WO2019089942A1 - Formulations for use in the transdermal delivery of proteasome inhibitors - Google Patents

Abstract

Topical formulations comprising pentadecalactone or a structurally similar C_10-20 lactone, and a hydrocinnamate and/or cinnamate proteasome inhibitor, such as PTTC, where the proteasome inhibitor is present in a concentration of between about 2 and about 10%, and methods for using the formulations to treat skin disorders associated with proteasome activity, such as rosacea, psoriasis, and acne, are disclosed.

Description

FORMULATIONS FOR USE IN THE TRANSDERMAL

DELIVERY OF PROTEASOME INHIBITORS

Field of the Invention

The invention is generally in the area of treatment of skin disorders, particularly skin disorders mediated by proteasomes, and specifically rosacea, psoriasis, and eczema.

Background of the Invention

Psoriasis is a skin disorder of unknown etiology. It is characterized by pain, itching, reduction of manual dexterity, and cosmetic problems such as prominent hand, leg, or facial lesions. Other skin conditions such as acne, seborrheic dermatitis, and skin damage caused by aging and/or photoaging, may manifest with similar symptoms and are often just as painful for their sufferers.

To date, there is no cure for psoriasis. Some patients opt for suppressive therapy, typically using biological agents such as the anti-TNF agents adalimumab, etanercept, and infliximab, the anti-interleukin (IL)- 12/23 antibody ustekinumab, and the anti-IL-17 antibody secukinumab, which are administered systemically rather than topically to the afflicted area. While patients generally see significant improvement, decreasing the severity and extent of psoriasis to a point at which it no longer substantially interferes with the patient's occupation, well-being, or personal or social life, there are substantial side-effects. These side-effects include a weakened immune system, liver damage, and the like.

Non-biological topical treatments have also been used, depending on the severity of the disease. The topical treatment of psoriasis uses emollients, keratolytic agents, coal tar, anthralin, corticosteroids of medium to strong potency, and calpotriene. However, these topical treatments have variable efficacy and may also have undesired side effects. Similar treatments have also been attempted for acne, seborrheic dermatitis, and skin damage caused by aging and/or photoaging, with equally limited success.

Accordingly, a need exists for an effective skin disorder treatment that avoids the disadvantages associated with the currently available topical or systemic treatments. More specifically, an effective treatment for psoriasis, acne, seborrheic dermatitis, and skin damage caused by aging and/or photoaging is needed that does not have the same disadvantages as currently available topical or systemic treatments.

U.S. Patent No. 8,809,283 to Jack Arbiser discloses certain compounds active as proteasome inhibitors, including pentaerythritol tetrakis(3,5-di-tert-butyl-4- hydroxyhydrocinnamate (PTTC) and other hydrocinnamates and cinnamates. PTTC and the other disclosed hydrocinnamates tend to be extremely hydrophobic, and as such, are difficult to formulate in a manner in which they can penetrate the skin in high concentrations. It would be advantageous to have topical formulations that enhance the bioavailability of PTTC and similar hydrocinnamates and cinnamates.

Summary of the Invention

Topical formulations comprising cinnamate and/or hydrocinnamate proteasome inhibitors are disclosed. The formulations include pentadecalactone or a structurally similar solvent, which solvent is effective at solubilizing the proteasome inhibitors. Methods for treating skin disorders by topically applying the formulations to affected skin are also disclosed.

Structurally similar solvents are C ₁₀-₂₀ lactones with one or two lactone moieties, optionally one or two ketone moieties, optionally, one or two Ci_-6 alkyl substituents, and optionally, one or two oxygen atoms in the ring.

In one embodiment, the compositions comprise between about 2 and about 10% by weight of the proteasome inhibitors, along with sufficient pentadecalactone or structurally similar solvent, and, optionally, other solvents, to solubilize the proteasome inhibitors.

In one aspect of this embodiment, the proteasome inhibitors compounds have one of the following formulas:

i) a hydrocinnamate compound selected from the group consisting of







cinnamate compound having one of the following formulas:

wherein W is selected from the group consisting of a methyl group, an alkyl group, a methylene group, an amine group, an acyl group, a carbonyl group, an oxygen atom, a sulfur atom, and wherein Xi to X₅ are independently selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an ether group, an alkyl group, an aryl group, a nitro group, a cyano group, a thiol group, a thioether group, an amino group, an amido group, and an OR group, where R is an ester of a dihydrocinnamate;

and analogs thereof wherein one or more of the hydrogen atoms on the phenyl ring in the dihydrocinnamate moiety in the listed compounds can be replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, alkyl, aryl, nitro, cyano, thiol, thioester, amino, and amido, in a pharmaceutically acceptable formulation for topical administration to the skin, and analogs of the compounds in (i) or (ii) wherein one to three of the hydrogen atoms on the aromatic ring in the dihydrocinnamate moiety is replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, Ci_-6 alkyl, C₆-io aryl, nitro, cyano, thiol, thioester, amino, and amido. These compounds, and analogs thereof, are described, for example, in U.S. Patent No. 8,809,283, the contents of which are hereby incorporated by reference for all purposes.

In one embodiment, the hydrocinnamate is PTTC, otherwise known as Irganox® 1010, having the chemical name pentaerythritol tetrakis (3-(3, 5-di-tert- butyl-4-hydroxyphenyl) propionate, and having the formula:

The present invention is based, in part, on the discovery that the proteasome inhibitors described above can be used to topically treat skin disorders associated with proteasome activity, but tend to be poorly soluble in conventional topical formulations, limiting the effective concentration of the compounds. After testing a variety of solvents, it was determined that pentadecalactone was the most effective solvent tested for solubilizing an effective amount of these compounds in topical formulations.

The formulations described herein can be applied to the skin, in any of a variety of topical formulations, such as lotions, cremes, and the like. When applied to the skin, the formulations can treat or prevent skin disorders associated with proteasome activity, and skin disorders associated with an inflammatory response, including those caused by microbial infection, in both humans and non-human animals.

Representative formulations include lotions, cremes, oil-in-water emulsions, water-in-oil emulsions, dispersions, and micelles. The formulations can optionally include, in addition to the pentadecalactone and proteasome inhibitors, specific carriers for topical administration.

In addition to the proteasome inhibitors described herein, the formulations can comprise one or more additional active agents. Representative additional active agents include, but are not limited to, anesthetics, anti-inflammatory agents, antimicrobial/anti-infective agents, anti-proliferative agents and combinations thereof.

The formulations described herein can be administered to the skin to treat skin disorders mediated by proteasomes, and/or which have an inflammatory component. The formulations are applied to the skin in an amount effective to treat or prevent the disorders. When administered with additional agents, the formulations can also provide anesthesia, prevent or treat inflammation, prevent unwanted cell proliferation and/or to provide treatment or prophylaxis of microbial infections.

Representative skin disorders that can be treated using the proteasome inhibitor formulations described herein, for example, by topical application of compositions including one or more proteasome inhibitor, and also optionally including an anti-inflammatory agent, include skin rosacea, eczema, dermatitis, acne, angiosarcoma, hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Kaposi's sarcoma, and the non-malignant diseases or conditions psoriasis, lymphangiogenesis, hemangioma of childhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis, tuberous sclerosis, pyogenic granulomas, recessive dystrophic epidermolysis bullosa, venous ulcers, molluscum contagious, seborrheic keratosis, and actinic keratosis. In one embodiment, the disorder is selected from the group consisting of skin rosacea, excema, dermatitis, and acne.

Representative microbial infections that can be treated or prevented using combinations of the proteasome inhibitors and a suitable antimicrobial agent include viral, fungal, and bacterial skin infections. Where an infection causes a disorder associated with an inflammatory component, the co-administration of anti-inflammatory agents and antimicrobials (i.e., antivirals, antib acted als, antifungals, antiparasitics, and the like), can be desirable. Other active agents, such as anti-proliferatives, anti-metabolites, VEGF inhibitors, prostaglandins, TGF-beta, mitomycin C, and antioxidants can also be added.

In one embodiment, the PTTC is combined with a vasoconstrictor, such as tetrahydrozoline or brimonidine. In one aspect of this embodiment, such a formulation can be used to treat skin disorders such as rosacea and psoriasis.

The present invention will be better understood with reference to the following detailed description.

Brief Description of the Drawings

Figure 1 is a chart showing the skin penetration of PTTC (i.e., intradermal delivery) using finite dosing, in terms of the average amount ^g) +/- a standard deviation (SD), in the epidermis (left side) and dermis (right side), by formulation number.

Detailed Description

The invention described herein relates to specific topical formulations of the proteasome inhibitors described herein, and methods for using the formulations described herein to treat skin disorders, including those mediated by proteasomes, those associated with an inflammatory component, and those associated with infections, including viral, bacterial, fungal, and parasitic skin infections.

The proteasome inhibitor can be administered alone or in combination with one or more additional active agents. Where the disorder is associated with a skin infection, the additional active agent can be an antibiotic, and where the disorder is associated with inflammation, or the patient has skin surgery which can result in inflammation, the additional active agent can be an anti-inflammatory agent.

The present invention will be better understood with reference to the following detailed description, and with respect to the following definitions.

Definitions

The term "an effective amount" refers to the amount of proteasome inhibitor, alone or in combination with one or more antibiotics, needed to eradicate the skin infection, and/or, in combination with an anti-inflammatory agent, to eradicate the bacterial cause and inflammatory symptoms associated with various skin disorders.

By "administering" is meant a method of giving one or more doses of a topical formulation as described herein to an animal (e.g., topical administration). The method of administration may vary depending on various factors, e.g., the components of the pharmaceutical composition, site of the potential or actual bacterial infection, bacteria involved, and severity of the actual bacterial infection.

By "bacteria" is meant a unicellular prokaryotic microorganism that usually multiplies by cell division.

By "skin bacterial infection" is meant the invasion of the skin in a host animal by pathogenic bacteria. For example, the infection may include the excessive growth of bacteria that are normally present in or on the body of an animal or growth of bacteria that are not normally present in or on the animal. More generally, a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host animal. Thus, an animal is "suffering" from an skin bacterial infection when an excessive amount of a bacterial population is present in or on the animal's skin, or when the presence of a bacterial population(s) is damaging the cells in the skin of the animal.

By "persistent bacterial infection" is meant an infection that is not completely eradicated through standard treatment regimens using antibiotics. Persistent bacterial infections are caused by bacteria capable of establishing a cryptic phase or other non- multiplying form of a bacterium and may be classified as such by culturing bacteria from a patient and demonstrating bacterial survival in vitro in the presence of antibiotics or by determination of anti -bacterial treatment failure in a patient.

As used herein, a persistent infection in a patient includes any recurrence of an infection, after receiving antibiotic treatment, from the same species more than two times over the period of two or more years or the detection of the cryptic phase of the infection in the patient. An in vivo persistent infection can be identified through the use of a reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate the presence of 16S rRNA transcripts in bacterially infected cells after treatment with one or more antibiotics (Antimicrob. Agents Chemother. 12:3288-3297, 2000).

Skin viral infections include infections caused by various herpes viruses, for example, those which occur with exposure to the Herpes simplex virus, shingles, and the human papilloma virus. Skin fungal infections include athlete's foot and jock itch.

By "chronic disease" is meant a disease that is inveterate, of long continuance, or progresses slowly, in contrast to an acute disease, which rapidly terminates. A chronic disease may begin with a rapid onset or in a slow, insidious manner but it tends to persist for several weeks, months or years, and has a vague and indefinite termination.

By "immunocompromised" is meant a person who exhibits an attenuated or reduced ability to mount a normal cellular or humoral defense to challenge by infectious agents, e.g., viruses, bacterial, fungi, and protozoa. Persons considered immunocompromised include malnourished patients, patients undergoing surgery and bone narrow transplants, patients undergoing chemotherapy or radiotherapy, neutropenic patients, HIV-infected patients, trauma patients, burn patients, patients with chronic or resistant infections such as those resulting from myelodysplastic syndrome, and the elderly, all of who may have weakened immune systems.

By "inflammatory disease" is meant a disease state characterized by (1) alterations in vascular caliber that lead to an increase in blood flow, (2) structural changes in the microvasculature that permit the plasma proteins and leukocytes to leave the circulation, and (3) emigration of the leukocytes from the microcirculation and their accumulation in the focus of injury. The classic signs of acute inflammation are erythema, edema, tenderness (hyperalgesia), and pain. Chronic inflammatory diseases are characterized by infiltration with mononuclear cells (e.g., macrophages, lymphocytes, and plasma cells), tissue destruction, and fibrosis. Non-limiting examples of inflammatory skin diseases include rosacea, skin inflammation following skin surgery, inflammation resulting from physical trauma to the skin, and dermatitis.

By "treating" is meant administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. To "prevent disease" refers to prophylactic treatment of a patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a patient already suffering from a disease to improve the patient's condition. Thus, in the claims and embodiments, treating is the administration to a mammal either for therapeutic or prophylactic purposes.

The term "pharmaceutically acceptable salt" is used throughout the specification to describe any pharmaceutically acceptable salt form of the proteasome inhibitors described herein. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Citric acid is a specific example of a suitable acid. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.

Pharmaceutically acceptable salts include also include complexes with amines, including ammonia, primary, secondary and tertiary amines. The amines can form salts or partial salts with one or more of the phenolic hydrogens.

The present invention satisfies an existing need for compounds that effective in treating skin disorders mediated by proteasomes, or associated with inflammation.

I. Proteasome inhibitors

In one embodiment of the invention, the proteasome inhibitors compounds have one of the following formulas:

i) a hydrocinnamate compound selected from the group consisting of



cinnamate compound having one of the following formulas:

wherein W is selected from the group consisting of a methyl group, an alkyl group, a methylene group, an amine group, an acyl group, a carbonyl group, an oxygen atom, a sulfur atom, and wherein X₁ to X₅ are independently selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an ether group, an alkyl group, an aryl group, a nitro group, a cyano group, a thiol group, a thioether group, an amino group, an amido group, and an OR group, where R is an ester of a dihydrocinnamate; and analogs thereof wherein one or more of the hydrogen atoms on the phenyl ring in the dihydrocinnamate moiety in the listed compounds can be replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, alkyl, aryl, nitro, cyano, thiol, thioester, amino, and amido, in a pharmaceutically acceptable formulation for topical administration to the skin, and analogs of the compounds in (i) or (ii) wherein one to three of the hydrogen atoms on the aromatic ring in the dihydrocinnamate moiety is replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, Ci_-6 alkyl, C₆-₁₀ aryl, nitro, cyano, thiol, thioester, amino, and amido. These compounds, and analogs thereof, are described, for example, in U.S. Patent No. 8,809,283, the contents of which are hereby incorporated by reference for all purposes.

In one embodiment, the hydrocinnamate is PTTC, otherwise known as Irganox® 1010, having the chemical name pentaerythritol tetrakis (3-(3, 5-di-tert- butyl-4-hydroxyphenyl) propionate, and having the formula:

Analogs of the compounds discussed above, wherein the compounds have multiple cinnamate or hydrocinnamate esters, include those wherein one or more of the esters is hydrolyzed to the free OH group (i.e., partial esters of PTTC and other proteasome inhibitors).

Complexes of the compounds described herein with albumin, such as human serum albumin (HSA), are also within the scope of the invention. Looking at the amino acid sequence of HSA, there are a number of ionizable groups: 1 16 total acidic groups (98 carboxyl and 18 phenolic OH) and 100 total basic groups (60 amino, 16 imidazolyl, and 24 guanidyl). These complexes can be formed by mixing the compounds with albumin, or by complexing albumin with the compounds described herein using a multivalent cation. Multivalent cations can, for example, can bridge phenolic groups on the compounds described herein, and phenolic groups on HSA.

Analogs also include hydrocinnamate and cinnamate esters of polyhydric alcohols like pentaerythritol, for example, pentaerythritol esters with 3,2, and 1 acyl groups. As used herein, an acyl group is an ester group with a Ci-2o alkyl, C2-20 alkenyl, ₂-2oalkynyl, or C₆ or C₁₀ aryl moiety.

The compounds described herein all include at least one aryl ring, and each ring can, independently, be further substituted with one or more substituents, as defined herein. Those skilled in the art will readily understand that incorporation of other substituents onto an aryl ring used as a starting material to prepare the compounds described herein, and other positions in the compound framework, can be readily realized. Such substituents can provide useful properties in and of themselves or serve as a handle for further synthetic elaboration.

Benzene rings can be substituted using known chemistry, including the reactions discussed below. For example, alkyl substituents can be added using friedel craft alkylation reactions. Biphenyl compounds can be synthesized by treating aryl phenylmagnesium bromides with copper salts, by the oxidative dehydrogenation of the aryl rings, or the dealkylation of toluene or other methyl-substituted aromatic rings.

Aryl rings can be nitrated, and the resulting nitro group on the aryl ring reacted with sodium nitrite to form a diazonium salt. The diazonium salt can be manipulated using known chemistry to form various substituents on a benzene ring.

Diazonium salts can be halogenated using various known procedures, which vary depending on the particular halogen. Examples of suitable reagents include bromine/water in concentrated HBr, thionyl chloride, pyr-ICl, fluorine and Amberlyst-A.

A number of other analogs, bearing substituents in the diazotized position, can be synthesized from the corresponding amino compounds, via the diazo intermediate. The diazo compounds can be prepared using known chemistry, for example, as described above.

Nitro derivatives can be reduced to the amine compound by reaction with a nitrite salt, typically in the presence of an acid. Other substituted analogs can be produced from diazonium salt intermediates, including, but are not limited to, hydroxy, alkoxy, fluoro, chloro, iodo, cyano, and mercapto, using general techniques known to those of skill in the art.

For example, hydroxy-aromatic analogues can be prepared by reacting the diazonium salt intermediate with water. Halogens on an aryl ring can be converted to Grignard or organolithium reagents, which in turn can be reacted with a suitable aldehyde or ketone to form alcohol-containing side chains. Likewise, alkoxy analogues can be made by reacting the diazo compounds with alcohols. The diazo compounds can also be used to synthesize cyano or halo compounds, as will be known to those skilled in the art. Mercapto substitutions can be obtained using techniques described in Hoffman et al., J. Med. Chem. 36: 953 (1993). The mercaptan so generated can, in turn, be converted to an alkylthio substitutuent by reaction with sodium hydride and an appropriate alkyl bromide. Subsequent oxidation would then provide a sulfone. Acylamido analogs of the aforementioned compounds can be prepared by reacting the corresponding amino compounds with an appropriate acid anhydride or acid chloride using techniques known to those skilled in the art of organic synthesis.

Hydroxy-substituted analogs can be used to prepare corresponding alkanoyloxy-substituted compounds by reaction with the appropriate acid, acid chloride, or acid anhydride. Likewise, the hydroxy compounds are precursors of both the aryloxy via nucleophilic aromatic substitution at electron deficient aromatic rings. Such chemistry is well known to those skilled in the art of organic synthesis. Ether derivatives can also be prepared from the hydroxy compounds by alkylation with alkyl halides and a suitable base or via Mitsunobu chemistry, in which a trialkyl- or triarylphosphine and diethyl azodicarboxylate are typically used. See Hughes, Org. React. (N.Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996) for typical Mitsunobu conditions.

Cyano- substituted analogs can be hydrolyzed to afford the corresponding carboxami do- substituted compounds. Further hydrolysis results in formation of the corresponding carboxylic acid- substituted analogs. Reduction of the cyano- substituted analogs with lithium aluminum hydride yields the corresponding aminomethyl analogs. Acyl- substituted analogs can be prepared from corresponding carboxylic acid-substituted analogs by reaction with an appropriate alkyllithium using techniques known to those skilled in the art of organic synthesis. Carboxylic acid- substituted analogs can be converted to the corresponding esters by reaction with an appropriate alcohol and acid catalyst. Compounds with an ester group can be reduced with sodium borohydride or lithium aluminum hydride to produce the corresponding hydroxymethyl-substituted analogs. These analogs in turn can be converted to compounds bearing an ether moiety by reaction with sodium hydride and an appropriate alkyl halide, using conventional techniques. Alternatively, the hydroxymethyl-substituted analogs can be reacted with tosyl chloride to provide the corresponding tosyloxymethyl analogs, which can be converted to the corresponding alkylaminoacyl analogs by sequential treatment with thionyl chloride and an appropriate alkylamine. Certain of these amides are known to readily undergo nucleophilic acyl substitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- or N- arylcarbamoyloxy-substituted compounds by reaction with N-alkyl- or N- arylisocyanates. Amino-substituted analogs can be used to prepare alkoxycarboxamido-substituted compounds and urea derivatives by reaction with alkyl chloroformate esters and N-alkyl- or N-arylisocyanates, respectively, using techniques known to those skilled in the art of organic synthesis.

Any of the aforementioned substituents can be present on any or all of the aromatic rings in the compounds described herein.

II. Pharmaceutical Compositions/Formulations

The pharmaceutical compositions and formulations described herein include proteasome inhibitor as described herein, a suitable carrier, and, optionally, one or more other active agents. The carrier includes, at least, pentadecalactone or a similar lactone solvent. Pentadecalactone (also known as oxacyclohexadecan-2-one) has the following structure:

In one embodiment, compounds which are structurally similar to pentadecalactone can also be used, in addition to or in place of pentadecalactone. Compounds which are structurally similar to pentadecalactone are defined herein as cyclic lactones with from 10 to 20 carbon atoms in the ring structure, where the rings can include one or two lactone moieties, and, optionally, one or two carbonyl moieties, where the rings can include one or two ether moieties, i.e., where a methylene group is replaced with an oxygen, and where one or two carbons can be substituted with a Ci_-6 alkyl group.

Representative examples of compounds which are structurally similar to pentadecalactone include Oxacyclohexadecan-2-one, l,9-Dioxacycloheptadecan-2- one, 1,9-dioxacycloheptadecan-lO-one, 10-decanolactone; Oxacyclopentadecan-2- one; Oxacyclododecan-2-one; l,8-Dioxacycloheptadecan-9-one; Oxacyclotridecan-2- one; Oxacyclotetradecan-2-one; l,5-Dioxacycloheptadecan-6-one, 1,8- Dioxacycloheptadecan-7-one, l,5-dioxacyclohexadecan-6-one, 1,8- dioxacyclohexadecane-9, 16-dione, l,8-dioxacyclooctadecan-9-one, 1,8- dioxacycloheptadecane-9, 17-dione, 1 ,6-dioxacycloheptadecan-7-one, oxacycloheptadecan-2-one, 1,10-dioxacyclononadecane-l 1, 19-dione, 1,9- dioxacyclooctadecane-10, 18-dione, oxacyclododecane-2,7-dione, oxacyclotetracosan- 2-one, l,6-dioxacyclooctadecan-7-one, l,4-dioxacycloheptadecan-5-one, 1,9- dioxacyclononadecane-2,8-dione, l,5-dioxacyclotetradecane-6,14-dione, oxacyclohexadecane-2, 16-dione, 1, 10-dioxacyclooctadecane-2,9-dione, 1,5- dioxacyclopentadecane-6, 15-dione, l,8-dioxacycloheptadecane-2,7-dione, 1,6- dioxacyclotetradecane-7, 14-dione, oxacyclopentacosan-2-one, 1,5- dioxacyclotridecane-6, 13-dione, oxacycloheptacosan-2-one, 1,5- dioxacyclohexadecane-6, 16-dione, oxacyclotricosan-2-one, oxacyclohexadecane- 2, 11-dione, oxacyclohexadecane-2, 10-di one, oxacyclopentadecane-2,10-dione, 1,5- dioxacycloheptadecane-6, 17-dione, 1 ,6-dioxacyclopentadecane-7, 15-dione, oxacyclotetradecane-2, 14-dione, l,8-dioxacyclohexadecane-2,7-dione, oxacyclododecane-2, 12-dione, oxacycloicosan-2-one, oxacyclononadecan-2-one, oxacyclohentriacontan-2-one, 1 , 10-dioxacyclooctadecane-2, 11 -dione, oxacyclohenicosan-2-one, oxacyclooctadecan-2-one, l,4-dioxacyclopentadecan-5- one, 1 , 8-dioxacyclooctadecane-9, 18-dione, 1 ,6-dioxacyclohexadecane-7, 16-dione, l,7-dioxacycloheptadecan-8-one, 1, 1 l-dioxacyclodocosane-2, 12-dione, (12S or 12R)- 12-methyl-oxacyclododecan-2-one; 1,11-dioxacycl oicosane-2, 10-di one, 1,4,7- trioxacyclopentadecane-8,15-dione, (15S or 15R)-15-methyl-oxacyclopentadecan-2- one, l,4,7-trioxacycloheptadecan-8-one, oxacyclotridecane-2,9-dione, 14-ethyl- oxacyclotetradecan-2-one, oxacyclotetradecane-2, 11-dione, 1,6-dioxacyclotridecane- 7, 13-dione, l,4,7-trioxacyclohexadecane-8, 16-dione, 1,11-dioxacyclohenicosane- 2, 10-dione, 1 ,7-dioxacyclohexadecane-8, 16-dione, 1 ,5-dioxacyclododecane-6, 12- dione, l, 12-dioxacyclodocosane-2, l 1-dione, l,4-dioxacyclopentadecane-5,15-dione, 1 , 10-dioxacyclodocosane-2,9-dione, 1 , 10-dioxacycloicosane-2,9-dione, 1,8- dioxacyclooctadecane-2,7-dione, oxacyclopentadecane-2, 11-dione, oxacyclotetradecane-2, 10-dione, l,8-dioxacycloicosane-9,20-dione, 1,6- dioxacyclooctadecane-7, 18-dione, l,6-dioxacyclononadecane-7, 19-dione, 13-ethyl- oxacyclotridecan-2-one, l,l l-dioxacycloicosane-2, 12-dione, 1,4- dioxacyclododecane-5,12-dione, and oxacycloundecane-2,8-dione. Such compounds, by virtue of their structural similarity, would be expected to have similar solubilizing properties with respect to the proteasome inhibitors described herein.

Proteasome Inhibitor Formulations

The proteasome inhibitor formulations described herein can be in any suitable form for topical administration which provides suitable solubility and bioavailability for the preoteasome inhibitors.

In one embodiment, the formulations comprising the proteasome inhibitors described herein are prepared under strictly controlled Good Manufacturing Practice (GMP) conditions, ensuring both the quality and uniformity of the materials while avoiding the requirement for reconstitution by the pharmacist, physician, or patient. Moreover, sufficiently stable formulations are amendable to commercial transportation and can dispensed and administered without concern that the active component will be unacceptably degraded.

In addition, suitably stable formulations can be dispensed for administration over an extended course of treatment, or packaged in single dose forms suitable for direct administration by a patient or physician without the effort or concern over reconstitution. The formulations are intended to be topically applied.

The topical formulations are typically in the form of lotions, cremes, ointments, emulsions, micelles, liquid drops, viscous solutions or gels, or solids. In some embodiments, they include only organic solvents, and in others, they include water, organic solvents, and emulsifiers/surfactants. In those formulations that include water, the water preferably has no physiologically harmful constituents. Typically purified or deionized water is used. The pH can be adjusted as desired by adding any physiologically acceptable pH adjusting acids, bases, or buffers to within the range of about 5.0 to about 7.0, more preferably from about 5.8 to about 6.8, more preferably about 6.0 to about 6.5, more preferably at a pH of about 6.2 to about 6.4, more preferably about 6.25 to about 6.35, or more preferably about 6.3. In alternative embodiments, the proteasome inhibitor compositions of the present invention can be adjusted to a pH in the range of 5.0 to about 6.0, or more preferably about 5.5 to about 5.95, or more preferably 5.6 to 5.9. Any of the aforementioned ranges can be used with any of the topical compositions of the present invention. Examples of acids include acetic, boric, citric, lactic, phosphoric, hydrochloric, and the like, and examples of bases include potassium hydroxide, sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, tromethamine, THAM (tris-hydroxymethylamino- methane), and the like. Salts and buffers include but are not limited to citrate/dextrose, sodium bicarbonate, ammonium chloride and mixtures of the aforementioned acids and bases. The pH is preferably adjusted by adding sodium hydroxide.

Representative formulations are described in detail below.

Formulations for Topical Administration

The proteasome inhibitor compositions suitable for topical administration to the skin can include one or more specific carriers, in addition to the pentadecalactone or structurally similar compound.

The amount of proteasome inhibitor topically supplied is typically between about 2 and about 10% by weight of the formulation, and is effective to treat or prevent a disorder mediated by proteasomes, or associated with inflammation, in the skin tissue.

More specifically, the concentration within the skin tissue is desired to be at least about 0.25 μ^, preferably at least about 1 μ^, and more preferably at least about 10 μ^. The amount of proteasome inhibitor actually supplied to the skin surface will almost always be higher than the tissue concentration. This reflects the penetration hold up of the proteasome inhibitor by the outer tissue layers of the skin and that penetration is, to some extent, concentration driven. Thus, supplying greater amounts to the exterior will drive more of the proteasome inhibitor into the tissues.

Where a series of applications are typically employed in a topical administration dosing regimen, it is possible that one or more of the earlier applications will not achieve an effective concentration in the skin tissue, but that a later application in the regimen will achieve an effective concentration. This is contemplated as being within the scope of topically applying proteasome inhibitor in an effective amount. However, generally a single application, such as consisting of one or two drops, provides a therapeutically effective concentration of the proteasome inhibitor within a tissue of the skin. Indeed, although dependent on the amount and form of the topical formulation, a single application will typically provide a therapeutically effective amount of the proteasome inhibitor within a tissue of the skin for at least about 2, more preferably about 4, more preferably about 8, more preferably about 12, and more preferably at least about 18 hours. As discussed above, the formulations may be topically administered to a variety of tissues, including the skin, to provide prophylaxis or treatment of skin disorders mediated by proteasomes.

Ointments

Ointments, which are essentially an oil-based delivery vehicle, are a well known compositions for topical administration. Common bases for the preparation of ointments include mineral oil, petrolatum and combinations thereof, but oil bases are not limited thereto. When used for ophthalmic administration, ointments are usually applied as a ribbon onto the lower skinlid. The disadvantage of ointments is that they can be difficult to administer, can be messy, and can be uncomfortable or inconvenient to the patient.

Optional Components

In addition to the additional active agents that can be used, the compositions can also contain one or more of the following: surfactants, adjuvants including additional medicaments, buffers, antioxidants, tonicity adjusters, preservatives, thickeners or viscosity modifiers, and the like. Additives in the formulation may desirably include sodium chloride, EDTA (disodium edetate), and/or BAK (benzalkonium chloride), sorbic acid, methyl paraben, propyl paraben, and chlorhexidine. Other excipients compatible with various routes of administration such as topical and parenteral administration are outlined in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990).

Additional optional components include Cetyl/Stearyl Alcohol mixture, Diethylene glycol monoethyl ether, PEG 400, Propylene glycol, Ethyl Acetate, Ethanol, Stearyl alcohol, Cetyl alcohol, Stearic acid, Whiskey Lactone, Octanoic acid, Ethyl Pyruvate, Triacetine, Butylene glycol, Caprylic/capric triglyceride (coconut triglyceride), Arachidyl Alcohol/ glucoside, Behenyl alcohol, Dimethylpolysiloxane, glycolic acid ethoxalate (Laureth 4 or 7), Gael (Glycolic Acid Ethoxylate Lauryl), Isopropyl Palmitate, Butyl Alcohol, N-Butanol, C₁₃.₁₄ Isoparaffin, Propylene Carbonate, Isoamyl Alcohol, Diethanolamine, Isopropyl Palmitate, Paraffin Oil, Polycarbophil, PVP, White Petrolatum, Diazolidinyl Urea Iodopropynyl Butylcarbamate, Diazolidinyl Urea Iodopropynyl Butylcarbamate, LETCO Cream, Isoamyl Alcohol, Propylene Carbonate, Ethyl Pyruvate, Isopropyl Palmitate, Caprylic/capric triglyceride (coconut triglyceride), 3 -Methyl gammalactone, Triacetine, Arachidyl Alcohol/glucoside, Butylene glycol, Behenyl alcohol, and benzyl alcohol.

Additional optional components include C 12-15 Alkyl Benzoate, Silica, Dimethicone, Tetrahydroxypropyl Ethylenediamine, Di-ethyl hexyl 2,6 Naphthalate, Cetearyl alcohol/glucoside, Aluminum Starch octenyl succinate, cyclopentasiloxane, Phenoxy ethanol, Steareth, glyceryl steareth, Citric Acid, Dimethicone, polyacrylamide, Arachidyl Alcohol/glucoside, Methyl paraben, Acrylates, such as C₁₀. 30 alkyl acrylates, Behenyl alcohol, Benzalkonium chloride, Boron Nitride, EDTA and salts thereof, ethylparaben, Laureth 7, propyl paraben, Sodium Hydroxide, Algae Extract, Hydrolyzed Wheat protein, Ganoderma Lucidum(mushroom), Lentinus Edodes extract, Ascorbic Acid/ Ascorbyl Glucoside, BHT, Cetearyl alcohol/glucoside, chlorphenesin, Ceteareth-20, Dimethicone/ Crosspolymer, glyceryl stearate, lsohexadecane, Myrtus communis leaf extract, Polyacrylamide, PPG- 15 Stearyl ether, Polyethylene polysorbate 20, 80, PTFE, Retinol, trisiloxane, shea butter, beeswax, Caprylyl glycol, Gamier New BB Cream, Phenoxyethanol, Propanediol, Palmitic acid, PEG- 100 stearate/PEG 20, Stearic acid/alcohol, sodium cocoyl glutamate, tocopheryl acetate, Geraniol, Linalool, Limonene, Citrol, Limonene, and other essential oils, Benzyl Salicylate, Hydrolyzed Soy Protein, Cinnamic acid, phloroglucinol trimethyl ether, Arachidyl Alcohol/ glucoside, C Alkyl Benzoate, chlorphenesin, Mg Aspartate, Phenoxyethanol, Phenyl trimethicone, K-Aspartate, Steareth 2/21, glyceryl steareth, sodium cocoyl glutamate, sarcosine, trisiloxane, Ammonium Aciylyoldimethyl, tetra hydroxy propyl ethylenediamine, tocopheryl acetate, Glycine Soja, polyisobutene, phenoxyethanol, mica, titanium dioxide, Calcium Pantethein sulfonate, Rice Bran, Passiflora Edulis, corn, Citrus, apricot kernel,Majorana Rosa Canina, Camelina sativa, Aloe barbadensis, lactose, methylparaben, and propyl paraben.

III. Optional Additional Active Agents

A further aspect of the present invention involves the above-mentioned use of additional active agents in combination with the proteasome inhibitor. A composition comprising proteasome inhibitor, an additional active agent, and the topically- acceptable carriers described herein, can advantageously simplify administration and allow for treating or preventing multiple conditions or symptoms simultaneously. The "additional medicaments," which can be present in any of the ophthalmic compositional forms described herein including fluid and solid forms, are pharmaceutically active compounds having efficacy in skin application and which are compatible with the proteasome inhibitor and with the skin.

Typically, the additional medicaments include anti-inflammatory agents including steroidal and non-steroidal anti-inflammatories, anti-allergic agents, antivirals, antifungals, anesthetics, and vasoconstrictors. These other medicaments are generally present in a therapeutically effective amount. These amounts are generally within the range of from about 0.01 to 5%, more typically 0.1 to 2%, for fluid compositions and typically from 0.5 to 50% for solid dosage forms.

Anesthetics

Representative anesthetics used in skin surgeries include tetracaine, lidocaine, marcaine, oxybuprocaine, benzocaine, butamben, dibucaine, pramoxine, proparacaine, proxymetacaine, cocaine, and Alpha-2 adrenergic receptor agonists such as Dexmedetomidine and Propofol.

Anti-inflammatories

Steroids are one of the most commonly used medications for decreasing skin inflammation. By inhibiting the breakdown of phospholipids into arachidonic acid, these agents act early on the inflammatory pathway. The most common side effects of this class of medications are cataract formation and glaucoma. Representative antiinflammatory agents used for dermal indications include cortisone, dexamethasone, fluocinolone, loteprednol, difluprednate, fluorometholone, prednisolone, medrysone, triamcinolone, rimexolone, and pharmaceutically-acceptable salts thereof.

Although nonsteroidal anti-inflammatory drugs have been used to treat inflammatory conditions, physicians should exercise caution when prescribing this class of medications.

Vasoconstrictors

Representative vasoconstrictor include ²⁵I- BOMe, amphetamines, AMT, antihistamines, caffeine, cocaine, DOM, LSA, methylphenidate, mephedrone, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, tetrahydrozoline, brimonidine, and pharmaceutically acceptable salts thereof. Preferred vasoconstrictors include tetrahydrozoline and its salts, for example, tetrahydrozoline hydrochloride, and brimonidine.

In one aspect of this embodiment, such a formulation can be used to treat skin disorders such as rosacea and psoriasis.

Although the data is not shown, a formulation comprising PTTC and tetrahydrozoline hydrochloride was applied to a patient with psoriasis, and no rebound was observed.

In some aspects of this embodiment, the PTTC and vasoconstrictors are used with topical formulations other than those described herein.

Combination Therapy

Because skin disorders are frequently associated with inflammation, it can be advantageous to co-administer the proteasome inhibitor with one or more antiinflammatory agents. One such combination includes both proteasome inhibitor and dexamethasone, which can be administered in the form of a suspension, or in the form of skin drops, for topical application. Another representative corticosteroid is loteprednol etabonate.

The combination therapy can be extremely useful in connection with steroid- responsive inflammatory skin conditions for which a corticosteroid is indicated and where bacterial infection or a risk of bacterial skin infection exists. The use of a combination drug product that includes a proteasome inhibitor and an anti-inflammatory agent is indicated where the risk of inflammation is high. Steroids are one of the most commonly used medications for decreasing skin inflammation. By inhibiting the breakdown of phospholipids into arachidonic acid, these agents act early on the inflammatory pathway. The most common side effects of this class of medications are cataract formation and glaucoma. Drugs such as loteprednol etabonate (Lotemax; Bausch + Lomb, Rochester, NY) carry a lower risk of increased IOP. Another new agent is difluprednate (Durezol; Sirion Therapeutics, Tampa, FL), which possesses even greater potency than the other available corticosteroids.

Combinations of Proteasome Inhibitors and Antimicrobial Agent

The proteasome inhibitors described herein can be administered before, during, or after administration of an antimicrobial agent, and an antimicrobial compound can be included in the proteasome inhibitor-containing compositions. The antimicrobials include antibiotics, antivirals, and antifungals.

Exemplary antibiotics include beta-lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, and temocillin), cephalosporins (e.g., cepalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin, cefmatozole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, BAL5788, and BAL9141), carbapenams (e.g., imipenem, ertapenem, and meropenem), and monobactams (e.g., astreonam); beta-lactamase inhibitors (e.g., clavulanate, sulbactam, and tazobactam); aminoglycosides (e.g., streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, dibekalin, and isepamicin); tetracyclines (e.g., tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, and doxycycline); macrolides (e.g., erythromycin, azithromycin, and clarithromycin); ketolides (e.g., telithromycin, ABT-773); lincosamides (e.g., lincomycin and clindamycin); glycopeptides (e.g., vancomycin, oritavancin, dalbavancin, and teicoplanin); streptogramins (e.g., quinupristin and dalfopristin); sulphonamides (e.g., sulphanilamide, para-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, and sulfathalidine); oxazolidinones (e.g., linezolid); quinolones (e.g., nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, and sitafloxacin); metronidazole; daptomycin; garenoxacin; ramoplanin; faropenem; polymyxin; tigecycline, AZD2563; and trimethoprim.

These antibiotics can be used in the dose ranges currently known and used for these agents, particularly when such are prescribed for treating skin disorders. Different concentrations may be employed depending on the clinical condition of the patient, the goal of therapy (treatment or prophylaxis), the anticipated duration, and the severity of the infection for which the drug is being administered. Additional considerations in dose selection include the type of infection, age of the patient (e.g., pediatric, adult, or geriatric), general health, and co-morbidity. Determining what concentrations to employ are within the skills of the pharmacist, medicinal chemist, or medical practitioner. Typical dosages and frequencies are provided, e.g., in the Merck Manual of Diagnosis & Therapy (17th Ed. MH Beers et al., Merck & Co.).

IV. Treatment of Skin Disorders

The proteasome inhibitors described herein are suitable for use in treating skin disorders mediated by proteasomes, and skin disorders associated with inflammation, including those resulting from a bacterial, viral or fungal infection.

Skin Disorders With an Inflammatory Component

Several skin disorders have an inflammatory component, and thus can be treated or prevented using the proteasome inhibitors described herein. Representative types of inflammatory skin disorders that can be treated using the proteasome inhibitors described herein, for example, by topical application of compositions including one or more proteasome inhibitor, and also optionally including an antiinflammatory agent, include rosacea, acne, psoriasis, and eczema.

Specific skin disorders are described in more detail below.

Rosacea In one embodiment, the dermal disorder to be treated or prevented is rosacea. Rosacea is a manifestation of rosacea that affects the skins and skinlids. Signs and symptoms generally consist of redness, irritation or burning of the skins.

Any of these conventional approaches can be combined with treatment using proteasome inhibitors.

Skin Disorders Caused by or Associated With Microbial Infection

Certain skin disorders have a microbial component, including viruses, bacteria, fungi, and parasites.

The proteasome inhibitor formulations of this invention can be used, along with an appropriate antimicrobial agent, to treat or prevent a variety of conditions associated with skin infection. The role of the proteasome inhibitor in this setting is to minimize damage associated with inflammation, while the antimicrobial agent is administered to address the underlying cause of the inflammation (i.e., the microbial infection).

For example, conditions of the eyelids, including blepharitis, blepharconjunctivies, meibomianitis, acute or chronic hordeolum, chalazion, dacryocystitis, dacryoadenities, and acne rosacea, are a few of the tissues and conditions that can be treated by topical application of the proteasome inhibitor and the antimicrobial agent.

The prevention of infection includes pre-operative treatment prior to surgery as well as other suspected infectious conditions or contact. Examples of prophylaxis situations include treatment prior to surgical procedures such as blepharoplasty. The compositions described herein, including a proteasome inhibitor and an appropriate antimicrobial agent specific for the type of microbial infection, can be used to treat or prevent an skin infection, and to prevent, minimize, or treat inflammation resulting from an skin infection.

Specific indications that can be treated or prevented include conditions of the eyelids, including blepharitis, blepharconjunctivies, meibomianitis, acute or chronic hordeolum, chalazion, dacryocystitis, dacryoadenities, and acne rosacea; and post operative infections.

The prevention of infection includes pre-operative treatment prior to surgery as well as other suspected infectious conditions or contact. Representative microbial species include one or more of the following organisms: Staphylococcus including Staphylococcus aureus and Staphylococcus epidermidis; Streptococcus including Streptococcus pneumoniae and Streptococcus pyogenes as well as Streptococci of Groups C, F, and G and Viridans group of Streptococci; Haemophilus influenza including biotype III (H. Aegyptius); Haemophilus ducreyi; Moraxella catarrhalis; Neisseria including Neisseria gonorrhoeae and Neisseria meningitidis; Chlamydia including Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae; Mycobacterium including Mycobacterium tuberculosis and Mycobacterium avium-intracellular complex as well as a typical mycobacterium including M. marinum, M. fortuitm, and M. chelonae; Bordetella pertussis; Campylobacter jejuni; Legionella pneumophila; Bacteroides bivius; Clostridium perfringens; Peptostreptococcus species; Borrelia burgdorferi; Mycoplasma pneumoniae; Treponema pallidum; Ureaplasma urealyticum; toxoplasma; malaria; and nosema.

Some of the more common genera found are Haemophilus, Neisseria, Staphylococcus, Streptococcus, and Chlamydia. Specific types of skin disorders that can be treated or prevented by the active agents-containing compositions include, but are not limited to, the following:

Blepharatis

Nonspecific conjunctivitis (NSC) has many potential causes, including fatigue and strain, environmental dryness and pollutants, wind and dust, temperature and radiation, poor vision correction, contact lens use, computer use and dry skin syndrome. Another cause relates to the body's innate reaction to dead cells, which can cause nonspecific conjunctivitis.

This type of infection can occur when a patient's lid disease causes mild conjunctivitis, and dead Staphylococcal bacteria from the lids fall onto the skin surface. The cells trigger an inflammatory hypersensitivity reaction on the already irritated skins. This inflammatory reaction against the dead cells can be treated using the proteasome inhibitors described herein, optionally in combination with another anti-inflammatory agent, to combat inflammation, and an antibacterial compound to address the underlying cause of the inflammation, namely, infection by living Staph bacteria. Aside from allergy, the combined causes of inflammation and infection are probably the most common origins of conjunctivitis. In fact, this combination is more common than all types of infection combined. The concentration of mast cells in the conjunctiva and the eyelids makes them prime targets for hypersensitivity reactions and inflammatory disease. A compromised skin surface cannot protect itself from bacteria with full efficacy. Although NSC patients may not have full-blown bacterial infections, their skins are susceptible to some bacterial disease components.

Unlike patients with allergic conjunctivitis, who are typically treated using steroids alone, or patients who need a strong antibiotic for bacterial disease, NSC patients can benefit from a combination treatment (active agents and an antiinflammatory agent) to battle inflammatory NSC.

IV. Methods of Treating or Preventing Inflammation Following Surgery

Following surgery, a patient may suffer from skin inflammation around the incision. Administration of a proteasome inhibitor as described herein, before, during, and/or after skin surgery, can minimize, prevent, or treat the inflammation.

Any and all of the disorders discussed above can be treated using the proteasome inhibitors described herein, alone or in combination with other active agents, such as anti-inflammatory agents, antimicrobials, and anesthetics, using appropriate compositions as described herein.

The present invention will be better understood with reference to the following non-limiting examples. In these examples, all of the percentages recited herein refer to weight percent, unless otherwise indicated.

Example 1: Representative PTTC Formulations

PTTC is a sterically hindered phenolic antioxidant, and is non-volatile and soluble in certain oils, fatty acids and fragrances. PTTC is available as an white to off-white powder, granules, pellets or crystals, and is a hygroscopic and odorless compound. PTTC has the following physiochemical properties:

Molecular Weight: 1176.5/1177.63 (hydroxyl cinnamate moiety)

Molecular Formula: C73H108O12

Solubility in water: Insoluble

Melting Point: 110° - 125°C

Calculated Log P (octanol -water): 23 pH value : 5.9

CAS Registry number : 6683-19-8

Chemical Name: Benzenepropanoic acid, 3,5-bis(l,l-dimethylethyl)-4- hydroxy-, 2,2-bis[[3-[3,5-bis(l,l-dimethylethyl)-4-hydroxyphenyl]-l- oxopropoxy]methyl] - 1 , 3 -propanediyl ester

Established name: IrganoxlOlO (also known as PTTC when used as an excipient)

Formulation Studies:

Solubility

A review of dermal products containing PTTC was performed to create a list of materials compatible with PTTC. The selected solubility-enhancing agents were tested and the results are listed in Table 1.

The procedure for performing these solubility studies was as follows:

Ten (10) ml or more of the solubilizing compound are exposed to an excess of

PTTC. Each mixture is exposed for a minimum of 18 to 24 hours in a glass container while mixing.

At the end of the exposure period the mixture was filtered through a suitable filter and the filtrate collected and submitted for HPLC analysis.

The results of the study are tabulated in Table 1.

Diethylene Glycol mono-ethyl ether 42.8

Cetyl alcohol 35.5

Octanoic acid 30.2

Ethyl Pyruvate 20.8

Triacetine 20.5

Butyl ene glycol 20.1

Caprylic/capric triglyceride (coconut triglyceride) 16.6

Steryl alcohol + Cetyl alcohol (1 : 1) + EtOH (1 :2) 16.3

Arachidyl Alcohol/ glucoside 14.7

Behenyl alcohol 12.8

Dimethylpolysiloxane 12.4

glycolic acid ethoxalate (Laureth 4 or 7 10.8

Gael (Glycolic Acid Ethoxylate Lauryl) 10.8

Isopropyl Palmitate 10.0

Butyl Alcohol 8.5

N-Butanol 3.9

CI 3- 14 Isoparaffin 3.3

Propylene Carbonate 1.8

Isoamyl Alcohol 1.4

Ethanol 0.8

Diethanolamine 0.4

The focus of formulation development was defining t le oil/non-aqueous base.

In order to perform this task, the highest solubility excipients were reviewed to identify their maximum allowable amount based on the FDA list of approved excipients. This amount noted for use in dermal products was then incorporated into the solubility spread sheet.

The FDA approved concentration data were then combined with the solubility data to calculate maximum potential PTTC solubility. Several solubilizing excipients were also combined to ensure that a 1% concentration or greater can be achieved. The results of this evaluation are included below in Table 2.

Table 2

Preliminary Formulation Development and Selection Considerations

Due to the high log P and the negligible aqueous solubility of PTTC, the focus of this work was on developing PTTC-containing hydrophobic ointments.

Physical and chemical stability of PTTC and the semi-solid formulations were used to discriminate between the developed ointments.

Keeping a very hydrophobic insoluble compound like PTTC in a dissolved form is critical to allow the active to reach the dermis/epi dermis layers and exert a beneficial effect. Any formulation incapable of keeping PTTC in dissolved form was therefore excluded from consideration. A simple but effective tactile quick test was performed for all developed formulations to evaluate grittiness.

The ability of a formulation to incorporate a high level of PTTC without becoming gritty or showing signs of separation played a key role in the selection of the formulation candidates.

Based on the solubility and the above criteria, three key excipients or excipient combinations were selected as the hydrophobic base for the formulations. The first three base selections were as follows:

- Cetyl Alcohol /Steryl Alcohol

- Ethyl Acetate

- Pentadecalactone

Formulation Direction 1 - Ethyl Acetate

Formulation # 1A Emulsion Formulation

Objective: PTTC has good solubility in ethyl acetate (EtOAc), so ethyl acetate was used as a primary ingredient in this formula. The other excipients in the formulation were paraffin oil, stearyl alcohol, laureth, glycerin and water. This was intended to be an oil-in-water (o/w) emulsion formula (refer to Table 3). Table 3

Formulation # 1A

Chemicals Percentage Rationale

Ethyl Acetate 31 solubilizer

Paraffin Oil 19 Oil phase

Stearyl alcohol 5 Surfactant (higher HLB)

Laureth 10 Surfactant (lower HLB)

Glycerin 5 Water phase

PTTC 1.55 active

Water 42.65 Water phase

PTTC was dissolved in Ethyl Acetate and incorporated in the oil phase. Separately, a water phase was created with appropriate surfactants. Under homogenization, an oil phase containing PTTC was added to the water phase.

Results: Phase separation was seen with an organic phase on the top and aqueous phase at the bottom. No difference was seen even after these phases were homogenized for more than 3 hours.

Formulation # IB: Ethyl Acetate as the Hydrocarbon base

Objective: Due to PTTC's negligible solubility in water, and the accompanying challenges with o/w emulsion formulations, a solvent mixture of ethyl acetate:diethylene glycol monoethyl ether: ethanol was evaluated as a formulation base. The formulation was created by mixing these components in a ratio of (31 :25:34) v/v and then adding PTTC (refer to Table 4). The equilibrium solubility was then determined by adding an excess of PTTC to the above vehicle phase. The equilibrium solubility was determined to be 240.67 mg/ml. Table 4

Formulation IB

Chemicals Percentage Rationale

Ethyl Acetate 27.8 solubilizer

Diethylene glycol 25.5 solubilizer

monoethyl ether

Ethanol 34.9 solubilizer

PTTC 11.8 active

Result: The results show that it is possible to formulate a PTTC solution with greater than 10% PTTC concentration, in the form of a clear solution, with no thickening. However, the formulation had a strong odor of ethyl acetate, and ethyl acetate is extremely volatile. Using this data point, other formulations were pursued with a goal of having a higher viscosity, and with a solvent which has less odor and less volatility, such that the formulation would remain in the applied area for a longer period of time.

Formulation # 1C: Efforts to Increase the viscosity

Objective: In order to thicken this formulation, various viscosifying agents were used. Table 5 identifies the viscosity-enhancing agents selected for this experiment, and indicates the preliminary results of these formulation attempts.

Table 5

Viscosity Enhancing Agents

From this information PVP at a concentration of 6% was selected for inclusion in the final formulation. Visually, this formulation seemed to be an improvement over the previous formulation in terms of viscosity. This formulation is identified in Table 6. The PTTC concentration was found to be 86.59 mg/ml.

Table 6

Formulation

Formulations Comprising Cetyl-Stearyl Alcohol

Formulation # 2A

Objective: Cetyl-Stearyl alcohol is commonly used in dermal formulations. The solubility data also indicated that this combination was in the top 5 of the solubilizing agents tested. The initial formulation incorporated 45% Cetyl-Stearyl alcohol mix (the maximum acceptable FDA range) in paraffin oil (52%) media with 3 % PTTC (refer to Table 7).

Process: The Cetyl-Stearyl Alcohol mixture comes as a waxy solid at room temperature. Hence, the temperature of the alcohol mixture was increased to 80- 100°C to bring it to the liquid phase. PTTC was then added, and mixed to achieve a clear solution at 80-100°C. Paraffin oil was then added.

Table 7 : Formulation 2 A

Results: After cooling, the formulation appeared homogenous, but was also very hard and waxy. As a result, the formulation was deemed to be pharmaceutically unacceptable, due to unusually high concentration of Cetyl-Stearyl alcohol.

Formulation #2B

Objective: To reduce the Cetyl-Stearyl alcohol concentration to result in a pharmaceutically-acceptable formulation.

Formulation #2B I (22.5% Cetyl Stearyl)

The formulation was prepared on the laboratory scale to identify the appropriate amount of Cetyl Stearyl alcohol mix to be added in the final formulation. The other ingredients included pentadecalactone, PEG 400, propylene glycol and diethylene glycol monoethyl ether (refer to Table 8). Paraffin oil was not selected in this formulation because of its negative effect on PTTC solubility.

(Note Pentadecalactone was used in this formulation, and is also one of the main formulation hydrocarbon bases and is discussed in the next section).

Process: First, the solids (both cetyl stearyl alcohol mix and Pentadecalactone) were melted at 80-100°C. The active was then added to the heated liquid. Separately, the glycols were added in the specific ratio and finally added to the active liquid mix.

Table 8

Formula #2B- I

Results: The formulation resulted in pharmaceutically elegant ointment structure with a slightly waxy feel.

Formulation #2B II (11.25% Cetyl Stearyl Alcohol)

The formulation was prepared in lab scale to identify the appropriate amount of cetyl stearyl alcohol mix to be added in the final formulation. The other ingredients were pentadecalactone, PEG 400, propylene glycol and diethylene glycol monoethyl ether (refer to Table 9). Paraffin oil was not selected in this formulation due to its negative effect on PTTC solubility.

Process: First, the solids (both cetyl stearyl alcohol mix and Pentadecalactone) were melted at 80-100°C. PTTC was then added to the heated liquid. Separately, the glycols were added in the specific ratio and finally added to the active liquid mix.

Table 9: Formula #2B- II

Results: The formulation resulted in pharmaceutically elegant ointment structure with 2B having more consistent creamy texture than 2A, which was waxier.

Additional modifications:

Objective: Both of these formulations were further modified by adding ethanol in varied ranges to determine the best consistency for the final formulation. Ethanol was used to attempt to modify the hard waxy texture into creamier texture and also for enhancing penetration of PTTC into the skin. Formula# 2B-I Ethanol (with 20% Dehydrated Ethanol): 20% ethanol by was added to the original formula described as 2B-I (refer to Table 10)

Table 10 - Formula # 2B-I EtOH

Process: The formula 2B-I (Table 8) was prepared separately and then ethanol was added to the formula and mixed.

Results: The final formulation looked consistent with Formula 2B-I and has a creamy feel.

Formula#2B-II EtOH (with 20% Dehydrated ethanol): 20% ethanol was added by weight to the original formula described as 2B-II

Table 11- Formula # 2B-II EtOH

Process: The formula 2B-II (Table 9) was prepared separately and then Ethanol was added to the formula and mixed.

Results: The formulation had a liquid feel, and hence was not be considered for further formulation studies.

Formulation # 3 - Pentadecalactone-based formulations

The solubility studies showed pentadecalactone to be one of the top 5 solubilizing agents. The maximum allowable concentration of pentadecalactone for dermal products is 8%. Additional components included ethanol, Peg 400 propylene glycol, and di ethylene glycol monoethyl ether (see Table 12).

Table 12 - Formula #3

Process: First the solids (both Cetyl Stearyl alcohol and pentadecalactone) were melted at 80-100°C, and PTTC was added to the heated liquid. Separately, the glycols were added in the specific ratio and added to the active liquid mix. Finally, ethanol was added to the mixture, mixed, and cooled to room temperature.

Results: The formulation was a clear solution. However, the initial goal was to formulate a cream-like product, so this formulation was considered unacceptable from a patient application perspective and was not further developed.

An alternative route of incorporating pentadecalactone into a final PTTC formulation was pursued by combining the thick and waxy Formulation # 2, with this clear thin Formulation # 3, in various ratios as shown in Table 13. Table 13 - Combination of #2A and #3

Based on these results, the 50 to 50 mixture was chosen for further evaluation, formulation is shown in Tablel4.

Table 14

Formula #3 032i

Process: First, the solids (both Cetyl Stearyl alcohol mix and pentadecalactone) were melted at 80-100°C, and PTTC was added to the heated liquid. Separately, the glycols were added in the specific ratio and added to the active liquid mix. Finally, ethanol was added to the mixture, mixed, and cooled to room temperature.

Results: This formulation met the criteria (i.e., had a creamy texture) and was selected as one of the final formulations. Isopropyl Myristate +Pentadecalactone formulation

Preliminary work (not shown) demonstrated very good solubility of PTTC in a 2: 1 mixture of isopropyl myrisitate and pentadecalactone. Therefore, this combination was incorporated into a Cetyl Stearyl alcohol-based formulation (see Table 15).

Table 15 - Formula 035

Process: Pentadecalactone and Isopropyl Myristate were added in a 1 :2 ratios and heated to 60-80°C. Half of the PTTC was added and mixed until a clear solution was formed. Then the portion of Cetyl-Stearyl alcohol was added and mixed well while maintaining the temperature between 60-80°C. The remainder of PTTC was then added to the above mixture while hot and observed for clear solution. The glycols were then added, mixed, and cooled to room temperature.

Results: This formulation was considered very attractive, as it was the first formulation to incorporate 10% PTTC by weight without losing its creamy, homogenous, and pharmaceutically-elegant property. Therefore, it was selected for the final formulation development. Formulation Combinations

Formulation #1C +Formulation #2B-I

With the goal of formulating a homogenous ointment, and having an enhanced PTTC penetration into the skin with ethyl acetate, a combination of various formulations was evaluated, as shown below in Table 16.

Table 16

Process: Formulation #1C and Formulation #2B-I were prepared separately. They were weighed in the ratio mentioned in Table 16 and mixed well.

Results: The ratio as defined by formulation 040B was selected as one of the final formulations.

Table 17 - Formula 040B

Note: This formula is purely theoretical and was used for calculation purpose to identify the composition Emulsifying Agents:

In general, emulsions were not pursued during this formulation work. However, a base cream formulation from Letco medical products was used for formulating purposes. This product is an emulsion cream, and contains the surfactant sodium laurel sulfate, as well as white petrolatum, Cetyl-Stearyl alcohol mix, propylene glycol, glycerin, diazolidinyl urea, iodopropynyl butyl carbamate and purified water.

LETCO Emollient Cream-Based Formulations

Formula: With the intent to incorporate PTTC in aqueous based cream (o/w emulsion), three formulations were attempted with 1%, 2 % and 4% PTTC in them.

033 I, 033 II & 038:

The LETCO cream base was heated to 80°C - 100°C, PTTC was then added, and the mixture was homogenized for 15 minutes at 15,000 RPM.

Results: The formulations looked homogenous with no apparent coarseness/grittiness. Hence, it was decided to move forward with 2% and 4% PTTC/LETCO cream in the final formulations.

Additive Selection: The following additional excipients were selected to provide additional formulation properties.

Antimicrobial(s) for preservative effectiveness.

Preservatives: For emollient cream -based formulation, no preservative was added. These formulations contain Diazolidinyl Urea Iodopropynyl Butylcarbamate as a preservative. For other formulations, the preservative benzyl alcohol was added at a concentration of 0.1%, though a range of from about 0.01 to about 1% can be used.

Thickening /thinning agents for enhanced product elegance. As indicated above, several of the initial formulations were too thick and required thinning. This was managed by lowering the concentrations of Cetyl-Stearyl alcohol and replacing the bulk of the formulation with materials such as mineral oil and or glycols. Skin penetration Enhancers: The primary penetration enhancer used in these formulations is ethanol. Formulation # contains ethyl acetate. While this was initially investigated as a solubility enhancing excipient, it can also improve skin penetration. Also, Isopropyl Myristate was included in one formulation as a penetration enhancer.

Formulation Composition

Based on the final review of the formulation data presented above, eight formulations were selected for further development. These formulations and their composition are tabulated in Table 18. A Stability Study was conducted to evaluate the stability of these formulations in a preliminary (one month room temperature (RT) and accelerated (higher temperature)) stability profile. In addition, these formulations were tested to determine their skin penetration profile.

The composition of these formulations is shown in Table 18.

Table 18 - Final formulations Ingredients Matrix

LETCO^® Emollient base cream

CSA Cetyl-Stearyl alcohol mix

EA Ethyl Acetate j

PDL Pentadecalactone

PEG -400 Polyethylene glycol ;

PPG Propylene glycol

Ethanol Ethanol Anhydrous 200 proof

PVP Polyvinyl pyrrolidone (Kollidon-25)

DEGMEE Diethylene glycol mono-ethyl ether

SLS Sodium Lauryl Sulphate

wPET White Petrolatum

IPBC lodopropynyl butylcarbamate

D-Urea Diazolidinyl urea

IPM Isopropyl Myristate :

Preservative Benzyl Alcohol/ yet to be decided ;

benzalconium chloride Stability Study: The primary stability evaluation focused on test the PTTC assay for the product and the physical attributes (appearance, color, consistency, signs of separation, viscosity). The samples were stored at 25°C, 40°C, and 55°C for 1 month.

Appearance: The appearance of the formulations was evaluated visually, noting whether the formulations appeared the be in the form of ointments or liquids, whether or not they had any grittiness, and whether the ointments appeared to be creamy, smooth, firm, and/or oily.

Table 19 - Comparison of Appearance in formulations at initial, 2 weeks at 55°C, and 1 month at 25°C, 40°C, and 55°C

048 White firm White firm White firm White firm White firm ointment with ointment ointment ointment ointment slight with slight with slight with slight with slight grittiness grittiness grittiness grittiness grittiness

049 White firm White firm White firm White firm White firm ointment with ointment ointment ointment ointment slight with slight with slight with slight with slight grittiness grittiness grittiness grittiness grittiness

050 White semi- White soft White soft White soft White soft creamy creamy creamy creamy creamy ointment with ointment ointment ointment ointment liquid feel with liquid with liquid with liquid with liquid oily feel, oily feel, oily feel, oily feel, oily

051 White semi- White semi- White semi- White White semi- creamy creamy creamy semi- creamy ointment with ointment ointment creamy ointment liquid feel, with liquid with liquid ointment with liquid oily feel, oily feel, oily with liquid feel, oily feel, oily

052 White thick White thick White White White Liquid liquid like, liquid like, Liquid with Liquid with with oily oily component component component separation separation separation seen seen seen

053 White soft White soft White soft Not tested White soft creamy creamy creamy creamy ointment, oily ointment, ointment, ointment, oily oily oily

Note: No sample was testec for 053 at 55° C 2 weeks' time point

TBT: To be tested Conclusion: Comparing the formulations between Initial and 1 month pull points, all the formulations except 052 showed acceptable stability in terms of the appearance of the formulations.

Viscosity (in cp reported @ 1.5 rpm): The intrinsic viscosities of the formulations were evaluated using a Helipath spindle (Brookfield DV-II viscometer). The viscosity of the initial time-point samples was determined at 1.5 rpm. The rest of the measurement were determined using the same parameters at 3 rpm. This test method was chosen due to the high viscosity of some of the original formulations, and so that the higher viscosity products (LETCO cream base) could be measured. The final three formulations, however, were the least viscous formulations.

PTTC Viscosity Test Procedure

Objective: To determine apparent viscosity of cream formulations by using Brookfield DV-II Viscometer Spindle No. T-F, T-D and T-E (Helipath Stand). Viscosity was measured in centipoise (cp) units.

Operating Methods and conditions: The test was run at room temperature in an open system. Operating procedures were SOP-SAS-057 Rev 4

Calibration method: Appropriate reference viscosity standards were used, depending on the range of viscosity of the test formulations. The RPM was selected at which the study was performed. The equipment was turned on and calibrated by running a standard material at that particular RPM, and the calibration was repeated.

Test method:

1) The Helipath unit was positioned approximately 114" from the top of the formulation by adjusting with the driving unit;

2) The test RPM was set, and the power switch was turned to the "on" position;

3) The appropriate "SPDL" value was entered,

4) The motor was turned "on" and allowed to spin 2-3 rounds before recording the viscosity;

5) The viscosity was measured at specific time intervals (every 5 seconds);

6) The three measurements at the middle of the run were considered for an average viscosity readout. Usually, the middle three readouts were stable; and

7) The viscosity read out and cps % were tabulated at specific time intervals. The results are shown below in Table 20.

Table 20: Comparison of viscosity in formulations at Initial, 1 month 25°C, 40°C, and 55°C

Conclusion: The intent of the study was to compare the viscosities across the eight (8) selected formulations. The viscosities of the formulations varied from 500,000 to less than 1000 cps. The results showed that out of the final 3 formulations, 051 is slightly more viscous than 052 and 053 formulations.

Content Assay (%): The PTTC content was evaluated, and the results are shown in Table 21 below. Table 21: Comparison of PTTC Assay in formulations at initial, 2 weeks at 55°C, and 1 month at 25°C, 40°C, and 55°C

*Discrepancy possibly due to non-homogeneity of the samples

Note: No sample was tested for 053 at 55°C 2 weeks' time point

Conclusion: The content assays at 25°C for all the formulations except 048 and 049 were within +5 % specifications. At 55°C (2&4 weeks), both LETCO formulations showed significant variability from the label claim. It is unclear why a lower-than-label claim (2%) was obtained for the "Initial" test point of formulation 048. The discrepancy could possibly be attributed to non-homogeneity of the samples due to reduced viscosity.

Stability Study Including 3 Best Formulations (051; 052; 053)

A further stability study was performed on the best three formulations from the first study. This study included a comparison of "Appearance" and PTTC Assay of the final three (3) formulations at 25°C and 40°C conditions. The appearance of the formulations 25°C and 40°C is tabulated in Tables 22 and 23, respectively. Table 22: Appearance at 25°C:

Table 23: Appearance at 40°C

Conclusions: The results indicate that 051 appearance was consistent throughout the 3-month stability study at both 25°C and 40°C conditions. 052 ointment started having separation of components at 1 month at the 40°C condition. The 053 formulation started having component separations at the 3 -month pull point at the 40°C condition.

The PTTC Content (% by weight) was assayed at 25°C and 40°C, and the results are shown in Tables 24 and 25, respectively.

Table 24: Assay at 25°C

Table 25: Assay at 40°C

Conclusion: The above stability results indicate that the PTTC Content Assays for all three formulations remained within ±5% from the target specification.

Dermal Penetration Studies: In this study, dermal patches were exposed to the eight (8) selected formulations. Subsequent to the exposure (treatment) the skin was peeled back in layers and analyzed for drug concentration. The depth and total concentration of drug into the dermal layers was evaluated. Intradermal Delivery of PTTC (Mercer University, Report)

Objective: The goal of this study was to develop and evaluate topical PTTC formulations which can achieve delivery into the dermis.

The study performed was a finite dose study, as opposed to an infinite dose study. The amount of formulation to be spread on the skin in an infinite dose study can be anywhere from 100mg-300mg/cm², whereas a finite dose study typically uses

10 mg/cm².

Methods

Instrumental setup

Vertical Franz diffusion cells were used for the in vitro permeation studies. Such cells, designed to model skin conditions in vivo, are known in the art, and described, for example, in Hererra et al;. "In Vitro Release Testing of PLGA

Microspheres with Franz Diffusion Cells," Dissolution Technologies, pp. 6-11 (May, 2012 ). These cells include a donor chamber, where a donor compound is introduced, a membrane, a heater/circuylator, a stir bar, a receptor chamber, and a sampling port. In these experiments, the donor chamber was exposed to room temperature (25°C) while the receptor chamber was maintained at 37°C. The membrane is excised skin, and maintaining the two chambers at these temperatures brings the excised skin temperature to around 32° C. The receptor compartment was covered with aluminum foil to minimize potential evaporation of formulation solvents during the study.

Skin preparation

Skin was provided from a tissue bank and stored at -80°C until use. The skin was thawed according to directions provided from the skin bank, cut into desired size pieces and mounted between the donor and receptor compartments with the stratum corneum facing up towards the donor. The skin was then equilibrated for 15 minutes prior to the experiment. A series of test formulations evaluated in this study is shown below in Table 26. Table 26: Composition of test formulations (percentage by weight)

PTTC Penetration Investigation

Removal of residual formulation from surface of skin

Following the completion of the sampling period, skin was removed from the Franz diffusion cell. A cotton swab soaked in acetonitrile was wiped across the surface of the skin. The procedure was repeated 3 times, removing any residual with a Kim wipe.

Drug recovery from skin

The epidermis was carefully peeled off from the dermis using forceps. The minced epidermis and minced dermis were placed in 6-well plates along with extraction fluid of acetonitrile and shaken overnight. The samples were then filtered and analyzed by FIPLC. Statistical Analysis

A student's T test was performed to investigate the significance between two groups Once-way analysis of variance (ANOVA) followed by a Tukey's test was used to investigate the significance between many groups. Statistical significance was determined at a p-value < 0.05. Statistical analysis was carried out using Microsoft Excel.

HPLC Method

Column: uBondapack™ C18 lOum 125A, 3.9 x 300mm

Mobile Phase: Gradient 95%- 100% ACN over 10 min; hold 100% ACN for 5 min; 100%-95% ACN over 5 min.

Run Time: 20 min

Retention Time: ~9.7 min

Flow Rate: 1.2 ml/min

Wavelength: 210-215 nm

The results, showing PTTC skin penetration in terms of μg/ml, are shown below in Table 27.

Table 27: PTTC Skin Penetration data reported in μg/ml

A chart showing the skin penetration of PTTC using finite dosing is shown in Figure 1. Conclusions: All formulations delivered PTTC into the dermis. This may be attributed to the use of several enhancers or co-solvents in the formulations. Formulation #053 was found to have significantly greater penetration of PTTC into epidermis and dermis compared to all other formulations tested, most likely due to the high percentage of PTTC (10%) present in this formulation.

Overall Conclusions

Based on the physicochemical characteristics of PTTC, in particular very high log P and negligible aqueous solubility, many semi-solid hydrophobic ointment-type formulations were developed and tested.

Physical and chemical stability of PTTC and the ointments were used to discriminate between the developed formulations.

The ability of the formulation to incorporate a high level of PTTC, without becoming gritty and eventually separate, played a key role in selecting the formulation candidates.

Ten percent (10%) was found to be at the high-end of PTTC loading capacity for the type of formulations that were developed without turning gritty, and potentially result in phase separation.

Skin Penetration: Skin penetration studies demonstrated the ability of PTTC to penetrate into the skin from formulations 053, 052, and 051. Greater levels of PTTC penetrated into the epidermis and dermis from formulation 053 in comparison to the other two lead formulations. This is most likely due to the higher level of PTTC in formulation 053.

PTTC Assay: When tested in various dermal formulations at 25°C, 40°C, and 55°C for three (3) months, no PTTC degradation compounds or reduction in PTTC concentrations.

Formulation Appearance: Review of the Appearance data at 25°C indicated that the three (3) selected formulations retain their initial appearance and consistency at all test points throughout the study.

Review of the Appearance data at 40°C however, does indicate some separation of the formulation components for Formulations 052 and 053. It should be noted that the 40°C stability condition is above the melting point of a key excipient in these formulations. As a results, it is recommended that for future stability studies, the 25°C and 30°C intermediate stability conditions are considered instead of the 40°C. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

Claims

1. A topical formulation for skin administration, comprising

a) a Cio-20 lactone with one or two lactone moieties, optionally one or two ketone moieties, optionally, one or two Ci_-6 alkyl substituents on any carbon in the ring, and optionally, one or two oxygen atoms in the ring, and

b) a proteasome inhibitor, or a pharmaceutically-acceptable salt thereof, in a carrier for administration to the skin, wherein the proteasome inhibitor is present in a concentration of between about 2 and about 10% by weight of the formulation, and has one of the following formulas:

i) a hydrocinnamate compound selected from the group consisting of

62

63

cinnamate compound having one of the following formulas:

wherein W is selected from the group consisting of a methyl group, an alkyl group, a methylene group, an amine group, an acyl group, a carbonyl group, an oxygen atom, a sulfur atom, and wherein X₁ to X₅ are independently selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an ether group, an alkyl group, an aryl group, a nitro group, a cyano group, a thiol group, a thioether group, an amino group, an amido group, and an OR group, where R is an ester of a dihydrocinnamate;

and analogs thereof wherein one or more of the hydrogen atoms on the phenyl ring in the dihydrocinnamate moiety in the listed compounds can be replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, alkyl, aryl, nitro, cyano, thiol, thioester, amino, and amido, in a pharmaceutically acceptable formulation for topical administration to the skin, and analogs of the compounds in (i) or (ii) wherein one to three of the hydrogen atoms on the aromatic ring in the dihydrocinnamate moiety is replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, Ci_-6 alkyl, C₆-io aryl, nitro, cyano, thiol, thioester, amino, and amido,

in a pharmaceutically acceptable formulation for topical administration to the skin.

2. The formulation of Claim 1, wherein the solvent is pentadecalactone.

3. The formulation of Claim 1, wherein the proteasome inhibitor is PTTC.

4. The formulation of Claim 1, wherein the formulation is in the form of a lotion, creme, ointment, drop, oil-in-water emulsion, water-in-oil emulsion, dispersion, or micelle.

5. The formulation of Claim 1, wherein the formulation comprises one or more additional components, selected from the group consisting of Cetyl/Stearyl Alcohol mixture, Diethylene glycol monoethyl ether, PEG 400, Propylene glycol, Ethyl Acetate, Ethanol, Stearyl alcohol, Cetyl alcohol, Stearic acid, Whiskey Lactone, Octanoic acid, Ethyl Pyruvate, Triacetine, Butylene glycol, Caprylic/capric triglyceride (coconut triglyceride), Arachidyl Alcohol/ glucoside, Behenyl alcohol, Dimethylpolysiloxane, glycolic acid ethoxalate (Laureth 4 or 7), Gael (Glycolic Acid Ethoxylate Lauryl), Isopropyl Palmitate, Butyl Alcohol, N-Butanol, C 13-14 Isoparaffin, Propylene Carbonate, Isoamyl Alcohol, Diethanolamine, Isopropyl Palmitate, Paraffin Oil, Polycarbophil, PVP, White Petrolatum, Diazolidinyl Urea Iodopropynyl Butylcarbamate, Diazolidinyl Urea Iodopropynyl Butyl carbarn ate, LETCO Cream, Isoamyl Alcohol, Propylene Carbonate, Ethyl Pyruvate, Isopropyl Palmitate, Caprylic/capric triglyceride (coconut triglyceride), 3-Methyl gammalactone, Triacetine, Arachidyl Alcohol/glucoside, Butylene glycol, Behenyl alcohol, and benzyl alcohol.

6. The formulation of Claim 1, further comprising one or more additional active agents, selected from the group consisting of anti-inflammatory agents, antimicrobial agents, anesthetics, and anti -proliferative agents.

7. The formulation of Claim 6, wherein the anti-inflammatory agent is a steroid.

8. The composition of Claim 6, wherein the antimicrobial agent is selected from the group consisting of amikacin, gentamycin, tobramycin, streptomycin, netilmycin, kanamycin, ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, enoxacin, sulfonamides, polymyxin, chloramphenicol, neomycin, paramomomycin, colistimethate, bacitracin, vancomycin, tetracyclines, rifampins, cycloserine, beta-lactams, cephalosporins, amphotericins, fluconazole, flucytosine, natamycin, miconazole, ketoconazole, corticosteroids, diclofenac, flurbiprofen, ketorolac, suprofen, comolyn, lodoxamide, levocabastin, naphazoling, antazoline, and pheniramimane.

9. A topical formulation comprising PTTC in a concentration of between about 2 and about 10% by weight and pentadecalactone in a concentration of between about 5 and about 8% by weight.

10. The topical formulation of Claim 1, further comprising a mixture of Cetyl and Stearyl alcohols, isopropyl myristate, diethylene glycol monoethyl ether, and PEG 400.

11. A method for treating skin disorders associated with proteasome activity, comprising administering to skin of a mammal a formulation of any of Claims 1-10.

12. The method of Claim 11, wherein the skin disorder is selected from the group consisting of skin rosacea, eczema, dermatitis, acne, angiosarcoma, hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Kaposi's sarcoma, and the non-malignant diseases or conditions psoriasis, lymphangiogenesis, hemangioma of childhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis, tuberous sclerosis, pyogenic granulomas, recessive dystrophic epidermolysis bullosa, venous ulcers, molluscum contagious, seborrheic keratosis, and actinic keratosis.

13. The method of Claim 11, wherein the disorder is selected from the group consisting of skin rosacea, excema, dermatitis, and acne.

14. The method of Claim 11, wherein the disorder is or results from an bacterial infection.

15. The method of Claim 1 1, wherein the composition further includes one or more additional active agents, selected from the group consisting of antiinflammatory agents, antimicrobial agents, anesthetics, and anti-proliferative agents.

16. The method of Claim 15, wherein the anti -inflammatory agent is a steroid.

17. The use of:

a) a Cio-20 lactone with one or two lactone moieties, optionally one or two ketone moieties, optionally, one or two Ci_-6 alkyl substituents on any carbon in the ring, and optionally, one or two oxygen atoms in the ring, and

b) a proteasome inhibitor, or a pharmaceutically-acceptable salt thereof, in a carrier for administration to the skin, wherein the proteasome inhibitor is present in a concentration of between about 2 and about 10% by weight of the formulation, and has one of the following formulas:

i) a hydrocinnamate compound selected from the group consisting of



70

cinnamate compound having one of the following formulas:

wherein W is selected from the group consisting of a methyl group, an alkyl group, a methylene group, an amine group, an acyl group, a carbonyl group, an oxygen atom, a sulfur atom, and wherein X₁ to X₅ are independently selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an ether group, an alkyl group, an aryl group, a nitro group, a cyano group, a thiol group, a thioether group, an amino group, an amido group, and an OR group, where R is an ester of a dihydrocinnamate;

and analogs thereof wherein one or more of the hydrogen atoms on the phenyl ring in the dihydrocinnamate moiety in the listed compounds can be replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, alkyl, aryl, nitro, cyano, thiol, thioester, amino, and amido, in a pharmaceutically acceptable formulation for topical administration to the skin, and analogs of the compounds in (i) or (ii) wherein one to three of the hydrogen atoms on the aromatic ring in the dihydrocinnamate moiety is replaced with a moiety selected from the group consisting of halogen, hydroxyl, ether, Ci_-6 alkyl, C₆-io aryl, nitro, cyano, thiol, thioester, amino, and amido,

in the preparation of a pharmaceutically acceptable topical formulation for treating skin disorders associated with proteasome activity.

18. The use of Claim 17, wherein the Cio-20 lactone is pentadecalactone.

19. The use of Claim 17, wherein the proteasome inhibitor is PTTC.

20. The use of Claim 17, wherein the skin disorder is selected from the group consisting of skin rosacea, eczema, dermatitis, acne, angiosarcoma, hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma, malignant melanoma and Kaposi's sarcoma, and the non-malignant diseases or conditions psoriasis, lymphangiogenesis, hemangioma of childhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis, tuberous sclerosis, pyogenic granulomas, recessive dystrophic epidermolysis bullosa, venous ulcers, molluscum contagious, seborrheic keratosis, and actinic keratosis.

21. The use of Claim 17, wherein the disorder is selected from the group consisting of skin rosacea, excema, dermatitis, and acne.

22. The use of Claim 17, wherein the disorder is or results from an bacterial infection.

23. The use of Claim 17, wherein the medicament is in the form of a lotion or a creme for direct administration to the skin.

24. The use of Claim 17, wherein the medicament further includes an antiinflammatory agent.

25. The use of Claim 17, wherein the medicament further comprises one or more additional active agents selected from the group consisting of anti-inflammatory agents, antimicrobial agents, anesthetics, and anti-proliferative agents.

26. The use of Claim 25, wherein the anti-inflammatory agent is a steroid.

27. The use of Claim 17, wherein the skin disorder to be treated is a microbial infection, and the medicament further comprises an antimicrobial compound.