Polymeric micellar nanocarriers for targeted epidermal delivery of the hedgehog pathway inhibitor TAK-441

Abstract

Disclosed are micellar compositions comprising hedgehog pathway inhibitors and their use in treating diseases, conditions or disorders of the skin, such as skin cancer, including basal cell carcinoma.

Description

[Technical Field]

[0001] Basal cell carcinoma (BCC) is one of the most common cancers worldwide, accounting for approximately 90% of all skin cancers, with an incidence of 100 per 100,000 in the UK and 884 per 100,000 in Australia (Madan et al., 2010; Staples et al., 2006). The primary causative factor is ultraviolet (UV) radiation (Couve-Privat et al., 2002; Daya-Grosjean and Sarasin, 2000). UV-B damage induces structural mutations from C to T (or CC to TT) in the DNA of epidermal basal cells (Athar et al., 2006). [Background technology]

[0002] The Hedgehog (HH) signaling pathway is critically involved in BCC progression. While highly active during embryonic development, it is inactive in most adult tissues, except for maintaining stem cell populations and controlling hair follicle and sebaceous gland growth (Athar et al., 2006). However, mutations in the Patched1 (PTCH1) and Smoothened (SMO) proteins result in loss of function in PTCH1 and gain of function in SMO. These functional changes, respectively, lead to activation of the GLI family of transcription factors (Dlugosz et al., 2012), resulting in the basal cell hyperproliferation seen in BCC (Roewert-Huber et al., 2007; Samarasinghe and Madan, 2012). PTCH1 inactivation has been proposed to be a necessary step in BCC progression (Gailani and Bale, 1997).

[0003] Patients with locally advanced BCC cannot undergo surgery or radiation therapy (Gould et al., 2014). As a result, drug therapies have been developed to inhibit SMO and prevent activation of the HH signaling pathway. Vismodegib, a "first-in-class" synthetic inhibitor of SMO (Robarge et al., 2009; Gould et al., 2014), was approved by the U.S. Food and Drug Administration (FDA) in 2012 for the treatment of metastatic or locally advanced BCC (Dlugosz et al., 2012). Sonidegib, another Hedgehog inhibitor, was also approved by the FDA in 2015 for the treatment of locally advanced BCC (Burness, 2015).

[0004] However, mutations in SMO can impair drug interactions and potentially lead to resistance to treatment. Even before the FDA approved vismodegib in 2012, the first case of acquired resistance to vismodegib treatment due to an SMO mutation (SMO-D473H) was reported in 2009 (Yauch et al., 2009). Sonidegib treatment in vismodegib-resistant patients was ineffective (Jain et al., 2017). Furthermore, resistance to sonidegib treatment due to mutations in the drug-binding site of SMO (SMO-Q476 and SMO-D473) has also been reported (Danial et al., 2016; Jain et al., 2017; Nguyen and Cho, 2022). Summary of the Invention [Problem to be solved by the invention]

[0005] In some embodiments, the presently disclosed subject matter provides a composition comprising a hedgehog pathway inhibitor and a polymeric surfactant. In certain embodiments, the hedgehog pathway inhibitor is also active against the vismodegib-resistant Smoothened receptor D473H mutant.

[0006] In some embodiments, the hedgehog pathway inhibitor is selected from TAK-441, vismodegib, salidegib / patidegib, glasdegib, sonidegib, taladegib (Env-101), and BMS-833923 (XL-139). In certain embodiments, the hedgehog pathway inhibitor comprises TAK-441.

[0007] In some embodiments, the polymeric surfactant is biocompatible and / or biodegradable. In some embodiments, the polymeric surfactant is selected from D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), mPEG-dihex-PLA, poloxamer, poly(e-caprolactone), poly(L-amino acid), and polyvalerolactone. In certain embodiments, the polymeric surfactant comprises D-α-tocopherol polyethylene glycol 1000 succinate (TPGS).

[0008] In some embodiments, the composition comprising a hedgehog pathway inhibitor and a polymeric surfactant comprises a micelle composition. In some embodiments, the micelle composition comprises spherical micelles having a diameter ranging from about 10 nm to about 100 nm. In certain embodiments, the micelle composition comprises spherical micelles having a diameter ranging from about 10 nm to about 15 nm.

[0009] In some embodiments, the composition comprises TPGS at a concentration ranging from about 5 mg / mL to about 300 mg / mL, hi certain embodiments, the composition comprises TPGS at a concentration of about 10 mg / mL.

[0010] In some embodiments, the composition contains TAK-441 in the range of about 100 to about 500 mg per gram of TPGS. In certain embodiments, the concentration of TAK-441 is in the range of about 100 to about 300 mg of TAK-441 per gram of TPGS.

[0011] In other embodiments, the composition further comprises a hydrogel. In some embodiments, the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose-based gel formers, and poloxamer-based gelling agents. In certain embodiments, the hydrogel is selected from HPC and HPMC.

[0012] In certain embodiments, the HPMC is selected from: (a) HPMC having a molecular weight of about 26 kDa, a methoxyl content ranging from about 19% to about 24%, and a hydroxypropoxyl content ranging from about 7% to about 12%; (b) HPMC having a molecular weight of about 10 kDa, a methoxyl content ranging from about 28% to about 30%, and a hydroxypropoxyl content ranging from about 7% to about 12%; and (c) combinations thereof.

[0013] In a more specific embodiment, the composition comprises about 0.25% (w / w) TAK-441; about 0.93% (w / w) TPGS; about 5% (w / w) HPMC having a molecular weight of about 26 kDa, a methoxyl content ranging from about 19% to about 24%, and a hydroxypropoxyl content ranging from about 7% to about 12%; and about 3% HPMC having a molecular weight of about 10 kDa, a methoxyl content ranging from about 28% to about 30%, and a hydroxypropoxyl content of about 7% to about 12%.

[0014] In some embodiments, the composition further comprises a rheology modifier. In some embodiments, the rheology modifier is selected from glycerol, low and high molecular weight celluloses, and sorbitol. In certain embodiments, the rheology modifier comprises glycerol.

[0015] In some embodiments, the composition further comprises a preservative. In some embodiments, the preservative is selected from sodium disulfite, benzyl alcohol, benzalkonium chloride, chlorobutanol, sodium benzoate, potassium sorbate, methylparaben, and propylparaben. In certain embodiments, the preservative comprises sodium disulfite.

[0016] In some embodiments, the composition retains about 90% to 100% of the hedgehog pathway inhibitor content after about 6 months of storage.

[0017] In another aspect, the presently disclosed subject matter provides a method of treating a disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway, comprising administering to a subject in need thereof a composition disclosed herein.

[0018] In some embodiments, the composition is administered topically. In certain embodiments, the composition is delivered to the skin. In certain embodiments, the composition is delivered to the viable epidermis of a subject. In certain embodiments, the composition is delivered to the upper dermis of a subject. In certain embodiments, topical administration results in minimal transdermal penetration.

[0019] In certain embodiments, the disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway comprises a skin disease, disorder, or condition. In certain embodiments, the skin disease, disorder, or condition comprises skin cancer. In more particular embodiments, the skin cancer comprises basal cell carcinoma. In even more particular embodiments, the subject has or is suspected of having locally advanced basal cell carcinoma. In even more particular embodiments, the subject has or is suspected of having metastatic basal cell carcinoma. In certain embodiments, the subject has not undergone surgical or radiation therapy. In more particular embodiments, administering the composition to the subject attenuates the progression of basal cell carcinoma. In certain embodiments, the basal cell carcinoma is associated with the vismodegib-resistant SMO mutant D473H.

[0020] In some embodiments, administering the composition to a subject prevents activation of the HH signaling pathway.

[0021] In certain embodiments, the disease, condition, or disorder is associated with a mutation in the Patched1 (PTCH1) protein. In particular embodiments, the mutation in the Patched1 (PTCH1) protein results in a loss of function of the PTCH1 protein. In certain embodiments, the disease, condition, or disorder is associated with a mutation in the Smoothened (SMO) protein. In particular embodiments, the mutation in the Smoothened (SMO) protein is associated with a gain of function of the SMO protein. In more particular embodiments, the loss of function of the PTCH1 protein or the gain of function of the SMO protein results in the activation of one or more GLI transcription factors. In even more particular embodiments, the activation of one or more GLI transcription factors results in hyperproliferation of basal cells associated with basal cell carcinoma.

[0022] In some embodiments, SMO is inhibited by administering the composition to a subject. In some embodiments, mutations in SMO cause resistance to treatment with a hedgehog pathway inhibitor. In certain embodiments, resistance to treatment with a hedgehog pathway inhibitor is accompanied by mutations in the drug binding site of SMO. In more specific embodiments, the drug binding site of SMO is SMO-Q476 and / or SMO-D473.

[0023] Having described certain aspects of the presently disclosed subject matter that are addressed in whole or in part by the presently disclosed subject matter, other aspects will become apparent as the description proceeds when taken in conjunction with the accompanying examples and drawings, as best described herein below.

[0024] The patent or application file contains at least one color drawing. Copies of any color drawing(s) in the patent or published patent application file will be provided by the Office upon request and payment of the necessary fee.

[0025] Having thus described the subject matter of the present disclosure in general terms, reference is now made to the accompanying drawings, which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0026] [Figure 1] The chemical structure of TAK-441 (MW 576.57 Da; logP 2.61; water solubility 81 μg / mL at pH 6.8) is shown ( Ohashi et al., 2012 ; Ishii et al., 2014 ). [Figure 2] 1 is a TEM image of an embodiment of a TAK-441 micelle formulation (3 mg / mL) of the present disclosure. [Figure 3] 1 is a rheogram of an embodiment of a TPGS micelle-based 3% HPC gel loaded with TAK-441 of the present disclosure; [Figure 4] Figures 4A, 4B, and 4C show the deposition amount and biodistribution of TAK-441 (micelle solution and micellar HPC gel formulations, n=6) in pig skin. (Figure 4A) Deposition amount of TAK-441 in pig skin; (Figure 4B) Distribution of TAK-441 in pig skin at an infinite dose; (Figure 4C) Distribution of TAK-441 in pig skin at a finite dose (**P<0.05, one-way ANOVA) (mean ± SD). [Figure 5] 1 is a rheogram of an embodiment of a TPGS micelle-based 3% HPMC gel loaded with TAK-441 of the present disclosure. [Figure 6] The stability of TAK-441-loaded micellar formulations is shown. The TAK-441 content in Formulation E and HPMC gel of Formulation E packaged in aluminum tubes (Nussbaum Kesswil AG, Switzerland) was quantified using UHPLC-MS / MS at different time points over a 6-month storage period (storage at 4°C). After 6 months, the TAK-441 content in the micellar solution was 79.62% of the initial value, while the TAK-441 content in the micellar gel was 91.86% of the initial value. This confirmed that the micelles were intact in the gel formulations. [Figure 7]Figures 7A, 7B, and 7C show the human skin deposition and biodistribution of TAK-441 (micelle-based HPMC gel formulation, n=6). (Figure 7A) Human skin deposition of TAK-441; (Figure 7B) Distribution of TAK-441 in human skin at an infinite dose; (Figure 7C) Distribution of TAK-441 in human skin at a finite dose (**P<0.05, one-way ANOVA) (mean ± SD). DETAILED DESCRIPTION OF THE INVENTION

[0027] The presently disclosed subject matter will now be described more fully with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it should be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0028] More specifically, in some embodiments, the presently disclosed subject matter provides a composition comprising a hedgehog pathway inhibitor and a polymeric surfactant. Representative hedgehog pathway inhibitors include, but are not limited to, vismodegib and sonidegib, and other hedgehog pathway inhibitors, particularly SMO inhibitors, currently undergoing clinical trials include, but are not limited to, IPI-926 (salidegib), BMS-833923 / XL139, PF-04449913 (glasdegib) and LY2940680 (taladegib). In some embodiments, the hedgehog pathway inhibitor is active against vismodegib-resistant Smoothened receptor D473H mutants.

[0029] In some embodiments, the hedgehog pathway inhibitor is selected from TAK-441, vismodegib, salidegib / patidegib, glasdegib, sonidegib, taladegib (Env-101), and BMS-833923 (XL-139). In certain embodiments, the hedgehog pathway inhibitor comprises TAK-441.

[0030] In some embodiments, the polymeric surfactant is biocompatible and / or biodegradable. In some embodiments, the polymeric surfactant is selected from D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), mPEG-dihex-PLA, poloxamer, poly(E-caprolactone), poly(L-amino acid), and polyvalerolactone. In certain embodiments, the polymeric surfactant comprises D-α-tocopherol polyethylene glycol 1000 succinate (TPGS).

[0031] In certain embodiments, the composition comprising a hedgehog pathway inhibitor and a polymeric surfactant comprises a micellar composition.

[0032] As used herein, the term "micelle" refers to an aggregate of surfactant molecules. Micelles form only when the concentration of surfactant is greater than the critical micelle concentration (CMC). Surfactants are amphiphilic, meaning they contain both hydrophobic and hydrophilic groups. Micelles can exist in a variety of shapes, including spheres, cylinders, and discs.

[0033] In certain embodiments, the micelle composition comprises spherical micelles having a diameter in the range of about 10 nm to about 100 nm, including 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nm. In certain embodiments, the micelle composition comprises spherical micelles having a diameter in the range of about 10 nm to about 15 nm, including about 10, 11, 12, 13, 14, and 15 nm.

[0034] Polymeric micelles can be used as nanocarriers for delivering poorly water-soluble, water-insoluble, or hydrophobic drugs, which can be solubilized in the hydrophobic core of the micelles. Therefore, micelles can be useful for improving the solubility and bioavailability of various hydrophobic drugs. Their small size (typically about 10 nm to about 100 nm, including about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nm) allows for efficient accumulation of the associated active moiety in target tissues. Micelles can be formed from one or more polymeric nonionic surfactants.

[0035] As described above, in certain embodiments, the surfactant comprises a tocopherol or a derivative thereof. Tocopherols are a type of methylated phenol, many of which have vitamin E activity. Tocopherol and its derivatives, such as esters, are widely used as vitamin supplements and antioxidants in the food industry and in many pharmaceutical compositions. Tocopherols include a variety of natural and synthetic compounds. Vitamin E α-tocopherol (chemical name: 2,5,7,8-tetramethyl-2-(4',8',12'-trimethyldecyl)-6-chromanol) is the most active, widely distributed in nature, and most widely studied. Other members of this class include β, γ, and δ tocopherols. Tocopherols exist in several isomeric forms, with the D and DL forms being the most widely available. As used herein, the term "tocopherol" includes all such natural and synthetic tocopherol or vitamin E compounds.

[0036] Any form or isomer of tocopherol and its derivatives, such as esters, can be used in accordance with the present disclosure. For example, α-tocopherol or α-tocopherol acetate, linoleate, nicotinate, or hemisuccinate, many of which are commercially available, can be used herein.

[0037] Tocopherol derivatives include chemical derivatives of vitamin E with various lengths of polyethylene glycol ester and ether linkages at various chemical sites. For example, derivatives can include vitamin E tocopherol polyethylene glycol succinate (TPGS) derivatives with PEG molecular weights of about 500-6000 Da, including about 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, and 6000 Da. In certain embodiments, the vitamin E polymer derivative is D-α-tocopherol polyethylene glycol 1000 succinate (TPGS).

[0038] More specifically, TPGS is a water-soluble derivative of vitamin E in which polyethylene glycol subunits are attached to the ring hydroxyls of the vitamin E molecule via succinic acid diesters. TPGS is a nearly odorless, waxy, amphiphilic substance with a molecular weight of approximately 1513. Due to its amphiphilic structure, TPGS forms stable micelles in aqueous vehicles with a hydrophile / lipophile balance (HLB) value of 13.2. TPGS is approved by the U.S. Food and Drug Administration (FDA) as a pharmaceutical excipient.

[0039] The tocopherol surfactants of the present disclosure can be used alone or in combination with other known surfactants, such as phospholipids, polysorbates, sorbitan esters of fatty acids, cetearyl glucoside or poloxamers, or other stabilizers, such as xanthan gum or propylene glycol alginate.

[0040] In certain embodiments, the composition comprises a concentration of TPGS having a range of about 5 mg / mL to about 300 mg / mL, including about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, and 300 mg / mL. In certain embodiments, the composition comprises a TPGS concentration of about 10 mg / mL.

[0041] In certain embodiments, the composition contains TAK-441 in the range of about 100 to about 500 mg per gram of TPGS, including about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, and 500 mg. In certain embodiments, the concentration of TAK-441 is in the range of about 100 to about 300 mg per gram of TPGS.

[0042] In other embodiments, the composition further comprises a hydrogel. In some embodiments, the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), cellulose-based gel formers, and poloxamer-based gelling agents. In certain embodiments, the hydrogel is selected from HPC and HPMC.

[0043] In certain embodiments, the HPMC is selected from: (a) HPMC having a molecular weight of about 26 kDa, a methoxyl content in the range of about 19% to about 24%, including 19, 20, 21, 22, 23, and 24%, and a hydroxypropoxyl content in the range of about 7% to about 12%, including about 7, 8, 9, 10, 11, and 12%; (b) HPMC having a molecular weight of about 10 kDa, a methoxyl content in the range of about 28% to about 30%, including 28, 29, and 30%, and a hydroxypropoxyl content in the range of about 7% to about 12%, including about 7, 8, 9, 10, 11, and 12%; (c) combinations thereof.

[0044] In a more specific embodiment, the composition comprises about 0.25% (w / w) TAK-441; about 0.93% (w / w) TPGS; about 5% (w / w) HPMC having a molecular weight of about 26 kDa, a methoxyl content ranging from about 19% to about 24%, and a hydroxypropoxyl content ranging from about 7% to about 12%; and about 3% HPMC having a molecular weight of about 10 kDa, a methoxyl content ranging from about 28% to about 30%, and a hydroxypropoxyl content ranging from about 7% to about 12%.

[0045] In some embodiments, the composition further comprises a rheology modifier. In some embodiments, the rheology modifier is selected from glycerol, low and high molecular weight cellulose, and sorbitol. In certain embodiments, the rheology modifier comprises glycerol.

[0046] In some embodiments, the composition further comprises a preservative. In some embodiments, the preservative is selected from sodium disulfite, benzyl alcohol, benzalkonium chloride, chlorobutanol, sodium benzoate, potassium sorbate, methylparaben, and propylparaben. In certain embodiments, the preservative comprises sodium disulfite. Exemplary preservatives further include, but are not limited to, sorbic acid, benzoic acid, methylparaben, propylparaben, methylchloroisothiazolinone, methylisothiazolinone, diazolidinyl urea, chlorobutanol, triclosan, benzethonium chloride, p-hydroxybenzoate, chlorhexidine, digluconate, hexadecyltrimethylammonium bromide, alcohol, benzalkonium chloride, boric acid, bronopol, butylparaben, calcium butylene acetate, calcium chloride, calcium lactate, carbon dioxide, cationic bentonite, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, and chlorocresol. Ingredients include methylparaben, chloroxylenol, citric acid monohydrate, cresol, dimethyl ether, ethylparaben, glycerin, hexetidine, imidurea, isopropyl alcohol, lactic acid, monothioglycerol, pentetic acid, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium benzoate, potassium metabisulfite, potassium sorbate, propionic acid, propyl gallate, propylene glycol, sodium acetate, sodium benzoate, sodium borate, sodium lactate, sodium sulfite, sodium propionate, xylitol, sulfur dioxide, carbon dioxide, and combinations thereof.

[0047] In certain embodiments, the composition retains about 90% to 100%, including about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100%, of the hedgehog pathway inhibitor content after about 6 months of storage, including 1, 2, 3, 4, 5, 6, 7, 8, and 9 months.

[0048] In other embodiments, the presently disclosed subject matter provides a method of treating a disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway, the method comprising administering to a subject in need of such treatment a composition of the present disclosure described herein.

[0049] In some embodiments, the composition is administered topically. In certain embodiments, the composition is delivered to, i.e., in relation to, the skin. In certain embodiments, the composition is delivered to the viable epidermis of a subject, i.e., the layer of skin just below the stratum corneum. In certain embodiments, the composition is delivered to the upper dermis of a subject. The dermis includes the papillary dermis, which is the top layer of the dermis, and the reticular dermis, which is the lower layer of the dermis found below the papillary dermis. In certain embodiments, topical administration results in negligible percutaneous penetration. Generally, percutaneous penetration involves the penetration of a therapeutic agent into the stratum corneum and passing through the deeper epidermis and dermis without drug accumulation in the dermal layer. Negligible percutaneous penetration may include drug accumulation in the dermal layer of about 0.001%, 0.01%, 0.1%, and 1%.

[0050] In certain embodiments, the disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway comprises a skin disease, disorder, or condition. In more particular embodiments, the skin disease, disorder, or condition comprises skin cancer. In more particular embodiments, the skin cancer comprises basal cell carcinoma. In even more particular embodiments, the subject has or is suspected of having locally advanced basal cell carcinoma. In even more particular embodiments, the subject has or is suspected of having metastatic basal cell carcinoma. In certain embodiments, the subject has not undergone surgical or radiation therapy. In more particular embodiments, administering the composition to the subject attenuates the progression of basal cell carcinoma. In certain embodiments, the basal cell carcinoma is associated with the vismodegib-resistant SMO mutant D473H.

[0051] In certain embodiments, administration of the composition to a subject prevents activation of the HH signaling pathway.

[0052] In certain embodiments, the disease, condition, or disorder is associated with a mutation in the Patched1 (PTCH1) protein. In certain embodiments, the mutation in the Patched1 (PTCH1) protein results in a loss of function of the PTCH1 protein. In certain embodiments, the disease, condition, or disorder is associated with a mutation in the Smoothened (SMO) protein. In certain embodiments, the mutation in the Smoothened (SMO) protein is associated with a gain of function of the SMO protein. In more specific embodiments, the loss of function of the PTCH1 protein or the gain of function of the SMO protein results in the activation of one or more GLI transcription factors. In even more specific embodiments, the activation of one or more GLI transcription factors results in hyperproliferation of basal cells associated with basal cell carcinoma.

[0053] In some embodiments, administering the composition to a subject inhibits SMO. In some embodiments, mutations in SMO cause resistance to treatment with hedgehog pathway inhibitors. In certain embodiments, resistance to treatment with hedgehog pathway inhibitors is accompanied by mutations in the drug binding site of SMO. In more specific embodiments, the drug binding site of SMO is SMO-Q476 and / or SMO-D473.

[0054] The compositions of the present disclosure can be administered as monotherapy or in combination with other therapies, including photodynamic therapy (PDT).

[0055] The term "combination" is used in the broadest sense to mean that a subject is administered at least two agents or treatments, e.g., a composition of the present disclosure and at least one other therapeutic agent or treatment. More specifically, the term "in combination" refers to the co-administration of two (or more) active agents, e.g., for the treatment of a single disease state. As used herein, the active agents may be administered in combination in a single dosage form, simultaneously in separate dosage forms, or alternately or sequentially on the same or different days in separate dosage forms. In one embodiment of the presently disclosed subject matter, the active agents are administered in combination in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., where it is desirable to vary the amount of one while maintaining the amount of the other). The single dosage form may include an additional active agent for the treatment of a disease state.

[0056] Additionally, the compositions of the present disclosure can be administered alone or in combination with adjuvants that enhance the stability of the composition, and can be administered alone or in combination with one or more therapeutic agents, in certain embodiments, to facilitate administration of pharmaceutical compositions containing them, increase solubility or dispersibility, enhance inhibitory activity, provide adjunctive therapy, etc., and include other active ingredients. Advantageously, such combination therapies utilize lower doses of conventional therapeutic agents, thus avoiding the toxicity and side effects that can occur when these agents are used as monotherapies.

[0057] The timing of administering the composition of the present disclosure and at least one additional therapeutic agent or treatment can be varied, as long as the beneficial effect of the combination of these drugs is achieved.Therefore, the phrase "in combination with" refers to administering the composition described herein and at least one additional therapeutic agent or treatment simultaneously, sequentially, or any combination thereof.Therefore, the subject who is administered the combination of the composition described herein and at least one additional therapeutic agent or treatment can administer the composition of the present disclosure and at least one additional therapeutic agent or treatment contemporaneously (i.e., simultaneously) or at different times (i.e., on the same day, sequentially, in either order, or on different days), as long as the combined effect of both drugs is achieved in the subject.

[0058] When administered sequentially, the agents can be administered within 1 minute, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, or more of each other. In other embodiments, sequentially administered agents can be administered within 1 day, 5 days, 10 days, 15 days, 20 days, or more of each other. When a compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each containing either the compound or the at least one additional therapeutic agent, or as a single pharmaceutical composition containing both agents.

[0059] When administered in combination, the effective concentration of each agent to induce a specific biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing for a reduction in the dose of one or more agents compared to the dose required when the agent is administered alone.The effects of multiple agents may be, but do not have to be, additive or synergistic.Agents may be administered multiple times.

[0060] In some embodiments, two or more agents may have a synergistic effect when administered in combination. As used herein, "synergy," "synergistic," "synergistically," and derivatives thereof, such as "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0061] Synergy can be expressed as a "synergy index (SI)", which can generally be determined by the method described in F.C. Kullet et al., Applied Microbiology 9, 538 (1961), from the ratio calculated as follows: Q a / Q A +B / Q B = Synergy Index (SI) During the ceremony, Q A is the concentration of component A acting alone that produced the endpoint for component A; Q a is the concentration of component A in the mixture that produced the endpoint; Q B is the concentration of component B acting alone that produced the endpoint for component B; Q b is the concentration of component B in the mixture that produced the endpoint.

[0062] In general, Q a / Q A and Q b / Q BA sum greater than 1 indicates antagonism; a sum equal to 1 indicates additive action; and a sum less than 1 indicates synergy. The lower the SI, the greater the synergistic effect of that particular mixture. Thus, a "synergistic combination" has greater activity than would be expected based on the observed activity of the individual components when used alone. Furthermore, a "synergistically effective amount" of a component refers to the amount of that component needed to elicit a synergistic effect, for example, with another therapeutic agent present in the composition.

[0063] As used herein, the term "treating" can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or signs of such disease, disorder, or condition. Prevention refers to preventing the occurrence of a disease, disorder, or condition, or its symptoms or signs, or worsening of its severity. Thus, the compounds of the present disclosure can be administered prophylactically to prevent or reduce the occurrence or recurrence of a disease, disorder, or condition.

[0064] In many embodiments, the "subject" treated by the methods of the present disclosure is desirably a human subject, although it is understood that the methods described herein are effective with respect to all vertebrate species intended to be encompassed by the term "subject." Thus, a "subject" can include a human subject for medical purposes, such as treatment of an existing disease or disorder or prophylactic treatment to prevent the onset of a disease or disorder, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include, but are not limited to, primates, such as humans, monkeys, apes, etc.; bovines, such as cows, oxen, etc.; ovines, such as sheep, etc.; ungulates, such as goats, etc.; porcines, such as pigs, boars, etc.; equines, such as horses, donkeys, zebras, etc.; felines (including wild cats and domestic cats); canines (including dogs); lagomorphs (including rabbits, hares, etc.); and rodents (including mice, rats, etc.). The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Furthermore, a "subject" can include a patient suffering from or suspected of suffering from a disease or disorder. Thus, the terms "subject" and "patient" are used interchangeably herein. The term "subject" also refers to an organism, tissue, cell, or collection of cells from a subject.

[0065] Generally, an "effective amount" of an active agent refers to the amount necessary to elicit a desired biological response. As will be understood by those skilled in the art, the effective amount of an agent can vary depending on factors such as the desired biological endpoint, the agent being delivered, the configuration of the pharmaceutical composition, and the drug target.

[0066] Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, a reference to "a subject" includes a plurality of subjects unless the context clearly dictates otherwise (e.g., multiple subjects), etc.

[0067] Throughout this specification and claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense unless the context otherwise requires. Similarly, the term "include" and its grammatical variations are intended to be non-limiting, and thus reference to items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0068] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, amounts, characteristics and other numerical values ​​used in the specification and claims are understood to be modified in all instances by the term "about," even if the term "about" is not explicitly stated in conjunction with a value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are not, and need not be, exact and can be approximated and / or increased or decreased as desired to reflect tolerances, conversion factors, rounding, measurement error, and the like, as well as other factors known to those of ordinary skill in the art depending upon the desired properties sought to be obtained by the presently disclosed subject matter. For example, when referring to a value, the term "about" can mean encompassing, in some embodiments, ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% variation from the stated amount, as variation appropriate for practicing the disclosed methods or utilizing the disclosed compositions.

[0069] Furthermore, when used in connection with one or more numerical values ​​or numerical ranges, the term "about" should be understood to refer to all such numerical values, inclusive of all numerical values ​​within the range, and to modify that range by extending the boundaries above and below the recited numerical values. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., integers including fractions thereof (e.g., recitation of 1 to 5 includes 1, 2, 3, 4, 5 and fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. [Example]

[0070] The following examples are included to provide guidance to those skilled in the art for practicing representative embodiments of the presently disclosed subject matter. In light of this disclosure and the general level of skill of those skilled in the art, those skilled in the art will appreciate that the following examples are intended to be illustrative only, and that numerous changes, modifications, and variations can be adopted without departing from the scope of the presently disclosed subject matter. The general description and specific examples that follow are intended for illustrative purposes only and are not to be construed in any way as limiting the ability to prepare compounds of the present disclosure by other methods.

[0071] Example 1 Formulation development and intradermal distribution in pig and human skin 1.1 Overview TAK-441 is a potent inhibitor of the Hedgehog pathway (IC 504.4 nm), which is active against the vismodegib-resistant Smoothened receptor D473H mutant and is intended for the treatment of basal cell carcinoma. This example describes the development of a micelle-based formulation of TAK-441 using D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and the investigation of its dermal delivery and distribution. Results showed that incorporation of TAK-441 into TPGS micelles increased its water solubility by approximately 40-fold. One embodiment, a TAK-441-loaded HPMC hydrogel of TPGS micelles, retained approximately 92% of its initial TAK-441 content even after 6 months of storage at 4°C. Finite-dose experiments using human skin demonstrated that this formulation resulted in significantly higher TAK-441 skin deposition after 12 hours compared to a control formulation without micelles (0.40 ± 0.11 μg / cm, respectively). 2 , 0.05±0.02μg / cm 2 Furthermore, no transdermal penetration was observed. The intradermal distribution profile showed that TAK-441 was delivered primarily to the viable epidermis and upper dermis. Delivery from the HPMC hydrogel formulation resulted in epidermal concentrations of TAK-441 exceeding the IC 50 It is several thousand times higher than the hydroxybenzoates, and transdermal penetration is nearly negligible, thereby reducing the risk of systemic side effects in vivo.

[0072] 1.2 Background TAK-441 is a potent inhibitor of the HH pathway (IC 50 4.4 nm; determined by luciferase reporter activity in NIH3T3 cells stably transfected with a Gli reporter construct) and is effective against the vismodegib-resistant SMO mutant D473H (Goldman et al., 2015; Ishii et al., 2014; Ohashi et al., 2012). Ishii et al. 50 reported an IC of 79 nm; whereas, IC of vismodegib 50The peak wavelength was 7100 nm. TAK-441 has a molecular weight of 576.57 Da (Figure 1), moderate lipophilicity (logP 2.61), and extremely low water solubility (81 μg / mL at pH 6.8) (Ohashi et al., 2012; Ishii et al., 2014). A phase I clinical trial investigated oral administration at doses ranging from 50 mg / day to a maximum daily dose (MFD) of 1600 mg / day (Goldman et al., 2015). Gli1 expression in skin biopsies was strongly suppressed at all doses, but various side effects were observed. All patients experienced at least one adverse event (AE); mild to moderate AEs included dysgeusia, fatigue, nausea, muscle cramps, and hyponatremia (Goldman et al., 2015). Approximately 35% of patients experienced serious AEs, including gastrointestinal disorders, neoplasms (progression of underlying disease), and hepatobiliary disorders. One death from cerebral hemorrhage in a patient with pancreatic cancer was assessed by the investigator as related to the study drug.

[0073] Topical administration of TAK-441 not only improves efficacy by better targeting the disease site, but also potentially improves treatment tolerability by reducing systemic side effects. Direct application to the disease site significantly reduces the required dose compared with oral administration, and by definition, also reduces systemic or "off-target" toxicity. However, topical drug therapy with TAK-441 must ensure sufficient cutaneous bioavailability, specifically, achieve supratherapeutic concentrations in the basal epidermis. Due to its poor water solubility, it is easier to formulate TAK-441 in a more lipophilic, more soluble system; however, the increased solubility and therefore formulation stability translates into lower thermodynamic activity and poorer partitioning into the stratum corneum.

[0074] Polymeric micelles are colloidal nanocarriers formed from polymeric surfactants that self-assemble in aqueous media at concentrations above the critical micelle concentration ( Lavasanifar et al., 2002 ). We previously demonstrated that methoxypoly(ethylene glycol)-di-hexyl-substituted-polylactic acid (mPEGhexPLA) micelles were used to develop aqueous formulations of several poorly water-soluble therapeutic agents for dermatological applications: econazole (Bachhav et al., 2011), tacrolimus (Lapteva et al., 2014a), cyclosporine (Lapteva et al., 2014b), retinoic acid (Lapteva et al., 2015), imiquimod (Lapteva et al., 2019), and spironolactone (Dahmana et al., 2021), enabling improved dermal delivery compared to existing approved formulations (Lapteva et al., 2014a, 2015, 2019). Furthermore, another study developed the first topical formulation of vismodegib using mPEGhexPLA micelles and demonstrated that therapeutically relevant amounts of the drug could be delivered to the epidermis and upper dermis using an intradermal distribution method ( Kandekar et al., 2019 ).

[0075] In other studies, we have used the copolymer d-α-tocopherol polyethylene glycol succinate 1000 (TPGS) (Kandekar et al., 2018), sirolimus (Quartier et al., 2021a), and co-formulations of econazole, terbinafine, and amorolfine (Gou et al., 2022). This biocompatible and biodegradable surfactant is an amphiphilic derivative of natural vitamin E and has received regulatory approval as a pharmaceutical excipient (Aggarwal et al., 2012). TPGS is FDA-approved as a pharmaceutical ingredient and is used as an excipient in various commercial products (Zhang et al., 2015; Vadlapudi et al., 2014). TPGS has also been approved as an active pharmaceutical ingredient (API) by the European Medicines Agency on July 24, 2009 (Vendrop®) for the treatment of vitamin E deficiency due to digestive malabsorption in pediatric patients with congenital or hereditary chronic cholestasis (Papas, 2021).

[0076] 1.3 Scope The objectives of this study were: (i) to investigate the feasibility of using TPGS micelles to overcome the inherently poor water solubility of TAK-441 and develop a stable aqueous formulation; (ii) to characterize the micelles in terms of drug content, size, and morphology; (iii) to develop a micelle-based hydrogel formulation that is easy to use for topical administration; (iv) to investigate the transdermal delivery of TAK-441 and measure its intradermal distribution in porcine skin after application of aqueous micellar and micelle-based hydrogel formulations and compare it with the results obtained with a non-micelle control formulation; and (v) to confirm the results using human skin.

[0077] 1.4 Materials and Methods 1.4.1 Material TAK-441 was kindly provided by Takeda Pharmaceutical Co., Ltd. (Japan). D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), formic acid (MS grade), isopentane, and Dulbecco's phosphate-buffered saline (DPBS). Hydroxypropyl methylcellulose (HPMC, approximately 26 kDa; methoxyl content 19–24%, hydroxypropoxyl content 7–12%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Low-molecular-weight HPMC-Methocel™ E5 Premium LV (approximately 10 kDa, methoxyl content 28–30%, hydroxypropoxyl content 7–12%) was procured from Dow Chemicals (Horgen, Switzerland). Hydroxypropyl cellulose (Klucel™ MF Pharm, HPC; MW approximately 850 kDa) and glycerol were purchased from Hanseler AG (Herisau, Switzerland). Bovine serum albumin (BSA) was purchased from Axon Lab (Baden-Dattwil, Switzerland). Acetone (analytical grade) and Nile Red dye were obtained from Acros Organics (Geel, Belgium). Methanol and acetonitrile (LC-MS grade) were purchased from Fisher Scientific (Reinach, Switzerland). PTFE membrane filters (0.22 μm) and Amicon Ultra 0.5 mL (5 kDa) filtration units were purchased from VWR (Nyon, Switzerland). Ultrapure water (Millipore Milli-Q Gard 1 Purification Pack Low Efficiency >18 MΩ cm; Zug, Switzerland) was used for formulation development and analysis. All other chemicals were of at least analytical grade.

[0078] 1.4.2 Analysis method TAK-441 was quantified using a Waters Acquity Core UHPLC® system equipped with a Xevo® TQ-MS tandem quadrupole detector. Isocratic separation was performed using an Acquity UHPLC® BEH C18 column (2.1 × 50 mm, 1.7 μm) coupled to an Acquity UHPLC® C18 VanGuard precolumn (2.1 × 5 mm, 1.7 μm) maintained at 25°C. The mobile phase consisted of a mixture of acetonitrile and water (75:25 v / v). The flow rate was 0.1 mL / min, and the injection volume was 5 μL. The TAK-441 peak was obtained at 1.7 minutes, with a runtime of 3.0 minutes. Mass spectrometric detection was performed by electrospray ionization in positive ion mode using multiple reaction monitoring (MRM). The detection settings for TAK-441 are shown in Table 1. The limit of detection (LOD) was 1.29 ng / mL, and the limit of quantification (LOQ) was 3.29 ng / mL. The UHPLC-MS / MS method was validated according to ICH guidelines.

[0079] [Table 1]

[0080] 1.4.3 Preparation of micelle formulations 1.4.3.1 Micellar solutions TPGS-based micelles of TAK-441 were prepared by the solvent evaporation method (Kandekar et al., 2019). Screening of surfactants and their concentrations was performed using microscale formulation techniques, which involve simultaneous multiple experiments using minimal amounts of drug and excipients. This process not only reduces material costs and the time required for excipient screening and formulation development, but also reduces drug exposure, which is beneficial when addressing cytotoxicity.

[0081] The candidate formulation was then scaled up to a lab-scale batch. Briefly, known amounts of TPGS and TAK-441 were dissolved in 2 mL of acetone to obtain a clear solution. This solution was slowly added to 4 mL of water under sonication (Branson Digital Sonifier S-450D). The acetone was then slowly removed using a rotary evaporator (Buchi RE 121 Rotavapor). The final volume was made up with water in a volumetric flask to obtain micellar formulations with TAK-441 and TPGS concentrations of 3 mg / mL and 10 mg / mL, respectively. After overnight equilibration, the micellar solution was centrifuged (Eppendorf Centrifuge 5804) at 10,000 rpm for 15 minutes to remove excess TAK-441, and the supernatant was carefully collected.

[0082] 1.4.3.2 Micelle gel In a preliminary study, TAK-441-TPGS micelles were incorporated into a 3% HPMC gel to investigate the transdermal delivery of TAK-441 from a semi-solid gel formulation. This formulation was compared with a control gel of identical composition except for the polymer surfactant. Based on these preliminary results, we decided to prepare a micelle-based HPMC gel with superior formulation properties for clinical application (see Section 1.5.1 for details).

[0083] 1.4.4 Characterization of micelle formulations 1.4.4.1 Determining Size The hydrodynamic diameter of the micelle (Z av ), polydispersity index (PDI), volume-weighted diameter and number-weighted diameter (d v and d n ) was measured using dynamic light scattering (DLS) with a Zetasizer HS 3000 (Malvern Instruments Ltd.; Malvern, UK). Measurements were performed at an angle of 90° and a temperature of 25°C. All values ​​were obtained after 10 measurements in triplicate.

[0084] 1.4.4.2 Form The micelle morphology was characterized using a transmission electron microscope (TEM) (FEI Tecnai G2 Sphera, Eindhoven, The Netherlands) using a negative staining method. Briefly, 5 μL of the micelle solution was dropped onto an ionized carbon-coated copper grid (0.3 Torr, 400 V, 20 seconds). The grid was then placed in a 100 μL drop of saturated aqueous uranyl acetate solution for 1 second, followed by another 100 μL drop for 30 seconds. Excess staining solution was removed and the grid was allowed to dry at room temperature before measurement.

[0085] 1.4.4.3 Measurement of TAK-441 content in micelles The amount of TAK-441 encapsulated in the micelles was quantified by UHPLC-MS / MS. To confirm complete disruption of the micelles and release of the incorporated drug, the formulations were diluted with acetonitrile and analyzed. The drug content, drug loading, and encapsulation efficiency were calculated using Equations 1-3:

number

[0086] 1.4.4.4 Viscosity measurement The viscosity of the micelle gels was measured using a Thermo Scientific™ HAAKE™ MARS™ rheometer. Measurements were performed at a constant temperature (25°C) using a rotating plate spindle at different shear rates. Measurements and post-measurement evaluation were performed using Thermo Scientific™ HAAKE™ RheoWin software.

[0087] 1.4.4.5 Evaluation of the stability of micelle formulations TAK-441 micelle aqueous and micelle-based HPMC gel formulations were prepared and stored for 6 months at 4° C. The formulations were evaluated to measure drug content at various time points (day 1, then monthly).

[0088] 1.4.5 In vitro skin delivery and biodistribution studies 1.4.5.1 Skin preparation Pig ears were purchased from a local slaughterhouse (CARRE; Rolle, Switzerland) immediately after slaughter. After washing with cold running water, skin samples approximately 0.8 mm thick were carefully collected from the external area of ​​the ear using a Zimmer air dermatome (Münsingen, Switzerland). Hair on the skin surface was removed with scissors. Disks corresponding to the permeable area were punched out (Berg & Schmid HK 500; Urdorf, Switzerland). Skin samples were frozen at -20°C and stored for up to 3 months. Before the experiment, skin samples were thawed at room temperature and rehydrated by placing them in 0.9% saline for 15 minutes.

[0089] Human skin samples were obtained immediately after surgery from the Department of Plastic, Cosmetic and Reconstructive Surgery at Geneva University Hospital (Geneva, Switzerland), adipose tissue was removed, and the skin was stored at −20°C. The donation was approved by the Central Committee for Research Ethics (CER:08-150 (NAC08-051); Geneva University Hospital).

[0090] 1.4.5.2 Micelle solution The experiment was conducted on a 2cm cross-sectional area 2 The measurements were performed using a standard two-compartment vertical (Franz-type) diffusion cell (Milian SA; Milian, Switzerland). The receptor compartment consisted of 10 mL of Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 containing 1% BSA to maintain sink conditions. The receiver compartment was maintained at 32°C–34°C. For the infinite dose condition, 200 μL of the TAK-441 micelle formulation (3 mg / mL) was applied to the surface of the skin sample (i.e., 300 μg of TAK-441 / cm of skin surface). 2 ), and for the limited dose, 20 μL of the micelle formulation (3 mg / mL) was applied (TAK-441 30 μg / skin surface cm). 2 As a control, a non-micelle formulation was used in which 3 mg / mL of TAK-441 was suspended in a 0.05% aqueous hydroxypropyl cellulose (HPC) solution.

[0091] TAK-441 had the lowest solubility in HPC. This characteristic minimized the risk of the suspension affecting drug delivery. Aliquots (1 mL) were withdrawn from the receiver compartment after 1, 4, and 12 hours and replaced with an equal volume of fresh medium. The samples were diluted with acetonitrile to precipitate BSA. After centrifugation at 10,000 rpm for 15 minutes, the permeated samples were analyzed by UHPLC-MS / MS.

[0092] After the experiment, excess formulation was removed from the skin surface using a validated washing method. The skin samples were cut into small pieces and immersed in 2 mL of methanol for 4 hours with stirring at room temperature to extract the deposited TAK-441. The extraction procedure was validated. The extracted samples were centrifuged at 10,000 rpm for 15 minutes, diluted, and filtered through a 0.22 μm PTFE filter before UHPLC-MS / MS analysis.

[0093] 1.4.5.3 Micelle gel TAK-441 micelles were incorporated into a 3% HPC gel to test the dermal delivery of TAK-441 from a semi-solid gel formulation to porcine skin. The composition of the control gel was the same except for the polymeric surfactant. The experiment was performed as described above (Section 1.4.5.2). For the infinite dose, 200 mg of micellar gel (2.88 mg TAK-441 / g of gel formulation; i.e., the gel contains 0.29% TAK-441) was applied to the skin surface (i.e., 288 μg of TAK-441 / cm of skin surface). 2 ), and for the limited dose, 20 mg of micellar gel was applied (TAK-441 28.8 μg / skin surface cm 2 ).

[0094] Similar experimental conditions were used for the micelle-based HPMC gel, which was used to test its delivery to human skin. At the infinite dose, 200 mg of micelle gel (2.5 mg TAK-441 / g gel formulation; i.e., the gel contains 0.25% TAK-441) was applied to the skin surface (i.e., 250 μg TAK-441 / skin surface cm 2), and for the limited dose, 20 mg of micellar gel was applied (TAK-441 25 μg / skin surface cm 2 The control gel had the same composition as the HPMC gel, except for TPGS.

[0095] After the experiment, a punch was used to cut the skin sample into pieces with a surface area of ​​0.785 cm 2 The inner disk has an area of ​​1.215 cm 2 The outer ring was then cut into small pieces, and the TAK-441 deposited in the tissue was extracted using a validated extraction method (Section 1.4.5.2) and quantified by UHPLC-MS / MS.

[0096] 0.785cm 2 The biodistribution of TAK-441 was measured as a function of skin depth using skin discs. These skin discs were flash-frozen in liquid nitrogen-cooled isopentane. For this procedure, skin samples were mounted with OCT on circular cork pieces, and a plastic O-ring was placed around the skin disc to avoid tissue compression and ensure a flat frozen sample. This process ensured the integrity of the thickness of various regions of the skin. The skin discs were then cryosectioned (Thermo Scientific™ CryoStar™ NX70; Reinach, Switzerland) to obtain 50-μm-thick sections from the stratum corneum to a depth of 400 μm. These lamellae, encompassing the stratum corneum, epidermis, and upper dermis, respectively, allowed the amount of TAK-441 to be determined as a function of skin location. Each lamella and the remaining dermis were individually extracted with 250 μL of methanol for 4 hours, and TAK-441 was quantified by UHPLC-MS / MS.

[0097] 1.4.6 Statistical analysis Data are expressed as mean ± SD. Outliers, determined using the Grubbs test, were discarded. Results were statistically evaluated using one-way analysis of variance (ANOVA) followed by Tukey's test or Student's t-test for multiple comparisons. The significance level was fixed at α = 0.05.

[0098] 1.5. Results and Discussion 1.5.1 Development and characterization of micelle formulations Formulations (AH) were prepared with a constant TPGS content (10 mg / mL) but different target TAK-441 loadings (100, 150, 200, 250, 300, 350, 400, and 500 mg of TAK-441 per gram of TPGS). The drug loading, drug content, and incorporation efficiency obtained for each formulation are shown in Table 2. Formulation E (2.97 ± 0.071 mg / mL) had the highest drug content.

[0099] [Table 2]

[0100] 1.5.1.1 Size Characterization TAK-441-loaded TPGS micelles were characterized and their size measured using DLS (Table 2). All TAK-441-loaded micelle formulations had a hydrodynamic diameter (Z av The volume-weighted diameter (d v ) measured values ​​were 10.55 to 12.29 nm, and the number-weighted diameter (d n ) were 8.74 nm to 10.51 nm. TEM micrographs of the optimized formulation (Formulation E) are shown in Figure 2, which reveal that the micelles were spherical in shape and had diameters of 10 nm to 15 nm. These dimensions were confirmed by DLS (Table 2).

[0101] 1.5.1.2 Development of HPC micelle gel Formulation E was used to obtain a final drug content of 2.88 mg. TAK-441 A 3% HPC gel was prepared with 1000 μg of gel formulation. The viscosity of the gel was measured at a shear rate of 0.01 s. -1 The viscosity was 283.5 Pas (Figure 3). As mentioned above, a 3% HPC gel containing TAK-441 was used as a control. This confirmed that the superiority of the micellar gel in the dermal delivery of TAK-441 was due specifically to the effect of the micelles.

[0102] 1.5.2 Evaluation of TAK-441 delivery in vitro 1.5.2.1 Transdermal delivery of TAK-441 from micellar solution in porcine skin This study was conducted to compare the skin deposition and transdermal permeation of TAK-441 from TPGS micellar solutions and a control formulation. These initial experiments examined the delivery of TAK-441 using porcine skin, one of the best surrogates for human skin (Dick and Scott, 1992; Schmook et al., 2001; Herkenne et al., 2006; Jacobi et al., 2007). The concentration of TAK-441 present in the receiver compartment was below the LOD of the UHPLC-MS / MS method, which indicates that the cumulative permeation amount after 12 hours of application of the formulation was <0.1 pg / cm. 2 As shown in Figure 4A, higher skin deposition was observed in the micellar solution groups (infinite dose and finite dose) compared to the control formulation. The deposition amounts of TAK-441 on pig skin from the micellar solution and the control formulation under infinite dose conditions were 1.44 ± 0.27 μg / cm, respectively. 2 and 0.41 ± 0.09 μg / cm 2 (p=0.015, one-way ANOVA; n=6) and 0.61±0.11 μg / cm at finite doses, respectively. 2 and 0.19 ± 0.052 μg / cm 2 (p = 0.029, one-way ANOVA; n = 6). Under infinite dose conditions, the concentration corresponding to the total amount of TAK-441 deposited in all skin samples after application of the micellar solution was an IC of 4.4 nM. 50 (Ohashi et al., 2012), and over 3,000 times higher under finite dose conditions.

[0103] 1.5.2.2 Cutaneous delivery of TAK-441 from micellar-HPC gel to porcine skin The amount of TAK-441 deposited on pig skin from micellar HPC gel and control HPC gel was 0.74±0.19μg / cm under infinite dose conditions. 2 and 0.12±0.05 μg / cm 2(p=0.002, one-way ANOVA; n=6) and 0.32±0.08 μg / cm at the finite dose, respectively. 2 and 0.03±0.01μg / cm 2 (p=0.002, one-way ANOVA; n=6). In pig skin, the TAK-441 concentration in skin samples 12 hours after delivery was higher in the micelle-treated groups (solution and gel) compared to the control formulation.

[0104] Biodistribution studies allowed us to determine the amount of TAK-441 deposited as a function of depth. Biodistribution at infinite and finite doses revealed that a higher amount of TAK-441 was present primarily in the target site, i.e., the epidermal region (Figure 4B and Figure 4C). Considering the amount of TAK-441 present in these small skin volumes, the estimated concentrations were higher than those estimated for the entire skin sample. Therefore, the TAK-441 concentrations achieved after application of the micelle-HPC gel at infinite and finite doses in the first lamellae from 0 μm to 50 μm were approximately equal to the IC 50 in the 50-100 μm region, the corresponding values ​​were >6800-fold and >3400-fold, respectively.

[0105] 1.5.2.3 Cutaneous Delivery of TAK-441 Using Micelle-HPMC Gels in Human Skin 1.5.2.3.1 Development of micelle-HPMC gels After obtaining promising results with pig skin, we decided to develop a micellar gel formulation of TAK-441 and test its delivery in human skin. However, because HPMC precipitates at relatively low temperatures (cloud point: approximately 39°C), this characteristic may cause stability issues (Greiderer et al., 2011). Therefore, we developed an HPMC-based micellar gel formulation (Table 3). The drug content was 2.5 mg. TAK-441 The viscosity of the gel was measured at a shear rate of 0.01 s -1at 646.9 Pas (Fig. 5), exhibiting shear thinning behavior and the advantage of good extensibility for ease of application (Brummer and Godersky, 1999; Kwak et al., 2015).

[0106] [Table 3]

[0107] 1.5.2.3.2 Determination of Skin Delivery and Biodistribution of TAK-441 from Micelle-HPMC Gels in Human Skin As seen in the pig skin study, the amount of TAK-441 that permeated human skin was also below the LOD of the UHPLC-MS / MS method after 12 hours of application. Higher TAK-441 deposition into the skin was observed with the micelle-HPMC gel formulation (Figure 7A). The deposition amounts under infinite dose conditions for the micelle-HPMC gel formulation and the control HPMC gel formulation were 1.17 ± 0.21 μg / cm, respectively. 2 and 0.22±0.07μg / cm 2 (p=0.002, one-way ANOVA; n=6) and under finite dose conditions, 0.40±0.11 μg / cm 2 and 0.05±0.02 μg / cm 2 (p=0.002, one-way analysis of variance; n=6).

[0108] Biodistribution at infinite and finite doses was similar to that observed in pig skin. Larger amounts of TAK-441 were again present in the epidermal region (Figures 7B and 7C). For example, the estimated TAK-441 concentrations achieved in the 0-50 μm region based on the amount of TAK-441 delivered by the micelle-HPMC gel at infinite and finite doses were IC , respectively. 50の over 22,000-fold and over 7,800-fold; whereas in the 50-100 μm region, the corresponding values ​​were IC 50 The results were over 9,400 and 3,100 times higher than those of the control group.

[0109] The micelle-HPMC gel outperformed the HPMC control gel in all skin delivery experiments. The distribution of TAK-441 in the HPMC gel was likely more uniform than in the HPMC control gel. TPGS micelles, containing TAK-441 in their lipophilic interior, form drug depots on the skin surface, promoting TAK-441 accumulation, particularly in intercluster regions and hair follicles (Kandekar et al., 2018; Lapteva et al., 2015, 2014b). TPGS micelles likely disintegrate upon contact with the lipophilic stratum corneum, thereby releasing the solubilized TAK-441 and making it available as a molecular dispersion.

[0110] Because TAK-441 is poorly water-soluble, such applications would result in local (super)saturation of TAK-441. High thermodynamic activity favors the formulation's partition from the aqueous environment into the skin (Hadgraft, 1999; Moser et al., 2001; Schwarb et al., 1999). As the concentration of TAK-441 present in the stratum corneum increases, the concentration gradient across the transport-limiting membrane increases, thus increasing flux. This increased flux is manifested at a macroscopic level by the greater amount of TAK-441 measured at each skin depth in the intradermal distribution profile.

[0111] Another important factor is water evaporation from the formulation at the skin surface. This factor becomes more important under finite-dose conditions and contributes to the formation of a supersaturated solution of TAK-441 (Cilurzo et al., 2015). Because nanocarriers have been shown to accumulate in and around hair follicles, the hair follicle route may play an enhanced role in the skin penetration of drugs applied using such delivery systems (Kandekar et al., 2018; Lapteva et al., 2015; Papakostas et al., 2011). Surfactants in micellar formulations may also act as penetration enhancers; indeed, we have used MS imaging to demonstrate that TPGS penetrates the epidermis (Quartier et al., 2021a, 2021b).

[0112] 1.6 Conclusion The results showed that TAK-441 can be incorporated into TPGS micelles, and that the micelle-HPMC gel formulation of TAK-441 significantly increased the IC of HH pathway inhibition. 50 The epidermal concentration achieved was several orders of magnitude higher than that achieved by conventional methods, confirming the feasibility of delivery to the skin. Considering that the occurrence of side effects is one of the factors that generally limits the use of HH inhibitors and restricts the clinical development of TAK-441, we considered the LOD (<1.29 ng / mL) of the highly sensitive UHPLC-MS / MS method, and the cumulative permeation amount (<0.1 pg / cm) was low. 2 Importantly, the TAK-441 penetration rate was significantly lower than that observed in healthy tissue (equivalent to 1000 mg / kg body weight). Thus, minimal skin penetration of TAK-441 may contribute to a reduced incidence of systemic side effects. It is clear that dermal delivery of TAK-441 from micelles to diseased skin may differ from that observed in healthy tissue and may depend on the type of lesion, which will require further in vivo investigation.

[0113] 1.7 Abbreviations AE side effects BCC Basal cell carcinoma BSA Bovine serum albumin cAMP cyclic adenosine monophosphate DPBS Dulbecco's Phosphate Buffered Saline GLI glioma-associated oncogene homolog GPR161 G protein-coupled receptor 161 HH Hedgehog HPC Hydroxypropyl Cellulose HPMC Hydroxypropyl methylcellulose KIF7 Kinesin family member 7 protein LOD (Limit of Detection) LOQ Limit of Quantification MFD maximum dose NR Nile Red p53 tumor suppressor protein p53 PC primary cilium PDI polydispersity index PKA Protein Kinase A PTCH1 Patched1 protein SMO Smoothened Receptor SUFU fusion homolog suppressor TEM Transmission Electron Microscope TPGS D-α-Tocopherol Polyethylene Glycol 1000 Succinate UHPLC Ultra High Performance Liquid Chromatography UV rays UV rays

[0114] References All publications, patent applications, patents, and other references mentioned in this specification are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although several patent applications, patents, and other references are referenced herein, it will be understood that such reference does not constitute an admission that any of these documents form part of the general knowledge in the art.

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[0116] Although the foregoing subject matter has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A composition comprising a hedgehog pathway inhibitor and a polymeric surfactant.

2. 2. The composition of claim 1, wherein the hedgehog pathway inhibitor is active against the vismodegib-resistant Smoothened receptor D473H mutant.

3. 3. The composition of claim 1 or claim 2, wherein the hedgehog pathway inhibitor is selected from TAK-441, vismodegib, salidegib / patidegib, glasdegib, sonidegib, taladegib (Env-101), and BMS-833923 (XL-139).

4. The composition according to any one of claims 1 to 3, wherein the polymeric surfactant is biocompatible and / or biodegradable. and claims:

5. The composition according to any one of claims 1 to 4, wherein the polymer surfactant is selected from D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), mPEG-dihex-PLA, poloxamer, poly(e-caprolactone), poly(L-amino acid), and polyvalerolactone.

6. The composition of any one of claims 1 to 5, wherein the composition comprising a hedgehog pathway inhibitor and a polymeric surfactant comprises a micellar composition.

7. The composition of claim 6, wherein the micellar composition comprises spherical micelles having diameters ranging from about 10 nm to about 100 nm.

8. The composition of any one of claims 1 to 7, comprising a TPGS concentration ranging from about 5 mg / mL to about 300 mg / mL.

9. 9. The composition of any one of claims 1 to 8, comprising a composition of TAK-441 having a range of about 100 mg to about 500 mg of TAK-441 per gram of TPGS.

10. 10. The composition of claim 9, wherein the concentration of TAK-441 ranges from about 100 mg to about 300 mg per gram of TPGS.

11. The composition of any one of claims 1 to 10, further comprising a hydrogel.

12. 12. The composition of claim 11, wherein the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), a cellulose-based gel former, and a poloxamer-based gelling agent.

13. The HPMC comprising: (a) HPMC having a molecular weight of about 26 kDa, a methoxyl content ranging from about 19% to about 24%, and a hydroxypropoxyl content ranging from about 7% to about 12%; (b) HPMC having a molecular weight of about 10 kDa, a methoxyl content ranging from about 28% to about 30%, and a hydroxypropoxyl content of about 7% to about 12%; and (c) Combinations thereof The composition of claim 12, wherein the composition is selected from:

14. about 0.25% (w / w) TAK-441; about 0.93% (w / w) TPGS; about 5% (w / w) HPMC having a molecular weight of about 26 kDa, a methoxyl content ranging from about 19% to about 24%, and a hydroxypropoxyl content ranging from about 7% to about 12%; 14. The composition of any one of claims 11 to 13, comprising about 3% HPMC having a molecular weight of about 10 kDa, a methoxyl content ranging from about 28% to about 30%, and a hydroxypropoxyl content of about 7% to about 12%.

15. The composition of any one of claims 11 to 14, further comprising a rheology modifier.

16. 16. The composition of claim 15, wherein the rheology modifier is selected from glycerol, low and high molecular weight celluloses and sorbitol.

17. The composition of any one of claims 11 to 16, further comprising a preservative.

18. 18. The composition of claim 17, wherein the preservative is selected from sodium disulfite, benzyl alcohol, benzalkonium chloride, chlorobutanol, sodium benzoate, potassium sorbate, methylparaben, and propylparaben.

19. 19. The composition of any one of claims 1 to 18, which retains about 90% to 100% of the content of the hedgehog pathway inhibitor after storage for about 6 months.

20. 20. A method for treating a disease, disorder or condition associated with the hedgehog (HH) signaling pathway, comprising administering to a subject in need thereof a composition of any one of claims 1 to 19.

21. 21. The method of claim 20, wherein the composition is administered topically.

22. 22. The method of claim 21, wherein the composition is delivered to the skin.

23. 22. The method of claim 21, wherein the composition is delivered to the viable epidermis of the subject.

24. 22. The method of claim 21, wherein the composition is delivered to the upper dermis of the subject.

25. 22. The method of claim 21, wherein topical administration results in minimal transdermal penetration.

26. 21. The method of claim 20, wherein the disease, disorder or condition associated with the Hedgehog (HH) signaling pathway comprises a skin disease, disorder or condition.

27. 27. The method of claim 26, wherein the skin disease, disorder or condition comprises skin cancer.

28. 28. The method of claim 27, wherein the skin cancer comprises basal cell carcinoma.

29. 29. The method of claim 28, wherein the subject has or is suspected of having locally advanced basal cell carcinoma.

30. 30. The method of claim 29, wherein the subject has or is suspected of having metastatic basal cell carcinoma.

31. 31. The method of claim 29 or claim 30, wherein the subject is not amenable to surgery or radiation therapy.

32. 32. The method of any one of claims 28-31, wherein administering the composition to the subject attenuates the progression of the basal cell carcinoma.

33. 29. The method of claim 28, wherein the basal cell carcinoma is associated with the vismodegib-resistant SMO mutant D473H.

34. 21. The method of claim 20, wherein administering the composition to the subject prevents activation of the HH signaling pathway.

35. 21. The method of claim 20, wherein the disease, condition or disorder is associated with a mutation in the Patched1 (PTCH1) protein.

36. 36. The method of claim 35, wherein the mutation in the Patched1 (PTCH1) protein results in a loss of function of the PTCH1 protein.

37. 21. The method of claim 20, wherein the disease, condition or disorder is associated with a mutation in the Smoothened (SMO) protein.

38. 38. The method of claim 37, wherein the mutation in the Smoothened (SMO) protein is accompanied by a gain of function of the SMO protein.

39. 39. The method of claim 36 or claim 38, wherein the loss of function of the PTCH1 protein or the gain of function of the SMO protein results in the activation of one or more GLI transcription factors.

40. 40. The method of claim 39, wherein activation of the one or more GLI transcription factors results in hyperproliferation of basal cells associated with basal cell carcinoma.

41. 21. The method of claim 20, wherein administering the composition to the subject inhibits SMO.

42. 38. The method of claim 37, wherein the mutation in SMO confers resistance to treatment with the hedgehog pathway inhibitor.

43. 43. The method of claim 42, wherein the resistance to treatment with the hedgehog pathway inhibitor is associated with a mutation in the drug binding site of SMO.

44. The method of claim 43, wherein the drug binding site of SMO is SMO-Q476 and / or SMO-D473.

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