, enzymatic oxidation, or carbonyldiimidazole), aldehyde and ketone reaction (e.g., hydrazine derivative, Schiff-base formation, or reductive amination), active hydrogen reaction (e.g., iodination reaction), photo-chemical reactions (e.g., psoralen compounds, aryl azides and halogenated aryl azides, benzophenones, anthraquinones), or cycloaddition reactions (e.g., chemoselective ligation such as click chemistry or Diels-Alder reaction).
In other embodiments, the nanostructure carrier is a metal-organic framework (MOF) nanoparticle. MOFs are a class of compounds comprising of metal ions or clusters (i.e., nodes) coordinated to multitopic organic ligands (i.e., linkers) to form multidimensional structures. They are a subclass of coordination polymers, with the special feature that they are often porous when assembled into a three dimensional structure. MOFs may be prepared to have low toxicity and/or be biocompatible. MOF-based nanostructured may be loaded with MBEH or a conjugate thereof and the MBEH or MBEH conjugate may be released within the subject.
An exemplary MOF for use as a nanostructured carrier is the zirconium-based metal-organic framework UiO-66 (
Methods of treating subjects in need of nanoparticulate monobenzone are also provided. Nanoparticulate monobenzone may be administered to the subject by any suitable route. In some embodiments, the nanoparticulate monobenzone is systemically administered Nanoparticulate monobenzone may be administered parenterally, e.g., injection, infusion or implantation, or enterally, e.g., orally.
As used herein, a “patient” may be interchangeable with “subject” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment. Suitable patients for the disclosed methods may include, for example mammals, such as humans, monkeys, dogs, cats, horses, rats, and mice. Suitable human patients include, for example, those who have melanoma or those who have been determined to be at risk for developing melanoma, or have related disorders. As used herein, a “subject in need of treatment” may include a patient having melanoma that is responsive to therapy with nanoparticulate monobenzone described herein.
As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of melanoma. As such, the methods disclosed herein encompass therapeutic administration. In some embodiments, treatment may result in the killing of cancer cells, such as melanoma tumor cells, or inhibition of the growth or proliferation of cancer cells. In some embodiments, treatment may result in increased T cell infiltration.
As used herein the term “effective amount” refers to the amount or dose of the nanoparticulate monobenzone, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed nanoparticulate monobenzone (e.g., as present in a pharmaceutical composition) for treating melanoma, whereby the effective amount causes the killing of cancer cells, inhibition of the growth or proliferation of cancer cells, or increases T cell infiltration in the patient.
An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques, and by observing results obtained under analogous circumstances. In determining the effective amount or dose of nanoparticulate monobenzone administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
The pharmaceutical compositions for use according to the methods as disclosed herein may include a single nanoparticulate monobenzone as an active ingredient or a combination of nanoparticulate monobenzones as active ingredients (e.g., a combination of nanoparticulate monobenzone having different labels, loadings of monobenzone conjugates, stabilizing or targeting agents). For example, the methods disclosed herein may be practiced using a composition containing nanoparticulate monobenzone with a single type of monobenzone conjugate. Alternatively, the disclosed methods may be practiced using a composition containing a nanoparticulate monobenzone with two or more types of monobenzone conjugates. In another non-limiting example, two or more nanoparticulate compositions differing in their loading of monobenzone conjugate, labels, stabilizing agents, or targeting agents can be combined.
The disclosed methods may be practiced by administering a first pharmaceutical composition comprising nanoparticulate monobenzone and administering a second, different pharmaceutical composition (e.g., a pharmaceutical composition comprising a different nanoparticulate monobenzone or different active pharmaceutical ingredient), where the first composition may be administered before, concurrently with, or after the second composition. As such, the first pharmaceutical composition and the second pharmaceutical composition may be administered concurrently or in any order, irrespective of their names.
As one skilled in the art will also appreciate, the disclosed pharmaceutical compositions can be prepared with materials (e.g., actives excipients, carriers, and diluents etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and/or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.
Pharmaceutical compositions comprising the nanoparticulate monobenzone may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal injection) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
The nanoparticulate monobenzone utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in liquid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary liquid dosage forms include, but are not limited to pharmaceutical compositions for parenteral administration, such as injectable liquids, injectable emulsions, or gels.
Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets
Alternatively, the nanoparticulate monobenzone utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended-release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.
The nanoparticulate monobenzone utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes an excipient, carrier, or diluent. For example, the excipient, carrier, or diluent may be selected from a group consisting of buffered physiologic salt solutions suitable for injection.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term that are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Monobenzyl ether of hydroquinone (MBEH), a phenolic compound and an FDA-approved drug, has been found to reduce subcutaneous melanoma growth (
As an FDA-approved treatment to induce skin depigmentation (
As demonstrated in the Examples, 100 nm liposomes are favorably taken up by tumor cells over normal tissue cells (
Chemotherapeutically, MBEH, MBEH conjugates, and other substitute phenols can target the melanogenic pathway, resulting in necrotic cell death of melanogenic cells. Experiments with C57BL/6 mice have also shown that topical application of MBEH prior to subcutaneous tumor challenge results in remarkable T cell infiltration into the tumors after two weeks (
Lauroyl-MBEH-loaded liposomes were synthesized with ˜10 mol % lauroyl MBEH incorporated into the DOPC lipid bilayer following previously established protocols12 and are stable at 4° C. in 20 mM HBS buffer (
The surface of the lauroyl-MBEH-loaded liposomal NPs can be modified with cholesterol-PEG(2000) (Chol-PEG(2K)) to prolong their stability in circulation and prevent opsonization. The Chol-PEG(2K) can simply be incorporated onto the surface of the liposomes post-extrusion (FIG. 7, 2nd step, top pathway). To enhance the specificity for melanoma cells, lauroyl-MBEH-loaded NPs can be modified with antibody R24 to the melanoma surface antigen GD3. The NPs will first be functionalized with commercially available Chol-PEG(2K)-DBCO (
The parent lauroyl-MBEH-loaded liposomal material ((
Synthesis and Characterization of 1-(p-Tolyl)-1-Dodecanone (Compound 1).
This synthesis was adapted from a published procedure for the synthesis of the tridecanoyl analog. [Yu P, Hu J, Zhou T-Y, Wang P, Xu Y-H. Synthesis, insecticidal evaluation of novel 1,3,4-thiadiazole chrysanthemamide derivatives formed by an EDCl/HOBt condensation. J. Chem. Res. 2011; 35(12):703-706] To a mixture of toluene (21.5 g, 234 mmol, 18 equiv) and aluminum trichloride (7.33 g, 55 mmol, 4.2 equiv) was added lauroyl chloride (2.84 g, 13 mmol, 1 equiv) dropwise in the ice bath. The reaction mixture was heated to reflux and stirred for overnight. After completion of the reaction, the mixture was poured into ice water. The pH of the mixture was adjusted to 5-6 with concentrated hydrochloric acid and extracted with benzene (3×30 mL). Inside a separatory funnel, the combined organics was successively washed with 10% aqueous sodium bicarbonate until the pH of the aqueous layer reached ˜7 and then with DI water. The isolated organic layer was dried over anhydrous MgSO4, filtered, and concentrated on a rotary evaporator to afford compound 1 as white crystals (2.7 g, 76% yield).
The structure and composition of compound 1 was confirmed by NMR spectroscopy and mass spectrometry. 1H NMR (500 MHz, CDCl3): δ 7.86 (2H, d, J=8.2 Hz), 7.25 (2H, d, J=8.0 Hz), 2.92 (2H, t, J=7.4 Hz), 2.40 (3H, s), 1.75-1.69 (2H, m), 1.26 (16H, br), 0.88 (3H, t, J=6.9 Hz). 13C NMR (126 MHz, CDCl3): δ 200.3, 143.6, 134.7, 129.2, 128.2, 38.5, 31.9, 29.6, 29.6, 29.5, 29.5, 29.43, 29.36, 24.5, 22.7, 21.6, 14.1. MS (ESI): Exact Mass Calcd for [C19H31O]+ (M+H+)=275.2. Found 275.2.
The synthesis of compound 2 was adapted from a published procedure for the synthesis of 1-(4-(bromomethyl)phenyl)propan-1-one. [Yang Y, Yu Y, Li X, Li J, Wu Y, Yu J, Ge J, Huang Z, Jiang L, Rao Y. Target elucidation by cocrystal structures of NADH-ubiquinone oxidoreductase of Plasmodium falciparum (PJNDH2) with small molecule to eliminate drug-resistant malaria. J. Med. Chem. 2017; 60(5):1994-2005] To a solution of compound 1 (2.72 g, 9.9 mmol, 1.0 equiv) in anhydrous benzene (20 mL) in a 100 mL round-bottom flask equipped with a magnetic stir bar were added 1.98 g (11.1 mmol, 1.1 equiv) of N-bromosuccinimide (NBS) and 168.8 mg (1.0 mmol, 0.1 equiv) of 2,2′-azobis(2-methylpropionitrile) (AIBN). A water-cooled reflux condenser was attached to the flask and the reaction mixture was placed under argon and heated to 70° C. when an aliquot of anhydrous MeCN (7 mL) was added to completely dissolve NBS. The temperature of the reaction mixture was then raised to 90° C. and kept refluxed at this temperature for 7 h. After cooled down, the reaction mixture was evaporated to dryness, redissolved in DCM (90 mL), and washed with DI water (90 mL). The organic layer was dried over anhydrous MgSO4, filtered, and evaporated to dryness before being purified by silica gel flash column chromatography (hexanes/EtOAc=30:1 v/v). The crude product was recrystallized in hexanes to yield compound 2 as white crystals (1.36 g, 39% yield).
The structure and composition of compound 2 was confirmed by NMR spectroscopy and mass spectrometry. 1H NMR (500 MHz, CDCl3): δ 7.94 (2H, d, J=8.2 Hz), 7.48 (2H, d, J=8.2 Hz), 4.50 (2H, s), 2.94 (2H, t, J=7.4 Hz), 1.74-1.71 (2H, m), 1.38-1.26 (17H, m), 0.88 (3H, t, J=6.9 Hz). 13C NMR (126 MHz, CDCl3): δ 199.9, 142.5, 136.9, 129.2, 128.6, 38.7, 32.2, 31.9, 29.7, 29.6, 29.50, 29.47, 29.36, 29.33, 24.34, 22.69, 14.12. MS (ESI+): Exact Mass Calcd for [C19H30BrO]+ (M+H+)=353.2 and 355.2. Found 353.2 and 355.2.
This synthesis was adapted from a published procedure for the synthesis of 1-(5-(benzyloxy)-2-hydroxyphenyl)ethanone. [Mays J R, Hill S A, Moyers J T, Blagg B S. The synthesis and evaluation of flavone and isoflavone chimeras of novobiocin and derrubone. Bioorg. Med. Chem. 2010; 18(1):249-66.] Potassium carbonate (534.8 mg, 3.87 mmol, 1 equiv) and compound 2 (1.36 g, 3.85 mmol, 1 equiv) were added in sequence to a solution of hydroquinone (424.7 mg, 3.86 mmol, 1 equiv) in acetone (10 mL) in a 100 mL round-bottom flask equipped with a magnetic stir bar. A water-cooled reflux condenser was attached to the flask and the reaction mixture was then heated to reflux for 48 h. After cooling to t, the reaction mixture was combined with saturated aqueous NaHCO3 solution (10 mL) and the resulting mixture was extracted with EtOAc (3×20 mL). The combined organic layers were washed with saturated aqueous NaCl solution (100 mL), dried over MgSO4, filtered, and concentrated to dryness. The residue was purified via flash column chromatography over silica gel (5:1 v/v hexanes/EtOAc) to give lauroyl MBEH (3) as a white amorphous solid (450 mg, 31%).
The structure and composition of compound 3 was confirmed by NMR spectroscopy and mass spectrometry. 1H NMR (CDCl3, 500 MHz): δ 8.10-7.96 (2H, m), 7.51-7.45 (2H, m), 6.90-6.84 (2H, m), 6.79-6.76 (2H, m), 5.14-5.02 (2H, m), 2.97-2.94 (2H, m), 1.74-1.72 (2H, m), 1.26 (16H, s, br), 0.89-0.86 (3H, m). 13C NMR (126 MHz, CDCl3): δ 200.3, 152.7, 149.9, 142.4, 136.6, 128.4, 127.2, 116.1, 116.0, 70.1, 38.7, 31.9, 29.6, 29.6, 29.5, 29.4, 29.3, 24.4, 22.7, 14.1. MS (ESI−): Exact Mass Calcd for [C25H33O3]− (M−H)−=381.2. Found 380.99. Exact Mass Calcd for dimer [C50H67O6]− (2M−H)−=763.49. Found 763.56.
DOPC-based liposomes can be synthesized in high yields (>90%) at room temperature (rt) due to the very low T. of DOPC lipid (−17° C.). The synthesized liposomes can be kept in a refrigerator and used for biological studies.
The DOPC lipid (31.5 mg, 42 μmol) was completely dissolved in HPLC-grade chloroform (1 mL) in a 50 mL round-bottom flask and then evaporated under reduced pressure by a rotary evaporator. The resulting lipid film was thoroughly dried on a Schlenk line for 12 h more before being combined with 20 mM sterile HBS (20 mM HEPES and 150 mM NaCl; corrected to pH 7.4 using HClaq (1 and 5 M) after the addition of NaHEPES salt to sterile saline; 4.0 mL). [Hong B J, Iscen A, Chipre A J, Li M M, Lee O-S, Leonard J N, Schatz G C, Nguyen S T. Highly stable, ultrasmall polymer-grafted nanobins (usPGNs) with stimuli-responsive capability. J. Phys. Chem. Let. 2018; 9(5):1133-1139] Using an Avanti Mini-Extruder, this turbid suspension was extruded at least 40 times through a 0.1-micron polycarbonate membrane (Avanti Polar Lipids 610005) at rt to give a clear solution of liposomes. [Onyango J O, Chung M S, Eng C-H, Klees L M, Langenbacher R, Yao L, An M. Noncanonical Amino Acids to Improve the pH Response of pHLIP Insertion at Tumor Acidity. Angew. Chem. Inter. Ed. 2015; 54(12):3658-3663] The solution (˜4 mL) contains liposomes with a mean hydrodynamic diameter of 124.4 nm (PDI=0.101) as determined by DLS measurements. [Lee S-M, Chen H, Dettmer C M, O'Halloran T V, Nguyen S T. Polymer-caged liposomes: a pH-responsive delivery system with high stability. J. Am. Chem. Soc 2007; 129(49):15096-15097] This solution (˜4 mL) was then passed through a Sephadex G-50 column (fraction range: 1,500-30,000 Da, 2 cm×7 cm) that was wet-packed and pre-equilibrated with 10-mM sterile PIPES solution (corrected to pH 7.4 using NaOHaq (1 and 5 M) after the addition of PIPES to sterile saline, 150 mM NaCl) to remove any left-over free DOPC lipid. The eluant was the same 10 mM PIPES solution. The liposome-containing elution was confirmed with a laser pointer and collected as a single fraction.
The phospholipid concentration in the liposome-containing fraction was measured using ICP-OES. Briefly, to prepare a sample for ICP-OES measurement, an aliquot (20 μL) of DOPC liposomes was diluted in nitric acid (c=0.5 mol L−1) to a total volume of 5 mL. This mixture was vortexed (1 min) thoroughly before being left to equilibrate for a minimum of 3 h before being subjected to ICP-OES analysis. The concentration of lipids in the purified liposome sample was determined to be 5.43 mM. [Fenzl C, Hirsch T, Baeumner A J. Liposomes with high refractive index encapsulants as tunable signal amplification tools in surface plasmon resonance spectroscopy. Anal. Chem. 2015; 87(21):11157-11163]
The lauroyl MBEH (4.5 mg, 12 μmol) and DOPC lipid (94.5 mg, 120 μmol) was completely dissolved in a 1:1 v/v mixture of HPLC-grade chloroform and EtOAc (2 mL) in a 50 mL round-bottom flask and then evaporated under reduced pressure by a rotary evaporator. The resulting lipid film was thoroughly dried on a Schlenk line for 12 h more before being combined with 20 mM sterile HBS (20 mM HEPES and 150 mM NaCl; corrected to pH 7.4 using HClaq (1 and 5 M) after the addition of NaHEPES salt to sterile saline; 8.0 mL). Lipid hydration was accomplished by vigorous vortexing on an Vortex Mixer (American Scientific Products, Charlotte, NC) for 5 min. [Hong B J, Iscen A, Chipre A J, Li M M, Lee O-S, Leonard J N, Schatz G C, Nguyen S T. Highly stable, ultrasmall polymer-grafted nanobins (usPGNs) with stimuli-responsive capability. J. Phys. Chem. Lett. 2018; 9(5):1133-1139] After being subjected to 10 freeze-thaw-under-sonication (˜10 min.) cycles, the resulting dispersion of lauroyl-MBEH-loaded liposomes was extruded through hydrophilic polycarbonate track-etched membranes with a stepping down extrusion method. Briefly, on a mini-extruder block the dispersion was extruded ten times through a two-stack membranes (800-nm pore-size), ten times through a stack of one 800-nm and one 400-nm membranes, ten times through a two stack 400-nm membranes, ten times through a stack of one 400-nm and one 200-nm membrane, ten times through a two-stack 200 nm membranes, ten times through a stack of one 200-nm and one 100-nm membrane, and then ten times through a two-stack 100-nm membranes. At the end, the initial vial and syringes were washed with an additional aliquot of HBS buffer (300 μL) and this wash was also extruded ten times through a two-stack 100-nm membranes. The combined solution (˜8 mL) was passed the incubated solution through a Sephadex G-50 column (fraction range: 1,500-30,000 Da, 2 cm×7 cm) that was wet-packed and pre-equilibrated with 10-mM sterile PIPES solution (corrected to pH 7.4 pH 7.4 using NaOHaq (1 and 5 M) after the addition of PIPES to sterile saline, 150 mM NaCl) to remove any free unloaded lauroyl MBEH and/or non-liposomal lipid rafts. The eluant was also 10-mM PIPES solution and the fractions was collected every 5 drops. The drug-loaded-liposome-containing fractions can be easily recognized by their turbidity and the most concentrated ones were combined together (˜4 mL). The hydrodynamic diameter (DH) of the lauroyl-MBEH-loaded liposomes is 81.9 nm (PDI=0.0034). The final concentration of lipids was 15.016 mM as determined by phosphorous ICP-OES. [Lee S-M, Chen H, Dettmer C M, O'Halloran T V, Nguyen S T. Polymer-caged liposomes: a pH-responsive delivery system with high stability. J. Am. Chem. Soc 2007; 129(49):15096-15097]
Preparation and Characterization of Lauroyl-MBEH-Loaded Liposomes Labeled with Rhod-PE.
This dye-labeled liposome was made following the above preparation procedure of the drug-loaded liposomes, except that the Rhod-PE was added. Briefly, Rhod-PE (1 mg, 0.8 μmol) was added to the solution of lauroyl MBEH (3 mg, 8 μmol) and DOPC lipid (63 mg, 80 μmol) in chloroform and EtOAc. Then the dye-labeled drug-loaded liposomes were prepared in 20 mM sterile HBS buffer. After purification, the solution contains liposomes with a mean hydrodynamic diameter of 90.63 nm (PDI=0.049) as determined by DLS (dynamic light scattering) measurements. The final concentration of lipids was 5.99 mM as determined by phosphorous ICP-OES (inductively coupled plasma optical emission spectroscopy). [Lee S-M, Chen H, Dettmer C M, O'Halloran T V, Nguyen S T. Polymer-caged liposomes: a pH-responsive delivery system with high stability. J. Am. Chem. Soc 2007; 129(49):15096-15097]
Monobenzone conjugates may be prepared by the addition of hydroquinone to a substituted (bromomethyl)phenyl as shown in Scheme 2 where R may be an alkyl, aryl, —X-alkyl, or —X-aryl where X is a linking group such as —C(═O)—, —C(═O)O—, —C(═O)N(H)—, —O—, —N(H)—, —N(alkyl)-, or a protected group thereof.
Proposed synthetic routes to several MBEH conjugates are shown in Schemes 3-6.
The present application claims priority to U.S. Provisional Patent Application No. 63/261,599, filed Sep. 24, 2021, the entire contents of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/077022 | 9/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63261599 | Sep 2021 | US |
